An epigenetic repressor TRIM66 dictates monogenic olfactory receptor expression, neural activity, and olfactory behavior

Domain: biology
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
Olfactory receptor gene choice is an intricate example of monogenic and monoallelic expression, where one out of over 1000 receptor genes is transcribed in each olfactory sensory neuron. This process involves expression of multiple olfactory receptor genes in immature neurons, followed by silencing of all but one receptor genes during maturation. However, the molecular identity of the repressors remains mysterious. Here, we discover TRIM66 as a key repressor. Multiple receptor genes are retained at low levels in most single mature OSNs after deletion of Trim66, leading to decreased expression of the vast majority of olfactory receptor genes. Mechanistically, TRIM66 can bind to, assembly, and repress olfactory receptor enhancers, thereby silencing extra olfactory receptor genes. Functionally, deletion of Trim66 lead to severe defects in the olfactory information processing and innate olfactory behaviors. Our study provides the missing link in understanding the transition from polygenic to monogenic olfactory receptor expression.

Introduction
To perceive the vast array of external and internal stimuli, higher organisms have evolved diverse receptors through gene expansion or gene regulation. For instance, the immune system generates an almost unlimited number of antigen receptors to detect various pathogens by a combination of multiple copies of the V, D, and J segments1. Similarly, alternative splicing from multiple exons of the clustered protocadherin (Pcdh) genes produces a highly diverse Pcdh receptor barcode in the nervous system2. Yet, the olfactory system utilizes hundreds to thousands of olfactory receptors by gene duplications and mutations during evolution to detect an infinity of odorants3–5. In addition to having the extraordinary receptor diversity, most systems achieve precision by expressing very few or even one receptor type in single cells. For example, lymphocytes deploy VDJ recombination to maintain the monoallelic and monogenic antigen receptor expression1. The stochastic selection and stabilization of Pcdh isoforms through epigenetic regulation in a neuron is crucial for self-recognition and dendritic self-avoidance2. Among these, the olfactory system faces the greatest challenge that each olfactory sensory neuron (OSN) randomly chooses one, and only one, gene from a large family (>1000 genes in mouse) of olfactory receptors for expression, also known as the “one-neuron-one-receptor” rule6.

Recent advances in understanding the mechanisms regulating the “one-neuron-one-receptor” rule have revealed a complex epigenetic regulatory system. The olfactory receptors are mainly comprised of two families of G protein-coupled receptors—odorant receptors (ORs) and trace amine-associated receptors (TAARs)6,7. In mouse, the majority of functional OR genes are clustered in all chromosomes except chromosomes 12 and 18, and Y, with some OR genes existing as singletons. The Taar genes are all located in chromosome 10, forming a single Taar gene cluster. Both OR and Taar genes are gradually decorated by constitutive heterochromatin marks—H3K9me3 and H4K20me3 in the process of OSN differentiation from olfactory stem cells8,9. Moreover, the marked OR and Taar genes aggregate to form the repressive heterochromatin environment that prevents expression of any receptor genes10,11. Olfactory receptor gene expression is initiated upon removal of its heterochromatin marks catalyzed by LSD1 (lysine-specific demethylase 1, also known as KDM1A), a histone demethylase transiently expressed in immature OSNs12. The expression of the olfactory receptor gene is further facilitated by over 60 olfactory receptor-specific enhancers9,13–18. The transcribed and translated olfactory receptor elicits a feedback signal to downregulate LSD1, which prevents demethylation and desilencing of additional receptor genes, thereby stabilizing the chosen receptor gene12,19. It is believed that the sophisticated regulation of LSD1 and the rare occurrence of the transcriptionally active olfactory enhancer hub with a receptor gene together contribute to the singular receptor expression14,20. Theoretical modeling results suggest that slow activation (5–10 days) of an olfactory receptor gene by LSD1, combined with the rapid feedback to turn off LSD1 (within 1 h), is required to achieve monogenic receptor expression21. However, this process may not be entirely efficient, as single-cell transcriptomic studies have shown that OSNs transiently co-express multiple olfactory receptor genes during the immature stage and then eliminate all but one as they mature22,23. This observation is further supported by the finding that multiple olfactory enhancer hubs coexist in single OSNs, providing the transcriptionally active environment conducive to the expression of multiple receptor genes18,24. At the same time, it raises another critical question: how does one receptor prevail, and how are the extra ORs silenced during OSN development? Researchers have proposed a “symmetry breaking” model where the most highly transcribed receptor mRNA competes for transcriptional resources with other relatively lowly expressed receptor mRNAs25. This positive feedback eventually leads to an asymmetric transcription pattern, with boosted expression of one receptor gene and repressed expression of extra receptor genes, thereby ensuring the singular olfactory receptor gene choice in mature OSNs. However, it is still unclear what specific transcriptional regulators are involved in the process of eliminating all but one olfactory receptor gene.

Expression of olfactory receptor is known to directly activate a highly transcribed transcription factor (TF) ATF526 through unfolded protein response (UPR)19,27, which upregulates Adcy3 and consequently depletes Lsd18,21. Here, we propose that ATF5 may promote the expression of transcriptional repressors to silence extra olfactory receptor genes, which has not been identified for decades. We characterized the direct targets of ATF5 via ChIP-seq experiments and identified epigenetic repressor—TRIM66. Knockout of the Trim66 gene, either globally or conditionally in the olfactory system, results in widespread downregulation of olfactory receptor genes. Single-cell sequencing data reveal that multiple olfactory receptor genes are co-expressed in single mature OSNs after the loss of Trim66, seemingly violating the “one-neuron-one-receptor” rule. Moreover, these olfactory receptor genes are co-expressed at low levels, which could give rise to extensive receptor gene downregulation. We further show that TRIM66 can bind to, assembly, and repress subset of OR enhancers, eventually inhibiting the expression of low-expressed olfactory receptor genes. We also provide evidence that deletion of Trim66 causes compromised olfactory information relay in the olfactory bulb and related olfactory behaviors, suggesting that singular olfactory receptor gene expression is important for accurate olfactory processing. Thus, our results identify the critical epigenetic repressor directly linking olfactory receptor expression to silencing, which mediates the transition of polygenic to monogenic receptor expression during OSN maturation.

Results
Identifying Trim66 as a candidate regulating olfactory receptor genes
Since ATF5 is the first TF responding to OR transcription and translation, we hypothesized that the downstream targets of ATF5 may include the potential repressors silencing extra OR genes. We performed ATF5 ChIP-seq experiments and analyzed the genome-wide DNA binding pattern of ATF5 in the mouse main olfactory epithelium (MOE) (Fig. 1a). We identified a total of 1049 ATF5-binding peaks (Fig. 1b and Supplementary Data 1a). This binding pattern of ATF5 is similar to published ChIP-seq data of its paralog, ATF4, in mouse fibroblasts28. ATF4 is the canonical TF in the PERK arm of UPR29; in the MOE, ATF5 was suggested to take over because of its much higher expression level19,27. Supporting this idea, 477 out of 1049 (45.5%) ATF5 peaks, and an even larger fraction (67%) of the top 100 peaks, overlapped with the published ATF4 peaks (Fig. 1b). Furthermore, the top DNA motif in ATF5 peaks is nearly identical to the one for ATF428 (Supplementary Fig. 1a). The potential compensatory effect of ATF4 may explain the observations that Atf5-knockout pups still developed some mature neurons19,27.

Gene-ontology (GO) analysis of ATF5 target genes revealed an enrichment of UPR-related genes and neuronal genes (Supplementary Fig. 1b and Supplementary Data 1b). These target genes include Atf5 itself (the second strongest peak, by q-values, Supplementary Fig. 1c), transmembrane ER proteins Tmbim6 (the strongest peak) and Herpud1, and eIF2α phosphatases Ppp1r15a/b, all of which also show ATF4-binding peaks28. Interestingly, both GO Biological Processes and GO Cellular Components are enriched in neuronal, and in particular axonal genes (Supplementary Fig. 1b), including Gap43 (Supplementary Fig. 1c), Tubb3, and Nrp1/2. In addition, ATF5 binds to genes encoding other important signaling molecules in the olfactory system, such as Gnal, Gnb1 (Supplementary Fig. 1c), Gnai2, Ano2, and Stom. On the other hand, we found ATF5 binding at the promoter of Rtp1—a chaperone crucial for transporting ORs from the endoplasmic reticulum (ER) to the cell surface30, which is consistent with in vitro results27. We observed two additional, stronger peaks 4 kb and 11 kb upstream of Rtp1—likely representing enhancers (Supplementary Fig. 1c), as well as a peak near the promoter of Rtp2—a nearby Rtp1 paralog. Therefore, the previously observed Atf5-dependency of Rtp1/2 expression19, which ensures the relief of UPR after the OR choice, can be explained by ATF5 directly activating Rtp1/2 transcription.

Next, to understand ATF5’s role in gene regulation, we inspected target genes encoding nuclear proteins. Similar to ATF4, ATF5 binds to UPR-related target genes such as TFs Cebpg and Ddit331,32. ATF5 also binds to genes encoding known TFs that are important for MOE development, such as Nhlh133 and Zbtb7b34. Interestingly, ATF5 targets include several epigenetic remodelers, such as Hdac9, Kdm6b, and Kdm5b. Other targets include TFs Bcl11a and Foxp1, and RNA-binding protein Rbm39. We chose a candidate from those ATF5 target genes encoding nuclear proteins, Trim66 (also known as Tif1d), after taking into account published fold changes of mRNA levels in Atf5 knockout mice27, which measure functional impacts of ATF5 on target gene expression (Fig. 1c). In particular, Trim66 belongs to a gene family that interacts with HP1 proteins and H3K9me3, and forms foci in and near chromocenters35, consistent with the idea that H3K9me3 silences OR genes in the MOE8. Trim66 showed reduced expression level by 38% after deletion of Atf5. Note that fold changes can be confounded by potential rescue by ATF4 and by the detection limit and nonlinearity of microarray data. Trim66 gene also contained multiple strong ATF5 peaks (Fig. 1d).

We next examined the expression pattern of the Trim66 gene. Based on bulk RNA-seq results of different cell populations in the MOE enriched by fluorescence-activated cell sorting (FACS), we found that Atf5 mRNA was high in early progenitors and immature/mature OSNs as previously reported19. Trim66 was highly expressed in late progenitors and immature/mature OSNs (Fig. 1e), consistent with the notion that they are downstream targets of ATF5. To verify the gene expression pattern, we performed in situ hybridization experiments on E16.5 mouse embryos (Fig. 1f) and the MOE from postnatal day 14 (P14) in the MOE of E16.5 embryos (Fig. 1f). Furthermore, consistent with the early, transient timing of Atf5 translation19, Trim66 was expressed early in the neuronal lineage and more enriched in progenitors or immature OSNs8 (Fig. 1g and Supplementary Fig. 1e). Immunostaining using antibody against TRIM66 also validated similar expression pattern of proteins with mRNAs (Fig. 1h).

Trim66 specifically regulates olfactory receptor gene expression
To test the potential role of Trim66 in olfactory receptor expression, we generated knockout mouse lines and examined the changes in receptor expression after deletion of Trim66. For Trim66, we first generated global knockout mice in which exons 2–10 were deleted by CRISPR-Cas9 genome editing (Fig. 2a,b). Exons 2–10 are shared by Trim66A_long and Trim66B_short as well as other known Trim66 isoforms (Fig. 1d). We designed specific probe targeting exons 4–9 of Trim66 and verified the absence of Trim66 mRNA signals in the homozygous Trim66 knockout mice (Fig. 2c). We also performed immunohistochemistry experiments to confirm the loss of TRIM66 proteins in the homozygous Trim66 knockout mice (Fig. 2d). As reported before, the birth rate of Trim66 knockout pups is in accordance with Mendel’s law after heterozygous crossing, while the body weight of Trim66−/− mice is significantly decreased compared with control littermates at P14–P2137.

Next, we analyzed the transcriptomic profiles of the MOEs dissected from control and Trim66 knockout mice by RNA-seq. Quantification analyses again validated loss of reads in exons 2–10 of Trim66 in Trim66 −/− mice (Supplementary Fig. 2a). Further, we found that exon 1 reconnected to exon 11 to generate truncated Trim66 isoforms in Trim66 −/− mice (Supplementary Fig. 2a). We then compared the global gene expression in Trim66 −/− mice and control mice. Using the criteria of q < 0.05 (q value is false discovery rate corrected p value) and fold change > 1.5-fold, we detected 1389 differentially expressed genes (DEGs) (Supplementary Fig. 2b). We firstly checked and verified expression changes of several markers for different cell types. The mRNA abundance of most mature OSN markers was decreased, whereas that of basal stem cell and immature OSN markers was increased (Supplementary Fig. 3a–d). To further evaluate the impact of Trim66 deletion on OSNs turnover, we performed immunohistochemistry for phospho-histone H3 (PH3) and Ki67, the two established markers of proliferating cells. Quantification of PH3 and Ki67 positive cells per unit length of the MOE revealed a significant increase of proliferating cell numbers in Trim66 −/− mice compared to those in controls (Supplementary Fig. 3e,f). In addition, measurement of MOE thickness showed a significant reduction in Trim66 −/− mice (Supplementary Fig. 3g), consistent with elevated epithelial turnover.

Surprisingly, olfactory receptor genes were profoundly misregulated, with 1064 out of 1389 (77%) DEGs were olfactory receptor genes (Fig. 2e and Supplementary Fig. 2b). For functional OR genes, the mRNA abundance of 962 and 54 out of 1137 (85% and 5%) was significantly decreased and increased in Trim66 −/− mice, respectively. For functional Taar genes, the mRNA abundance of 8 and 3 out of 14 (57% and 21%) was significantly decreased and increased in Trim66 −/− mice, respectively (Fig. 2e and Supplementary Figs. 2b, 4a, b). Therefore, the majority of olfactory receptor genes showed decreased mRNA abundance, leading to the fact that a few olfactory receptor genes showed high mRNA abundance. For instance, five OR genes, Olfr1507, Olfr464, Olfr446, Olfr51, and Olfr957, occupied 33% of OR gene profiles in Trim66 −/− mice in contrast to 3% in control mice. Taar2 and Taar6 occupied 84% of Taar gene profiles in Trim66 −/− mice, in contrast to 25% in control mice (Fig. 2f).

We then selected 15 representative receptor genes whose mRNA abundance was significantly decreased or increased, including four class I ORs (Olfr571, Olfr686, Olfr578, and Olfr549), 7 class II ORs (Olfr464, Olfr981, Olfr1015, Olfr959, Olfr3, Olfr1463, and Olfr1507), and four Taars (Taar6, Taar7b, Taar7d, and Taar9), to conduct in situ hybridization experiments. We quantified cells with positive mRNA expression signals and observed an increase in the number of OSNs expressing Olfr571, Olfr464, Olfr1507, and Taar6, as well as a decrease in OSNs expressing Olfr686, Olfr578, and Olfr549, Olfr981, Olfr1015, Olfr959, Olfr3, Olfr1463, Taar7b, Taar7d, and Taar9, which was in accordance with the RNA-seq results (Fig. 2g, h and Supplementary Fig. 4c, d). To further validate these findings, we performed immunohistochemistry analysis on the MOE using specific antibodies against one class I OR (Olfr552), one class II OR (Olfr1507), and two TAAR (TAAR4 and TAAR5) proteins. Again, we observed that the changes in the numbers of OSNs expressing receptor proteins were consistent with the RNA-seq results (Supplementary Fig. 4e, f). We further analyzed the correlation between receptor mRNA abundance in Trim66 knockout bulk RNA-seq data and the number of cells expressing each gene or protein based on in situ hybridization and immunohistochemistry data (Fig. 2h and Supplementary Fig. 4d, f). We observed a strong positive correlation between the changes in total mRNA abundance and the changes of the cell numbers after Trim66 knockout (Supplementary Fig. 4g). This analysis suggests that Trim66 could regulate the expression probability of olfactory receptor genes.

Notably, we observed reduced Adcy3 mRNA abundance in Trim66 −/− mice (Supplementary Fig. 3a). Since the Adcy3 gene is also the target of UPR in OSNs19, we assessed if UPR is involved in the regulation of olfactory receptor genes by Trim66 deletion. The ATF5 protein levels were not significantly changed in Trim66 −/− mice (Supplementary Fig. 4h, i). Our bulk RNA-seq results also revealed no significant changes in the Lsd1 gene in Trim66 −/− mice compared to controls (Supplementary Fig. 4j). Therefore, the UPR pathway may not contribute to the effects on olfactory receptor gene expression in Trim66 knockout mice.

We next examined the cell numbers and projection patterns of OSN subpopulations by crossing Trim66 −/− mice with various genetically modified mice. The total cells and projection intensities in glomeruli of class I OR subpopulations labeled in J-gVenus transgenic mice17 were markedly reduced after loss of Trim66 (Fig. 2i, m). Olfr73 (also known as MOR174-9) is a class II OR that is expressed in the dorsal MOE. OSNs expressing Olfr73 labeled in Olfr73-ires-GFP mice were completely eliminated in the absence of Trim66 (Fig. 2j, n). As a result, the axonal projection of Olfr73-expressing OSNs to the olfactory bulb (OB) disappeared (Fig. 2j). On the other hand, OSNs expressing Olfr1507 (also known as MOR28), a class II OR in the ventral MOE, showed increased cell numbers as well as increased axonal projection intensities in the Olfr1507-ires-tdTomato; Trim66 −/− mice. We also observed an increased number of tdTomato-positive glomeruli in the Trim66 knockout background compared to controls, suggesting axon mistargeting of Olfr1507-expressing OSNs (Fig. 2k, o). This will be discussed in more detail later. For TAAR OSN subpopulations, we utilized the previously generated transgenic mouse lines, TAAR enhancer 1-GFP and TAAR enhancer 2-tdTomato, in which the GFP and tdTomato reporter genes are driven by TAAR enhancer 1 and TAAR enhancer 2, respectively9. We bred those two mouse lines, which label distinct subsets of TAAR OSNs, with the Trim66 knockout mouse line. The numbers of both GFP- and tdTomato-positive OSNs were similar between Trim66 −/− and control mice (Fig. 2p, q). This is consistent with the observation that the sum of the mRNA abundance of the Taar gene family, measured as the total TPM of all Taar genes, was not significantly changed after Trim66 deletion (Supplementary Fig. 4b). However, the Taar gene expression pattern did change to biased Taar2 and Taar6 expression in Trim66 −/− mice (Fig. 2f and Supplementary Fig. 4b). As a result, the axonal projection pattern of TAAR OSNs in the OB of Trim66 −/− mice indeed changed (Fig. 2l). In all, the above findings obtained from the genetically modified mice validated the RNA-seq results (Supplementary Figs. 2b and 5a, b). To examine whether the zonal expression patterns of OR and Taar genes are altered in Trim66 −/− mice, we performed in situ hybridization for Olfr1507, Olfr571, and Taar6 (Supplementary Fig. 5c–e). Olfr1507 is normally expressed in zone 5, and its zonal expression remained largely unchanged in Trim66 −/− mice (Supplementary Fig. 5c). In contrast, Olfr571, which is normally confined to zone 2, exhibited ectopic expression in zone 1 (dorsal MOE) in Trim66 −/− mice (Supplementary Fig. 5d). Likewise, Taar6, which is usually expressed in the ventral MOE, showed expanded expression into more dorsal regions in Trim66 −/− mice (Supplementary Fig. 5e). These results suggest that the loss of Trim66 can lead to disrupted zonal distribution in a subset of OR and Taar genes, potentially contributing to the defects in glomerular targeting.

Since Trim66 is broadly expressed across tissues, we aimed to generate Trim66 conditional knockout (cKO) mice. We constructed a mouse line carrying a floxed allele of Trim66 by flanking the exons 2–10 with loxP sites. This floxed Trim66 line (Trim66 flox) was bred with two Cre-driver mouse lines, Foxg1-cre and Goofy-cre (Fig. 2a, b). Foxg1 is expressed in progenitor cells throughout the olfactory placode and telencephalic neuroepithelium at early embryonic stages38, while Goofy is specifically expressed in immature and mature OSNs from embryonic day (E) 11.5 to adulthood39. We validated the loss of the Trim66 gene and TRIM66 protein in the MOE of Foxg1-cre; Trim66 f/f and Goofy-cre; Trim66 f/f mice (Supplementary Figs. 6a–c and 7a–c). Similar to Trim66 global knockout mice, the two lines of Trim66 cKO mice showed decreased mRNA abundance of mature OSN markers and increased mRNA abundance of markers for basal stem cells and immature OSNs (Supplementary Figs. 6d and 7d). The majority of olfactory receptor genes, including ORs and Taars, also showed significantly decreased mRNA abundance in the two Trim66 cKO lines (Supplementary Figs. 6e, f and 7e, f). Most importantly, we found that the changes of olfactory receptor genes were highly consistent between Trim66 conditional and global knockout mice (Pearson correlation coefficient r = 0.93, p < 2.2 × 10−16 for Foxg1-cre; Trim66 flox vs. Trim66 global knockout, Pearson correlation coefficient r = 0.94, p < 2.2 × 10−16 for Goofy-cre; Trim66 flox vs. Trim66 global knockout, Supplementary Figs. 6g, 7g). Together, those results suggest that Trim66 is functional in immature and mature OSNs to maintain proper olfactory receptor gene expression.

Loss of Trim66 induces polygenic receptor expression in mature OSNs
In order to understand how Trim66 regulates olfactory receptor genes in single cells of different cell types, we performed single-cell RNA-seq analyses on the MOE of Trim66 knockout mice (Fig. 3a). For Trim66 knockout mice, we obtained 16,662 cells from Trim66 −/− mice and 10,103 cells from control mice after discarding low-quality cells. The number of detected genes in each cell were similar between Trim66 −/− and control mice (total = 21,981 vs. 20,769, median = 3366 vs. 3064). We then used uniform manifold approximation and projection (UMAP), a nonlinear dimensionality-reduction technique, on the transcriptomic profiles of the single cells. This approach resolved 11 distinct clusters based on the expression of known cell-type-specific markers, including the main cell types found in the MOE: HBCs, GBCs, INPs, immature OSNs, mature OSNs, GC-D cells, microvillous cells, and sustentacular cells (Fig. 3b and Supplementary Fig. 8a). Further, cells from Trim66 −/− and control mice distributed in all the clusters (Supplementary Fig. 8b). We also found that the percentage of mature OSNs was lower, while the percentages of basal immature cells were higher in Trim66 −/− mice (Supplementary Fig. 8c), consistent with the changes of cell makers observed in RNA-seq results (Supplementary Fig. 3a–d). The decrease in the number of mature OSNs was not due to the enhanced cell death, as the apoptotic cells did not significantly increase in Trim66 −/− mice (Supplementary Fig. 8d). Next, we evaluated the expression of olfactory receptor genes in individual immature and mature OSNs by setting the threshold of unique molecular identifiers (UMIs) (Supplementary Fig. 8e). We calculated the percentages of cells expressing single and multiple receptor genes in immature and mature OSNs. In control mice, the vast majority of immature OSNs expressed multiple olfactory receptors (77% multi-receptor expressing vs. 21% singular receptor expressing), while the majority of mature OSNs expressed a single olfactory receptor (18% multi-receptor expressing vs. 81% singular receptor expressing). This observation aligns with previous reports that OSNs transiently co-express multiple olfactory receptors during immature stage22,23. In Trim66 −/− mice, most immature OSNs still expressed multiple olfactory receptors (83% multi-receptor expressing vs. 13% singular receptor expressing). However, a large fraction of mature OSNs also continued to express multiple olfactory receptors (88% multi-receptor expressing vs. 7% singular receptor expressing), seemingly violating the “one-neuron-one-receptor” rule in mature OSNs after Trim66 deletion (Fig. 3c and Supplementary Fig. 9a). This was not due to differences in sequencing depths, as the UMI summation appeared indistinguishable between control and Trim66 −/− OSNs (Supplementary Figs. 8f, 9b, c). Further, the changes of receptor gene expression were consistent between bulk and single-cell RNA-seq data (Supplementary Fig. 8g). Lastly, the pseudotime analyses showed that the development trajectories from GBC, INP, immature OSN to mature OSN were normal in the absence of Trim66 (Supplementary Fig. 8h). Therefore, loss of Trim66 specifically induces polygenic receptor expression in mature OSNs.

