Heritable changes in chromatin contacts associated with transgenerational susceptibility to diet-induced insulin dysregulation and obesity

Domain: neuroscience
Published: 2026-02-12
Editorial outcome: major_revision

Abstract
Effects of prenatal environmental exposures can be transmitted across generations through the germline without DNA mutations, an example of transgenerational epigenetic inheritance. Understanding how such inheritance occurs remains a central unanswered question in biology. Here we show that gestational exposure of mice to the environmental obesogen tributyltin produces heritable changes in chromatin interactions within the Ide gene encoding insulin-degrading enzyme in male primordial germ cells. These altered contacts persist through the F3 generation and are accompanied by reduced hepatic Ide expression, hyperinsulinemia, hyperglycemia and hyperleptinemia, resembling the phenotype of Ide-deficient mice that are predisposed to adult-onset, diet-induced obesity. The formation of new chromatin contacts, suppression of Ide expression, and associated metabolic phenotypes occur only in males. These findings reveal a plausible molecular mechanism by which environmental exposures induce location-specific, three-dimensional changes in chromatin structure that transmit susceptibility to metabolic disorders to subsequent unexposed generations in mammals.

Introduction
Evidence is accumulating that transgenerational epigenetic inheritance (TEI) may negatively impact the health of mammals after gestational exposure of their ancestors to toxic substances, but without direct exposure of the affected generations. Several mechanisms have been proposed to explain mammalian TEI1,2—namely, changes in DNA methylation3, histone methylation4, histone retention5,6, or transmission of small noncoding RNA species to germ cells7. However, whereas significant epigenetic alterations have been observed in germ cells or somatic cells in multiple generations after the initial exposure to toxicants, specific toxicant-induced epigenetic changes that persist across the post-exposure generations and explain the inherited phenotypes have not been clearly identified in the genome of the germline cells. For example, many studies, including ours8, showed that genome-wide DNA methylation profiles were altered after ancestral chemical exposures, but whether the persistent presence of such differentially methylated regions—which are apparently resistant to the two waves of global DNA demethylation events during the early phase of mammalian germ cell development—is required for TEI or not remains to be determined8,9. Contributions of histone modifications or noncoding RNA species to TEI also await identification of specific and persistent epigenetic changes that are directly responsible for the inherited phenotypes10.

Our preceding studies suggested that stable alterations of higher-order chromatin structures might provide a unifying theory to explain mammalian transgenerational inheritance8,11. Support for such a model requires the identification of persistently altered regions of higher-order chromatin structure that are passed to subsequent generations. Therefore, we sought to identify regions of persistently altered chromatin interactions (CIs) in PGCs from mice exposed to the environmental obesogen tributyltin (TBT) (F1) and unexposed (F2, F3) generations and associate these specific changes with the transgenerational susceptibility to obesity phenotypes observed. These alterations in CIs are likely to represent changes in higher-order chromatin structure, that can be inferred from the changed CIs.

Here, we used an established, highly reproducible murine model of male-specific transgenerational susceptibility to diet-induced obesity to show that exposure to TBT elicited heritable changes in chromatin interactions (CIs) in primordial germ cells (PGCs) and that such CIs may contribute to the transgenerational metabolic phenotypes. New CIs were formed within the Ide gene encoding insulin-degrading enzyme in the directly exposed PGCs, then stably maintained in PGCs of the subsequent (unexposed) two generations. Concomitantly, Ide mRNA expression was decreased in the livers of male descendants from the exposed dams. These males were hyperinsulinemic and hyperglycemic, phenocopying Ide-deficient mice that are predisposed to adult-onset, diet-induced obesity. Our results provide a plausible molecular mechanism underlying the TEI of male-specific predisposition to obesity caused by gestational exposure of mice to TBT, a representative environmental obesogen. They also provide an entry point for future studies aimed at understanding how environmental exposures can cause location-specific changes in the 3D chromatin structure to influence physiology across multiple generations in mammals.

Results
Ancestral TBT exposure led to a transgenerational predisposition to increased WAT mass
In the new transgenerational experiment 4 (T4) detailed here, effects of ancestral TBT exposure throughout gestation were confirmed to be much stronger in male versus female F2 and F3 generation C57BL/6J mice. This confirms our previous transgenerational experiments denoted as T112, T28, and T313 and another study using OG2 C57BL/6J mice14. Salient findings included increased WAT depot size, more overall body fat and in some cases, increased body weight (Fig. 1). After the diet was changed from a standard chow diet (SD) to a higher fat diet (HFD) at 5 weeks of age, only at 11 weeks did male F2 animals ancestrally exposed to TBT accumulate significantly more WAT than controls (Fig. 1a); body weight did not differ between groups. In contrast, F3 male animals did not show increased body weight or fat mass when switched to the HFD at 5 weeks (Supplementary Fig. 1). We hypothesized that HFD challenge had started too soon compared with previous experiments8,13. Therefore, we switched sibling DMSO- and TBT-group F3 animals that had been maintained on the SD to HFD at 17 weeks of age. TBT-group F3 males rapidly accumulated body weight and fat mass compared with controls; these differences became statistically significant at 19 (body fat) or 20 weeks (body weight) (Fig. 1b). No effects of ancestral TBT exposure on fat accumulation were observed in females (Fig. 1c, d).

Ancestral TBT exposure caused transgenerationally stable changes in chromatin interactions in the genome of male PGCs
To determine whether CIs were stably altered after ancestral TBT exposure, we performed Hi-C seq of PGCs isolated from E13.5 embryonic gonads of F1–F3 mice, pooled by litter and sex. Approximately 20,000 FACS-enriched PGCs from each group were subjected to Hi-C sequencing, data generation and analysis (Supplementary Fig. 2). Successful detection of a known set of TADs around the HoxD gene cluster15 demonstrated the validity of our Hi-C seq data (Supplementary Fig. 2i, j).

PGCs in F1 embryos were directly exposed to TBT while the embryos were within the treated F0 dams. Those isolated from F2 or F3 embryos were not exposed. PGCs in F2 embryos became gametes producing F3 animals, which showed a transgenerational predisposition to diet-induced obesity8,12–14. The global profiles of chromatin contacts were well conserved in PGC genomes across sex, F0 exposure to TBT, or F1–F3 generations (Supplementary Fig. 3a). The median distance between two chromatin contacts was ~1 megabase (Supplementary Fig. 3b), which agrees with the previously reported size of TADs in the mouse genome (880 kb)16.

Applying a strict criterion of detection (differential chromatin interaction scores >3.0), we identified 20 autosomal differential chromatin interactions (DCIs) conserved between F1 and F2 male PGCs and only one DCI in chromosome 19 that was conserved across all generations (F1–F3) of male PGCs (Supplementary Fig. 3c).

The DCI score plots shown in Fig. 2a demonstrate a region containing significant DCIs—which were gained in the TBT group compared to the DMSO group—well conserved in chromosome 19 of male PGCs across F1–F3 generations (chr19:36,920,000-37,420,000; blue horizontal bar) but not in female PGCs. Formation of new CIs in this region in the genome of the TBT-group PGCs was confirmed by direct visual inspection of normalized chromatin contact matrix data of male PGCs isolated from F1–F3 embryonic testes although these new contacts became weaker in F3 embryonic testes (Fig. 2b). In contrast, there were no changes in CIs in this region among female PGCs across generations after ancestral TBT exposure compared with vehicle controls (Supplementary Fig. 4a). Visual inspection of DCIs for the whole chromosome 19 of male PGCs isolated from the F2 embryos identified only a single region displaying discernible DCIs (Supplementary Fig. 4b, top panel), which was confirmed to be identical to the region described above (Supplementary Fig. 4b, zoom-in panels). Detailed examination of this region revealed three DCIs—namely, two small DCIs around chr19:36,920,000-37,020,000 (DCI1) and chr19:37,220,000-37,420,000 (DCI2) and a large DCI (DCI-3) spanning over the two small DCIs (Fig. 2c and Supplementary Fig. 4b). The nested structure involving these three DCIs convincingly supports TBT-induced formation and transgenerational persistence of these DCIs. To increase our confidence that the two small DCIs (DCI-1 and DCI2) involved in the longer-range DCI (DCI-3) were not problematic genomic regions, we confirmed that DCI1-3 are not flagged by the ENCODE blacklist17, which are genomic regions unsuitable for deep sequencing-based analyses due to unusual structures. We also confirmed that DCI1-3 are not involved in any segmental duplication as detected by SDquest18, eliminating the possibility that DCI1 and DCI2 are deep sequencing artifacts stemming from significantly similar sequences locating nearby. Interestingly, DCI2 contained the Ide gene, which encodes insulin-degrading enzyme (Fig. 2b, c). Hepatic Ide is responsible for the majority of insulin clearance19. These results demonstrated the formation of transgenerational, germline-transmitted alterations in CIs after exposure of pregnant F0 female mice to the obesogen, TBT.

Ancestral TBT exposure induced CI formation in the Ide gene in male livers
To determine whether the DCIs identified in PGCs were also found in the Ide gene in somatic tissues, we sought to examine CTCF binding status in F3 livers because liver is the primary site of Ide expression and F2 PGCs give rise to F3 descendants. Five CTCF binding sites predicted by the JASPAR transcription factor binding site database lie near the CI (A–E) together with two known CTCF binding sites (F, G) (Chr19:37,320,000 in mm10) (Fig. 3a). ChIP-qPCR analysis was performed on CTCF or RAD21 chromatin pulldown samples. We observed increased CTCF binding in four regions (B, C, F, G) within the Ide gene in male livers and infer that these may form a small chromatin loop (Fig. 3b). These findings were reproduced with different primer sets (Supplementary Fig. 5). No significant enrichment was noted in TBT female samples (Fig. 3c). We did not detect enrichment of RAD21 binding on the Ide gene (Fig. 3d, e).

We assessed the CTCF binding profile of the whole chromosome 19 (61.7 Mb) using ChIP-seq and found that it was largely identical between the DMSO and TBT groups of the F3 male livers (Supplementary Fig. 6a). Exposure-specific differences were not readily detected in 17.6 Mb or 4.4 Mb windows around the Tnks2-Myof genes, which did not contain very strong CTCF binding sites (Supplementary Fig. 7b, c). However, focused analysis of a ~1 Mb region spanning from Tnks2 to Exo6 revealed differential CTCF binding sites (DCBSs) as well as preserved binding sites between the DMSO and TBT groups (Supplementary Fig. 6a). One DCBS observed near the first exon of the Btaf1 gene was greater in the DMSO group than the TBT group (Supplementary Figs. 6a and 7d–f; blue wedge) whereas three DCBSs within the Cpeb3 gene were greater in the TBT group than the DMSO group (Supplementary Figs. 6 and 7d, f red wedges). Seven relatively strong CTCF binding sites were preserved between the DMSO and TBT groups (Supplementary Figs. 6 and 7d–h). The intra-Cpeb3 DCBSs, which were stronger in the TBT group than the DMSO group, may contribute to the centromeric (left-side) boundary of the dTAD that contains the whole Ide gene (Supplementary Fig. 6b, red triangle) while the preserved CTCF binding sites near the Hhex gene may form the other (telomeric, right-side) boundary (Supplementary Figs. 6 and 7d–g). The DCBS near Exon I of Btaf1 is in the close vicinity of a well-preserved CTCF binding site at the Fgfbp3 gene (Supplementary Figs. 6 and 7d–f). It is tempting to speculate that CTCF bound to the Btaf1 site may interact with CTCF bound to the Fgfbp3 site in the liver of normal adult males, blocking the accessibility of the Fgfbp3 site from interacting with other CTCF. When CTCF binding to the Btaf1 site is lost by the ancestral exposure to TBT, CTCF at the Fgfbp3 site may be released and can interact with the CTCF bound to the Hhex area, forming a large and new dTAD (Supplementary Fig. 6b, blue triangle). To elucidate the molecular mechanisms regulating Ide gene expression in adult mouse liver and the effects of the ancestral exposure to TBT, future studies will be necessary to characterize detailed protein-protein interactions between the CTCF proteins bound to the DNA regions identified in the current study. The binding of CTCF within the Ide gene was not detected by our ChIP-seq. Presumably, these interactions were too weak for detection by ChIP-seq, although ChIP-PCR had sufficient sensitivity to quantify them.

Ancestral TBT exposure led to a transgenerational, male-specific hyperinsulinemia
Formation of transgenerationally persistent, novel CIs in male PGCs within the Ide gene (Fig. 2 and Supplementary Figs. 3 and 4) prompted us to hypothesize that expression of Ide and neighboring genes Kif11 and Hhex mRNAs might be affected by ancestral TBT exposure. Strikingly, significant decreases in Ide mRNA expression were found in the livers of F2 and F3 adult males (Fig. 3f) but not in skeletal muscle (Supplementary Fig. 8a, gonadal WAT (Supplementary Fig. 8b), spleen, or brain (Supplementary Fig. 21a, b) or in females (Supplementary Fig. 8g, j, m). Notably, Ide mRNA expression was also decreased in F3 male livers from the previously published T3 experiment13 (Supplementary Fig. 9), indicating that this phenotype is reproducible across experiments. Strikingly, expression of Kif11 and Hhex mRNAs was upregulated in male livers (Fig. 3g, h) but not in skeletal muscle (Supplementary Fig. 8b, c), gWAT (Supplementary Fig. 8e, f) or in females (Supplementary Fig. 8h, I, k, l, n, o). In contrast, the expression of genes upstream of Ide such as March5, Cpeb3, Btaf1, and Fgfbp3 was largely unchanged in either males (Supplementary Fig. 19) or females (Supplementary Fig. 20). There was a slight increase in expression of hepatic Cpeb3 expression in males (Supplementary Fig. 19d) whereas expression of Btaf1 in male gWAT was slightly decreased (Supplementary Fig. 19h). These data are consistent with a model in which the altered TAD structures in and around Ide lead to decreased Ide expression accompanied by increased Hhex and Kif11 expression in liver.