To validate the increased number of mature OSNs with polygenic olfactory receptor expression in Trim66 −/− mice, we selected several receptors showing frequent co-expression patterns in single-cell RNA-seq data (Fig. 3d). We designed specific RNA probes for the receptor genes and performed two-color fluorescent in situ hybridization (FISH) to examine the co-expression of fluorescein-labeled genes (green, Olfr525 or Taar2/6 pool or Olfr3/549/959 pool) and Cy3-labled genes (red, Olfr332/446/874 pool or Olfr332/446/525/874 pool) in single cells. Among these receptor genes, the mRNA abundance of Olfr525, Taar2, and Taar6 increased, while that of Olfr3, Olfr549, and Olfr959 decreased in Trim66 −/− mice based on the bulk RNA-seq results. In all three pairs of FISH experiments, we observed significantly increased numbers of cells co-expressing multiple receptors in the MOE of Trim66 −/− mice compared to control mice (Fig. 3e, f). Moreover, the cells showing polygenic receptor expression were almost exclusively located in the apical MOE, where mature OSNs reside. Additionally, the increased numbers of Olfr1507 glomeruli in the Olfr1507-ires-tdTomato; Trim66 −/− mice compare to control mice could also be due to other co-expressed olfactory receptors in Olfr1507-expressing OSNs (Fig. 2k and Supplementary Fig. 9d).

The decreased mRNA abundance of most olfactory receptor genes could result from the decreased number of mature OSNs (Supplementary Fig. 3a–d and Supplementary Fig. 8c) as well as reduced expression levels in single OSNs. Thus, we next investigated how olfactory receptor gene expression was regulated by analyzing the single-cell data in detail. For cells with singular OR gene expression, the averaged OR gene levels in immature and mature OSNs were lower in Trim66 −/− mice than in control mice (Fig. 3g). For cells with multiple OR gene expression, we ranked the top three OR genes based on the expression levels in each immature and mature OSN. We found that the top one OR gene expression levels were lower, while the top two and three OR gene expression levels were higher in Trim66 −/− mice than in control mice (Fig. 3h). Therefore, knockout of Trim66 led to lower and more even receptor expression levels. This raised the question of why a small subset of OR genes exhibited increased mRNA abundance in Trim66 −/− mice. We found that both the OR genes with increased and decreased mRNA abundance showed decreased expression levels in Trim66 −/− mice (Fig. 3i). However, there were more OSNs containing OR genes with increased mRNA abundance and less OSNs containing OR genes with decreased mRNA abundance in Trim66 −/− mice compared to control mice (Fig. 3j). As a result, the OR genes with decreased mRNA abundance were lowly expressed and were selected in fewer number of cells, while the OR genes with increased mRNA abundance were still lowly expressed but were selected in greater number of cells after the loss of Trim66 (Fig. 3k). We also quantified the signal intensities of in situ hybridization results for Olfr549, Olfr981, Taar6, and Taar7d, and immunohistochemistry results for Olfr552, Olfr1507, TAAR4, and TAAR5 in Trim66 +/− and Trim66 −/− mice (Fig. 2h and Supplementary Fig. 4d, f). Quantification analyses of single-cell signals showed consistent results with our single-cell RNA-seq data. mRNA levels of Olfr549, Olfr981, Taar6, Taar7d, and protein levels of Olfr552, TAAR4, and TAAR5 were significantly reduced, while Olfr1507 protein levels were increased upon Trim66 deletion (Supplementary Fig. 10a–d).

TRIM66 can bind to and repress olfactory enhancers
To explore the mechanisms of olfactory receptor gene regulation by Trim66, we attempted to investigate the specific characteristics of the upregulated and downregulated receptor genes in Trim66 mice. Both upregulated and downregulated receptor genes were situated in nearly all of the olfactory receptor gene clusters across different chromosomes (Supplementary Fig. 11a). The percentages of upregulated and downregulated receptor genes were also comparable among olfactory receptors from different classes or expression zones40 (Supplementary Fig. 11b). The significantly changed receptor genes did not show correlations with the expression levels of receptor genes (Supplementary Fig. 11c). We observed weak positive correlations between changes of receptor genes after knockout of Trim66 and changes of receptor genes from progenitors to mature OSNs (Supplementary Fig. 11d, e). We next wondered if the genomic locations were distinct for upregulated and downregulated receptor genes.

Dozens of olfactory receptor-specific enhancers have been successively identified in the receptor gene clusters9,13–18. So we examined the distances between olfactory receptor genes and the closest enhancers. Interestingly, we found that the significantly upregulated receptor genes were much closer to the olfactory enhancers than downregulated or unchanged receptor genes detected in Trim66 knockout mice (Fig. 4a). This observation suggests that TRIM66 can potentially bind to the olfactory enhancers and repress the enhancer networks.

We then performed ChIP-seq experiments using a specific antibody against TRIM66, and found that TRIM66 indeed bound to a subset of olfactory enhancers. We identified 1514 TRIM66 peaks (Fig. 4b and Supplementary Data 2a). TRIM66 primarily bound to transcription start sites with an enrichment of Pol II, transcription factors, and RNA-binding proteins (Supplementary Data 2b). Interestingly, 54 (3.6%) of TRIM66 peaks was near olfactory receptor genes (Supplementary Data 2a), the top of which ranked 79/1514 (Supplementary Data 2a), in contrast to only 8 (0.8%) of ATF5 peaks (Supplementary Data 1a) with a top ranking of 414/1049. In particular, many of these peaks overlapped with the known olfactory enhancers (Fig. 4b and Supplementary Data 1a, 2a) (top ranks: 78/1514 in TRIM66, in contrast to 414/1049 in ATF5), which were reported to form an inter-chromosomal network that jointly activates olfactory receptor genes14. TRIM66 exhibited a very similar binding pattern across olfactory enhancers, with Sifnos and Rhodes being the highest, which partly correlated with published DNase-seq peak strengths41 (Fig. 4b).

Considering that TRIM66 is known as an epigenetic repressor, we hypothesized that TRIM66 could silence the olfactory enhancer networks. If this hypothesis is correct, knockout of Trim66 should increase the accessibility of the olfactory enhancers. To test this idea, we performed ATAC-seq experiments on the whole MOE of Trim66 knockout mice. Due to the fact that immature OSNs have greater chromatin accessibility in enhancers and there are more immature OSNs in Trim66 −/− mice, we also purified Olfr1507 OSNs from Olfr1507-ires-tdTomato; Trim66 knockout mice to perform ATAC-seq (Supplementary Fig. 12a). Approximately 45% of Olfr1507-expressing OSNs were mature OSNs in both Trim66 +/− and Trim66 −/− mice (Supplementary Fig. 12b, c). We found that the overall accessibility of open chromatins was indistinguishable between Trim66 −/− and control mice (Fig. 4c). However, the olfactory enhancers indeed showed significantly increased accessibility in Trim66 −/− and Olfr1507-ires-tdTomato; Trim66 −/− mice compared to those in the control mice, on average by 105.5 and 221.4%, respectively (Fig. 4d, e). Next, we sought to determine if the increased accessibility of enhancers resulted from enhanced active enhancers. Inactive enhancers are associated with repressive marks such as H3K9me3, H3K79me3, and H3K27me3, while active enhancers are characterized by the presence of H3K4me1 and H3K27ac modifications13,14. We sorted Olfr1507-expressing OSNs from Olfr1507-ires-tdTomato; Trim66 knockout mice and performed Cleavage Under Targets and Tagmentation (CUT&Tag) experiments for H3K27ac, H3K4me1, H3K27me3, and H3K9me3. We found that active H3K27ac and H3K4me1 modifications on olfactory enhancers were generally increased, on average by 212.6 and 105.3%, respectively, including the H enhancer—the closest olfactory enhancer to the Olfr1507 gene cluster, while the repressive H3K27me3 and H3K9me3 modifications were decreased after knockout of Trim66, on average by 25.2 and 17.5%, respectively (Fig. 4f, g and Supplementary Fig. 13a–g). Taken together, the above results support our hypothesis that TRIM66 globally represses the olfactory enhancer networks to ensure transcription of very few olfactory receptor genes.

To determine whether TRIM66 directly regulates heterochromatin marks to repress OR genes, we examined H3K9me3 enrichment on OR gene bodies. H3K9me3 levels were similar in Trim66 −/− mice compared to controls (Supplementary Fig. 13h). Consistently, the mRNA abundance of G9a (Kmt1c) and Glp (Kmt1d), the two methyltransferases responsible for H3K9me3 modification42, were unaffected in Trim66 −/− mice (Supplementary Fig. 13i, j). These findings suggest that TRIM66 may not directly modulate the heterochromatin marks on OR genes to regulate their repression levels.

On the other hand, the appearance of several polycomb-group genes in ATF5’s target list raised another possibility that PRC2 and the histone methylation H3K27me3 may be responsible for receptor silencing—a hypothesis proposed by several previous studies43–46. We ruled it out by ChIP-seq of two PRC2 components, the H3K27me3 methyltransferase EZH2 and the GLI-Kruppel zinc-finger transcription factor YY1. These two proteins did not bind to olfactory enhancers (Fig. 4b) or olfactory receptor genes. Therefore, the silencing of olfactory receptor genes and enhancers is likely not mediated by EZH2 and YY1.

Trim66 knockout causes a reduction in trans enhancer interactions
Next, we asked if TRIM66 regulates the interaction of olfactory enhancers to form enhancer networks or enhancer hubs. We performed in situ Hi-C experiments in the MOE of Trim66 +/− and Trim66 −/− mice (Supplementary Fig. 14a, b). We first examined the global genomic architecture. The A/B compartment strength showed a slight decrease but without significance in Trim66 −/− mice compared to controls (Supplementary Fig. 14c, d). The overall composition of topologically associating domains (TADs) in the MOE were similar between Trim66 +/− and Trim66 −/− mice (Supplementary Fig. 14e, f). Zoomed-in genomic views showed extensive inter-chromosomal interactions (38.3% of total Hi-C contacts) and OSN-specific trans contacts between OR gene clusters (0.19% of all inter-chromosomal contacts) in Trim66 +/− mice, consistent with a previous study20. Interestingly, the inter-chromosomal interactions were significantly reduced to 33.3% of total Hi-C contacts, and the trans contacts between OR gene clusters were significantly reduced to 0.11% of all inter-chromosomal contacts in Trim66 −/− mice (Fig. 5a, b and Supplementary Fig. 14a). Strikingly, we found a much stronger reduction in trans interactions between olfactory enhancers, i.e., 0.65‱ of all inter-chromosomal contacts in Trim66 +/− mice to 0.093‱ of all inter-chromosomal contacts in Trim66 −/− mice (Fig. 5c–f and Supplementary Fig. 14a). Further analyses of all Hi-C contacts in olfactory enhancers revealed that the trans and long-range cis olfactory enhancer interactions were strongly decreased but the short cis interactions remained unchanged (Fig. 5g, h). The observed changes of trans contacts after Trim66 deletion resembled those in Lhx2 and Ldb1 knockout mice20. Taken together, our results suggest that TRIM66 not only exerts the inhibitory effect on enhancer hubs, but also regulates the assembly of enhancer hubs.

Trim66 −/− mice show abnormal olfactory processing and innate olfactory behaviors
To explore whether the disrupted singular olfactory receptor expression in the MOE affects olfactory information processing, we focused on detecting electrophysiological activities in the OB and animal olfactory behaviors in Trim66 knockout mice. First, we examined whether loss of Trim66 alters spontaneous activity in the OB. We recorded neural activity from mitral/tufted cells (M/Ts) in the OB of awake, head-fixed mice (Fig. 6a), analyzing different frequency bands of local field potentials (LFPs), including theta (2–12 Hz), beta (15–35 Hz), low gamma (36–65 Hz), and high gamma (66–95 Hz) (Fig. 6b and Supplementary Fig. 15a, b). LFP oscillations reflect the neural activity of surrounding cell populations, and OB LFPs are closely linked to odor chemistry, olfactory learning, and discrimination. The LFP data revealed that Trim66 knockout significantly reduced spontaneous LFP activity, particularly in the Beta band (Supplementary Fig. 15c, d), highlighting a critical role for Trim66 in maintaining basic oscillatory patterns within the OB neural network. Furthermore, we assessed the effect of Trim66 knockout on odor-evoked LFP responses, focusing on the Beta and High gamma bands, which are crucial for odor perception and discrimination. In Trim66 +/− mice, odor stimulation significantly enhanced Beta responses and decreased High gamma responses (Fig. 6b–d and Supplementary Fig. 15e–h). In Trim66 −/− mice, while a similar trend was observed, the amplitudes of both Beta and High gamma responses were markedly attenuated (Fig. 6b–d and Supplementary Fig. 15e–h). These findings indicate that Trim66 knockout not only reduces specific frequency bands of neural network activity but also potentially disrupts odor information processing in the OB.

To further investigate the impact of Trim66 knockout on M/Ts activity, we recorded single-unit activity from M/Ts in the OB (Fig. 6a and Supplementary Fig. 16a, b). The results showed that Trim66 knockout significantly increased the mean spontaneous firing rate of M/Ts (Supplementary Fig. 16c–e), underscoring the importance of Trim66 in regulating neuronal excitability. Moreover, in vivo recordings of M/Ts responses to different odor stimuli revealed that Trim66 −/− mice exhibited a significantly lower percentage of odor-responsive cells for both excitatory and inhibitory reactions (Supplementary Fig. 16f–h), indicating impaired odor representation ability.

For excitatory neurons, the odor-evoked firing frequency in Trim66 knockout mice was significantly increased, while the inhibitory effect in inhibitory neurons was markedly reduced (Fig. 6e–h). This disruption in the excitation-inhibition balance in the OB may directly contribute to impaired olfactory function.

Thus, we next conducted buried food pellet tests and olfactory learning experiments (Go/No-go task, GNG) to evaluate the impact of Trim66 knockout on olfactory behaviors (Fig. 7a, c). The data showed that Trim66 −/− mice took significantly longer to find buried food pellets compared to Trim66 +/− mice, indicating compromised olfactory detection ability (Fig. 7b). In the GNG task (Fig. 7d), Trim66 −/− mice exhibited a pronounced decline in olfactory recognition ability, with significantly weaker discrimination of both S+ and S− odors compared to Trim66 +/− mice (Fig. 7e and Supplementary Fig. 17a, b). These behavioral outcomes align with the electrophysiological data, suggesting that Trim66 deletion not only affects neural activity but also impairs olfactory function. Since Trim66 is also expressed in the OB, albeit with a much lower expression level than in the MOE (Supplementary Fig. 17c), we performed the behavioral tests in Trim66 cKO mice (Goofy-cre; Trim66 flox). We found that Goofy-cre; Trim66 f/f mice exhibited impairments in odor detection and performance in the GNG task (Supplementary Fig. 17d, e), in analogy to the behavioral defects observed in Trim66 global knockout mice. Taken together, our results strongly suggest that Trim66 is critical for precise olfactory information processing and olfaction-related behaviors by regulating the singular olfactory receptor gene expression.

Methods
Animals
All mouse experiments received approval from the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine and the Institutional Animal Care and Use Committee at the Department of Laboratory Animal Science under animal protocol number A-2022-082. The mice were housed under standard conditions with temperatures of 18–23 °C, humidity levels of 40–60%, and a 12-h light/dark cycle. They had access to rodent chow and water ad libitum. Both male and female mice were utilized in the experiments. Ai6 (Lsl-ZsGreen, #007906) and Ai9 (Lsl-tdTomato, #007909) mouse lines were obtained from Jackson Laboratory. Trim66 global knockout, Trim66 flox/flox, Olfr1507-ires-tdTomato, TAAR enhancer 1-GFP, and TAAR enhancer 2-tdTomato were generated in our laboratory. Other mouse lines were kindly shared by other labs, including Foxg1-Cre (Dr. Zunyi Zhang), Goofy-Cre and J-gVenus (Dr. Junji Hirota), Omp-ires-Cre (Dr. Catherine Dulac), Ascl1-creERT2 (Dr. Zhiyong Liu), Ngn1-creERT2 (Dr. Lisa Goodrich), and Olfr73-ires-GFP (Dr. Sandeep Robert Datta). Both male and female mice were included in this study. All experimental mice sexes and ages are shown in Supplementary Table 1. Strains used and genotyping primers are listed in Supplementary Table 2.

The Trim66 global knockout mouse line was created by CRISPR/Cas9 technology. There are four known Trim66 transcripts in NCBI. We chose to delete exons 2–10 that are shared in the four transcripts and the two novel isoforms (Trim66A_long and Trim66B_short) we identified in this study. The targeted sequences for exons 2–10 of Trim66 were 5’-GCACCAGGAGATTGCCACCC-3’ and 5’-TCCCTAGACCAAGACAGACC-3’. The sgRNAs were transcribed in vitro and were microinjected with Cas9 mRNA into the fertilized eggs of C57BL/6J mice. Fertilized eggs were transplanted to obtain positive F0 mice, which were confirmed by PCR and sequencing. A stable F1 generation mouse line was obtained by mating positive F0 generation mice with C57BL/6J mice. We eventually established Trim66 knockout mouse line with a 15,667 bp deletion (mm10, chr7:109,471,203–109,486,869). Note that we found the insertion of an additional 22 bp sequence: ACTAACAGAAGAACCCGTTGTG.

The Trim66 conditional knockout mouse line, Trim66 flox was also created by CRISPR/Cas9 technology. Two lox sequences flanking exons 2–10 of Trim66 were inserted through homologous recombination. The homologous recombination vector (donor vector) was constructed and co-injected with sgRNAs and Cas9 mRNA into the fertilized eggs of C57BL/6J mice. The targeted sequences for exons 2–10 of Trim66 were 5’-GCACCAGGAGATTGCCACCC-3’, 5’-GAAGAGTCATGGTGGTCTCC-3’, 5’-TCCCTAGACCAAGACAGACC-3’, and 5’-AGTCCTTTGGCTCAAAACCC-3’. We confirmed the flanked genomic sequence (mm10, chr7:109,471,203–09,486,869) of the floxed Trim66 allele by PCR. By crossing the Trim66 flox line with two Cre transgenic mouse lines, Foxg1-cre and Goofy-cre, conditional knockout of Trim66 can be achieved in the early development of the olfactory epithelium and in immature/mature OSNs.

The Olfr1507-ires-tdTomato knock-in mouse line was generated by Cyagen Biosciences (Suzhou) Inc. A targeting vector was designed to insert “ires-tdTomato-ires-Cre-WPRE-BGH-pA” cassette into the 3’ untranslated region (after the ~50 bp downstream of the translational termination site) of the Olfr1507 locus. The Cas9 protein, sgRNA (sgRNA: 5’-TGATAAGTGCTTAGGATTCT-3’), and the targeting vector were co-injected into fertilized eggs obtained from C57BL/6J wild-type mice. The injected embryos were cultured in KSOM medium overnight, and those that developed to the two-cell stage were transferred into the oviduct of pseudopregnant ICR female mice. The F0 founder mice were identified by PCR analysis and Sanger sequencing, which were bred to wild-type mice to test germ-line transmission and F1 animal generation. The genotype of F1 mice was also confirmed by PCR.

Published data
ATF4 ChIP-seq reads28 were downloaded from the SRA (SRR407511/2/3 for the sample with a total of 63,528,233 single-end 37 or 39 bp reads, and SRR407514/5/6 for the control with a total of 61,967,262 single-end 37 or 39 bp reads). For each condition, the three replicates were pooled for analysis. MOE DNase-seq reads41 were downloaded from the SRA (SRR1031022 with a total of 134,845,528 paired-end 51+51 bp reads). MOE RNA-seq reads36 were downloaded from the ENA (ERR225908/09/10/11/12/13 with a total of 278,435,883 paired-end 75+75 bp reads). The six replicates were pooled for analysis. Atf5-knockout microarray data27 were downloaded from the GEO (GSE37609). For each condition, the log expression values from the three replicates were averaged.

In situ hybridization
In situ hybridization analysis of mouse MOE was performed as described before50. Anti-sense cRNA riboprobes labeled with digoxigenin (DIG) and fluorescein were prepared to detect mRNA expression of Atf5, Trim66, Taars, and ORs in single and two-color in situ hybridization experiments. For single-color in situ hybridization experiments, riboprobes labeled with DIG against Atf5, Trim66, Taars, class I ORs, and class II ORs were used. Primary anti-DIG antibody conjugated with alkaline phosphatase (AP) and NBT/BCIP substrate were incubated to observe mRNA signals. Single-color images were taken using a light microscope. For two-color in situ hybridization experiments, riboprobes labeled with DIG and riboprobes labeled with fluorescein were used. The signal of fluorescein-labeled riboprobes was first detected by primary anti-fluorescein antibody conjugated with peroxidase (POD) and further amplification using the Cy3 TSA fluorescence kit (APExBIO, K1051-100). Subsequently, 10% H2O2 was added for 30 min to inactivate POD, followed by fixing the signal with 4% paraformaldehyde (PFA) for 2 h. The signal of DIG-labeled riboprobes was detected by primary anti-DIG antibody conjugated with POD and amplification using the fluorescein TSA fluorescence kit (APExBIO, K1048). Two-color in situ hybridization images were taken using a Leica TCS SP8 confocal microscope. For each mouse, we collected the tissue on 20–25 slides, with each slide containing five MOE sections. To quantify the number of positive cells, three mice (n = 3) and at least three intact MOE sections at similar anatomical positions per mouse were used for each genotype. For in situ hybridization of Olfr549, Olfr981, Taar6, and Taar7d, chromogenic signal intensities were quantified at the single-cell level using ImageJ in both Trim66 +/− and Trim66 −/− mice. We manually annotated receptor-positive cells and background regions to measure signal intensities. The chromogenic signal intensities of each cell were determined by subtracting the background signals. The raw counts have been provided in the source data. The probe information and primers used to generate probes are summarized in Supplementary Tables 3 and 4, respectively.

Immunohistochemistry
Coronal MOE sections (14-μm thickness) were obtained from 4% PFA-fixed and freshly frozen P14 to P60 mice. Freshly frozen MOE sections were fixed with 4% PFA in phosphate-buffered saline (PBS) for 10 min at room temperature. This step was skipped for 4% PFA-fixed sections. The sections were washed with PBS three times (5 min each) and incubated with permeable buffer (0.3% Triton X-100 in PBS) containing 5% donkey serum for 30 min. Primary antibodies against TRIM66 (OriGene, TA339923), OMP (Abcam, ab93127), GAP43 (Abcam, ab16053), SOX2 (ABclonal, A0561), KRT14 (ABclonal, A15069), Caspase-3 (Cell Signaling Technology, 9662), TAAR4 (homemade), TAAR5 (homemade), Olfr552 (Osenses, OSR00204W), and ATF5 (Santa Cruz Biotechnology, sc-99205X) were used at 1:4000, 1:1000, 1:1000, 1:1000, 1:500, 1:500, 1:500, 1:5000, 1:5000, 1:5000, 1:5000, and 1:500 dilution in incubation buffer (1% bovine serum albumin, 0.01% sodium azide, 0.3% Triton X-100 in PBS). Primary antibody incubations were performed at 4 °C for one overnight. The sections were then rinsed three times (5 min each) in PBS and incubated at 37 °C for 30 min with different fluorophore-conjugated secondary antibodies (diluted at 1:1000), including Donkey Anti-Rabbit IgG conjugated to Alexa Fluor® 488 (Jackson ImmunoResearch, 711-545-152), Donkey Anti-Rabbit IgG conjugated to Cyanine CyTM3 (Jackson ImmunoResearch, 711-165-152), Donkey Anti-Guinea Pig IgG conjugated to Alexa Fluor® 488 (Jackson ImmunoResearch, 706-545-148), and Donkey Anti-Guinea Pig IgG conjugated to Cyanine CyTM3 (Jackson ImmunoResearch, 706-165-148). Slides were rinsed three times (5 min each) and coverslipped using mounting medium containing DAPI (SouthernBiotech). Images were taken using a Leica TCS SP8 confocal microscope. For immunohistochemistry experiments, both the number of positive cells and the fluorescence intensity of individual cells were quantified using procedures similar to those employed in the in situ hybridization analysis. Additionally, for quantification of ATF5 signals, as well as positive cell numbers for Ki67, and PH3, we employed the Trainable WEKA Segmentation plugin in ImageJ. Specifically, ATF5-positive cells were identified by WEKA segmentation. The fluorescence signal intensities of ATF5 in each cell were determined by subtracting the background signals. For quantification of Ki67 and PH3 positive cell numbers, positive cells were automatically detected via WEKA Segmentation in the MOE regions. The cell numbers were then normalized to the lengths of the MOE regions. The raw counts have been provided in the source data.

Whole-mount olfactory bulb imaging
The olfactory bulbs of mice at P14-P30 were exposed by removing the bones and were placed on a 35 mm dish. Dorsal surfaces (Olfr73-ires-GFP, J-gVenus, TAAR enhancer 1-GFP, and TAAR enhancer 2-tdTomato mice) or ventral surfaces (Olfr1507-ires-tdTomato mice) of the olfactory bulbs were placed under the microscope. Fluorescent reporter signals were visualized and imaged by an Olympus BX53 microscope.

Bulk RNA-seq
mRNAs from the whole MOE tissues were extracted and purified with a Cell/Tissue total RNA isolation kit V2 (Vazyme, RC112-01). Libraries were prepared with Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme, NR604-02). Trimmomatic (version 0.39) was used to filter out low-quality reads. Reads were mapped to the mouse reference genome (mm10) with Hisat2 (version 2.1.0). Raw read counts mapped to genes were calculated using featureCounts from the Subread package (version 2.0.1). DEG analyses were performed using the DESeq2 (version 1.40.1) package with the default settings. Significantly changed genes were identified with an adjusted p value cutoff of 0.05.

Single-cell RNA-seq
Tissue dissociation and cell purification. The MOE tissues were dissected and transported in a sterile culture dish with 10 ml 1× Dulbecco’s Phosphate-Buffered Saline (DPBS; Thermo Fisher, 14190144) on ice to remove the residual tissue storage solution, then minced on ice. We used the dissociation enzyme, 0.25% Trypsin (Thermo Fisher, 25200-072) and 10 μg/mL DNase I (Sigma, 11284932001) dissolved in PBS with 5% fetal bovine serum (FBS; Thermo Fisher, SV30087.02) to digest the tissues. The MOE tissues were dissociated at 37 °C with a shaking speed of 50 rpm for about 40 min. We repeatedly collected the dissociated cells at an interval of 20 min to increase cell yield and viability. Cell suspensions were filtered using a 40-μm nylon cell strainer, and red blood cells were removed by 1× Red Blood Cell Lysis Solution (Thermo Fisher, 00-4333-57). Dissociated cells were washed with 1× DPBS containing 2% FBS. Cells were stained with 0.4% Trypan blue (Thermo Fisher, 14190144) to check the viability on Countess® II Automated Cell Counter (Thermo Fisher). Each of the solutions above contained transcriptional inhibitors (5 μg/mL Actinomycin D, 10 μg/mL Anisomycin, and 10 μM Triptolide, Sigma)51.

Library preparation and sequencing. Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation, so that each cell was paired with a bead in a Gel Beads-in-emulsion (GEM). After exposure to the cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA synthesis, each cDNA molecule was tagged on the 5’ end (the 3’ end of a messenger RNA transcript) with UMI and cell label indicating its cell of origin. Briefly, 10× beads that were then subject to second-strand cDNA synthesis, adapter ligation, and universal amplification. Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3’ end of the transcripts linked with the cell barcode and UMI. All the remaining procedures, including the library construction, were performed according to the standard manufacturer’s protocol (CG000206 Rev D). Sequencing libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). The libraries were sequenced on NovaSeq 6000 (Illumina) using 2×150 chemistry.

Single-cell RNA-seq data processing. Reads were processed using the Cell Ranger (version 6.1.2) pipeline with default parameters. FASTQs generated from Illumina sequencing output were aligned to the mouse genome (GRCm38, mm10) using the STAR algorithm52. Next, Gene-Barcode matrices were generated for each individual sample by counting UMIs and filtering non-cell-associated barcodes. Finally, we generate a gene-barcode matrix containing the barcoded cells and gene expression counts. This output was then imported into the Seurat R toolkit (version 4.3.0) for downstream analysis53,54. The different filtering thresholds are suitable for Trim66 knockout data. For Trim66 knockout data, cells with less than 700 transcripts, or less than 200 genes, or more than 15000 transcripts, or more than 10% of mitochondrial gene expression were filtered out as low-quality cells; genes expressed in less than three cells were excluded. The normalized data (NormalizeData function in Seurat package) after merging the different datasets was performed for extracting a subset of variable genes. Next, we performed principal component analysis (PCA) and reduced the data to the top 30 PCA components after scaling the data with the vars.to.regress parameter to remove the mitochondrial variables. Then we used the Harmony package to integrate the different data. And the integrated data was displayed on a 2D map produced with uniform manifold approximation and projection (UMAP) using harmony embeddings (1–20)55.

The clusters were obtained using the FindNeighbors and FindClusters functions with the resolution set to 0.9 for the Trim66 knockout, resulting in 25 clusters. The cell types were annotated by overlapping the cluster markers found using the FindAllMarkers function with the known cell-type marker genes. The following cell types were annotated with selected marker genes listed below: HBCs (Krt5, Krt14, Trp63, Cxcl14, Sox2, Meg3); GBCs (Ascl1, Sox2, Lgr5, Hes6, Cxcr4, Ezh2, Neurog1, Neurod1, Tmprss4, Kit, Mki67, Top2a); INPs (Neurog1, Neurod1, Neurod2, Top2a, Mki67, Lhx2, Ebf1); immature OSNs (Ablim1, Drd4, Dbn1, Dpysl5, Crmp1, Ppp2cb, Marcksl1, Atf5, Gnas, Hdac2, Gng8, Dpysl3, Gap43, Stmn1, Stmn2, Ebf2, Lhx2, Cbx8, Trib3); mature OSNs (Cngb1, Adcy3, Slc17a6, Cnga4, Cnga2, Gnal, Omp, Gng13, Stoml3, Ebf2, Cbx8, Rtp1); sustentacular cells (Cyp2g1, Cyp1a2, Notch2, Notch3, Hes1, Hes5, Gpx6, Ermn, Sox2, Hey1, Sult1c1); olfactory ensheathing glia (S100b, Plp1, Mpz, Alx3); olfactory microvillar cells (Coch, Ascl3, Cftr, Hepacam2, Sox9, Trpm5); GC-D cells (Ms4a4c, Ms4a6c, Ms4a7, Pde2a, Car2, Cnga3); immune cells (Cd3d, Cd3e, Cd8a, Cd4, Il7r, Fgfbp2, Fcgr3a, Cx3cr1, Cd19, Cd79a, Ms4a4a); blood cells (Ptprc, Hba-a1, Mzb1, Sdc1, Cd14, S100a12, Clec10a, C1qa, C1qb, C1q)49,56–64.

Single-cell neuron subpopulation analysis. For identifying the OSNs identity, we set different thresholds for analysis. Mature OSNs expressing a single OR (UMI ≥ 2) were identified based on the OR that they expressed, while immature OSNs may simultaneously express several OR genes (UMI ≥ 1) at low levels.

ChIP-seq. Cells were dissociated from the mouse MOE as previously described22. Briefly, dissected MOE was dissociated by the Papain Dissociation System (Worthington) at 37 °C for 15 min and trituration for 5–15 times with a cut P1000 pipette tip. Cells were filtered by a 40-μm strainer (Falcon). After spinning at 400×g for 2 min, cells were resuspended in DMEM (Gibco). ChIP-seq was performed on dissociated MOE cells with a ChIP-IT High Sensitivity Kit (Active Motif) and an S2 focused-ultrasonicator (Covaris) (settings: frequency sweeping, intensity = 3, duty cycle = 2%, cycles per burst = 200, 15 cycles of 1 min on and 1 min off). The final DNA product was eluted into ~60 μL. Libraries were prepared with an NEBNext Ultra DNA Library Prep Kit (New England Biolabs) and 11–18 cycles of PCR amplification, and then size-selected with PCR Clean DX magnetic beads (Aline Biosciences) (0.55× to remove long fragments, and 0.9× to remove short fragments). Libraries were quantified by a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and a High Sensitivity DNA Analysis Kit on a Bioanalyzer 2100 (Agilent), and then sequenced on a MiSeq or HiSeq2500 (Illumina). ChIP-seq and DNase-seq reads, including those from published data, were mapped to the mouse reference genome GRCm38.p4 (downloaded from the GENCODE M10 release) with bwa 0.7.15 (mem with default parameters), samtools 1.3.1, and sambamba 0.6.3. RNA-seq reads were mapped to the mouse reference genome mm10 (downloaded from Illumina iGenomes) with tophat 2.0.11. ChIP-seq peak were called with macs2 2.1.0.20140616 with “-B” to generate read pileups. Overlaps between peaks were identified with bedtools 2.26.0. DNA motifs were identified (findMotifsGenome.pl: “-size 200 -mask”) and visualized (motif2Logo.pl: “-pdf -ns 1000 -bit”) with Homer 4.8.3. GO analysis was conducted with ClusterProfiler 4.8.1 (after removing peaks that are not on reference chromosomes). Antibodies used for ChIP-seq include ATF5 (Santa Cruz Biotechnology, sc-99205X), TRIM66 (Sigma-Aldrich, HPA027420), EZH2 (Cell Signaling Technology, 5246), EZH2 (Active Motif, 33902), and YY1 (Santa Cruz Biotechnology, sc-1703X).

ATAC-seq. ATAC-seq was performed as previously described with minor modifications65. We used 800,000 MOE cells for library construction with the TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) and applied the index from the TruePrep® Index Kit V2 for Illumina (Vazyme, TD202). The libraries were sequenced on the NovaSeq 6000 (Illumina) using 2×150 chemistry. Reads were trimmed using Trimmomatic (version 0.39), then mapped to the mouse reference genome (mm10) using Bowtie 2 (version 2.4.1) with the “very-sensitive –X 2000” parameters. Significant ATAC-seq peaks were identified using Genrich (version 0.6.1) with the “-j -y -r -e chrM” parameters and setting the threshold of p value less than 0.05. The olfactory regulatory peaks were visualized in a multi-region track using the UCSC genome browser.

Cleavage under targets and tagmentation (CUT&Tag). CUT&Tag was performed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903). The MOE from Olfr1507-ires-tdTomato; Trim66 −/− or Olfr1507-ires-tdTomato; Trim66 +/− mice was dissected and dissociated. Then, more than 10,000 tdTomato-positive cells were sorted by flow cytometry. The sorted tdTomato-positive cells were incubated with 10 μl pre-washed ConA beads and 2 μg primary antibodies against H3K27ac (Millipore, 17-683), H3K4me1 (Abcam, ab8895), H3K27me3 (Millipore, 07-449), and H3K9me3 (Abcam, ab8898) at 4 °C overnight. The secondary antibody (1:100) was incubated at room temperature for 1 h, and then 2 μl pA/G-Tnp with 98 μl dig-300 buffer was added to incubate at room temperature for 1 h. Subsequently, 10 μl 5× TTBL with 40 μl dig-300 buffer was added to incubate at 37 °C for 1 h. After the above fragmentation step, 5 μl Proteinase K, 100 μl Buffer L/B, and 20 μl DNA Extract Beads were added to each sample and incubated at 55 °C for 10 min. The libraries were amplified by 14 cycles, and the products were purified using DNA Clean Beads (Vazyme, N411). The libraries were sequenced on NovaSeq 6000 (Illumina) using 2×150 chemistry. Reads were mapped to the mouse reference genome (mm10) using Bowtie 2 (version 2.5.1) with the “--local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700” parameters. The olfactory enhancer peaks were visualized in a multi-region track using the UCSC genome browser and deeptools (version 3.5.5).

In situ Hi-C. The MOE from Trim66 −/− or Trim66 +/− mice was dissected and dissociated. The live cells were fixed with 1% formaldehyde for 10 min and quenched with 0.2 M glycine for 5 min at room temperature. The cells were then washed twice with 100 μL HBSS containing 0.1% BSA. The pelleted cells were resuspended in 100 μL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630, and protease inhibitors) and incubated on ice for 30 min. After centrifugation at 3000×g for 5 min at 4 °C, the pelleted nuclei were washed once with 100 μL of ice-cold 1× NEB Buffer 3.1. Chromatin was solubilized in 20 μL of 0.3% SDS and incubated at 65 °C for 10 min, followed by quenching with 5 μL of 10% Triton X-100 and incubation at 37 °C for 10 min. The nuclei were digested with 50 U DpnII in a total volume of 50 μL at 37 °C for 3 h in a Thermomixer with interval shaking. DpnII was then inactivated by incubation at 65 °C for 20 min. For biotin labeling, 3 μL of 1 mM biotin-14-dATP, 0.3 μL of 10 mM dGTP, 0.3 μL of 10 mM dTTP, 0.3 μL of 10 mM dCTP, and 10 U Klenow were added. The reaction was performed at 37 °C for 1.5 h in a Thermomixer. Subsequently, 60 μL of ligation mixture (33.6 μL water, 12 μL 10× NEB T4 DNA ligase buffer, 12 μL 10% Triton X-100, 1.2 μL 10 mg/mL BSA, and 1.2 μL 2000 U/μL T4 DNA ligase) was added, and the reaction was incubated at 25 °C for 4 h in a Thermomixer. Crosslinks were reversed and DNA was purified. The purified DNA was sheared to 300–500 bp using a Covaris M220. End repair, dA-tailing, and adapter ligation were then performed using the Rapid Plus DNA Lib Prep Kit V2 (ABclonal). Biotin-labeled DNA fragments were enriched using 50 μL of Dynabeads MyOne Streptavidin C1 (Invitrogen). Sequencing libraries were prepared on beads, followed by six to eight cycles of PCR amplification. Fragments ranging from 300 bp to 1000 bp were size-selected. All libraries were sequenced on a NovaSeq X Plus platform following the manufacturer’s instructions.

Hi-C data processing and data analysis. The in situ Hi-C data were processed following the 4DN Hi-C data processing pipeline (https://data.4dnucleome.org/resources/data-analysis/hi_c-processing-pipeline). Briefly, sequencing reads were aligned to the mm10 reference genome with BWA (version 0.7.17). Valid Hi-C contacts were extracted by removing PCR duplicates and short cis contacts within 1 kb using pairtools (version 1.0.1). Hi-C contact matrices were then generated by binning the genome at multiple resolutions and normalized using matrix balancing implemented in cooler (version 0.9.2). To account for variations in sequencing depth and alignment efficiency, contact frequencies were normalized by dividing the binned interaction counts by the total number of Hi-C contacts at the specified resolution. Contact matrices were visualized using Python libraries including pandas, seaborn, and matplotlib. Aggregate pileup analyses were conducted using the pileup function from the coolpuppy package (version 1.1.0). A/B compartment profiles were derived from ICE-normalized intrachromosomal contact maps at 500 kb resolution by converting them into observed-over-expected (O/E) matrices. Principal component analysis (PCA) was performed, and PC1 values were interpreted based on GC content. Regions with positive PC1 scores, typically gene-rich, were assigned as compartment A, while regions with negative PC1 scores were assigned as compartment B. Compartment strength was assessed and visualized by saddle plots in cooltools (version 0.5.4). TADs were identified by calculating insulation scores via the diamond insulation algorithm implemented in cooltools, with a window size of 125 kb and a bin size of 25 kb.

Surgery for microelectrode implantation. Before 16-channel microelectrode array (Stablohm 675, 0.0013” diameter, insulated with HFV-Natural, CWF) implantation surgery, mice were anesthetized by pentobarbital sodium (i.p. 90 mg/kg) to a deep anesthesia state. Then, the anesthetized mice were transferred to stereotaxic apparatus (RWD Life Science, China). Erythromycin eye ointment was applied to protect the eyes. The hair between the ears and eyes was removed, and the exposed craniofacial skin was sterilized using iodophor. The incisor bar and double ear rods were adjusted until the bregma and lambda were in the same horizontal plane. According to the mouse brain atlas, a craniotomy window over the olfactory bulb was made with a dental drill, and the dura was carefully removed. Two miniature stainless-steel screws inserted into the skull served as ground and reference electrodes. The microelectrode was slowly and accurately lowered into the target areas (AP, 4.0 mm; ML, 1.0 mm; DV, 1.8–2.0 mm) under stereotaxic guidance, with continuous signal monitoring to ensure optimal placement. The microelectrode was secured with dental cement for stability. To allow head-fixation during recording, a custom-designed headplate was attached to the skull using dental cement, which was also used to secure the implant to the bone. Mice were returned to their home cages and allowed to recover for at least one week.

In vivo electrophysiology recordings. After mice were recovered from surgery, in vivo recordings were conducted to obtain spontaneous and odor-evoked spikes and local field potentials (LFPs). Awake mice were head-fixed via the attached headplate, with an odor port positioned in front of the nose for odor delivery. A set of six different odorants, including isoamyl acetate, 2-heptanone, trimethylamine, 1-nonanol, muscone, and spermidine were diluted in mineral oil at a concentration of 0.01% v/v. The six odors were presented in a random order for 2 s with an inter-trial interval of 20 s (total of 15 trials each odor). Both the LFPs and extracellular spike activities were recorded using an electrophysiological recording system (NeuroLego System, Jiangsu Brain Medical Technology Co., Ltd). For spike recording, the microelectrode signal was sent to a preamplifier and amplified by a 16-channel amplifier. Processed signals were sampled at rate of 30 kHz using the NeuroLego electrophysiological recording system. For LFPs recordings, the signals were collected by a microelectrode and amplified by an amplifier (bandpass filtered at 0.1–300 Hz, sampled at 1000 Hz). Odor stimulation event markers were recorded alongside the spike/LFPs data via the same recording system.

Buried food assay. A buried pellet test was performed to assess general olfactory detection, as described previously66. Briefly, Male and female mice were fasted for 24 h prior to the test. Before the test, mice were habituated for 5 min in a test cage (48 cm × 34 cm × 18 cm). Mice were introduced individually into a clean cage containing clean bedding (3 cm) with a 1.5 g food pellet buried 1 cm deep in a random corner of the cage. The time taken by the mouse to retrieve the food pellet (the time at which the mouse uncovers the food pellet and grasps it with its front paws or teeth) was recorded. If the mouse failed to find the food pellet within 5 min, the test was terminated and the latency to find the pellet was recorded as 300 s. The visible pellet test was identical to the buried pellet test except that the food was placed on the top of the bedding.

Go/no-go behavioral tasks. The mice were deprived of water for 24 h before the experiment. The experiment was divided into two phases: training phase (Go/Go, GG) and testing phase (Go/no-go, GNG). During the experiment, the infrared sensor detected head entries into the hole, triggering the start of a test, and an odor was then delivered through the odor port. In the GG phase, two odors (isoamyl acetate and 2-heptanone) were chosen for training. If the mouse licked the waterspout during a specific time window of odor presentation (regardless of whether isoamyl acetate or 2-heptanone was presented), it received a reward. In the GNG phase, only one of the odors corresponded to a reward trial (S+), so the mouse needs to discriminate the odors to obtain a reward. During the S+ odor presentation, licking within the time window was rewarded with a water droplet (Hit), absence of licking results in no reward (Miss). The S− odor is associated with no reward. Licking or no licking in response to S− within the time window was regarded as a false alarm (FA) or correct rejection (CR). Odors were administered for 2 s per trial, with the final 0.5 s designated for the time window. The correct response was calculated as: (number of Hit trials + number of CR trials)/total number of trials. Once the mouse learned to distinguish the two odors (with an accuracy rate of over 80%), they were moved onto the next stage. The whole experiment contained a total of three odor discrimination tasks, specifically: isoamyl acetate vs. 2-heptanone, trimethylamine vs. 1-nonanol, muscone vs. spermidine. In these three tasks, isoamyl acetate, trimethylamine, and muscone served as the S+, while 2-heptanone, 1-nonanol, and spermidine were served as the S−. All odorants were dissolved in mineral oil at a concentration of 0.01% v/v. A total of 120 trials were conducted each day, with equal numbers of S+ and S− trials.

Statistics and reproducibility
Age- and sex-matched mice were randomly assigned for the experiments. No animals were excluded from statistical analyses, and the investigators were not blinded in the studies. All statistical tests were performed in R. A significance cutoff of p value of 0.05 was used for all analyses and p values were adjusted for multiple comparisons where relevant (denoted as padj in the text, figures, and figure legends). Multiple types of statistical hypothesis tests were employed, and the number of biological replicates varied across experiments. Specific statistical tests are described for each experiment in the accompanying figures, figure legends, and method details. No methods were used to predetermine sample sizes. No data were excluded from the analyses.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The sequencing data generated in this study have been deposited in the GEO database under accession code GSE277322. All data were available in the main text and in the supplementary materials. Source data are provided with this paper.