No differences in fasting blood glucose levels were observed in either males (Fig. 4a) or females (Supplementary Fig. 10a) prior to diet challenge, as we previously reported8. However, plasma insulin levels in TBT group males were significantly higher (Fig. 4b), in accord with the decreased hepatic Ide mRNA expression (Fig. 3f) whereas no significant differences were observed in females (Supplementary Fig. 10b). We calculated homeostasis model assessment of insulin resistance (HOMA-IR) to estimate insulin resistance and found that TBT-group male descendants showed higher potential for insulin resistance than did DMSO descendants (Fig. 4c). Females showed no differences (Supplementary Fig. 10c). After 6 weeks of HFD diet, TBT male descendants were hyperglycemic (Fig. 4d) and hyperinsulinemic (Fig. 4e), in addition to having increased fat content (Fig. 1a, b). Females showed no effects (Supplementary Fig. 10d, e). HOMA-IR index was strikingly increased in the male (Fig. 4f) but not female (Supplementary Fig. 10f) TBT animals, suggesting a high likelihood of insulin resistance compared with vehicle controls.

To address whether insulin secretion or insulin breakdown was responsible for increased insulin levels in TBT-group males, we measured C-peptide (Fig. 4g, h) and calculated C-peptide:insulin ratios (Fig. 4i, j). Analysis of C-peptide levels revealed that there were no differences between DMSO and TBT group males (Fig. 4g) or females before HFD challenge (Supplementary Fig. 10g), or females after HFD challenge (Supplementary Fig. 10h). C-peptide levels increased ~50% in DMSO and TBT-group males after HFD challenge, indicating that HFD led to increased insulin secretion (Fig. 4h). Measurement of the ratios of C-peptide:insulin indicated that increased insulin levels observed in TBT-group males before and after HFD were likely the result of impaired insulin clearance resulting from decreased IDE production (Fig. 4i, j). Females were unaffected (Supplementary Fig. 10i, j). In accord with our previous publications8,13, plasma leptin levels were strongly increased in TBT-group males (Supplementary Fig. 11a), but not females, after HFD challenge (Supplementary Fig. 11b).

Methods
Chemicals and reagents
TBT, dexamethasone, isobutylmethylxanthine, and insulin were purchased from Sigma-Aldrich (St. Louis, MO). Rosiglitazone (ROSI) was purchased from Cayman Chemical (Ann Arbor, MI). Embryoid Body Dissociation Kit (#130-096-348) was purchased from Miltenyi Biotec (North Rhine-Westphalia, Germany). Zombie Red Dye (#77475) was purchased from BioLegend (San Diego, CA). PE Mouse anti-SSEA-1 (#560142) and Alexa-Fluor 647 Mouse anti-CD61 (#563523) were purchased from BD Biosciences (Franklin Lakes, NJ). Blood glucose meter kits (BG1000) were purchased from Clarity Diagnostics (Boca Raton, FL). Mouse Leptin ELISA Kit (#90030), Mouse C-peptide ELISA kit (#80954) and mouse insulin ELISA kit were purchased from Crystal Chem (Elk Grove Village, IL, USA). Arima-Hi-C Kit (A510008) was purchased from Arima Genomics (San Diego, CA). Ultra-pure formaldehyde (#18508) was purchased from Ted Pella Inc. (Redding, CA). MinElute Reaction Cleanup Kit (#28206) was purchased from Qiagen (Hilden, Germany).

Animal maintenance and exposure
C57BL/6J mice were purchased from the Jackson Laboratory (Sacramento, CA) and housed in micro-isolator cages in a temperature-controlled room (26°C) with a 12 h light/dark cycle. Water and food were provided ad libitum unless otherwise indicated. Animals were treated humanely and with regard for the alleviation of suffering. At the moment of euthanasia, each mouse was assigned a code, known only to a lab member not involved in the dissection process. All tissue harvesting was performed with the dissector blinded to which groups the animals belonged. Group sizes were based on our prior experiments and a prior power analysis
For this new transgenerational experiment, denoted as T4, we purchased 50 male and 148 female C57BL/6J mice (5 weeks of age). The number of mice used was based on a priori power analysis aimed at detecting a 10% difference in body fat with sufficient statistical power. We also have a stringent selection criterion for litter size, requiring litters of 6–8 pups with at least two of each sex (one for phenotypic analysis and another for breeding the next generation. Female mice (74 females per treatment group) were randomly assigned to the different F0 treatment groups and exposed via drinking water to 50 nM TBT or 0.1% DMSO vehicle (both diluted in 0.5% carboxymethyl cellulose in water to maximize solubility), for 7 days prior to mating as we have described8,13. One male was housed with two vehicle or 50 nM TBT exposed F0 females per cage to breed during the dark cycle (6 PM to 6 AM). Vaginal plug appearance was defined as embryonic day (E) 0.5. Treatment was removed during mating, then resumed for F0 females after copulation plugs detected (and males removed) then maintained until pups were born (Supplementary Fig. 11). This TBT concentration was chosen based on our previous studies8,12,13,31 and is five fold lower than the established no observed adverse effect level (NOAEL)32. While chemicals were administered to the dams throughout pregnancy, sires were never exposed to the treatment. No statistically significant differences were observed in the number of pups or the sex ratio per litter among the different groups (Supplementary Fig. 12). It should be noted that F2 descendants were exposed to TBT as germ cells in the exposed F1 embryos. F3 descendants were not exposed to TBT.
From each generation, we randomly chose only one male and one female per litter for endpoint analysis and another one male and one or two females per litter for breeding to produce the next generation (Supplementary Fig. 12). Animals selected for breeding the next generation were randomly chosen from the entire group of animals which passed the litter size criterion (6–8 pups, ≥2 of each sex per litter) and after eliminating the smallest and largest remaining animals (outliers) at weaning. Since our experiments used individuals from n=16 litters for each treatment group and outliers were not included in the pool from which random animals were selected, there should not be any outlier effects. There were insufficient animals in the F1 generation to both breed the F2 generation and analyze phenotypes in the diet challenge, so we only bred the F1 animals. To randomize the breeding process as much as possible, we did not breed siblings and never bred females from the same litter with the same male. Control animals were bred to each other, and TBT-exposed animals were bred to each other. There was no effect of treatment on litter size or sex ratio (Supplementary Fig. 13) as in our prior experiments. Each pregnancy was derived from a single male as demonstrated by pedigree analysis (Supplementary Fig. 17). All crosses were virgin crosses which was intended to eliminate any confounding effects of multiparous vs. virgin dams.

Diet challenge and body composition analysis
Animals from control and treatment groups were maintained on a standard diet (SD) (PicoLab 5053; 24.5% Kcal from protein, 13.1% Kcal from fat, and 62.3% Kcal from carbohydrates) from weaning onward. In diet challenge experiments, F2 (14 males and 14 females for each group) and F3 (15 males and 15 females for each group) were switched to higher fat diet (HFD) (PicoLab Rodent Chow 5058, 23.2% kcal from protein, 21.6% kcal from fat, and 55.2% kcal from carbohydrates) whereas control groups (F2: 15 males and 15 females; F3: 12 males and 12 females) were maintained on the SD (PicoLab Rodent Chow, 5053). Body weight and body composition were measured weekly for each animal using EchoMRI™ Whole Body Composition Analyzer, which provides lean, fat and water content information. Total water weight includes free water mainly from the bladder and water contained in lean tissue. Littermates of the animals chosen for HFD analysis were used for breeding the next generation in every case; therefore, the animals chosen to breed the F2 and F3 generations were never exposed to HFD challenge. The choice of different times for the HFD experiment was based on an attempt to shorten the length of the experiment. While we typically used 12 weeks (See ref. 13) for the diet challenge, we shortened this to 5 weeks for this experiment based on another study of ours (Chamorro-Garcia et al. 2018) that only examined phenotypes in F131. F2 descendants started diet challenge at 5 weeks of age for 8 weeks when a significant fat content increase was confirmed and persisted. F3 descendants starting the diet challenge at week 5 had not become significantly fatter by week 17. Therefore, we switched a group of reserved siblings of F3 animals to the HFD at week 17 for 5 weeks. Fat content was significantly increased in this group by week 19. Mice were fasted for 12 h prior to euthanasia and tissue collection.
Blood was collected via the saphenous vein at week 4 and week 12 (before and after diet challenge) for F2, and at weeks 4, 12, and 22 for F3. Blood was collected into heparinized tubes, then centrifuged for 15 min at 2000 × g at 4°C. The resulting plasma was transferred to a clean tube and preserved at −80°C. Animals were euthanized by isoflurane exposure followed by cardiac exsanguination after 4 h fasting. Blood was drawn into a heparinized syringe and centrifuged for 15 min at 2000 × g at 4°C. The resulting plasma was transferred to a clean tube and preserved at −80°C. We measured plasma leptin levels to confirm the previously reported phenotypes8. Inguinal white adipose tissue (iWAT), gWAT, pancreas, spleen, liver, interscapular brown adipose tissue (iBAT), and soleus muscle were flash frozen in liquid N2 then stored at −80°C for subsequent analysis. Feces were freshly collected from animals prior to, and during the diet challenge at week 4 and week 12 for F2, week 4, 12, and 22 for F3, and stored at −80°C.

PGC isolation
A randomly-selected subset of pregnant females was euthanized 13 days (E13.5) after vaginal plug detection. E13.5 embryos were isolated from euthanized pregnant dams, and E13.5 gonads containing primordial germ cells (PGCs) were isolated using a Leica MZ 9.5 Binocular Stereo Microscope. Gonads were identified and sexed by their characteristic morphology at E13.5 (Supplementary Fig. 14a, b), and sex was verified by PCR33. Primer sequences are given in Supplementary Table 1. Gonads from same-gender embryos in each litter were pooled prior to tissue dissociation. Gonads were enzymatically digested using the Embryoid Body Dissociation Kit (Miltenyi Biotec). Next, total dissociated gonad cells were stained with Zombie Red Dye (BioLegend), PE Mouse anti-SSEA-1 (BD Pharmingen), and Alexa-Fluor 647 Mouse anti-CD61 (BD Pharmingen). Primordial germ cells were purified based on the expression of Zombie Red-/SSEA-1+/CD61+ using BD FACS Aria II Cell Sorter (BD Bioscience). Somatic gonad cells were purified based on the expression of Zombie Red-/SSEA-1-/CD61. The gating strategy and purity of the isolated cells is shown in Supplementary Fig. 15.

Hi-C data generation
Five litters of each group with ~20,000 PGCs (Zombi Red-/SSEA-1+/CD61+) were designated to proceed for Hi-C sample preparation using Arima Hi-C Kit following the manufacturer’s instructions for low-input Hi-C sequencing. This low-input protocol supported quantitative determination of topologically associating domains (TADs) from 10,000 human cells (Arima Genomics Application Note: “Unlock Low-Input 3D Genome Analysis with the Arima-Hi-C Kit”, Arima Genomics), which was confirmed by our current study (Supplementary Fig. 2a–d). Briefly, cells were fixed with formaldehyde (Ted Pella) to crosslink chromatin contacts. Genomic DNA was isolated from the fixed cells and digested using a restriction enzyme cocktail. The digested 5’-overhanging ends were filled with biotinylated nucleotides, and spatially proximal digested ends were ligated. Proximally ligated DNA fragments, which capture chromatin contacts, were purified, fragmented by sonication, and enriched using streptavidin-conjugated beads. Illumina sequencing libraries were synthesized from the solid phase-captured DNA fragments using the Swift Biosciences® Accel-NGS® 2S Plus DNA Library Kit (Swift). Libraries were sequenced using an Illumina NovaSeq 6000 deep sequencer to obtain 150+150 nt paired-end FASTQ reads.
Prior to Hi-C analysis of PGCs, we verified by PCR that each pool was exclusively comprised of male or female embryos. Supplementary Fig. 16 shows a table of X and Y chromosome counts from the Hi-C reads. Y chromosome counts for males were well balanced among all samples, intra- or intra-condition categories. Female samples showed very small but non-zero Y chromosome counts due to known strong similarities in the genomic DNA sequences between the X and Y chromosomes; these counts would not affect the interpretation of the Hi-C data. The X chromosome counts were approximately twice greater in females than males, which is expected, and well-balanced intra- and inter-conditions. Therefore, the numbers of Hi-C reads mapped to sex chromosomes were consistent with expectations.

Identification of differential topologically associating domains (dTADs) and genes
After adapter sequences and low-quality reads (<30) were removed using the Trim Galore! tool, FASTQ reads were subjected to Hi-C seq analysis using the Hi-C Pro tool34. The Hi-C Pro quality control plots showed that at least 300 million valid interaction pairs were generated for each group of embryos (>60 million valid pairs per individual embryo) with greater than 60% long (>20 kb) cis interactions, indicating successful generation of sufficient amounts of high-quality Hi-C data (Supplementary Fig. 2e–h). Using these data, we were able to reproduce a known TAD profile surrounding the HoxD gene cluster15, which was demonstrated using a contact map (Supplementary Fig. 2i) and a directionality index plot (Supplementary Fig. 2j). Locations of chromatin contact boundaries and the differential chromatin interaction (DCI) scores between TBT versus DMSO vehicle groups were calculated using the BART-3D software tool35. Distributions of TADs and dTADs were visualized using the OmicCircos36 and the HiTC37 R/Bioconductor tools. For visualization, DCI scores in chromosome 19 were smoothened with 40 kbp bins and rolling averaged with 11 bins.