Code availability
All code is available in the GitHub data repository database: https://github.com/YaleiKong/Trim66_project. We have linked our GitHub repository to Zenodo and obtained a https://doi.org/10.5281/zenodo.17132541.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review comments: Nature Communications # NCOMMS-25-16212-T\nIn the present manuscript, Bao et al. identify TRIM66 as a novel transcriptional regulator responsible for establishing the\nsingular expression of olfactory receptor genes, commonly referred to as the “one-neuron one-receptor” rule. Each olfactory\nsensory neuron (OSN) is thought to express a single functional allele of a single olfactory receptor gene from the extensive\nrepertoire of either odorant receptors (ORs) or trace amine-associated receptors (TAARs). However, recent advances in\nsingle-cell RNA sequencing have revealed that OSNs transiently co-express multiple olfactory receptor genes during their\nimmature stage and subsequently eliminate all but one as they mature. The regulatory mechanisms underlying this selective\nsilencing remain unknown. In this study, the authors have successfully identified a regulatory factor that links the transition\nfrom polygenic (in immature OSNs) to monogenic expression (in mature OSNs). The manuscript is technically sound and\npresents potentially significant insights, with several conclusions supported by convincing data. However, I would like\nto request that the authors address a several major issues, which I will outline below.\nMajor points\n1) The present study demonstrates that TRIM66, an epigenetic repressor, binds to OR enhancers and that its absence\nincreases the accessibility of the OR enhancers in Trim66-KO mice, suggesting that TRIM66 represses the enhancer hub\nand contributes to the singular expression of OR/TAAR genes. However, given that Trim66 is a downstream gene of ATF5\nand that the expression level of Adcy3 is significantly reduced in Trim66-KOs (Extended Data Figs. 2c, 4d, 5d), the\npossibility of secondary effects due to insufficient negative-feedback signaling via the UPR cannot be ruled out. in Trim66-\nKO mice. Such effects may contribute to the failure to repress multiple OR gene expression. To clarify this point, I\nrecommend evaluating this possibility by assessing ATF5 protein levels via immunohistochemistry in Trim66-KO mice\n(since the mRNA level remains unchanged) and by analyzing Lsd1 (Kdm1a) expression levels.\n2) In addition, the loss-of-function experiments demonstrate that Trim66 is necessary for the singular expression but do not\nestablish its sufficiency in this process. To provide conclusive evidence for the critical role of Trim66 in the singular olfactory\nreceptor gene expression, it is essential to conduct both loss-of-function and gain-of-function experiments. I believe that\nincorporating such experiments would significantly enhance the scientific impact of this study. For example, forced\nexpression of Trim66 in immature/mature OSNs in the Trim66 KO background could help determine whether Trim66 alone is\nsufficient to drive singular OR gene expression.\n3) As described by the authors, single-cell RNA-seq results show decreases in the mRNA levels of olfactory receptor gene\nper cell. On the other hand, the signal intensities of ISH for OR RNA and IHC for OR protein do not seem to have changed\nmuch. It is necessary to quantitatively analyze that the expression level (mRNA and protein). Related to this, the IHC images\nof wt and KO are not convincing at all, because the background signals in the wt sections are largely higher than in the KO\nimages (these images appear to be biased by image processing); Fig.2d, Extended Data Figs. 2d, 2f, 3e, 4b, 5b, and 6d.\nThese images should be re-taken under the same imaging conditions.\n4) In this study, the authors primarily analyze histone modification levels at OR enhancers. However, given that TRIM66\ninteracts with HP1 proteins and H3K9me3, the H3K9me3 levels in the OR gene bodies should also be\nexamined/mentioned. Additionally, the double knockout of G9a (Kmt1c) and GLP (Kmt1d), which are required for H3K9me3\nmodification, has been reported to decrease overall OR gene expression while increasing the expression of certain OR\ngenes (Lyons DB et al., Cell Rep. 2014), a phenotype that appears similar to that of Trim66 KO, though the authors do not\ncite this paper. To clarify whether Trim66 KO shares the same mechanistic basis, I believe it is important to assess this\npossibility.\n5) It is intriguing that abnormal sexual behavior is observed in Trim66 KO mice. However, given that the vomeronasal\nsystem plays a major role in regulating sexual and social behaviors in mice, further analysis is needed to substantiate these\nfindings. If the authors choose to retain these results in Figure 6 (Figures 6f–i), it would be essential to analyze the\nexpression of vomeronasal receptors in the vomeronasal organ (VNO) to determine whether the singular expression is\ndisrupted, as well as to conduct physiological function analyses of the VNO. However, incorporating data on the\nvomeronasal system may dilute the primary focus of this study. To maintain clarity and coherence, I recommend that the\nsection on sexual and social behavior experiments be removed from the manuscript.\n6) Some aspects of the data interpretation, particularly in the Extended Data, are difficult to follow due to insufficient details\nin the experimental methods. Notably, the methodology for quantifying the number of expressed cells is not described,\nmaking it unclear how each data point was obtained (e.g., whether each point represents data from an individual animal or\ndata per unit area). For instance, in Extended Data Figure 6d, there is substantial variation in the number of apoptosis-\npositive cells, but the meaning of each data point remains unclear. To improve clarity, the authors should explicitly describe\nthe method used to obtain each data point. For the quantification of apoptosis-positive cells, I recommend acquiring multiple\nsections from similar positions in the olfactory epithelium and presenting the data as either the sum of the slides or the\nnumber of positive cells per unit area. This approach would help minimize inter-individual variability. The details of these\nexperimental methods should be submitted as the Extended Methods.\n7) The ISH analysis of Trim66 in Goofy-Cre; Trim66 fl/fl cKO mice showed little expression of Trim66 (Extended Data Fig.\n5a), with no apparent differences in Trim66 expression between cKO and global KO. The authors stated that Trim66 is\nhighly expressed in late progenitors and in immature/mature OSNs, whereas Goofy-Cre-mediated excision of Trim66 occurs\nin immature OSNs, preserving Trim66 expression in late progenitors. Alternatively, the results presented in Fig. 1e may be\ninaccurate. Unfortunately, as mentioned above, the IHC results (Extended Data Fig. 5a) do not allow for a definitive\nconclusion on this issue. It is essential to rigorously assess whether Trim66 is truly knocked out after the immature OSN\nstage. To clarify the expression profile of Trim66, a co-expression analysis using OSN differentiation markers (e.g., double-\nlabel ISH or a combination of IHC and ISH with probes for Ascl1, Neurog1, Neurod1, Gap43, and OMP) is required. These\nadditional analyses are necessary to address the concerns raised above.\n8) Two factors influence OR expression levels: the amount of mRNA per cell and the number of expressing cells (i.e.,\nprobability of choice). While this study primarily focuses on mRNA levels, little consideration is given to the probability of\nchoice. To provide a more comprehensive analysis, the authors should clarify the correlation between total mRNA levels\nand the number of expressing cells. Additionally, a discussion on expression probability should be included to determine\nwhether Trim66 contributes to the regulation of expression probability.\n9) In the Discussion, the authors mention to the results of Fig. 1-4 but not Fig 5 and 6. Additional discussion on these is\nrequired.\nMinor points\n10) Abstract, page 2, line 31: The phrase “very low level” may not be entirely accurate, as the single OR expression levels in\nTrim66-/- OSNs exhibit broad distribution. Please consider revising this wording for precision.\n11) Abstract, page 2, lines 36–37: The statement “with broader implications for other monoallelic or monogenic expression\nsystems” appears to be an overgeneralization. The singular expression of immune receptor genes, for example, is governed\nby a different mechanism involving gene rearrangement. Please refine this statement accordingly.\n12) Introduction, page 3, line 60: The phrase “the functional OR genes are clustered” is not entirely accurate, as some OR\ngenes exist as singletons. Please revise this sentence to reflect this nuance.\n13) Page 4, line 70, and page 16, line 343: The phrase “also called Greek Islands” should be removed, as not all OR\nenhancers belong to Greek Islands (e.g., H, P, and J elements).\n14) Results, page 6, lines 121–123: The statement, “This similarity may explain the observations that Atf5-knockout pups still\ndeveloped some mature neurons and that a small fraction of those pups survived into adulthood,” is debatable. The\nmaturation of some OSNs may result from upregulated OR genes, and the survival of a small fraction of Trim66 KO pups\nmay depend on maternal care. To support the latter claim, it would be necessary to verify whether these pups can reach\nadulthood through artificial lactation. Otherwise, please revise this statement accordingly.\n15) Page 7, line 153: The phrase “by indirect consequences of having fewer mature neurons in the knockout mice” is\nunclear. It seems that the reduced expression of Trim66 in Atf5 KO results in fewer mature neurons. Please clarify and revise\nthis sentence for accuracy.\n16) Page 9: A description of the general phenotype of Trim66-KO mice should be included. Relevant details such as body\nweight, survival rate, growth rate, and fertility should be provided.\n17) Throughout the text and figure legends, please specify the age of the mice analyzed.\n18) Page 10, line 209: The phrase “increase in OSNs expressing” should be revised to “increase in the number of OSNs\nexpressing” for clarity.\n19) Page 11, line 234: The sentence “the expression levels of all Tarr genes were not significantly changed” is inaccurate,\nas the expression levels of individual Tarr genes show significant changes (some being upregulated, others downregulated,\nwith three exceptions). Please revise this statement to accurately reflect the observed data.\n20) Page 14, lines 307–309: The statement “Moreover, the cells showing polygenic receptor expression were almost\nexclusively located in the apical MOE, where mature OSNs reside” presents an intriguing observation. Since multiple OR\ngenes are co-expressed in both immature and mature OSNs in Trim66-KO mice, please elaborate on why co-expression\nwas predominantly observed in mature OSNs and discuss the potential underlying mechanisms.\n21) Page 14, lines 309–311: The authors state, “Additionally, the increased numbers of Olfr1507 glomeruli in the Olfr1507-\nires-tdTomato; Trim66 -/- mice compared to control mice could also be due to other co-expressed olfactory receptors in\nOlfr1507-expressing OSNs.” I suggest mentioning the zonal expression of Olfr1507, as OR expression in an ectopic\nzone could result in multiple glomerular projections.\n22) Page 14, line 314: The reference (Extended Data Fig. 6c and Extended Data Fig. 2c-f) should be corrected to (Extended\nData Fig. 2c-f).\n23) Methods, page 24, line 522-524 and Acknowledgements: Please specify the origin of the mouse lines used (e.g., gifted\nfrom XX or JAX accession#). Additionally, any gifted animal lines and materials should be properly acknowledged in the\nAcknowledgements section.\n24) Page 26, line 579: The full name of the DIG abbreviation should be written where it the first place it appears; line 670.\n25) Page 27: Correct the name of the antibodies, e.g., Alexa Fluo 488 should be Alexa Fluor® 488.\n26) Page32, ChIP experiment: The catalog number for the anti-TRIM66 antibody is missing and should be provided.\n27) References: The authors should ensure all references are correctly cited and formatted. For instance: #31 is missing a\ntitle and journal name, #51 appears to be duplicated with #38, #60 lacks a page number.\n28) Figures 2b: The timing of Trim66 deletion is misaligned with the Cre expressions in cKO mice. Please revise the figure\naccordingly.\n29) Figure 4 Legends: The following portion in panel (g) overlaps with (f), “and each experiment was performed on Olfr1507\nOSNs sorted from 1 Olfr1507-ires-tdTomato; Trim66 -/- mouse or from 3-4 Olfr1507-ires-tdTomato; Trim66 +/- mice.”\n30) Extended Data Figures and Tables, p83, Foxg1-cre;Trim66 flox bulk RNAseq: Please confirm and explicitly state the\nnumber of sexes and animals used in Extended Data Figures and Tables.\n31) Extended Data Fig. 3a and its legend: In the inner circle, data should be categorized as follows: significantly upregulated\ngenes (red), significantly downregulated genes (green), and not significantly changed genes (grey). Please revise the figure\naccordingly.\n32) Extended Figs. 4 and 5: These figures should be formatted consistently (i.e.,presented in the same order).\n33) Extended Data Fig. 4 legend, line 1279: “in 2a” should be “in Fig. 2a”.\n34) Extended Data Fig. 6e: The vertical scale is not labeled as a percentage. Please add the appropriate label.\n35) Extended Data Fig. 6g: The legend states, “Pearson correlation coefficient (r) and p-value are shown” (lines 1334–1335),\nbut these values are not displayed in the figure. Please ensure they are included.", "reviewer_2": "The monogenic, monoallic expression of odorant receptor (OR) genes by olfactory sensory neurons (OSNs) is a leading\nexample and model of mechanisms allowing tissues – especially neural tissues - to produce multiple subtypes of key cells.\nThe main conclusion from this study is that Trim66 is a repressor of OR and trace amine-associated receptor (TAAR) gene\nexpression during the differentiation of OSNs. The authors provide evidence that the singularity of OR expression in OSNs is\ndisturbed in the absence of Trim66, that Trim66 can bind at enhancer and promoters of OR genes, and that binding at\nenhancers correlates with OR mRNA abundance differences when Trim66 is absent. The consequent disruptions in OR\nexpression patterns lead to differences in OSN projections to olfactory bulb glomeruli, in the physiology of olfactory bulb\nneurons, and altered olfactory behaviors.\nThe authors interpret their data in a way that emphasizes the role of Trim66 in determining the level of expression of OR and\nTAAR genes. Yet the level of expression is not directly measured. To do so requires quantifying the rate of transcription\n(transcripts produced per unit time). Instead, what is measured is the abundance of each mRNA species. This alternative\napproach is convenient, high throughput, and very common, but only sometimes reflects the level of expression. For\nexample, in cases where differences in cell type abundance occur, which is clearly true after KO of Trim66, measures of\nmRNA abundance are not sufficient to conclude that level of expression is affected. Instead of assuming that measures of\nmRNA abundance directly reflect levels of gene expression, one must use evidence, typically multiple lines of evidence, to\nreach the conclusion that gene expression differs. To their credit, the authors generate data leading toward this conclusion.\nIt should be described as a conclusion, not as an assumption.\nWhy should the authors be so strict in their descriptions? Thinking that mRNA abundance differences equal different levels of\ngene expression is a trap that leads to silly or impossible conclusions. For example, on line 317 the authors write, “the\naveraged OR gene levels in immature and mature . . .” Obviously, genes do not have levels but 2 copies per somatic cell.\nAnother example begins on line 323. “We found that both the upregulated and downregulated OR genes showed decreased\nexpression levels in Trim66 -/- mice” This sentence contradicts itself. If an OR gene is upregulated then its expression\ncannot be less.\nThe manuscript would be more accurate and greatly improved by using the term mRNA abundance or transcript abundance\nrather than gene expression level. Similarly, use increases or decreases in mRNA abundance rather than upregulation or\ndownregulation in both the text and the figures. The Discussion should include an explicit argument that differences in\nOR/TAAR mRNA abundance between WT and KO mice are due to two factors. (1) Differences in the relative abundance of\ncell types in the OE, especially between immature and mature OSNs. (2) Reduced effectiveness of the feedback mechanism\ninvolving Trim66 that helps suppress expression of more than one OR gene. This leads to less than normal amounts of the\nmajor OR mRNA and continued expression of other OR genes.\nOther major criticisms\n1. The turnover of OSNs is not adequately investigated given that the manuscript presents evidence that turnover is\nelevated. For example, increases in Ascl1+ GBCs, in HBCs, in and immature OSNs in the KO all point toward increased\nturnover. The measure of apoptosis is not normalized to the extent of epithelium inspected (e.g., blinded counts per unit of\nlength or volume of tissue) and absent is a measure of basal cell proliferation; the best of which is phospho-histone 3\nimmunoreactivity. The depth/thickness of the OE should also be measured. If turnover is increased then the OE will be\nthinner in KO mice.\n2. Fig 2h, m; Fig 3f, xFig 6, etc. Cell count data must be normalized to the area or volume counted. ORs are a special case.\nBecause ORs are expressed only in zones rather than across the entire OE, the only way to equitably count cells expressing\nthem is to measure the area between the outermost positive cells in the field of view and count all positive cells within that\nregion of OE. Do TAAR cell counts in the same manner.\n3. 277-296; 313-329 and the figures that support these sections. To properly identify OSNs expressing multiple OR genes,\ndifferences in the sensitivity of mRNA detection of these single cell RNA-seq data need to be normalized. An OSN in which\nmRNAs expressed by 1,500 genes are detected is 3X less likely to identify mRNAs from multiple OR genes than an OSN in\nwhich mRNAs encoded by 4,500 genes are detected.\n4. Line 429 and affiliated figures. Were these done with conditional KO mice? Lack of controls for motivation, locomotion,\nand other factors that can affect such behaviors.\nMinor issues.\nA. Lines 233-2347. States that “all” TAARs were not changed after Trim66 KO, but the data in xFig3 show that most TAARs\nwere substantially affected. These mRNA abundance measures are no substitute for cell count data.\nB. Fig 1e. Clarify whether these data are bulk or single cell RNA-seq data.\nC. Fig 1f. These images are not representative of the OE as they feature a fold or tip of a turbinate.\nD. Fig 1h. The decrease in OE thickness from left to right across these images indicates that this piece of OE is probably\nnear the edge of the OE where it meets respiratory epithelium. At these edges the distribution of cell types is not\nrepresentative of OE. It has many fewer mature OSNs and more immature OSNs, especially at PD14.\nE. 220-222. Where is evidence supporting the statement that Olfr73 expression is absent?\nF. Line 1328. Provide correct description of UMI.", "reviewer_3": "It has been more than three decades since the discovery of the one neuron-one receptor rule in olfactory sensory neurons\n(OSNs). The molecular mechanisms underlying the intricate process of monogenic and monoallelic expression of OR genes\nhave been actively researched but are still not fully understood. Immature OSNs express multiple ORs but during maturation,\nall OR genes except for one allele are silenced. In this study, Bao et al. discovered TRIM66 as a key repressor for this\nselective silencing. They found that knocking out the TRIM66 gene in mice resulted in decreased expression of ORs and\ncoexpression of multiple OR genes in mature OSNs. Additionally, they found that deletion of Trim66 led to severe defects in\ninformation processing in the olfactory bulb, as well as impaired innate olfactory behaviors. The experiments are well-\ndesigned, conducted, and presented. The results are intriguing, and the conclusions are convincing. We have only minor\nconcerns.\n1. Title. To make the grammar correct, I would suggest revising it to “An epigenetic repressor … dictates …”.\n2. Page 8. Line169-171. It is stated that Trim66 was more enriched in progenitors or immature OSNs. However, antibody\nstaining (Fig. 1h) seems to show Trim66 is highly expressed in the mature OSNs (more apical layers) than in the immature\nOSNs. Even in Fig. 1g, the in situ hybridization signal seems abundant across mature and immature OSN layers. This\nstatement needs clarification or revision.\n3. Line 212. In Fig. 2h, more details are needed on how the positive cells were quantified from three mice. For each mouse,\nthe number of sections and their locations in the nasal cavity should be described. Do zonal distributions of these receptor\ngenes change in Trm66-/- mice?\n4. Fig. 3e. It seemed that colocalization also exists in Trim66+/- section. One red cell (in the middle of the image) seemed\nboth red and green (lower level). It might be helpful to show enlarged images. Fig. 3f, it would be helpful to describe how\nmany sections and their locations for the quantification.\n5. 5. Supplementary Tables are not cited in order. Table 4 was not mentioned in the text.\n6. Line 412. Suggest using “reduces” or “decreases” instead of “inhibits” in the sentence.\n7. Line 418. “underscoring the importance of Trim66 in regulating neuronal excitability”. The effect of Trim66 knockout on the\nbaseline firing rates and excitatory/inhibitory responses to odors is likely indirect via modulating OR gene expressions in\nOSNs. It is not clear how Trim66 knockout leads to the OB neuronal activity changes. The authors should discuss this point\nin more depth.", "reviewer_4": "The authors use ChIP-seq to identify binding sites for Atf5, thereby identifying candidate transcriptional targets of the OR-\ninduced unfolded protein response. This analysis identified Atf5 sites proximal to numerous genes that are relevant to OSN\ndifferentiation and function, providing useful insights into the mechanism by which the OR-induced UPR signal regulates\nneuronal maturation and proper OR folding. The authors focus their analysis on one of these targets, Trim66, based upon its\ndownregulation in published Atf5 KO datasets and its membership in a gene family that regulates chromatin structure. The\nauthors show that Trim66 RNA expression peaks in the Atf5-expressing population of OSN progenitors, although it also\nappears to be abundant in earlier progenitors at both the RNA and protein levels. Analysis of Trim66 knockout mice reveals\nextensive widespread changes in OR and Taar gene expression by both RNA-seq and IHC, with most receptor genes\ndownregulated and a small number getting upregulated. Notably, the authors also observe reduced numbers of mature cells\nand reduced expression of mature markers, as well as a concomitant increase in progenitor cells / markers. Single cell\nRNAseq analysis reveals that in the Trim66 KO most mature OSNs co-express multiple ORs per cell, which is confirmed by\nISH, and that OR expression levels are lower for the top OR and higher for additional OR (consistent with co-expression).\nImportantly, this reduced expression is observed even for the ORs that were identified as overexpressed by bulk RNA-seq -\nthese ORs have increased expression because they are expressed by more cells, but at lower levels per cell. The authors\ngo on to observe that the more frequently chosen (upregulated in bulk) OR genes are located near OR enhancers elements.\nUsing ChIPseq, they show that Trim66 binds to many OR enhancers, and they observe that these enhancers show\nincreased accessibility and increased active histone modifications in Trim66 knockout MOEs.The authors go on to show that\nmitral and tufted cells in the olfactory bulb have altered electrophysiological properties in Trim66 KO mice. Consistent with\nimpaired olfactory function, these mice also show behavioral defects in multiple modalities, including finding buried food\npellets, social behaviors, and olfactory learning. Based upon these findings the authors propose that Trim66 is required to\nsilence OR enhancer hubs, aside from the hub associated with the chosen OR gene.