Chromatin immunoprecipitation-quantitative polymerase chain reaction
Chromatin immunoprecipitation (ChIP) was performed using the method by Abcam and optimized with the established method we previously described38. Briefly, 50 mg of liver tissue that had been snap-frozen in liquid N2 were thawed on ice in cold PBS and dispersed into single cell suspensions using a 100-μm cell strainer (#22363549; Fisher Brand, PA). Cells were washed twice with PBS containing protease inhibitor cocktail (#ab201111; Abcam, Cambridge, UK), then resuspended and fixed at room temperature for 10 minutes with 1% paraformaldehyde (Fisher Chemical, PA) in DMEM, followed by an ice-cold phosphate-buffered saline wash, and then quenched for 5 min with 125 mM glycine at room temperature. Fixed cells were washed, collected by centrifugation, then resuspended in phosphate-buffered saline at 107 cells/mL. To isolate nuclei, cell pellets were lysed at 4°C for 10 min with a gentle detergent recipe consisting of 50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 0.25% Triton X-100, Protease Inhibitor Cocktail (#ab201111; Abcam, Cambridge, UK). Nuclei were recovered by centrifugation at 8000 × g for 15 min, washed for 10 min at room temperature (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitors (#ab201111; Abcam, Cambridge, UK), and lysed in 300 μL nuclear lysis buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, protease inhibitors (#ab201111; Abcam, Cambridge, UK). Chromatin samples were prepared by sonicating in 0.5 mL thin-walled polymerase chain reaction tubes (BrandTech, CT) using a QSonica Q800R2 (QSonica, CT) with the following settings: 30 son/30 soff, amplitude 40% repeated for 30 minutes. Triton X-100 (1%) was added to sonicated lysates prior to high-speed, cold centrifugation to remove debris. A total of 5 μg DNA was immunoprecipitated with preblocked protein A/G Dynabeads (ThermoFisher Scientific, MA) complexed to 2.5 μg antibody (anti-CTCF, ab128873, anti-RAD21, ab217678, or Isotype IgG control, ab171870, Abcam, Cambridge, UK). Beads were washed three times with LiCl buffer (50 mM HEPES-KOH, pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% Nonidet P-40, 0.7% Na-deoxycholate) and once with Tris-EDTA buffer plus 50 mM NaCl. To release chromatin from beads, pelleted beads were resuspended in elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 1% sodium dodecyl sulfate) and incubated at 65°C for 30 min. Cross-link reversal was performed overnight at 65°C. DNA samples were purified using Qiaquick PCR Cleanup kit (#28106, Qiagen, Germantown, MD) following RNase A (0.2 mg/mL, 2 h, 37°C) and proteinase K (0.2 mg/mL, 2 h, 55°C) treatment. Input DNA content was determined by spectrophotometry (Nanodrop, ThermoFisher Scientific, MA). For analysis of candidate loci, real-time PCR was performed using SYBR™ Green PCR Master Mix (ThermoFisher Scientific, MA) on a Roche LightCycler 480 II (Roche, Switzerland) according to the recommended protocol. Enrichment of the ChIP target was presented as a fold difference between specific Ab-immunoprecipitated samples and the immunoprecipitated total input with an IgG control. Primer sequences of the examined loci are listed in Supplementary Table 1. Multiple primer sets were tested for each site. For sites B and C, 2 of 2 primer sets showed significant enrichment. For binding site F, 1 of 3 primer sets showed enrichment and for site G, 2 of 3 showed enrichment.

Quantitative real-time reverse transcriptase polymerase chain reaction (qPCR)
Tissue that had been previously snap-frozen in liquid N2 was cut into ~20 mg pieces and lysed with Trizol following the manufacturer’s recommended protocol (ThermoFisher Scientific, MA); total RNA was recovered after isopropanol precipitation (Fisher Chemical, PA). Complementary DNA was synthesized from 5 μg total RNA using SuperScript IV First-Strand Synthesis System (ThermoFisher Scientific, MA) according to the manufacturer’s instructions. Gene expression was assessed with real-time quantitative polymerase chain reaction (qPCR) using SYBR™ Green PCR Master Mix (ThermoFisher Scientific, MA) on a Roche LightCycler 480 II (Roche, Switzerland). Primer sequences of the examined genes were listed in Supplementary Table 1. Cycle threshold values were quantified as the second derivative maximum using LightCycler software (Roche, Switzerland). The 2−ΔΔCt method39 was used to analyze RT-qPCR data and determine relative quantification corrected for primer efficiency. Ide expression was normalized to the housekeeping gene, GAPDH, and compared to DMSO descendants’ group. Error bars represent the SEM from 15 to 17 biological replicates, calculated using standard propagation of error.

Measurement of insulin and C-peptide
C-peptide serum levels were measured by EIA (Crystal Chem #80954; Elk Grove Village, IL, USA) at two different time points (before and after diet challenge) in plasma from blood samples drawn after overnight (12 h) fasting. Insulin levels were measured by EIA (Crystal Chem #90080; Elk Grove Village, IL, USA) in plasma from blood samples drawn after overnight (12 h) fasting.

ChIP-seq analysis
ChIP-seq deep sequencing libraries were prepared from 10 ng purified genomic DNA fragments co-precipitated with chromatin enriched for CTCF (ab128873, Abcam Cambridge, UK and ChIP-IT Express 53008, Active Motif, Carlsbad, CA USA) using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Cat# E7103S) and sequenced with Illumina NovaSeq X Plus to generate 150+150 paired-end reads. FASTQ reads were subjected to quality assessment, adapter sequence removal, and filtering (Phred score >20) using FastQC. The filtered FASTQ files were aligned to the GRCm38/mm10 mouse reference genome using STAR40 with spliced alignment disabled by “--alignIntronMax 1 –alignEndsType EndToEnd” options as we previously described41. The resulting BAM files were sorted and subjected to the extraction of uniquely mapped reads using SAMBAMBA42. From the deduplicated BAM files, normalized bigWig files were generated using deepTools43 (bamCoverage) with binSize=10, ignoreForNormalization=chrM, ChrX, for the mm10 genome size. The bigWig data were visualized using the Integrative Genomics Viewer44 for direct assessment of CTCF ChIP-seq profiles of chromosome 19. Peaks corresponding to the repetitive sequences were identified using RepeatMasker45 and excluded from the analysis.

ddPCR CNV analysis
Genomic DNA was isolated from snap-frozen liver tissue of F2 and F3 offspring using the Quick-DNA Miniprep Plus Kit (Zymo Research, D3025). DNA quality was confirmed by NanoDrop 2000 UV/Vis Spectrophotometer (Thermo Scientific), and 200 ng total gDNA per sample was sent to the Van Andel Institute Genomics Core (Grand Rapids, MI) for droplet digital PCR (ddPCR) analysis. Copy number of the Ide locus was measured using primers within the Ide gene and normalized to Kif11, which lies outside the reported CNV region. Raw CNV values returned by the core were analyzed by one-way ANOVA to test for group differences, and copy number values were plotted against Ide expression levels measured in the same samples. Results are shown in Supplementary Figs. 22 and 23.

Ethics Statement
All experiments were conducted in compliance with all relevant ethical regulations. Animal procedures conducted in this study were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Experiments utilizing chemicals, biohazardous materials and recombinant DNA were approved by the Institutional Biosafety Committees at UCI and MGB.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, by using a mosue model, the authors reveal that exposure to the obesogen tributyltin (TBT) induces\nheritable chromatin interaction changes in primordial germ cells (PGCs). New interactions within the Ide gene are formed in\ndirectly exposed PGCs, persisting in subsequent unexposed generations. This leads to decreased Ide mRNA expression in\ndescendants' livers, resulting in male-specific hyperinsulinemia, hyperglycemia, and increased fat mass. These findings\npropose a molecular basis for transgenerational obesity predisposition due to gestational exposure to an environmental\nobesogen, offering insights for future studies on chromatin structure alterations across generations in mammals. The\ndifference between control and experimental groups are significant and figures are well organized. I listed some\ncomments below for the authors to consider before publication.\n1. In the authors' prior research, insulin levels remained unchanged in F4 descendants of DMSO- and TBT-treated animals\nat 33 weeks of age. However, the authors observed significantly higher plasma insulin levels in TBT group males compared\nto the DMSO control. Could the variance be attributed to the different ages of mice used in the experiments?\n2. Consider relocating Extended Data Figures 8 and 9 to the main figures, as they provide crucial insights into the increased\ninsulin levels observed in TBT-group males both before and after high-fat diet (HFD), suggesting a potential link to impaired\ninsulin clearance.\n3. The background in the introduction section should be more comprehensive, given the recently rapid growth of\nintergenerational/transgenerational inheritance reported in mammals.\n4. This authors are suggest to discuss the current finding into context, for example, recent reports using a model of BPA-\ninduced obesity also found multi-generational inheritance in mice, similarly reveals the contribution of heritable long-range\nchromatin interactions, which is synergized with altered DNA methylation and RNA/RNA modifications (PNAS 2022 PMID:\n36469784; Nat Rev Endocrinol 2023 PMID: 36792712)", "reviewer_2": "1) The topic of transgenerational epigenetic inheritance and molecular mechanisms involved is an interesting topic and role\nof chromatin structure is a critical mechanism to consider. The observations provided and the data presentation and\nexperimental design are novel and appropriate for consideration of publication. This is one of the first studies to demonstrate\nthe impact of chromatin structure alterations in epigenetic transgenerational inheritance, so is also appropriate for\npublication.\n2) The data presented, analyses, and statistical considerations were also appropriate and well described in the Methods and\nResults sections. No suggested modifications are required in the Results or Methods sections.\n3) The primary issue with the manuscript is the complete bias of the authors in the discussion and perspective provided for\nthe manuscript. The authors state in lines 22-25, 36-38 of the Abstract, 46-49 Introduction, and 235-242 Discussion that no\nprevious studies provide mechanistic information on the phenomenon of epigenetic transgenerational inheritance. Over\n5,000 publications on the topic exist and over 2,000 specific publications address the\nmechanistic aspects of transgenerational inheritance from drosophila to humans. The combined actions of different epigenetic facts such as DNA\nmethylation, histone modifications and ncRNA in this process has been demonstrated. However, the author states these\nstudies are not relevant and the current study somehow reveals the mechanistic aspect of transgenerational inheritance in\nthe text referenced above. This is not accurate and intentionally disregards the extensive literature on the topic. Although this\nlanguage may have prompted the editors to review the paper, in its current form is extremely misleading. Such a\nmisrepresentation to the previous literature and the true context of the data presented requires revision to consider for\npublication.\n4) The term “chromatin contacts” or “chromatin interactions” (CI) is also poor vocabulary and misleading. Out of over 100,000\nmanuscripts the reference to “chromatin structure” is used to reflect the 3D loops or TADs in reference to chromatin, while the\nterms chromatin interactions and contacts are reflected in the over 20,000 studies to binding of ncRNA, histone modifications\nprompting protein interactions, or DNA methylation promoting various chromatin remodeling\nproteins to allow the chromatin\nstructure to alter. Therefore, the terminology used for chromatin interactions (CI), or contacts are misleading and should be\n“chromatin structure” in reference to TADs as presented. This vocabulary needs to be corrected throughout the manuscript to\nput the paper in a better context and less misleading interpretations. Clearly no epigenetic element, including chromatin\nstructure acts independently, such that DNA methylation, ncRNA and histone modifications are required to get any defined\nchromatin structure. This needs to be thoroughly revised throughout the manuscript.\n5) Although the manuscript requires a better vocabulary, context, and inclusion of all epigenetic factors to be accurate and\nnon-biased, this reviewer feels the manuscript can be thoroughly revised to present the data obtained and put into a\nthoughtful and more accurate context.", "reviewer_3": "Chang and colleagues report in this manuscript that exposure to tributyltin (TBT) causes heritable changes in chromatin\ninteractions (CIs) in PGCs in F1s, which can be stably maintained in PGCs and the liver of the subsequent unexposed F3\ngeneration. In the only DCI identified to be persistently maintained across three generations, the downregulation of Ide in the\nliver was shown to correlate with the hyperglycemic and hyperinsulinemic phenotype in F3 mice. The authors concluded\nthat CI changes may represent a molecular mechanism underlying the transmission of the transgenerational predisposition\nto obesity due to gestational exposure to an environmental obesogen. While the idea that changes in chromatin\nconfiguration underlie transgenerational epigenetic inheritance is of great interest, it is not completely novel, and the\ndata shown in this report are largely correlative.\nGeneral:\nThis study raises more questions than answers. For example, why was only one DCI maintained in F3 while 20 ICs were\ndetected in F1 and F2? Why are only males affected? Why were the DCIs detected in PGCs maintained in the liver but not in\nother organs? Are other genes in the only DCI dysregulated? If so, what are the contributions of those genes? Are those\nDCIs detected in PGCs maintained in sperm? This reviewer understands that these may be topics for future studies. The\nlarge gaps between DCIs and the observed phenotype leave the study purely correlative and hard to believe.\nSpecifics:\n  Validation of CI formation in the Ide gene was conducted using ChIP-qPCR, which can be biased. Wouldn’t it be better to\nuse ChIP-seq? ChIP-seq would allow for the validation of the Hi-C data as well.\n  The title and the text all mentioned obesity as the phenotype, but the data showed no significant changes in either body\nweight or composition. The changes detected reflect glucose metabolic disorders.\n  Given that significant reprogramming occurs during the meiotic phase of spermatogenesis (NCB, 2023, 25:1520), some of\nthe CI changes may be reset and thus disappear in sperm. Are those detected DCIs present in sperm as well?", "reviewer_4": "This manuscript describes experiments to examine whether 3-D genome interactions as detected by HiC could potentially\nbe involved, or associated with multigenerational phenotypes observed in offspring of mice treated with TBT. Overall the\nmanuscript is very clearly written considering the challenge often associated with communicating inter/transgenerational\nliterature. The phenotyping data appear well done. Overall the data are correlative without a test of causality or replication.\nThe phenotypes themselves raise no concerns and are in keeping with obesity-prone developmental programming, and\ntherefore are important. The genomic data and experimental design raise a number of key questions that leave the impact of\nthe work unclear. These issues are of high importance given that the entire field currently suffers from the legacy of such\ncritiques. It is therefore important at this level of publication that the experiments, data and design rise up to a definitive level.