\nThe author's specific findings are well supported by the data, with appropriate controls, analytical approaches, and use of\nstatistics. However, the authors should be more cautious in interpreting some of their findings. Two points in particular need\nto be addressed:\n1) The authors note increased numbers of immature OSNs in the Trim66 KO mice across several modalities. This\ncomplicates the interpretation of ATAC-seq and ChIP-seq findings as reported in figure 4. As the authors note in the\ndiscussion, immature cells have been observed to have greater ATAC-seq accessibility of OR enhancers, thus the\nincreased signal observed in the KO could reflect the change in cell type composition rather than a change in the mature\ncells in the KO. The purified olfr1507 positive cells could potentially suffer from this same issue (ie containing more cells in a\nmore immature state in the KO). The authors could support this point by performing similar experiments on purified mature\ncells, for example using OMPgfp. While this would be a valuable experiment, I think that the authors could adequately\naddress this issue in the text of the current manuscript.\n2) The enhancer-hub model proposed by the authors to synthesize their results substantially overreaches the data\npresented, and ignores alternative explanations for their findings. For example, it is possible that the Trim66 KO OSNs fail to\nform chromatin hubs entirely, which might be expected to result in co-expression of ORs at low levels just as they observe. If\nthe authors wish to use this framework, they must provide evidence that trim66 ko mice form hubs, using an approach such\nas Hi-C or DNA-FISH.\nIn addition to these major points, I have provided questions and comments related to specific panels and/or analyses below.\nThese issues aside, this work provides important new insights into the mechanism of the OR induced feedback signal by\nidentifying the targets of Atf5, and it identifies a Trim66 as required for regulation of OR gene choice and the Greek Island\nenhancers. These are highly significant findings for the field of olfaction, and more generally for our understanding of the\nmolecular mechanisms that govern gene choice mechanisms.\n#####\nAdditional comments:\n2f: The sorting of ORs/Taars in this panel is unclear. Are they sorted by expression level in the KO?\n4c: The labeling of this figure is confusing. The text suggests that the authors are plotting signal over Trim66 peaks, not\ngenes.\n4f,g and extended data 7h,i: Analysis of heterochromatin marks should also examine regions flanking the enhancer\nelements since these marks may be depleted within the body of the regulatory element. This could take the form of either\nexpanding the width of the element or considering flanking regions separately\nExtended Data 4g & 5g: This plot would be much more useful in evaluating the similarity between these results if it showed\nall ORs and highlighted the ORs by significance in each data set."}, "author_response": "We sincerely thank all the reviewers for their positive comments and their valuable\nsuggestions on our manuscript. In response to those comments, we have added several\nadditional pieces of data, and edited the text accordingly. Please refer to our point-by-\npoint response below for detailed information. We also summarize the main changes in\nthe revised manuscript, including:\n1) reviewer 1 major point #1 and #4: analyses of other factors known to regulate\nolfactory receptor gene expression in Trim66 knockout mice, including protein\nexpression level of ATF5 (UPR pathway) and gene expression levels of G9a and Glp\n(H3K9 methyltransferases);\n2) reviewer 1 major point #3: quantification of gene and protein expression levels of\nreceptors in the in situ and immunohistochemistry experiments, respectively;\n3) reviewer 1 major point #6, reviewer 2 major point #2, and reviewer 3 minor point\n#3: detailed quantification methods were provided;\n4) reviewer 1 major point #7: co-expression analyses of Trim66 and different OSN\ndifferentiation markers;\n5) reviewer 1 major point #8 and reviewer 2 main comments: analysis of correlation\nbetween receptor mRNA levels and probability of receptor gene choice, as well as\ndiscussion on potential factors contributing to receptor expression changes after Trim66\ndeletion;\n6) reviewer 1 minor point #21 and reviewer 3 minor point #3: analysis of zonal\ndistribution changes of receptor genes in Trim66 -/- mice;\n7) reviewer 2 main comments: changes of “level of expression” and\n“upregulation/downregulation” to “mRNA abundance” and “increased/decreased\nmRNA abundance” in description of the bulk RNA-seq results;\n8) reviewer 2 major point #1: analyses of the turnover rates of OSNs;\n9) reviewer 2 major point #3: further normalized analyses of single cell RNA-seq data;\n10) reviewer 4 major point #2: Hi-C analyses of enhancer interaction in Trim66\nknockout mice;\n11) reviewer 4 minor point on CUT & Tag data: showing the flanking regions of the\nenhancer elements.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn the present manuscript, Bao et al. identify TRIM66 as a novel transcriptional\nregulator responsible for establishing the singular expression of olfactory receptor\ngenes, commonly referred to as the “one-neuron one-receptor” rule. Each olfactory\nsensory neuron (OSN) is thought to express a single functional allele of a single\nolfactory receptor gene from the extensive repertoire of either odorant receptors (ORs)\nor trace amine-associated receptors (TAARs). However, recent advances in single-cell\nRNA sequencing have revealed that OSNs transiently co-express multiple olfactory\nreceptor genes during their immature stage and subsequently eliminate all but one as\nthey mature. The regulatory mechanisms underlying this selective silencing remain\n1\nunknown. In this study, the authors have successfully identified a regulatory factor that\nlinks the transition from polygenic (in immature OSNs) to monogenic expression (in\nmature OSNs). The manuscript is technically sound and presents potentially significant\ninsights, with several conclusions supported by convincing data. However, I would like\nto request that the authors address a several major issues, which I will outline below.\nMajor points\n1) The present study demonstrates that TRIM66, an epigenetic repressor, binds to OR\nenhancers and that its absence increases the accessibility of the OR enhancers in\nTrim66-KO mice, suggesting that TRIM66 represses the enhancer hub and contributes\nto the singular expression of OR/TAAR genes. However, given that Trim66 is a\ndownstream gene of ATF5 and that the expression level of Adcy3 is significantly\nreduced in Trim66-KOs (Extended Data Figs. 2c, 4d, 5d), the possibility of secondary\neffects due to insufficient negative-feedback signaling via the UPR cannot be ruled out\nin Trim66-KO mice. Such effects may contribute to the failure to repress multiple OR\ngene expression. To clarify this point, I recommend evaluating this possibility by\nassessing ATF5 protein levels via immunohistochemistry in Trim66-KO mice (since\nthe mRNA level remains unchanged) and by analyzing Lsd1 (Kdm1a) expression levels.\nResponse: We thank the reviewer for the critical thinking. We performed\nimmunohistochemistry staining using ATF5 antibody (Santa Cruz Biotechnology, sc-\n99205X) to evaluate the protein expression levels. Our results showed that the ATF5\nprotein levels remain unchanged between Trim66 +/- and Trim66 -/- mice. Our bulk\nRNA-seq results also revealed no significant changes of Lsd1 (Kdm1a) gene in Trim66\n-/- mice compared to controls. Therefore, we think that the UPR may not contribute to\nthe effects on olfactory receptor gene expression in Trim66 knockout mice. The new\nanalyses and results are provided in the new Supplementary Fig. 4h-4j and shown\nbelow.\nSupplementary Fig. 4: Trim66 knockout leads to decreased mRNA abundance of\nmost olfactory receptor genes. h, Detection of ATF5 proteins in Trim66 knockout\nmice by Immunohistochemistry. Nuclei are labeled by DAPI. Scale bar, 20 μm. i, Violin\nplot depicting mean ATF5 signal intensity of individual cells. The results were\n2\nquantified from images taken from all zones of the MOE. Each dot represents a cell,\nand N represents the number of ATF5 positive cells counted for each genotype. n = 3\nmice. j, mRNA abundance of Lsd1 (TPM) in Trim66 knockout bulk RNA-seq are\nshown.\n2) In addition, the loss-of-function experiments demonstrate that Trim66 is necessary\nfor the singular expression but do not establish its sufficiency in this process. To provide\nconclusive evidence for the critical role of Trim66 in the singular olfactory receptor\ngene expression, it is essential to conduct both loss-of-function and gain-of-function\nexperiments. I believe that incorporating such experiments would significantly enhance\nthe scientific impact of this study. For example, forced expression of Trim66 in\nimmature/mature OSNs in the Trim66 KO background could help determine whether\nTrim66 alone is sufficient to drive singular OR gene expression.\nResponse: We sincerely thank the reviewer for suggesting the gain-of-function\nexperiments to validate TRIM66’s sufficiency in singular OR expression. While we\nfully acknowledge the value of this experiment, generating a Trim66-overexpressing\nmouse model in the Trim66 knockout background would require an extremely long\ntimeline, i.e., around one year to make transgenic mouse line and half a year to cross\nwith Trim66 knockout mice. Viral delivery is an alternative approach for gene rescue\nor overexpression. Unfortunately, virus infection in OSNs has been proven very\ninefficient. For example, Williams et al. (Williams et al., Molecular Therapy, 2017)\ntested multiple AAV serotypes in the mouse MOE and observed that even AAV9,\nwhich has the highest specificity (86% of transduced cells being OSNs), infected very\nlimited number of OSNs. As shown in the representative figure below, AAV9 only\ninfected a few cells out of the whole OMP-positive mature OSNs.\nWilliams et al., Molecular Therapy, 2017, Fig. S6: (a) Representative image shows\na fixed coronal section of OE from control mice 3 weeks after treatment with AAV9-\n3\nmCherry. Coronal section was immunostained for cytokeratin-5 to reveal horizontal\nbasal cells and olfactory marker protein (OMP) to reveal mature OSNs.\nIn another study, Belfort et al. (Belfort et al., Frontiers in Neuroscience, 2025) utilized\nfour relatively efficient AAV serotypes (AAV1, AAV7, AAV-DJ/8, and AAV-rh10) to\ninfect the MOE, and identified 8,970 mature OSNs by snRNA-seq. However, only 143\nof these cells (~1.6%) were successfully transduced, suggesting the extremely low\ninfection efficiency of existing viral tools in the MOE (see the figure below).\nBelfort et al., Frontiers in Neuroscience, 2025, Fig. 2: (D) Cell type distribution with\ntotal cell counts. The x-axis is the cell type with each bar’s color reflecting the\ncorresponding color on the UMAP, and the y-axis is the absolute count of the respective\ncell types. (I) Total AAV+ mOSN cell counts.\nThus, the current technical limitations make gain-of-function experiments challenging\nat this stage. Instead, we have explicitly addressed this limitation in the discussion\nsection (lines 539-545 of the revised manuscript) and proposed future studies using\noverexpression mouse models to validate TRIM66’s function. The revised text is shown\nbelow:\n“While our loss-of-function data demonstrate that TRIM66 is necessary for maintaining\nsingular OR gene expression, we did not assess whether TRIM66 is sufficient for this\nprocess. Due to technical challenges—such as poor viral transduction efficiency in the\nMOE and the lack of a Trim66-overexpressing model—we were unable to perform\ngain-of-function experiments. Further studies using mouse models with forced\nexpression of Trim66 in immature/mature OSNs could help determine whether Trim66\nalone is sufficient to drive singular OR gene expression.”\n3) As described by the authors, single-cell RNA-seq results show decreases in the\nmRNA levels of olfactory receptor gene per cell. On the other hand, the signal\nintensities of ISH for OR RNA and IHC for OR protein do not seem to have changed\nmuch. It is necessary to quantitatively analyze that the expression level (mRNA and\nprotein). Related to this, the IHC images of wt and KO are not convincing at all, because\nthe background signals in the wt sections are largely higher than in the KO images\n(these images appear to be biased by image processing); Fig.2d, Extended Data Figs.\n4\n2d, 2f, 3e, 4b, 5b, and 6d. These images should be re-taken under the same imaging\nconditions.\nResponse: Thank you for the suggestion. For your first concern about signal\nquantification, we have quantified the signal intensities of ISH results for Olfr549,\nOlfr981, Taar6, and Taar7d, and IHC results for Olfr552, Olfr1507, TAAR4, and\nTAAR5 in Trim66 +/− and Trim66 −/− mice. Quantification analyses of single cell signals in both ISH and IHC results are consistent with our single-cell RNA-seq data.\nmRNA levels of Olfr549, Olfr981, Taar6, Taar7d, and protein levels of Olfr552,\nTAAR4, and TAAR5 were significantly reduced, while Olfr1507 protein levels were\nincreased upon Trim66 deletion. The new analyses and results are provided in the new\nSupplementary Fig. 10a-10d and shown below.\nTrim66 knockout mice. a, Quantification of in situ hybridization chromogenic signal\nintensities for Olfr549, Olfr981, Taar6, and Taar7d in Trim66 +/− and Trim66 −/−\nmice. The representative images are shown in Fig. 2g and Supplementary Fig. 4c. n =\n6\n3 mice. b, Single-cell RNA-seq expression levels of the receptor genes shown in (a).\nc, Quantification of immunohistochemistry fluorescence signal intensities for Olfr1507,\nOlfr552, TAAR4, and TAAR5 in Trim66 +/- and Trim66 −/− mice. The representative\nimages are shown in Supplementary Fig. 4e. n = 3 mice. d, Single-cell RNA-seq\nexpression levels of the receptors shown in (c). Each dot represents an individual cell.\nComparison of two groups was carried out using the Mann-Whitney U test (a, c) and\nunpaired Student’s t test (b, d) in R. *p < 0.05, **p < 0.01, ***p < 0.001.\nWe have added these results in lines 365-372 of the revised manuscript. The revised\ntext is shown below:\n“We also quantified the signal intensities of in situ hybridization results for Olfr549,\nOlfr981, Taar6, and Taar7d, and immunohistochemistry results for Olfr552, Olfr1507,\nTAAR4, and TAAR5 in Trim66 +/− and Trim66 −/− mice (Fig. 2h and\nSupplementary Fig. 4d, 4f). Quantification analyses of single cell signals showed\nconsistent results with our single-cell RNA-seq data. mRNA levels of Olfr549, Olfr981,\nTaar6, Taar7d, and protein levels of Olfr552, TAAR4, and TAAR5 were significantly\nreduced, while Olfr1507 protein levels were increased upon Trim66 deletion\n(Supplementary Fig. 10a-10d).”\nTo address the reviewer’s second concern about background differences, we have\ncarefully checked the old images and acquired new representative images under the\nsame staining and imaging conditions to ensure consistency across genotypes. The old\nIHC images that showed different background signals in Fig. 2d, Extended Data Figs.\n2d, 2f, 3e, 4b, 5b, 6d of the previous manuscript have been replaced by the new images\nin Fig. 2d, Supplementary Figs. 3b, 3d, 4e, 6b, 7b, and 8d of the revised manuscript.\nThe figures are also shown below.\n7\n4) In this study, the authors primarily analyze histone modification levels at OR\nenhancers. However, given that TRIM66 interacts with HP1 proteins and H3K9me3,\nthe H3K9me3 levels in the OR gene bodies should also be examined/mentioned.\nAdditionally, the double knockout of G9a (Kmt1c) and GLP (Kmt1d), which are\nrequired for H3K9me3 modification, has been reported to decrease overall OR gene\nexpression while increasing the expression of certain OR genes (Lyons DB et al., Cell\nRep. 2014), a phenotype that appears similar to that of Trim66 KO, though the authors\ndo not cite this paper. To clarify whether Trim66 KO shares the same mechanistic basis,\nI believe it is important to assess this possibility.\nResponse: We thank the reviewer for this insightful suggestion. We analyzed H3K9me3\nCUT&Tag data and observed similar H3K9me3 levels on OR gene bodies in Olfr1507-\nires-tdTomato; Trim66 knockout mice (the new Supplementary Fig. 12h).\nFurthermore, Trim66 knockout bulk RNA-seq revealed unchanged expression levels of\nG9a (Kmt1c) and Glp (Kmt1d) (the new Supplementary Fig. 12i, 12j). These data\nindicate that TRIM66 may not directly modulate the heterochromatin marks on OR\ngenes to regulate their repression levels. We have added this point in lines 433-439 of\nthe revised manuscript and cited the reference (Lyons DB et al., Cell Rep. 2014).\nSupplementary Fig. 12: Loss of Trim66 impairs the maintenance of epigenetic\nsignatures at olfactory enhancers. h, H3K9me3 signals on all functional OR genes in\nOlfr1507 OSNs sorted from Olfr1507-ires-tdTomato; Trim66 knockout mice. i-j,\nmRNA abundance (TPM) of G9a (i) and Glp (j) from Trim66 knockout bulk RNA-seq\ndata are shown.\n5) It is intriguing that abnormal sexual behavior is observed in Trim66 KO mice.\nHowever, given that the vomeronasal system plays a major role in regulating sexual\nand social behaviors in mice, further analysis is needed to substantiate these findings.\nIf the authors choose to retain these results in Figure 6 (Figures 6f–i), it would be\nessential to analyze the expression of vomeronasal receptors in the vomeronasal organ\n(VNO) to determine whether the singular expression is disrupted, as well as to conduct\nphysiological function analyses of the VNO. However, incorporating data on the\nvomeronasal system may dilute the primary focus of this study. To maintain clarity and\ncoherence, I recommend that the section on sexual and social behavior experiments be\nremoved from the manuscript.\nResponse: We sincerely thank the reviewer for this constructive comment. We agree\nthat the expression of vomeronasal receptor genes could be changed in Trim66 -/- mice,\nwhich may lead to defects of sexual and social behaviors. Following the reviewer’s\nrecommendation, we have removed the sexual and social behavior experiments from\nthe revised manuscript. In the future, we plan to analyze the effects of Trim66 on\nvomeronasal receptor gene expression and could incorporate the behavioral data as a\nnew story.\n9\n6) Some aspects of the data interpretation, particularly in the Extended Data, are\ndifficult to follow due to insufficient details in the experimental methods. Notably, the\nmethodology for quantifying the number of expressed cells is not described, making it\nunclear how each data point was obtained (e.g., whether each point represents data from\nan individual animal or data per unit area). For instance, in Extended Data Figure 6d,\nthere is substantial variation in the number of apoptosis-positive cells, but the meaning\nof each data point remains unclear. To improve clarity, the authors should explicitly\ndescribe the method used to obtain each data point. For the quantification of apoptosis-\npositive cells, I recommend acquiring multiple sections from similar positions in the\nolfactory epithelium and presenting the data as either the sum of the slides or the\nnumber of positive cells per unit area. This approach would help minimize inter-\nindividual variability. The details of these experimental methods should be submitted\nas the Extended Methods.\nResponse: We have revised the Methods section to clarify the quantification procedures.\nFor each mouse, we collected the tissue on 20–25 slides, with each slide containing five\nMOE sections. To quantify the number of positive cells, three mice (n = 3) and at least\nthree intact MOE sections at similar anatomical positions per mouse were used for each\ngenotype. In the quantification figures, each data point represents the total number of\npositive cells counted in one MOE section. The details have been added in lines 683-\n690 and 715-723 in the Methods section of the revised manuscript.\n7) The ISH analysis of Trim66 in Goofy-Cre; Trim66 fl/fl cKO mice showed little\nexpression of Trim66 (Extended Data Fig. 5a), with no apparent differences in Trim66\nexpression between cKO and global KO. The authors stated that Trim66 is highly\nexpressed in late progenitors and in immature/mature OSNs, whereas Goofy-Cre-\nmediated excision of Trim66 occurs in immature OSNs, preserving Trim66 expression\nin late progenitors. Alternatively, the results presented in Fig. 1e may be inaccurate.\nUnfortunately, as mentioned above, the IHC results (Extended Data Fig. 5a) do not\nallow for a definitive conclusion on this issue. It is essential to rigorously assess whether\nTrim66 is truly knocked out after the immature OSN stage. To clarify the expression\nprofile of Trim66, a co-expression analysis using OSN differentiation markers (e.g.,\ndouble-label ISH or a combination of IHC and ISH with probes for Ascl1, Neurog1,\nNeurod1, Gap43, and OMP) is required. These additional analyses are necessary to\naddress the concerns raised above.\nResponse: We thank the reviewer for raising this concern. To clarify the expression\nprofile of Trim66, we performed co-expression analysis using ISH for Trim66\ncombined with IHC for GAP43 (immature OSN marker) and OMP (mature OSN\nmarker), as well as reporter staining in Ascl1-CreERT2; lsl-tdTomato (label early\nneuronal progenitor) and Ngn1-CreERT2; lsl-ZsGreen (label late neuronal progenitor)\nmice injected with tamoxifen. These experiments showed that Trim66 is not expressed\nin Ascl1-positive early neuronal progenitors, but is expressed in Ngn1-positive late\nneuronal progenitors and immature/mature OSNs. The new analyses and results are\n10\nprovided in the new Supplementary Fig. 1e and shown below. These findings are\nconsistent with transcriptomic analyses of different cell populations in the MOE (Fig.\n1e) .\nSupplementary Fig. 1: ATF5 can bind to known regulatory genes. e, Detection of\nTrim66 by in situ hybridization (Green color) combined with immunohistochemistry\nfor GAP43 (immature OSN marker) and OMP (mature OSN marker), as well as\nreporter staining in Ascl1-CreERT2; lsl-tdTomato (early neuronal progenitor marker)\nand Ngn1-CreERT2; lsl-ZsGreen (late neuronal progenitor marker, pseudo colored in\nred) mice injected with tamoxifen. White arrows indicate cells co-expressing Trim66\nand ZsGreen; yellow arrows indicate ZsGreen positive cells without Trim66 expression.\nScale bar, 20 μm.\nIn terms of the knockout efficiency of Trim66 global and Goofy-cre conditional\nknockout mice, we checked the expression pattern of Goofy gene in different cell\npopulations. The transcriptomic analyses of different cell populations in the MOE\nrevealed that Goofy is expressed not only in immature and mature OSNs but also in late\nprogenitor cells (see Figure 1 for reviewers below). This explains why Trim66 is\nnearly undetectable in Goofy-Cre; Trim66 f/f mice, resembling global Trim66 knockout\nmice. Taken together, these results confirm that Trim66 gene is mainly expressed in\nlate neuronal progenitors and immature/mature OSNs, and Trim66 gene is almost\ncompletely depleted in both Trim66 -/- (global knockout) and Goofy-Cre; Trim66 f/f\n(cKO) mice.\ncell types of MOE. RNA-seq data is provided by Stavros Lomvardas’ group, n = 2 mice.\n8) Two factors influence OR expression levels: the amount of mRNA per cell and the\nnumber of expressing cells (i.e., probability of choice). While this study primarily\nfocuses on mRNA levels, little consideration is given to the probability of choice. To\nprovide a more comprehensive analysis, the authors should clarify the correlation\nbetween total mRNA levels and the number of expressing cells. Additionally, a\ndiscussion on expression probability should be included to determine whether Trim66\ncontributes to the regulation of expression probability.\nResponse: To address this point, we analyzed the correlation between OR and Taar\ngenes mRNA abundance (TPM) in Trim66 knockout bulk RNA-seq data and the\nnumber of cells expressing each gene based on in situ hybridization and\nimmunohistochemistry data. As shown in the new Supplementary Fig. 4g, we\nobserved a strong positive correlation between the changes of total mRNA levels and\nthe changes of the cell numbers after Trim66 knockout (Pearson’s r = 0.94, p = 1.5e-\n09). This suggest that changes in expression probability also contribute to receptor gene\nexpression regulation.\nmost olfactory receptor genes. g, Correlation analysis between mRNA abundance\n(TPM) from bulk RNA-seq and the number of expressing cells determined by in situ\nhybridization (from Fig. 2h and Supplementary Fig. 4d) and immunohistochemistry\n(from Supplementary Fig. 4f) for OR and Taar genes in Trim66 knockout mice. OR\nand Taar gene names in italics and non-italics represent quantified positive cells by in\nsitu hybridization and immunohistochemistry, respectively. Red and green indicate\ngenes with increased and decreased mRNA abundance in Trim66 knockout bulk RNA-\nseq, respectively. Pearson correlation coefficient (r) and p value are shown.\nAccording to the reviewer’s suggestion, we have added this point on expression\nprobability in lines 226-231 of the revised manuscript. The revised text is shown below:\n“We further analyzed the correlation between receptor genes mRNA abundance in\nTrim66 knockout bulk RNA-seq data and the number of cells expressing each gene or\nprotein based on in situ hybridization and immunohistochemistry data (Fig. 2h and\nSupplementary Fig. 4d, 4f). We observed a strong positive correlation between the\nchanges of total mRNA abundance and the changes of the cell numbers after Trim66\nknockout (Supplementary Fig. 4g). This analysis suggests that Trim66 could regulate\nthe expression probability of olfactory receptor genes.”\nIn addition, we have integrated this point with a similar comment raised by Reviewer 2\nabout the potential effect of changes in immature/mature cell populations on receptor\nexpression. The revised Discussion in lines 579-588 of the revised manuscript is also\nshown below:\n“The altered mRNA abundance of ORs and Taars observed in Trim66 -/- mice is likely\ninfluenced by three key mechanisms. First, the disruption of Trim66 leads to a change\nin the relative proportions of immature and mature OSNs in the MOE, which potentially\nalters the overall transcriptomic landscape. Second, the loss of Trim66 impairs the\nregulatory mechanism that normally ensures singular OR/Taar gene expression at high\nlevels in mature OSNs. As a result, multiple OR/Taar genes are co-expressed within\nindividual mature OSN at low levels in Trim66 -/- mice. Third, the numbers of cells\nexpressing most OR/Taar genes (i.e., probability of choice) are decreased in Trim66 -\n/- mice, suggesting that TRIM66 also regulates the probability of receptor gene choice.\nTogether, these mechanisms contribute to the widespread downregulation of OR and\nTaar gene expression in Trim66-deficient mice.”\n9) In the Discussion, the authors mention to the results of Fig. 1-4 but not Fig 5 and 6.\nAdditional discussion on these is required.\nResponse: According to the reviewer’s suggestion, we have added discussion on Figs.\n5 and 6 in lines 590-603 of the revised manuscript. The revised text is shown below:\n13\n“In Trim66 -/- mice, we observed increased neuronal activity in the OB, as indicated by\nelevated spontaneous firing rates of M/T cells and altered odor-evoked responses. This\nphenotype is very likely a direct consequence of disrupted singular olfactory receptor\ngene expression in OSNs. In the absence of Trim66, mature OSNs co-express multiple\nORs, leading to aberrant axonal projections that converge onto multiple glomeruli. As\na result, individual odors may activate a broader set of glomeruli, subsequently\nrecruiting a larger population of M/T cells and contributing to the observed\nhyperactivity in the OB. In addition, other possibilities may also contribute to the\naltered OB neuronal activity. First, Trim66 may regulate the expression of axon\nguidance or targeting molecules that directly influence OSN axonal projection patterns.\nSecond, since our electrophysiology recordings were performed in Trim66 global\nknockout mice, we cannot exclude the possibility that Trim66 may have functional roles\nwithin the OB itself. Although Trim66 is expressed in the OB at much lower levels than\nin the MOE, its loss may affect the intrinsic excitability of OB neurons or interfere with\nthe development of local circuits, such as connections between M/T cells and\ninterneurons, thereby affecting the overall network activity.”\nMinor points\n10) Abstract, page 2, line 31: The phrase “very low level” may not be entirely accurate,\nas the single OR expression levels in Trim66-/- OSNs exhibit broad distribution. Please\nconsider revising this wording for precision.\nResponse: We agree with the reviewer’s suggestion. We have revised this sentence in\nlines 27-29 of the revised manuscript. The revised text is shown below:\n“Here, we discover TRIM66 as a key repressor. Multiple receptor genes are retained at\nlow levels in most single mature OSNs after deletion of Trim66, leading to decreased\nexpression of the vast majority of olfactory receptor genes.”\n11) Abstract, page 2, lines 36–37: The statement “with broader implications for other\nmonoallelic or monogenic expression systems” appears to be an overgeneralization.\nThe singular expression of immune receptor genes, for example, is governed by a\ndifferent mechanism involving gene rearrangement. Please refine this statement\naccordingly.\nResponse: We have revised this sentence in lines 32-34 of the revised manuscript. The\nrevised text is shown below:\n“Our study provides the missing link in understanding the transition from polygenic to\nmonogenic olfactory receptor expression.”\n12) Introduction, page 3, line 60: The phrase “the functional OR genes are clustered”\nis not entirely accurate, as some OR genes exist as singletons. Please revise this\n14\nsentence to reflect this nuance.\nResponse: We have revised this sentence in lines 58-60 of the revised manuscript. The\nrevised text is shown below:\n“In mouse, majority of functional OR genes are clustered in all chromosomes except\nchromosomes 12 and 18, and Y, with some OR genes exist as singletons. The Taar\ngenes are all located in chromosome 10 forming a single Taar gene cluster.”\n13) Page 4, line 70, and page 16, line 343: The phrase “also called Greek Islands”\nshould be removed, as not all OR enhancers belong to Greek Islands (e.g., H, P, and J\nelements).\nResponse: Thank you for pointing this out. We have removed the phrase “also called\nGreek Islands” to avoid misclassification.\n14) Results, page 6, lines 121–123: The statement, “This similarity may explain the\nobservations that Atf5-knockout pups still developed some mature neurons and that a\nsmall fraction of those pups survived into adulthood,” is debatable. The maturation of\nsome OSNs may result from upregulated OR genes, and the survival of a small fraction\nof Trim66 KO pups may depend on maternal care. To support the latter claim, it would\nbe necessary to verify whether these pups can reach adulthood through artificial\nlactation. Otherwise, please revise this statement accordingly.\nResponse: We have revised this sentence in lines 119-121 of the revised manuscript.\nThe revised text is shown below:\n“Furthermore, the top DNA motif in ATF5 peaks is nearly identical to the one for ATF4\n(Supplementary Fig. 1a). The potential compensatory effect of ATF4 may explain the\nobservations that Atf5-knockout pups still developed some mature neurons.”\n15) Page 7, line 153: The phrase “by indirect consequences of having fewer mature\nneurons in the knockout mice” is unclear. It seems that the reduced expression of\nTrim66 in Atf5 KO results in fewer mature neurons. Please clarify and revise this\nsentence for accuracy.\nResponse: We appreciate the reviewer’s comment. We have deleted this statement to\navoid confusion. The revised sentence in lines 151-152 of the revised manuscript is\nshown below:\n“Note that fold changes can be confounded by potential rescue by ATF4 and by the\ndetection limit and nonlinearity of microarray data.”\n15\n16) Page 9: A description of the general phenotype of Trim66-KO mice should be\nincluded. Relevant details such as body weight, survival rate, growth rate, and fertility\nshould be provided.\nResponse: We appreciate the reviewer’s suggestion. As we stated in the original\nmanuscript, we observed defects of sexual and social behaviors in Trim66 -/- mice.\nTherefore, the fertility of Trim66 -/- mice is greatly impaired. We crossed Trim66 +/-\nmale with Trim66 +/- female mice to generate homozygous mice for our experiments.\nAs suggested by the reviewer, the defects in sexual and social behaviors of Trim66 -/-\nmice may be complicated by the potential role of Trim66 in the VNO system. Thus, we\nhave not included the description of fertility in the manuscript. On the other hand, a\nprevious study (Chen et al., Nature Communications, 2019) has reported that the birth\nrate of Trim66 +/- and Trim66 -/- pups was in accordance with Mendel’s law after\nheterozygote crossing. The body weight of Trim66 -/- mice was significantly decreased\ncompared with littermates at the age of 2 or 3 weeks. Following the reviewer’s\nsuggestions, we have included the brief description in lines 180-183 of the revised\nmanuscript.\n17) Throughout the text and figure legends, please specify the age of the mice analyzed.\nResponse: We thank the reviewer for the suggestion. The ages of the mice analyzed\nhave now been specified throughout the main text and figure legends where relevant.\n18) Page 10, line 209: The phrase “increase in OSNs expressing” should be revised to\n“increase in the number of OSNs expressing” for clarity.\nResponse: We have revised this sentence in lines 217-221 of the revised manuscript.\nThe revised text is shown below:\n“We quantified cells with positive mRNA expression signals and observed an increase\nin the number of OSNs expressing Olfr571, Olfr464, Olfr1507, and Taar6, as well as\na decrease in OSNs expressing Olfr686, Olfr578, and Olfr549, Olfr981, Olfr1015,\nOlfr959, Olfr3, Olfr1463, Taar7b, Taar7d, and Taar9, which was in accordance with\nthe RNA-seq results.”\n19) Page 11, line 234: The sentence “the expression levels of all Tarr genes were not\nsignificantly changed” is inaccurate, as the expression levels of individual Tarr genes\nshow significant changes (some being upregulated, others downregulated, with three\nexceptions). Please revise this statement to accurately reflect the observed data.\nResponse: We appreciate the reviewer’s comments. We have revised this sentence in\nlines 258-260 of the revised manuscript. The revised text is shown below:\n16\n“This is consistent with the observation that the sum of the expression levels of the Taar\ngene family, measured as the total TPM of all Taar genes, was not significantly changed\nafter Trim66 deletion.”\n20) Page 14, lines 307–309: The statement “Moreover, the cells showing polygenic\nreceptor expression were almost exclusively located in the apical MOE, where mature\nOSNs reside” presents an intriguing observation. Since multiple OR genes are co-\nexpressed in both immature and mature OSNs in Trim66-KO mice, please elaborate on\nwhy co-expression was predominantly observed in mature OSNs and discuss the\npotential underlying mechanisms.\nResponse: We thank the reviewer for the thoughtful comment. As shown in our single-\ncell RNA-seq data from Trim66 knockout mice, multiple OR genes are co-expressed in\nboth immature and mature OSNs after Trim66 knockout. However, the expression\nlevels of these co-expressed OR/Taar genes in immature OSNs are extremely low. In\ncontrast, their expression levels in mature OSNs are relatively higher than those in\nimmature OSNs, allowing for more reliable detection by in situ hybridization (Fig. 3h;\nsee also figure below). Therefore, the apparent restriction of multiple ORs/Taars\nexpression to mature OSNs likely reflects the limitation of detection methods rather\nthan a true biological absence in immature OSNs. To more accurately assess ORs/Taars\nco-expression in immature OSNs located in the basal region of the MOE, more sensitive\ntechniques may be required.\nFig. 3: Trim66 knockout leads to expression of multiple olfactory receptors at low\nlevels in mature OSNs. h, Violin plot showing the receptor gene expression levels in\nimmature OSNs and mature OSNs that express multiple olfactory receptors obtained\nfrom Trim66 knockout single-cell RNA-seq data. The expression of receptor genes\nranked in the top 3 levels are shown.\n21) Page 14, lines 309–311: The authors state, “Additionally, the increased numbers of\nOlfr1507 glomeruli in the Olfr1507-ires-tdTomato; Trim66 -/- mice compared to\ncontrol mice could also be due to other co-expressed olfactory receptors in\nOlfr1507-expressing OSNs.” I suggest mentioning the zonal expression of Olfr1507,\nas OR expression in an ectopic zone could result in multiple glomerular projections.\nusing in situ hybridization. As shown in the new Supplementary Fig. 5c (see also\nfigure below), Olfr1507 is primarily expressed in zone 5, and this zonal expression\npattern remains largely preserved in Trim66 -/- mice. Therefore, we believe that the\nmultiple glomerular projections of Olfr1507 OSNs is largely due to multiple receptors\nexpression.\nSupplementary Fig. 5: Zonal expression pattern of ORs and Taars in Trim66\nknockout mice. c, In situ hybridization for Olfr1507 on coronal sections of the MOE.\nOlfr1507 is primarily expressed in zone 5 in both genotypes, and its zonal distribution\nremains largely preserved in Trim66 -/- mice. Scale bar, 500 μm.\nIn addition, we also examined the zonal expression of two upregulated gene, Olfr571\nand Taar6. Olfr571, which is normally confined to zone 2, exhibited ectopic expression\nin zone 1 (dorsal MOE) in Trim66 -/- mice. Likewise, Taar6, which is usually restricted\nto the ventral MOE, showed expanded expression into more dorsal regions in Trim66 -\n/- mice. These results suggest that Trim66 regulates the zonal distribution of a subset of\nOR and Taar genes. These images are provided in the new Supplementary Fig. 5d-e\nand shown below.\nknockout mice. d–e, In situ hybridization for Olfr571 (d) and Taar6 (e). Both receptor\ngenes show ectopic expression in the dorsal MOE following Trim66 deletion. Scale\nbars, 500 μm (d,e, left) and 100 μm (d,e, right).\nWe have added this point on zonal expression in lines 264-273 of the revised\nmanuscript. The revised text is shown below:\n“To examine whether the zonal expression patterns of OR and Taar genes are altered\nin Trim66 -/- mice, we performed in situ hybridization for Olfr1507, Olfr571, and Taar6\n(Supplementary Fig. 5c-5e). Olfr1507 is normally expressed in zone 5, and its zonal\nexpression remained largely unchanged in Trim66 -/- mice (Supplementary Fig. 5c).\nIn contrast, Olfr571, which is normally confined to zone 2, exhibited ectopic expression\nin zone 1 (dorsal MOE) in Trim66 -/- mice (Supplementary Fig. 5d). Likewise, Taar6,\nwhich is usually expressed in the ventral MOE, showed expanded expression into more\ndorsal regions in Trim66 -/- mice (Supplementary Fig. 5e). These results suggest that\nthe loss of Trim66 can lead to disrupted zonal distribution in a subset of OR and Taar\ngenes, potentially contributing to the defects in glomerular targeting.”\n22) Page 14, line 314: The reference (Extended Data Fig. 6c and Extended Data Fig.\n2c-f) should be corrected to (Extended Data Fig. 2c-f).\nResponse: We appreciate the reviewer’s careful reading. We have re-examined our\nmanuscript and confirm that the original citation is correct. Extended Data Fig. 2c–f\n(updated to Supplementary Fig. 3a-3d in the revised version) presents results from\nTrim66 bulk RNA-seq and IHC staining analyses, while Extended Data Fig. 6c\n(updated to Supplementary Fig. 8c) shows that the proportions of mature OSNs were\n19\ndecreased in single-cell RNA-seq data. Thus, the two figures are both relevant to the\nstatement. Below, we provide the original description from the manuscript along with\nthe corresponding figure panels for your reference.\n“The downregulation of most olfactory receptor genes could be resulted from the\ndecreased number of mature OSNs (Extended Data Fig. 6c and Extended Data Fig.\n2c-f) as well as reduced expression levels in single OSNs.”\nExtended Data Fig. 6 | Trim66 knockout leads to expression of multiple olfactory\nreceptors at low levels in mature OSNs. c, Stacked bar plot comparing the proportions\nof various MOE cell types in the single-cell RNA-seq data of Trim66 knockout mice.\nHBCs, horizontal basal cells; GBCs, globose basal cells; INPs, immediate neuronal\nprecursors; iOSNs, immature OSNs; mOSNs, mature OSNs; SUSs, sustentacular cells;\nGC-D, guanylate cyclase-D; MVs, microvillous cells. UD, undefined.\ndifferent cell types in MOE. c, Changes in marker genes for different cell types in the\nMOE of Trim66 knockout mice. The Y-axis represents normalized TPM of marker\ngenes. Marker genes validated in immunohistochemistry experiments (d-f) are\nhighlighted in red. d, Immunohistochemistry of OMP (top) and GAP43 (bottom) in\nTrim66 knockout mice. Scale bar, 20 μm. e, The numbers and thickness ratios of OMP-\nand GAP43-positive cells in 300 μm MOE. The thickness ratio is calculated as the\nthickness of positive cells (yellow line in d) divided by the thickness of the entire MOE\n(white line in d). n = 3 mice. f, Immunohistochemistry of KRT14 (top) and SOX2\n(bottom) in Trim66 knockout mice. Scale bar, 20 μm.\n23) Methods, page 24, line 522-524 and Acknowledgements: Please specify the origin\nof the mouse lines used (e.g., gifted from XX or JAX accession#). Additionally, any\ngifted animal lines and materials should be properly acknowledged in the\nAcknowledgements section.\nResponse: We have added the origins of all mouse lines and Acknowledgements in\nlines 612-618 and 1155-1159 of the revised manuscript, respectively. The revised text\nis shown below:\n“Ai6 (Lsl-ZsGreen, #007906) and Ai9 (Lsl-tdTomato, #007909) mouse lines were\nobtained from Jackson Laboratory. Trim66 global knockout, Trim66 flox/flox,\nOlfr1507-ires-tdTomato, TAAR enhancer 1-GFP, and TAAR enhancer 2-tdTomato\nwere generated from our laboratory. Other mouse lines were kindly shared by other\nlabs, including Foxg1-Cre (Dr. Zunyi Zhang), Goofy-Cre and J-gVenus (Dr. Junji\nHirota), Omp-ires-Cre (Dr. Catherine Dulac), Ascl1-creERT2 (Dr. Zhiyong Liu), Ngn1-\ncreERT2 (Dr. Lisa Goodrich), and Olfr73-ires-GFP (Dr. Sandeep Robert Datta).”\nAcknowledgements：\nWe thank Dr. Zunyi Zhang for sharing the Foxg1-Cre line, Dr. Junji Hirota for sharing\nthe Goofy-Cre and J-gVenus lines, Dr. Catherine Dulac for sharing the Omp-ires-Cre\nline, Dr. Zhiyong Liu for sharing the Ascl1-creERT2 line, Dr. Lisa Goodrich for sharing\nNgn1-creERT2, and Dr. Sandeep Robert Datta for sharing the Olfr73-ires-GFP line.”\n24) Page 26, line 579: The full name of the DIG abbreviation should be written where\nit the first place it appears; line 669.\nResponse: We have added the full term for the abbreviation “DIG (digoxigenin)” when\nfirst mention in line 670 in the revised manuscript.\n25) Page 27: Correct the name of the antibodies, e.g., Alexa Fluo 488 should be Alexa\nFluor® 488.\nforms in lines 708-713, including changing “Alexa Fluo 488” to “Alexa Fluor® 488”\nand ensuring consistent and accurate naming of Cy3-conjugated antibodies. The revised\ntext is shown below:\n“Including Donkey Anti-Rabbit IgG conjugated to Alexa Fluor® 488 (Jackson\nImmunoResearch, 711-545-152), Donkey Anti-Rabbit IgG conjugated to Cyanine\nCyTM3 (Jackson ImmunoResearch, 711-165-152), Donkey Anti-Guinea Pig IgG\nconjugated to Alexa Fluor® 488 (Jackson ImmunoResearch, 706-545-148), and\nDonkey Anti-Guinea Pig IgG conjugated to Cyanine CyTM3 (Jackson ImmunoResearch,\n706-165-148).”\n26) Page32, ChIP experiment: The catalog number for the anti-TRIM66 antibody is\nmissing and should be provided.\nResponse: We have added the catalog number for the anti-TRIM66 antibody used in\nthe ChIP experiment in the revised manuscript. In addition, we have updated the\nMethods section to include catalog numbers and detailed information for all antibodies\nused in the ChIP-seq experiments in lines 831-833. The revised text is shown below:\n“Antibodies used for ChIP-seq include ATF5 (Santa Cruz Biotechnology, sc-99205X),\nTRIM66 (Sigma-Aldrich, HPA027420), EZH2 (Cell Signaling Technology, 5246),\nEZH2 (Active Motif, 33902), and YY1 (Santa Cruz Biotechnology, sc-1703X).”\n27) References: The authors should ensure all references are correctly cited and\nformatted. For instance: #31 is missing a title and journal name, #51 appears to be\nduplicated with #38, #60 lacks a page number.\nResponse: We sincerely thank the reviewer for carefully checking the references and\npointing out these issues. We have revised the reference list in the same format.\n28) Figures 2b: The timing of Trim66 deletion is misaligned with the Cre expressions\nin cKO mice. Please revise the figure accordingly.\nResponse: We have revised Fig. 2b to accurately reflect the timing of Cre expression\nand Trim66 deletion (see figure below).\ngenes. b, Schematic illustration of the genetic manipulation of Trim66 expression\nduring OSN differentiation. For conditional knockout mice, Trim66 flox mice were\ncrossed with Foxg1-cre mice to obtain mice with Trim66 knockout in the early\ndevelopment of the MOE. Similarly, crossing Trim66 flox mice with Goofy-cre mice\nresulted in Trim66 knockout in both mature and immature OSNs. HBCs, horizontal\nbasal cells; GBCs, globose basal cells; iOSNs, immature OSNs; mOSNs, mature OSNs.\n29) Figure 4 Legends: The following portion in panel (g) overlaps with (f), “and each\nexperiment was performed on Olfr1507 OSNs sorted from 1 Olfr1507-ires-tdTomato;\nTrim66 -/- mouse or from 3-4 Olfr1507-ires-tdTomato; Trim66 +/- mice.”\nResponse: We have revised the legend of Fig. 4. The description was kept in Fig. 4f\nlegend and this same sentence was deleted in the Fig. 4g legend.\n30) Extended Data Figures and Tables, p83, Foxg1-cre;Trim66 flox bulk RNAseq:\nPlease confirm and explicitly state the number of sexes and animals used in Extended\nData Figures and Tables.\nResponse: Sorry for the mistake. We have confirmed and corrected the number of sexes\nand animals used in the Supplementary Table 3. The revised table is shown below:\nSex Age Number\nTrim66 knockout bulk RNA-seq\n6 males and 4 females P28 Homo = 5, Het = 5\nFoxg1-cre; Trim66 flox bulk RNA-seq\n4 males and 4 females P30 Homo = 4, Het = 4\n31) Extended Data Fig. 3a and its legend: In the inner circle, data should be categorized\nas follows: significantly upregulated genes (red), significantly downregulated genes\n23\n(green), and not significantly changed genes (grey). Please revise the figure accordingly.\nResponse: We have revised the figure and legend following the reviewer’s suggestion.\nThe new Supplementary Fig. 4a (original Extended Data Fig. 3a) is also shown\nbelow.\nSupplementary Fig. 4: Trim66 knockout leads to downregulation of most olfactory\nreceptor genes. a, The Circos plot depicting changes of functional ORs and Taars in\nTrim66 -/- vs. Trim66 +/- mice. The outer circle shows the chromosomal locations of\nORs and Taars, blue represents class II ORs, red represents class I ORs, and yellow\nrepresents Taars. In the inner circle, red indicates significantly increased mRNA\nabundance of receptor genes, green indicates decreased mRNA abundance of receptor\ngenes, and grey indicates receptor genes without significant changes.\n32) Extended Figs. 4 and 5: These figures should be formatted consistently\n(i.e.,presented in the same order).\nResponse: We have formatted the two figures in the same order. They were provided\nas the new Supplementary Figs. 6 and 7 in the revised manuscript.\n33) Extended Data Fig. 4 legend, line 1279: “in 2a” should be “in Fig. 2a”.\nResponse: We have corrected this typo.\n34) Extended Data Fig. 6e: The vertical scale is not labeled as a percentage. Please add\nthe appropriate label.\nupdated panel now appears as Supplementary Fig. 8e in the revised manuscript (see\nalso figure below).\nSupplementary Fig. 8: Trim66 knockout leads to expression of multiple olfactory\nreceptors at low levels in mature OSNs. e, Percentage of mature OSNs expressing 0,\n1, or multiple ORs thresholded by the unique molecular identifiers (UMIs) required for\nan OR gene to be considered “detected” in Trim66 +/- mice.\n35) Extended Data Fig. 6g: The legend states, “Pearson correlation coefficient (r) and\np-value are shown” (lines 1334–1335), but these values are not displayed in the figure.\nPlease ensure they are included.\nResponse: We have revised the figure to include the Pearson correlation coefficient r =\n0.78 and p < 2.2e-16, and the panel now appears as Supplementary Fig. 8g in the\nrevised manuscript (see also figure below).\nreceptors at low levels in mature OSNs. g, Pearson correlation of normalized counts\nratio (the percentage of normalized counts in homozygous mice among the sum of\nnormalized counts in heterozygous and homozygous mice) of functional OR genes\nobtained from Trim66 knockout single-cell RNA-seq (X-axis) and Trim66 knockout\nbulk RNA-seq (Y-axis). Pearson correlation coefficient (r) and p value are shown.\nReviewer #2 (Remarks to the Author):\nThe monogenic, monoallic expression of odorant receptor (OR) genes by olfactory\nsensory neurons (OSNs) is a leading example and model of mechanisms allowing\ntissues – especially neural tissues - to produce multiple subtypes of key cells. The main\nconclusion from this study is that Trim66 is a repressor of OR and trace amine-\nassociated receptor (TAAR) gene expression during the differentiation of OSNs. The\nauthors provide evidence that the singularity of OR expression in OSNs is disturbed in\nthe absence of Trim66, that Trim66 can bind at enhancer and promoters of OR genes,\nand that binding at enhancers correlates with OR mRNA abundance differences when\nTrim66 is absent. The consequent disruptions in OR expression patterns lead to\ndifferences in OSN projections to olfactory bulb glomeruli, in the physiology of\nolfactory bulb neurons, and altered olfactory behaviors.