\nThe authors are lauded for the work invested. My significant concerns in order they arose:\n- Can the authors please show a pedigree of the experiment including all animals and color code perhaps which animals\nwere used for what experiments? It is unclear why so many mice were originally ordered and what the number of biological\nreplicates is for the Hi-C. Is it an n=1 pooled from many? Paternal variation appears not to have been eliminated by having an\nequal number of DMSO and TBT pregnancies derived from each male. Is this true? The fact that paternal\nintergenerational effects exist and that they have been suggested to potentially elicit CTCF-dependent, should be\nmentioned. Similarly, given that virgin/primiparous mothers lactate and behave differently than multi-parous mothers, were\nthese all virgin crosses?\n- How are animals chosen? Are they the most median-like for their respective group (eg. the most average sex-specific body\nweight for the litter? Randomized sampling, when overall sample number is low, ‘needlessly’ includes outliers at a\nfrequency that ‘randomly’ may impact one group more then another. This chance is reduced as sample number increases.\nSimilarly, it provides a robust measure of control to experiments for a field that is often criticized for such design (eg. inter-\n/transgenerational effects).\n- What is the rationale for the different times of HFD treatment in F2 and F3. Given the early timepoint associated with HFD\nin F2, does this mean the F3 are derived from F2 that also experienced HFD?\n- It sounds like the animals in Fig 1b were also exposed to HFD at 5w of age but this is not indicated on the graph. HFD is\nwell known to have substantially different effects when applied early vs late, and HFD creates substantial metabolic effects\nirrespective of weight-gain.\n- The metrics used to conclude that the Hi-C profiles are globally similar with exception of these detected DCI’s are\ninsufficiently self-critiquing. There is no evidence of intra-condition variability against which to gauge inter-condition\nvariability.\n- X and Y chromosome mapping counts across samples should be shown given the pooling approach.\n- Embryonic sufficiency and phenotypes co-vary with the position of each embryo relative to blood supply, with those\nanimals closest to the arterial supply being larger than those more distal. In animals that undergo natural birth, this\ninformation is lost and inaccessible. In animals that are taken at fetal stages however this variable can be controlled for. Was\nembryonic position accounted for?\n- One alternate explanation for constellation of results specifically at the Ide locus is a genomic structural rearrangement.\nSome translocations/inversions, and in particular genomic duplications, can trigger the same pattern of HiC results and\nlongevity of phenotypic changes. This reviewer is not an expert in SDquest; is the tool robust enough to rule out that a\nsubset of animals within a pooled replicate contain an SV? Even a minor fraction of such events should yield a robust and\nreproducible signal in HiC (and phenotypes). This could be approached by examining even two independent biological\nreplicates from distinct families/pedigrees (assuming this is not a striking hotspot event). Again, are there replicates (from\ndistinct pedigrees) that could help rule this out? alternatively, if the authors still have DNA from other tissues of the specific\nF2 and F3 animals involved, a methylation array analysis, WES, DNA microarrays could all be used to assess copy number\nacross individual animals to rule this in or out presumably without enormous costs (or even in technical replicates of the\npooled samples).\n- Do the authors discount potential roles for the many other DCI’s observed that didn’t survive the approach?\n- Given the issues above, the fairly strong implications about causality should be toned down (or eliminated until causality is\ntested) and replaced with alternate explanations / discussions of mechanisms through which these changes would be\nexpected to be repeatedly induced and how they might survive (or be saved/protected) through epigenome erasure and\ndevelopment. Do the authors suggest that these changes are maintained through toti- / pluripotency? Do they have\ncorroborative evidence for instance using FISH that these contacts are indeed increased?\n- Early post-weaning period is a phase of life still considered development and overlapping with substantial endocrine\nfluctuations, final maturation of the gonads, as well as puberty/menarchy. HFD appears to have been administered during\nthis time period in one set animals (F2, if I understand the methods correctly). HFD is well known to impact 5-8 w old mice\nvery differently than adult mice. Also, mice immediately post-weaning have just moved from mixed milk/chow to chow-only\ndiet and their metabolic tissues are undergoing a related wave of environmentally triggered terminal maturation and their\nmicrobiome is also highly variable between animals. Was there a rationale I am missing to start an intervention given the\nhigh degree of unmeasured variability and developmental rearrangement that is happening in this time window? With all\ndue respect, this is truly unfortunate for an experiment focused on variation/plasticity. All figures/text should make clear that\nF2 underwent 2 interventions. The authors are applauded for the transparency in the report.\nMinor\n- No DMSO/TBT legend on Ext data 10, unless I missed it."}, "author_response": "We appreciate the reviewer’s careful assessments of our prior and current studies. As the\nreviewer noted, hyperinsulinemia is frequently associated with aging, which may contribute to\ninsulin resistance in the elderly. Marmentini et al (PMC8150109). meticulously compared insulin\nmetabolism of male C57BL/6 mice – which is the same strain we used in our past and current\nstudies – between 3 and 18 months of age and found aging-associated increase in plasma\ninsulin and decrease in insulin clearance. Strikingly, their study identified a decrease in hepatic\nIde expression and IDE enzymatic activity as a major contributor to the observed age-\nassociated hyperinsulinemia. Therefore, in our prior study, it is likely that the IDE-dependent\nhepatic insulin clearance was diminished in both DMSO- and TBT-group male C57BL/6J mice at\n33-weeks of age, thus obscuring the TBT effects on hepatic IDE expression. We also noticed\nthat coprophagy could have also affected fasting plasma insulin level in our prior experiments,\nwhich has been addressed in our current study. As we continue efforts to optimize the animal\nexperiment protocol, effects of F0 TBT-exposure on fasting plasma insulin levels in the male\ndescendants is becoming evident. We added a brief discussion on this matter in the revision.\n2. Consider relocating Extended Data Figures 8 and 9 to the main figures, as they provide\ncrucial insights into the increased insulin levels observed in TBT-group males both before and\nafter high-fat diet (HFD), suggesting a potential link to impaired insulin clearance.\nWe have incorporated male data from Extended data Figures 8 and 9 into Figure 4 at kept the\nfemale data as a new Extended data Fig. 10.\n3. The background in the introduction section should be more comprehensive, given the\nrecently rapid growth of intergenerational/transgenerational inheritance reported in mammals.\nWe have attempted to increase the comprehensiveness of the background although the space\nallowed for this part in the manuscript is limited due to the journal’s formatting rules.\n4. These authors are suggested to discuss the current finding into context, for example, recent\nreports using a model of BPA-induced obesity also found multi-generational inheritance in mice,\nsimilarly reveals the contribution of heritable long-range chromatin interactions, which is\nsynergized with altered DNA methylation and RNA/RNA modifications (PNAS 2022 PMID:\n36469784; Nat Rev Endocrinol 2023 PMID: 36792712)\nWe thank the reviewer for this suggestion and have incorporated both references into our\ndiscussion.\nReviewer #2 (Remarks to the Author):\n1) The topic of transgenerational epigenetic inheritance and molecular mechanisms involved is\nan interesting topic and role of chromatin structure is a critical mechanism to consider. The\nobservations provided and the data presentation and experimental design are novel and\nappropriate for consideration of publication. This is one of the first studies to demonstrate the\nimpact of chromatin structure alterations in epigenetic transgenerational inheritance, so is also\nappropriate for publication.