\nThe authors interpret their data in a way that emphasizes the role of Trim66 in\ndetermining the level of expression of OR and TAAR genes. Yet the level of expression\nis not directly measured. To do so requires quantifying the rate of transcription\n(transcripts produced per unit time). Instead, what is measured is the abundance of each\nmRNA species. This alternative approach is convenient, high throughput, and very\ncommon, but only sometimes reflects the level of expression. For example, in cases\nwhere differences in cell type abundance occur, which is clearly true after KO of\nTrim66, measures of mRNA abundance are not sufficient to conclude that level of\nexpression is affected. Instead of assuming that measures of mRNA abundance directly\nreflect levels of gene expression, one must use evidence, typically multiple lines of\nevidence, to reach the conclusion that gene expression differs. To their credit, the\nauthors generate data leading toward this conclusion. It should be described as a\nconclusion, not as an assumption.\nWhy should the authors be so strict in their descriptions? Thinking that mRNA\nabundance differences equal different levels of gene expression is a trap that leads to\nsilly or impossible conclusions. For example, on line 317 the authors write, “the\naveraged OR gene levels in immature and mature . . .” Obviously, genes do not have\nlevels but 2 copies per somatic cell. Another example begins on line 323. “We found\nthat both the upregulated and downregulated OR genes showed decreased expression\nlevels in Trim66 -/- mice” This sentence contradicts itself. If an OR gene is upregulated\nthen its expression cannot be less.\nabundance or transcript abundance rather than gene expression level. Similarly, use\nincreases or decreases in mRNA abundance rather than upregulation or downregulation\nin both the text and the figures. The Discussion should include an explicit argument\nthat differences in OR/TAAR mRNA abundance between WT and KO mice are due to\ntwo factors. (1) Differences in the relative abundance of cell types in the OE, especially\nbetween immature and mature OSNs. (2) Reduced effectiveness of the feedback\nmechanism involving Trim66 that helps suppress expression of more than one OR gene.\nThis leads to less than normal amounts of the major OR mRNA and continued\nexpression of other OR genes.\nResponse: We thank the reviewer for this important and constructive comments. We\nagree that mRNA abundance is not a direct measure of transcriptional activity and that\ncell type composition can strongly influence the observed transcript levels. In response,\nwe have replaced “level of expression” and “upregulation/downregulation” with\n“mRNA abundance” and “increased/decreased mRNA abundance” in the bulk RNA-\nseq analyses of the revised manuscript, respectively.\nTo address the reviewer’s second concern about factors influencing receptor gene\nchanges, we have modified Discussion section to include an explicit explanation. In\naddition, we have integrated this point with a similar comment raised by Reviewer 1\npoint 8 about the potential effect of changes in receptor gene choice on receptor\nexpression. The revised Discussion in lines 579-588 of the revised manuscript is also\nshown below:\n“The altered mRNA abundance of ORs and Taars observed in Trim66 -/- mice is likely\ninfluenced by three key mechanisms. First, the disruption of Trim66 leads to a change\nin the relative proportions of immature and mature OSNs in the MOE, which potentially\nalters the overall transcriptomic landscape. Second, the loss of Trim66 impairs the\nregulatory mechanism that normally ensures singular OR/Taar gene expression at high\nlevels in mature OSNs. As a result, multiple OR/Taar genes are co-expressed within\nindividual mature OSN at low levels in Trim66 -/- mice. Third, the numbers of cells\nexpressing most OR/Taar genes (i.e., probability of choice) are decreased in Trim66 -\n/- mice, suggesting that TRIM66 also regulates the probability of receptor gene choice.\nTogether, these mechanisms contribute to the widespread downregulation of OR and\nTaar gene expression in Trim66-deficient mice.”\nOther major criticisms\n1. The turnover of OSNs is not adequately investigated given that the manuscript\npresents evidence that turnover is elevated. For example, increases in Ascl1+ GBCs, in\nHBCs, in and immature OSNs in the KO all point toward increased turnover. The\nmeasure of apoptosis is not normalized to the extent of epithelium inspected (e.g.,\nblinded counts per unit of length or volume of tissue) and absent is a measure of basal\n27\ncell proliferation; the best of which is phospho-histone 3 immunoreactivity. The\ndepth/thickness of the OE should also be measured. If turnover is increased then the\nOE will be thinner in KO mice.\nResponse: We appreciate the reviewer’s insightful comment. In response, we\nperformed additional experiments to more rigorously assess OSNs turnover in Trim66\n-/- mice. We conducted immunohistochemistry for both phospho-histone H3 (PH3) and\nKi67, two established markers of proliferating cells. We quantified the number of\npositive cells per unit length of the MOE. Indeed, Ki67 and PH3 positive cells showed\na significant increase in the Trim66 -/- mice compared to those in controls. These results\nsuggest enhanced basal cell proliferation in Trim66 -/- mice (the new Supplementary\nFig. 3e, 3f; see also figure below). We also measured the thickness of the MOE and\nfound that the MOE was thinner in Trim66 -/- mice (the new Supplementary Fig. 3g;\nsee also figure below).\nSupplementary Fig. 3: Trim66 knockout leads to changes in marker genes for\ndifferent cell types in MOE. e, Immunohistochemistry analyses for Ki67 (top) and\nPH3 (bottom) in Trim66 knockout mice. Scale bar, 20 μm. f, The numbers of Ki67 (left)\nand PH3 (right) positive cells per mm in each region of interest (ROI) of the MOE.\nEach dot represents one ROI. n = 3 mice. g, Bar plot showing average MOE thickness\nin 300 μm regions of the MOE. Each dot represents one region. n = 3 mice.\nThe details have been added in lines 194-200 in the Results section of the revised\nmanuscript. The revised text is shown below:\n“To further evaluate the impact of Trim66 deletion on OSNs turnover, we performed\nimmunohistochemistry for phospho-histone H3 (PH3) and Ki67, the two established\nmarkers of proliferating cells. Quantification of PH3 and Ki67 positive cells per unit\nlength of the MOE revealed a significant increase of proliferating cell numbers in\nTrim66 -/- mice compared to those in controls (Supplementary Fig. 3e, 3f). In addition,\nmeasurement of MOE thickness showed a significant reduction in Trim66 -/- mice\n(Supplementary Fig. 3g), consistent with elevated epithelial turnover.\nCaspase-3 positive cells to the length of the MOE, providing a more accurate and\nquantitative comparison between genotypes (Supplementary Fig. 8d; see also figure\nbelow). The figure and the related legend have been updated in the revised manuscript.\nSupplementary Fig. 8: Trim66 knockout leads to expression of multiple olfactory\nreceptors at low levels in mature OSNs. d, Representative images of cleaved\nCaspase-3 expression in Trim66 knockout mice using specific antibodies (left).\nQuantification of Caspase-3 positive cells per mm of MOE (right). Each dot represents\none MOE section. n = 3 mice. Scale bar, 20 μm.\n2. Fig 2h, m; Fig 3f, xFig 6, etc. Cell count data must be normalized to the area or\nvolume counted. ORs are a special case. Because ORs are expressed only in zones\nrather than across the entire OE, the only way to equitably count cells expressing\nthem is to measure the area between the outermost positive cells in the field of view and\ncount all positive cells within that region of OE. Do TAAR cell counts in the same\nmanner.\nResponse: We thank the reviewer for this thoughtful suggestion. We have revised\nthe Methods section to clarify the quantification procedures. For each mouse, we\ncollected the tissue on 20–25 slides, with each slide containing five MOE sections.\nTo quantify the number of positive cells, three mice (n = 3) and at least three intact\nMOE sections at similar anatomical positions per mouse were used for each\ngenotype. In the quantification figures, each data point represents the total number of\npositive cells counted in one MOE section. The details have been added in lines 683-\n690 and 715-723 in the Methods section of the revised manuscript. Cells expressing\nTAARs were quantified in the same manner. As a note, this quantification method has been\ncommonly utilized by most labs (Ressler et al., Cell, 1993 from Linda Buck’s lab at the\nFred Hutchinson Cancer Center; Fuss et al., Cell, 2007 from Peter Mombaerts’ lab at\nthe Max Planck Research Unit; Fleischmann et al., Neuron, 2008 from Richard Axel’s\nlab at Columbia University; Johnson et al., PNAS, 2012 from Gilad Barnea’s lab at\nLomvardas’ lab at Columbia University; Iwata et al., Nature Communications, 2017\nand Enomoto et al., Communications Biology, 2019 from Junji Hirota’s lab at Tokyo\nInstitute of Technology; Cichy et al., Current Biology, 2019 from Thomas Bozza’s lab\nat Northwestern University.). The raw counts have been provided in the source data.\n3. 277-296; 313-329 and the figures that support these sections. To properly identify\nOSNs expressing multiple OR genes, differences in the sensitivity of mRNA detection\nof these single cell RNA-seq data need to be normalized. An OSN in which\nmRNAs expressed by 1,500 genes are detected is 3X less likely to identify mRNAs from\nmultiple OR genes than an OSN in which mRNAs encoded by 4,500 genes are detected.\nResponse: We thank the reviewer for raising this important concern. To address this\nissue, we reanalyzed our single-cell RNA-seq data using the following strategies. To\navoid the impact of sequencing saturation from individual cells, we first extracted cells\nwith more than 10,000 detected UMIs. These filtered cells were then downsampled to\n10,000 UMIs per cell. We subsequently calculated the percentages of cells expressing\nsingle and multiple receptor genes in immature and mature OSNs. In Trim66 +/- mice,\nthe vast majority of immature OSNs expressed multiple olfactory receptors (77% multi-\nreceptor expressing vs. 21% singular receptor expressing), while the majority of mature\nOSNs expressed a single olfactory receptor (17% multi-receptor expressing vs. 80%\nsingular receptor expressing). In Trim66 -/- mice, most immature OSNs still expressed\nmultiple olfactory receptors (87% multi-receptor expressing vs. 10% singular receptor\nexpressing). However, a large fraction of mature OSNs also continued to express\nmultiple olfactory receptors (94% multi-receptor expressing vs. 5% singular receptor\nexpressing). The data is shown below in Supplementary Fig. 9a. This observation is\nconsistent with our analyses in the original manuscript.\nFurthermore, we examined the number of UMIs and the number of detected genes in\nimmature and mature OSNs that express either no receptor, single receptor, or multiple\nreceptors. We found that these results were comparable between Trim66 +/- and\nTrim66 -/- mice (see Supplementary Fig. 9b, 9c below), suggesting that mRNA\ndetection sensitivity is similar across two sequencing conditions. Together, these\nadditional analyses support the conclusion that the increased frequency of OSNs\nexpressing multiple receptors is not due to differences in mRNA detection sensitivity,\nbut rather reflects true biological changes in Trim66 -/- mice.\nreceptors in mature OSNs. a, To avoid the impact of sequencing saturation from\nindividual cells, we extracted cells with more than 10,000 detected UMIs. These filtered\ncells were then downsampled to 10,000 UMIs per cell. We subsequently calculated the\npercentages of cells expressing single and multiple receptor genes in immature and\nmature OSNs. b-c, Genes and transcripts detection in immature (b) and mature (c)\nOSNs. Top: number of genes detected per cell that express no receptor, single receptor,\nor multiple receptors. Bottom: number of unique molecular identifiers (UMIs) per cell\nthat express no receptor, single receptor, or multiple receptors. The numbers of detected\ngenes and UMIs were comparable between Trim66 +/- and Trim66 -/- mice, suggesting\nthat mRNA detection sensitivity is similar across two sequencing conditions.\n31\n4. Line 429 and affiliated figures. Were these done with conditional KO mice? Lack of\ncontrols for motivation, locomotion, and other factors that can affect such behaviors.\nResponse: We appreciate the reviewer’s insightful comments.\n(1) The buried food test was conducted using both global Trim66 knockout mice and\ncKO mice (Goofy-Cre; Trim66 flox/flox), as shown in Fig. 7a, 7b and Supplementary\nFig. 16d (see also figure below).\n(2) To control for potential confounding factors such as motivation and locomotor\nactivity, we performed the surface food test as controls, in which the food pellet was\nplaced on top of the bedding. In this assay, both Trim66 -/- (Goofy-cre; Trim66 f/f) and\nTrim66 +/- (Goofy-cre; Trim66 f/+) mice showed comparable latency in locating the\nfood, indicating that motor coordination, motivational state, and basic sensory function\nwere intact (see also figure below).\nFig. 7: Olfactory behaviors are compromised in Trim66 knockout mice. a,\nSchematic illustration of the buried food test. A 1.5 g pellet of regular chow was buried\n1 cm beneath the surface of the fresh bedding at a random corner of a clean test cage.\nb, The time to find the food that was either buried (left) or placed on surface (right) in\nTrim66 knockout mice was quantified. Trim66 +/-, n = 12 mice; Trim66 -/-, n = 11 mice.\nmice. d, The time to find the food that was either buried (left) or placed on surface\n(right) in Goofy-cre; Trim66 flox mice was quantified. Goofy-cre; Trim66 f/+, n = 9\nmice; Goofy-cre; Trim66 f/f, n = 12 mice.\nMinor issues.\nA. Lines 233-234. States that “all” TAARs were not changed after Trim66 KO, but the\ndata in xFig3 show that most TAARs were substantially affected. These mRNA\nabundance measures are no substitute for cell count data.\nResponse: We appreciate the reviewer’s comments. We have revised this sentence in\nlines 258-260 of the revised manuscript. The revised text is shown below:\n“This is consistent with the observation that the sum of the mRNA abundance of the\nTaar gene family, measured as the total TPM of all Taar genes, was not significantly\nchanged after Trim66 deletion.”\nB. Fig 1e. Clarify whether these data are bulk or single cell RNA-seq data.\nResponse: The data shown in Fig. 1e are from bulk RNA-seq performed on FACS-\nsorted different cell populations from the MOE. We have revised the figure legend\n(lines 1199-1201) as below:\n“mRNA abundance (represented as transcript per million, TPM) of Atf5, Trim66 in\ndifferent cell types of MOE. Bulk RNA-seq data on FACS-sorted different cell\npopulations is provided by Stavros Lomvardas’ group.”\nC. Fig 1f. These images are not representative of the OE as they feature a fold or tip of\na turbinate.\nResponse: We thank the reviewer for the critical observation. The morphologies of the\nMOE vary in different lateral to medial sections. We extracted several representative\nimages of Atf5 mRNA expression from Allen Brain Atlas database showing different\nmorphologies of MOE (see Figure 2 for reviewers below). The images in our\nmanuscript resembled one of the sections with the MOE enlarged to show the\nmorphology.\nAllen Brain Atlas database illustrate the morphological differences from the lateral to\nmedial MOE of E15.5 mouse embryos.\nD. Fig 1h. The decrease in OE thickness from left to right across these images indicates\nthat this piece of OE is probably near the edge of the OE where it meets respiratory\nepithelium. At these edges the distribution of cell types is not representative of OE.\nIt has many fewer mature OSNs and more immature OSNs, especially at PD14.\nResponse: To address this concern, we have now replaced the image in Fig. 1h with a\nnewly acquired image from a central region of the MOE (see also figure below), where\nthe tissue architecture and cell type distribution better reflect the typical structure of the\nMOE at postnatal day 14 (PD14).\nFig. 1: Trim66 that is regulated by ATF5 is promising candidate for olfactory\nreceptor gene regulation. h, Immunohistochemistry of TRIM66 protein in the MOE\nof Omp-ires-cre; ZsGreen flox mice at postnatal day 14. ZsGreen signals represent\n34\nmature OSNs, while red fluorescence signals represent TRIM66 counterstaining.\nNuclei are labeled by DAPI. Scale bar, 20 μm.\nE. 220-222. Where is evidence supporting the statement that Olfr73 expression is absent?\nResponse: We have now added the proper figure reference in lines 245-246 of the\nrevised manuscript. The revised text is shown below:\n“OSNs expressing Olfr73 labeled in Olfr73-ires-GFP mice were completely eliminated\nin the absence of Trim66 (Fig. 2j, 2n).”\nFig. 2: Trim66 global knockout leads to decreased mRNA abundance of most\nolfactory receptor genes. j, Representative images of positive cells in the MOE (left)\nand whole-mount fluorescent images of the olfactory bulb (right) in Olfr73-ires-GFP;\nTrim66 knockout. n, The numbers of OSNs expressing Olfr73 was quantified. n = 3\nmice.\nF. Line 1328. Provide correct description of UMI.\nResponse: We have revised the text in the figure legend for Supplementary Fig. 8e\n(previously Extended Data Fig. 6e) as shown below:\n“e, Percentage of mature OSNs expressing 0, 1, or multiple ORs thresholded by the\nunique molecular identifiers (UMIs) required for an OR gene to be considered “detected”\nin Trim66 +/- mice.”\nReviewer #3 (Remarks to the Author):\nIt has been more than three decades since the discovery of the one neuron-one receptor\nrule in olfactory sensory neurons (OSNs). The molecular mechanisms underlying the\nintricate process of monogenic and monoallelic expression of OR genes have been\nactively researched but are still not fully understood. Immature OSNs express multiple\nBao et al. discovered TRIM66 as a key repressor for this selective silencing. They found\nthat knocking out the TRIM66 gene in mice resulted in decreased expression of ORs\nand co-expression of multiple OR genes in mature OSNs. Additionally, they found that\ndeletion of Trim66 led to severe defects in information processing in the olfactory bulb,\nas well as impaired innate olfactory behaviors. The experiments are well-designed,\nconducted, and presented. The results are intriguing, and the conclusions are convincing.\nWe have only minor concerns.\n1. Title. To make the grammar correct, I would suggest revising it to “An epigenetic\nrepressor … dictates …”.\nResponse: We thank the reviewer for the helpful suggestion regarding the title.\nFollowing the recommendation, we have revised the title to:\n“An epigenetic repressor TRIM66 dictates monogenic olfactory receptor expression,\nneural activity, and olfactory behavior.”\n2. Page 8. Line169-171. It is stated that Trim66 was more enriched in progenitors or\nimmature OSNs. However, antibody staining (Fig. 1h) seems to show Trim66 is highly\nexpressed in the mature OSNs (more apical layers) than in the immature OSNs. Even\nin Fig. 1g, the in situ hybridization signal seems abundant across mature and immature\nOSN layers. This statement needs clarification or revision.\nResponse: We thank the reviewer for critical observation. As shown in Fig. 1g, the in\nsitu hybridization signals of Trim66A_long isoform are present in both mature and\nimmature OSNs. However, the in situ hybridization signals of Trim66B_short isoform\nare more abundant in immature OSNs. To address the discrepancy of antibody staining,\nwe carefully examined the staining results and acquired additional images from\nmultiple fields across the MOE. We indeed found TRIM66 protein expression in both\nmature and immature OSNs across different regions, yet the staining intensities\nbetween immature and mature OSNs may vary in different sections. We have replaced\nthe original image with another representative one that shows consistent expression\npattern with the in situ results (Fig. 1h; see also figure below).\nreceptor gene regulation. h, Immunohistochemistry of TRIM66 protein in the MOE\nof Omp-ires-cre; lsl-ZsGreen mice at P14. ZsGreen signals represent mature OSNs,\nwhile red fluorescence signals represent TRIM66 counterstaining. Nuclei are labeled\nby DAPI. Scale bar, 20 μm.\n3. Line 212. In Fig. 2h, more details are needed on how the positive cells were\nquantified from three mice. For each mouse, the number of sections and their locations\nin the nasal cavity should be described. Do zonal distributions of these receptor genes\nchange in Trm66 -/- mice?\nResponse: We have revised the Methods section to clarify the quantification procedures.\nFor each mouse, we collected the tissue on 20–25 slides, with each slide containing five\nMOE sections. To quantify the number of positive cells, three mice (n = 3) and at least\nthree intact MOE sections at similar anatomical positions per mouse were used for each\ngenotype. In the quantification figures, each data point represents the total number of\npositive cells counted in one MOE section. The details have been added in lines 683-\n690 and 715-723 in the Methods section of the revised manuscript.\nTo address this point, we examined the zonal expression pattern of Olfr1507 using in\nsitu hybridization. As shown in the new Supplementary Fig. 5c (see also figure below),\nOlfr1507 is primarily expressed in zone 5, and this zonal expression pattern remains\nlargely preserved in Trim66 -/- mice. Therefore, we believe that the multiple glomerular\nprojections of Olfr1507 OSNs is largely due to multiple receptors expression.\nSupplementary Fig. 5: Zonal expression pattern of ORs and Taars in Trim66\nknockout mice. c, In situ hybridization for Olfr1507 on coronal sections of the MOE.\nOlfr1507 is primarily expressed in zone 5 in both genotypes, and its zonal distribution\nremains largely preserved in Trim66 -/- mice. Scale bar, 500 μm.\nIn addition, we also examined the zonal expression of two upregulated gene, Olfr571\nand Taar6. Olfr571, which is normally confined to zone 2, exhibited ectopic expression\n37\nin zone 1 (dorsal MOE) in Trim66 -/- mice. Likewise, Taar6, which is usually restricted\nto the ventral MOE, showed expanded expression into more dorsal regions in Trim66 -\n/- mice. These results suggest that Trim66 regulates the zonal distribution of a subset of\nOR and Taar genes. These images are provided in the new Supplementary Fig. 5d-e\nand also shown below.\nSupplementary Fig. 5: Zonal expression pattern of ORs and Taars in Trim66\nknockout mice. d–e, In situ hybridization for Olfr571 (d) and Taar6 (e). Both receptor\ngenes show ectopic expression in the dorsal MOE following Trim66 deletion. Scale\nbars, 500 μm (d,e, left) and 100 μm (d,e, right).\nWe have added this point on zonal expression in lines 264-273 of the revised\nmanuscript. The revised text is shown below:\n“To examine whether the zonal expression patterns of OR and Taar genes are altered\nin Trim66 -/- mice, we performed in situ hybridization for Olfr1507, Olfr571, and Taar6\n(Supplementary Fig. 5c-5e). Olfr1507 is normally expressed in zone 5, and its zonal\nexpression remained largely unchanged in Trim66 -/- mice (Supplementary Fig. 5c).\nIn contrast, Olfr571, which is normally confined to zone 2, exhibited ectopic expression\nin zone 1 (dorsal MOE) in Trim66 -/- mice (Supplementary Fig. 5d). Likewise, Taar6,\nwhich is usually expressed in the ventral MOE, showed expanded expression into more\ndorsal regions in Trim66 -/- mice (Supplementary Fig. 5e). These results suggest that\nthe loss of Trim66 can lead to disrupted zonal distribution in a subset of OR and Taar\ngenes, potentially contributing to the defects in glomerular targeting”\n4. Fig. 3e. It seemed that colocalization also exists in Trim66+/- section. One red cell\n(in the middle of the image) seemed both red and green (lower level). It might be helpful\n38\nto show enlarged images. Fig. 3f, it would be helpful to describe how many sections\nand their locations for the quantification.\nResponse: We thank the reviewer for pointing this out. Upon re-examination of the\nimages, we found that the red and green signals in the Trim66 +/- section had identical\nmorphology and spatial overlap, indicating that the apparent colocalization was likely\ndue to signal bleed-through caused by strong red fluorescence signals (see figure below,\ntop). To address this, we replaced the image in Fig. 3e with a new image taken under\nsettings with minimize channel crosstalk (the new Fig. 3e, see also figure below,\nbottom).\nIn terms of quantification method for Fig. 3f, it is the same as we described in our\nresponse to your point #3. Specifically, we quantified the number of double positive\ncells across the entire MOE. We have added the details in the Methods section.\n5. Supplementary Tables are not cited in order. Table 4 was not mentioned in the text.\nResponse: We have revised the manuscript to ensure that all Supplementary Tables are\ncited in order. Supplementary Table 4, which summarizes the quantification results of\ntwo-color in situ hybridization, is now added to source data.\n6. Line 412. Suggest using “reduces” or “decreases” instead of “inhibits” in the sentence.\nResponse: Thank you for the suggestion. We have revised the sentence in lines 487-\n489 and replaced “inhibits” with “reduces” to be more accurate. The revised text is\nshown below:\n39\n“These findings indicate that Trim66 knockout not only reduces specific frequency\nbands of neural network activity but also potentially disrupts odor information\nprocessing in the OB.”\n7. Line 418. “underscoring the importance of Trim66 in regulating neuronal\nexcitability”. The effect of Trim66 knockout on the baseline firing rates and\nexcitatory/inhibitory responses to odors is likely indirect via modulating OR gene\nexpressions in OSNs. It is not clear how Trim66 knockout leads to the OB neuronal\nactivity changes. The authors should discuss this point in more depth.\nResponse: According to the reviewer’s suggestion, we have added discussion on OB\nneuronal activity changes in lines 590-603 of the revised manuscript. The revised text\nis shown below:\n“In Trim66 -/- mice, we observed increased neuronal activity in the OB, as indicated by\nelevated spontaneous firing rates of M/T cells and altered odor-evoked responses. This\nphenotype is very likely a direct consequence of disrupted singular olfactory receptor\ngene expression in OSNs. In the absence of Trim66, mature OSNs co-express multiple\nORs, leading to aberrant axonal projections that converge onto multiple glomeruli. As\na result, individual odors may activate a broader set of glomeruli, subsequently\nrecruiting a larger population of M/T cells and contributing to the observed\nhyperactivity in the OB. In addition, other possibilities may also contribute to the\naltered OB neuronal activity. First, Trim66 may regulate the expression of axon\nguidance or targeting molecules that directly influence OSN axonal projection patterns.\nSecond, since our electrophysiology recordings were performed in Trim66 global\nknockout mice, we cannot exclude the possibility that Trim66 may have functional roles\nwithin the OB itself. Although Trim66 is expressed in the OB at much lower levels than\nin the MOE, its loss may affect the intrinsic excitability of OB neurons or interfere with\nthe development of local circuits, such as connections between M/T cells and\ninterneurons, thereby affecting the overall network activity.”\nReviewer #4 (Remarks to the Author):\nThe authors use ChIP-seq to identify binding sites for Atf5, thereby identifying\ncandidate transcriptional targets of the OR-induced unfolded protein response. This\nanalysis identified Atf5 sites proximal to numerous genes that are relevant to OSN\ndifferentiation and function, providing useful insights into the mechanism by which the\nOR-induced UPR signal regulates neuronal maturation and proper OR folding. The\nauthors focus their analysis on one of these targets, Trim66, based upon its\ndownregulation in published Atf5 KO datasets and its membership in a gene family that\nregulates chromatin structure. The authors show that Trim66 RNA expression peaks in\nthe Atf5-expressing population of OSN progenitors, although it also appears to be\n40\nabundant in earlier progenitors at both the RNA and protein levels. Analysis of Trim66\nknockout mice reveals extensive widespread changes in OR and Taar gene expression\nby both RNA-seq and IHC, with most receptor genes downregulated and a small\nnumber getting upregulated. Notably, the authors also observe reduced numbers of\nmature cells and reduced expression of mature markers, as well as a concomitant\nincrease in progenitor cells / markers. Single cell RNA-seq analysis reveals that in the\nTrim66 KO most mature OSNs co-express multiple ORs per cell, which is confirmed\nby ISH, and that OR expression levels are lower for the top OR and higher for additional\nOR (consistent with co-expression). Importantly, this reduced expression is observed\neven for the ORs that were identified as overexpressed by bulk RNA-seq - these ORs\nhave increased expression because they are expressed by more cells, but at lower levels\nper cell. The authors go on to observe that the more frequently chosen (upregulated in\nbulk) OR genes are located near OR enhancers elements. Using ChIP-seq, they show\nthat Trim66 binds to many OR enhancers, and they observe that these enhancers show\nincreased accessibility and increased active histone modifications in Trim66 knockout\nMOEs.The authors go on to show that\nmitral and tufted cells in the olfactory bulb have altered electrophysiological properties in\nTrim66 KO mice. Consistent with impaired olfactory function, these mice also show\nbehavioral defects in multiple modalities, including finding buried food pellets, social\nbehaviors, and olfactory learning. Based upon these findings the authors propose that\nTrim66 is required to silence OR enhancer hubs, aside from the hub associated with the\nchosen OR gene.\nThe author's specific findings are well supported by the data, with appropriate controls,\nanalytical approaches, and use of statistics. However, the authors should be more\ncautious in interpreting some of their findings. Two points in particular need to be\naddressed:\n1) The authors note increased numbers of immature OSNs in the Trim66 KO mice\nacross several modalities. This complicates the interpretation of ATAC-seq and ChIP-\nseq findings as reported in figure 4. As the authors note in the discussion, immature\ncells have been observed to have greater ATAC-seq accessibility of OR enhancers, thus\nthe increased signal observed in the KO could reflect the change in cell type\ncomposition rather than a change in the mature cells in the KO. The purified olfr1507\npositive cells could potentially suffer from this same issue (ie containing more cells in\na more immature state in the KO). The authors could support this point by performing\nsimilar experiments on purified mature cells, for example using OMPgfp. While this\nwould be a valuable experiment, I think that the authors could adequately address this\nissue in the text of the current manuscript.\nResponse: We thank the reviewer for raising this important point. Unfortunately, Omp\ngene and Trim66 gene are located in the same chromosome (chr7), preventing\nacquisition of Trim66 homozygous knockout alleles in Omp-ires-GFP or Omp-ires-cre;\nlsl-reporter mice. On the other hand, we aimed to examine the proportion of mature\nfound that approximately 45% of Olfr1507-tdTomato cells were OMP positive in both\nOlfr1507-ires-tdTomato; Trim66 +/- and Olfr1507-ires-tdTomato; Trim66 -/- mice (the\nnew Supplementary Fig. 11f, 11g; see also figure below). The comparable proportions\nof mature OSNs in Olfr1507-tdTomato cells may be explained by increased Olfr1507\ncells in Trim66 -/- mice. Therefore, our ATAC-seq and CUT&Tag experiments\nperformed in purified Olfr1507 cells could partly address the reviewer’s concern. In\naddition, we have added a sentence to explain this limitation in lines 409-414 of the\nrevised manuscript. The revised text is shown below:\n“To test this idea, we performed ATAC-seq experiments on the whole MOE of Trim66\nknockout mice. Due to the fact that immature OSNs have greater chromatin\naccessibility in enhancers and there are more immature OSNs in Trim66 -/- mice, we\nalso purified Olfr1507 OSNs from Olfr1507-ires-tdTomato; Trim66 knockout mice to\nperform ATAC-seq. Approximately 45% of Olfr1507-expressing OSNs were mature\nOSNs in both Trim66 +/- and Trim66 -/- mice (Supplementary Fig. 11f, 11g).”\nSupplementary Fig. 11: Correlation analysis between changes in olfactory\nreceptor genes and chromosome locations, zonal expression pattern, and\nexpression levels. f, Immunostaining for OMP (green) in the MOE of Olfr1507-ires-\ntdTomato; Trim66 knockout mice. Nuclei are counterstained with DAPI (blue). g,\nQuantification of the proportion of OMP positive cells among tdTomato positive cells\nin both genotypes. Each dot represents a section. Data are presented as mean ± SEM.\nComparison of different groups was carried out using unpaired Student’s t test (g).\n2) The enhancer-hub model proposed by the authors to synthesize their results\nsubstantially overreaches the data presented, and ignores alternative explanations for\ntheir findings. For example, it is possible that the Trim66 KO OSNs fail to form\nchromatin hubs entirely, which might be expected to result in co-expression of ORs at\nlow levels just as they observe. If the authors wish to use this framework, they must\n42\nprovide evidence that trim66 ko mice form hubs, using an approach such as Hi-C or\nDNA-FISH.\nResponse: We thank the reviewer for this insightful comment. To directly address this\npossibility, we performed in situ Hi-C in the MOE of Trim66 +/- and Trim66 -/- mice.\nWe observed that 1) A/B compartment strength was slightly decreased but without\nsignificance after deletion of Trim66; 2) the TAD composition was similar between\nTrim66 +/- and Trim66 -/- mice; 3) the global interchromosomal contacts were\ndiminished in Trim66 -/- mice; 4) the trans contacts between OR clusters were\ndecreased in a similar degree as the global interchromosomal contacts; 5) deletion of\nTrim66 resulted in stronger reduction in trans interactions between olfactory enhancers\nthan that in interchromosomal and trans OR cluster interactions. Therefore, our results\nsuggest that TRIM66 not only exerts the inhibitory effect on enhancer hubs as proposed\nin our original manuscript, but also regulates the assembly of enhancer hubs. Both\nmechanisms could contribute to low expression levels of multiple olfactory receptor\ngenes in mature OSNs of Trim66 -/- mice. We have now included these Hi-C data (Fig.\n5 and Supplementary Fig. 13; see also figures below) and clarified this point in the\nResults and Discussion sections (lines 447–468 and 562-568) of the revised manuscript.\nAgain, we want to thank the reviewer for the great suggestion to reveal more\nmechanistic insights of TRIM66’s function.\ncomparison of interactions of OR gene clusters between the MOE of Trim66 -/- (lower\ntriangle heatmap) and Trim66 +/- (upper triangle heatmap) reveals widespread\nreductions in Hi-C contacts. Right, the difference heatmap depicts changes in contact\nfrequency between Trim66 -/- and Trim66 +/-. OR gene clusters are arranged by\ngenomic position and highlighted by a wide color bar, while a thin color bar indicates\n44\nchromosome identity. b, The pileup plot, displaying 500 kb bins of OR gene clusters\nexcluding olfactory enhancers, reveals a reduction in trans contacts after Trim66\nknockout. c, Left, comparison of olfactory enhancers (50 kb bins), shown as upper and\nlower triangle heatmaps for Trim66 +/- and Trim66 -/-, respectively. Right, the heatmap\nshows the differential contact frequencies observed between Trim66 -/- and Trim66 +/-\nmice. Olfactory enhancers are arranged by genomic position, with the color bar\nindicates chromosome identity. d, The pileup plot reveals strong focal contacts between\nolfactory enhancers, which are reduced in Trim66 -/- mice. e-f, Pairwise views of OR\ngene clusters reveal extensive interchromosomal interactions that are reduced in Trim66\n-/- mice. Trans interactions between olfactory enhancers are indicated by arrowheads.\ng, For each olfactory enhancer, the fraction of total Hi-C contacts made with other\nolfactory enhancer located in cis at short range (< 5 Mb, green), in cis at long range (>\nC contacts across all olfactory enhancers between Trim66 -/- and control mice. Data are\nshown as mean ± SEM. Comparison of two groups was carried out using two-sided\npaired Wilcoxon signed-rank test (h).\nSupplementary Fig. 13: Trim66 knockout caused reduction in trans enhancer\ninteractions. a, This table provides a summary of all Hi-C experiments described in\nthe manuscript, grouped by biological replicate. For each replicate, it reports the total\nnumber of Hi-C contacts, the total number of interchromosomal contacts, the trans\n45\ninteraction between OR gene clusters, and the trans interaction between olfactory\nenhancers with ±25 kb flanking regions. b, Comparison of Hi-C contact decay curves\nbetween Trim66 +/- and Trim66 -/- mice. c, Pairwise views of genome-wide saddle\nplots illustrating interaction patterns between Trim66 +/- and Trim66 -/- mice. d,\nSum of saddle strength within extent ≤ 10, reflecting that the overall compartmentalization\nstrength is lower in Trim66 -/- mice. Yet the change is not significant (p = 0.08). e,\nPileup plot showing aggregated TAD domain scores for Trim66 +/- and Trim66 -/- mice.\nf, Boxplot comparing TAD domain scores between Trim66 +/- and Trim66 -/- mice.\nOnly outlier points are displayed outside the whiskers.\nIn addition to these major points, I have provided questions and comments related to\nspecific panels and/or analyses below.\nThese issues aside, this work provides important new insights into the mechanism of\nthe OR induced feedback signal by identifying the targets of Atf5, and it identifies a\nTrim66 as required for regulation of OR gene choice and the Greek Island enhancers.\nThese are highly significant findings for the field of olfaction, and more generally for\nour understanding of the molecular mechanisms that govern gene choice mechanisms.\nAdditional comments:\n2f: The sorting of ORs/Taars in this panel is unclear. Are they sorted by expression\nlevel in the KO?\nResponse: In Fig. 2f (see also figure below), ORs and Taars are sorted from highest to\nlowest by their mRNA abundance (expression levels) in Trim66 -/- mice. We have\nclarified this in the figure legend of the revised manuscript. The revised figure legend\nis shown below:\n“Fig. 2: Trim66 global knockout leads to downregulation of most olfactory receptor\ngenes. f, Bar plot illustrating relative mRNA abundance of five most highly\nexpressed ORs and Taars in Trim66 knockout RNA-seq results. ORs and Taars are\nranked from highest to lowest by their mRNA abundance in the Trim66 -/- mice.”\n46\n4c: The labeling of this figure is confusing. The text suggests that the authors are\nplotting signal over Trim66 peaks, not genes.\nResponse: We appreciate the reviewer for pointing this out. We have corrected the\nlabeling in the Fig.4c (see also figure below).\nFig. 4: TRIM66 can bind to and repress the olfactory enhancers. c, Heatmap\nanalysis of the enrichment of ATAC-seq signals in Trim66 knockout mice. Peaks are\ncentered by their summits and sorted by score mean value per region. n = 2 mice.\n4f,g and extended data 7h,i: Analysis of heterochromatin marks should also examine\nregions flanking the enhancer elements since these marks may be depleted within the\nbody of the regulatory element. This could take the form of either expanding the width\nof the element or considering flanking regions separately.\nResponse: Following the reviewer’s valuable suggestion, we expanded the window of\nCUT & Tag results (H3K27me3, H3K9me3, H3K27ac, and H3K4me1) on Olfr1507\nOSNs to include 5 kb flanking regions on both sides of the olfactory enhancers. For the\ntwo active H3K27ac and H3K4me1 enhancer marks, the signals of the enhancer\nelements were higher than the flanking regions. The signals of H3K27ac and H3K4me1\nin the enhancers were further increased after Trim66 deletion. For the two\nheterochromatin marks, we agree with the reviewer that the H3K27me3 signals were\noverall very low in both of the enhancer elements and the flanking regions. And the\nH3K9me3 signals were higher in the enhancer elements than the flanking regions in the\npurified Olfr1507 OSNs. Neverthelss, both H3K9me3 and H3K27me3 signals were\n47\nslightly reduced in the enhancers after Trim66 deletion. The updated figures are now\npresented in the new Supplementary Fig. 12a-12e of the revised manuscript (see also\nfigure below).\nSupplementary Fig. 12: Loss of Trim66 impairs the maintenance of epigenetic\nsignatures at olfactory enhancers. a, UCSC Genome Browser view of CUT&Tag\nresults for H3K27ac, H3K4me1, H3K27me3, and H3K9me3 within ±5 kb of 63 class\nII OR enhancers, the J element, TAAR enhancer 1, and TAAR enhancer 2, based on\nOlfr1507 OSNs sorted from Olfr1507-ires-tdTomato; Trim66 knockout mice. n = 2;\neach experiment was performed using Olfr1507 OSNs isolated from one Olfr1507-ires-\ntdTomato; Trim66 -/- mouse or from 3–4 Olfr1507-ires-tdTomato; Trim66 +/- mice. b-\ne, Signal profiles of CUT&Tag data shown in (a), highlighting enrichment of H3K27ac\n(b), H3K4me1 (c), H3K27me3 (d), and H3K9me3 (e) within ±5 kb of the\ncorresponding enhancers.\nExtended Data 4g & 5g: This plot would be much more useful in evaluating the\nsimilarity between these results if it showed all ORs and highlighted the ORs by\nsignificance in each data set.\nResponse: We thank the reviewer for the helpful suggestion. To better illustrate the\nsimilarity between the Trim66 knockout and cKO (Foxg1-cre; Trim66 flox and Goofy-\ncre; Trim66 flox) datasets, we have now updated the plots to include log FC of all\n2\nfunctional OR genes. OR genes that show statistically significant changes in Trim66\nknockout or cKO dataset are now highlighted in red. The updated figures are now\npresented in Supplementary Fig. 6g and 7g of the revised manuscript (see also figure\nbelow).\nsystem also leads to downregulation of most olfactory receptor genes. g, Pearson\ncorrelation of log FC of the significantly functional ORs obtained from Trim66\n2\nknockout bulk RNA-seq results (Y-axis) and Foxg1-cre; Trim66 flox (X-axis). OR\ngenes that show statistically significant changes in Trim66 knockout or Foxg1-cre;\nTrim66 flox dataset are highlighted in red. Pearson correlation coefficient (r) and p\nvalue are shown.\nSupplementary Fig. 7: Trim66 knockout in immature OSNs and mature OSNs\nresults in downregulation of most olfactory receptor genes. g, Pearson correlation\nof log FC of the significantly functional ORs obtained from Trim66 knockout bulk\nstatistically significant changes in Trim66 knockout or Goofy-cre; Trim66 flox dataset\nare highlighted in red. Pearson correlation coefficient (r) and p value are shown.\nReviewer #1 (Remarks to the Author):\nReview comments to Nat Commun #5904533_R1\nI have reviewed the revised manuscript and carefully evaluated the authors’ responses\nto my previous comments. I appreciate the effort the authors have made to improve the\nmanuscript. Most of my concerns have been adequately addressed, and nearly all issues\nhave been resolved, with the exception of comment #2. I still believe that both loss-of-\nfunction and gain-of-function experiments are essential to provide definitive evidence\nfor the critical role of Trim66 in regulating singular olfactory receptor gene expression.\nHowever, since the related concern in comment #1 has been addressed satisfactorily, I\nwill leave the final decision regarding this point to the Editor’s discretion.\nA few minor issues remain or have newly arisen in the revised version. These do not\nrequire further re-review.\nResponse: We sincerely thank Reviewer #1 for the constructive comments and positive\nfeedback.\nMinor Points:\n1. p62, Figure 5b\nThere is a discrepancy regarding the bin size: the figure indicates 100 kb, while the\nfigure legend states 500 kb. Please clarify which value is correct.\nResponse: We thank the reviewer for pointing out this discrepancy. The bin size in Fig.\n5b is 100 kb, which is correct. We have corrected the figure legend accordingly.\n2. Typographical Errors\n• Page 25, line 551: \"Bcl11\" should be corrected to \"Bcl11b\".\n• References on pages 45–47: Entries #21, #27, #43, and #48 list “P Natl Acad Sci USA”\nand should be corrected to “Proc Natl Acad Sci USA”.\nResponse: We have corrected the typo “Bcl11” to “Bcl11b” and revised the journal\nabbreviation in references #21, #27, #43, and #48 from “P Natl Acad Sci USA” to “Proc\nNatl Acad Sci USA”.\n3. Figure 7f\nI suggest revising the illustration to reflect that the formation of the enhancer hub is\nalso incomplete in Trim66 -/- mice. This would be more consistent with the revised\ndescription in the results section of the in situ Hi-C data:\n“While in Trim66 -/- mice, both the assembly of enhancer hubs and repressive\nmechanisms are disrupted. As a result, multiple dispersed enhancers remain active and\ncompete for transcriptional resources, resulting in polygenic receptor expression in\nmature OSNs.”\nhub is also incomplete in Trim66 -/- mice (Fig. 7f; see also figure below). This change\nis now consistent with the revised description of the in situ Hi-C data in the Results\nsection. We thank Reviewer #1 for the constructive comments.\nFig. 7: Olfactory behaviors are compromised in Trim66 knockout mice. f, Proposed\nmodel for singular olfactory receptor gene expression regulated by TRIM66. In wild-\ntype mice, multiple olfactory receptor genes are transcribed by multiple active enhancer\nhubs assembled under the regulation of TRIM66 in immature OSNs, which is followed\nby TRIM66-mediated silencing of enhancer hubs to suppress receptor gene expression.\nUltimately, one enhancer hub escapes silencing and amplifies transcription of its linked\nolfactory receptor gene. The mechanism ensures and maintains singular olfactory\nreceptor gene expression in mature OSNs, enabling normal olfactory perception and\nolfaction-related behavior. While in Trim66 -/- mice, both the assembly of enhancer\nhubs and repressive mechanisms are disrupted. As a result, multiple dispersed\nenhancers remain active and compete for transcriptional resources, resulting in\npolygenic receptor expression in mature OSNs. Consequently, the olfactory behaviors\nare compromised if the “one-neuron-one-receptor” rule is violated.\nReviewer #2 (Remarks to the Author):\nThis revision sufficiently responds to my criticisms.\nResponse: We sincerely thank Reviewer #2 for the constructive comments and positive\nfeedback.\nReviewer #3 (Remarks to the Author):\nThe authors have done an excellent job in addressing the critiques from the initial\nreview. I have no further concerns.\nReviewer #3 (Remarks on code availability):\nthe quality of the codes.\nResponse: We sincerely thank Reviewer #3 for the constructive comments and positive\nfeedback.\nReviewer #4 (Remarks to the Author):\nThe revisions made by the authors address my concerns with the original manuscript.\nI recommend publication pending some minor revisions noted below.\nWe are sincerely grateful to Reviewer #4 for the insightful suggestions.\nRegarding my first major point related to chromatin accessibility, I acknowledge that\nthe location of Omp and Trim66 on the same chromosome does prevent addressing this\nissue as I proposed. The additional analysis of Olfr1507-expressing cells, together with\nthe textual changes noting this limitation, adequately addresses this issue.\nRegarding my second major point, I am very to see the Hi-C experiments and analyses\nadded to the revised version. The data and analyses are appropriate, and they support\nthe conclusions that the authors draw. I do note one small issue, which is that the error\nbars described in figure legend for figure 5 are not visible in the panels in my version.\nResponse: We thank the reviewer for the positive comments. In the revised version,\nwe have corrected the figure legend of Fig. 5. Specifically, we removed the description of\n“Data are shown as mean ± SEM”, because no error bars are displayed in the\ncorresponding panels. This figure display follows Monahan et al., Nature, 2019.\nThe reduction in enhancer contacts observed by Hi-C is quite striking, and it is very\ninteresting to observe that the absence of a repressor would lead to loss of enhancer\nhubs. Indeed, I think this authors should include a more detailed discussion of these\nfindings, particularly in regards to how loss of enhancer hubs together with loss of\nenhancer repression might lead to the OR co-expression phenotype the authors observe.\nResponse: According to the reviewer’s suggestion, we have added discussion on how\nloss of enhancer hubs together with loss of enhancer repression might lead to the OR\nco-expression phenotype in lines 616-621 of the revised manuscript. The revised text\nis shown below:\n“In Trim66 -/- mice, our Hi-C data revealed a striking impairment in the formation of\nenhancer hubs, resulting in the dispersion of individual enhancers. How TRIM66\nmediates enhancer interactions, however, remains an open question. In addition, the\n3\nrepressing networks on enhancers are also diminished in the absence of Trim66. As a\nresult, multiple dispersed and active enhancers compete for transcriptional resources,\nleading to polygenic receptor expression in mature OSNs of Trim66 -/- mice.”\nReviewer #4 (Remarks on code availability):\nI reviewed code for ATAC-seq, bulk RNA-seq, and Hi-C, but I did not install or run it.\nThe code mostly describes established workflows and is reasonably straight forward to\nfollow. There are useful, but not extensive, comments. A readme file exists, but lacks\nany content.\nResponse: We have updated the readme file with brief instructions and clarified key\nsteps in the code comments to improve clarity and usability.\n4"}]

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