\nWe thank the reviewer for these positive comments.\n2) The data presented, analyses, and statistical considerations were also appropriate and well\ndescribed in the Methods and Results sections. No suggested modifications are required in the\nResults or Methods sections.\nWe also thank the reviewer for these supportive comments.\n3) The primary issue with the manuscript is the complete bias of the authors in the discussion\nand perspective provided for the manuscript. The authors state in lines 22-25, 36-38 of the\nAbstract, 46-49 Introduction, and 235-242 Discussion that no previous studies provide\nmechanistic information on the phenomenon of epigenetic transgenerational inheritance. Over\n5,000 publications on the topic exist and over 2,000 specific publications address the\nmechanistic aspects of transgenerational inheritance from drosophila to humans.\nAs the reviewer points out, many mechanisms have been discussed in the literature with varying\nstrength of evidence in multiple organisms. However, with respect to environmentally caused\nnon-genetic inheritance of disorders in mammals, there are very few studies that have\nconvincingly demonstrated a heritable mechanism underlying the specific phenotypes. Whereas\nchanges in DNA methylation or histone modifications may contribute to the transgenerational\nphenotypes, no differential epigenetic marks of these types have been identified at the same\nloci in the same tissues across generations. The lack of such demonstration has been a major\nproblem in this field of research despite the large numbers of publications on this subject.\nContrary to this review comment, we carefully attempted to present our assessment on the\ncurrent status of research on mechanisms of transgenerational epigenetic inheritance (TEI)\nwithout introducing bias. To us, it sounds rather biased to state that the mechanisms of TEI have\nbeen established, or are even becoming evident, based on thousands of publications on this\nspecific subject. Thus, to address this critique, we added the following to the introduction and\nqualified throughout the manuscript that we are referring to TEI in mammals:\n“Thus, despite a large number of previously published studies proposing a wide variety\nof mechanistic insights into the phenomenon of epigenetic transgenerational inheritance\n(TEI), transgenerationally preserved changes in conventional epigenetic marks (such as\nDNA methylation or histone modifications) at specific loci are yet to be identified in\nmammals. The lack of this critical evidence has been a major obstacle to establish TEI\nas an alternative mode of inheritance in mammals.”\nThe combined actions of different epigenetic facts such as DNA methylation, histone\nmodifications and ncRNA in this process has been demonstrated. However, the author states\nthese studies are not relevant and the current study somehow reveals the mechanistic aspect of\ntransgenerational inheritance in the text referenced above. This is not accurate and intentionally\ndisregards the extensive literature on the topic. Although this language may have prompted the\neditors to review the paper, in its current form is extremely misleading. Such a\nmisrepresentation to the previous literature and the true context of the data presented requires\nrevision to consider for publication.\nAs noted above, combined action of different epigenetic phenomena has been suggested to\nunderlie transgenerational inheritance. However, it remains to be demonstrated that the same\nmarks, be they DNA methylation or histone modifications, are present in the same genes at the\nsame locations from generation to generation. Rather the opposite has been shown – namely,\nwhereas changes in DNA methylation, histone modification and ncRNA expression have been\nshown to exist from generation to generation, they are rarely, if ever the same marks at the\nsame loci. The extensive studies of Skinner and colleagues showed the absence of the same\nmarks across generations although, changes are observed in different places in different\ngenerations.\nTo address this critique, we rephrased our discussion to the following:\n“Associations of several different epigenetic events with TEI have been proposed in\npreceding studies. However, whether such associations – either as independent events\nor in combination – are relevant to the mechanistic basis of TEI has not yet been\nestablished. To provide mechanistic supports for mammalian TEI, evidence is required\nthat such epigenetic events are preserved across generations at specific loci and likely\naffect expression of genes that are directly relevant to the observed TEI phenotypes.”\n4) The term “chromatin contacts” or “chromatin interactions” (CI) is also poor vocabulary and\nmisleading. Out of over 100,000 manuscripts the reference to “chromatin structure” is used to\nreflect the 3D loops or TADs in reference to chromatin, while the terms chromatin interactions\nand contacts are reflected in the over 20,000 studies to binding of ncRNA, histone modifications\nprompting protein interactions, or DNA methylation promoting various chromatin remodeling\nproteins to allow the chromatin structure to alter. Therefore, the terminology used for chromatin\ninteractions (CI), or contacts are misleading and should be “chromatin structure” in reference to\nTADs as presented. This vocabulary needs to be corrected throughout the manuscript to put the\npaper in a better context and less misleading interpretations. Clearly no epigenetic element,\nincluding chromatin structure acts independently, such that DNA methylation, ncRNA and\nhistone modifications are required to get any defined chromatin structure. This needs to be\nthoroughly revised throughout the manuscript.\nChromatin “structure” is a hypothetical model developed based on interpretation of various data\nfor understanding genomic phenomena. Chromatin “contacts” or “interactions” are direct\noutcomes of assays based on the proximity ligation technique, which is also known as\nchromosome conformation capture. Hi-C sequencing is a typical application of the proximal\nligation technique, and its outcome can only describe chromatin contacts or chromatin\ninteractions. Ironically, our manuscript submitted to another Nature journal received a review\ncritique that chromatin “structure” is rather inadequate for our manuscript because the data\npresented in it shows chromatin “contacts or interactions.” We prefer sticking with the more\nprecise terminology (i.e., contacts/interactions) and have adjusted the discussion to make our\nposition clear and address the comments of this reviewer.\n5) Although the manuscript requires a better vocabulary, context, and inclusion of all epigenetic\nfactors to be accurate and non-biased, this reviewer feels the manuscript can be thoroughly\nrevised to present the data obtained and put into a thoughtful and more accurate context.\nWe have made revisions throughout the manuscript to remove any phrases that may be\ninterpreted as our bias while still indicating that we favor a model in which the new chromatin\ncontacts in and around the Ide gene play a prominent role in the observed phenotypes.\nReviewer #3 (Remarks to the Author)\nChang and colleagues report in this manuscript that exposure to tributyltin (TBT) causes\nheritable changes in chromatin interactions (CIs) in PGCs in F1s, which can be stably\nmaintained in PGCs and the liver of the subsequent unexposed F3 generation. In the only DCI\nidentified to be persistently maintained across three generations, the downregulation of Ide in\nthe liver was shown to correlate with the hyperglycemic and hyperinsulinemic phenotype in F3\nmice. The authors concluded that CI changes may represent a molecular mechanism underlying\nthe transmission of the transgenerational predisposition to obesity due to gestational exposure\nto an environmental obesogen. While the idea that changes in chromatin configuration underlie\ntransgenerational epigenetic inheritance is of great interest, it is not completely novel, and the\ndata shown in this report are largely correlative.\nGeneral:\nThis study raises more questions than answers. For example, why was only one DCI\nmaintained in F3 while 20 ICs were detected in F1 and F2?\nIn this study, we began with a high stringency selection criterion and found that only a single\nDCI was maintained in F1-F3 generation PGCs. When the stringency was lowered, or only F1\nand F2 generations were considered, more DCIs became evident. Instead of presenting all such\nlow-stringency DCIs and stopping there, in this study we decided to take a deeper look at the\nsingle DCI (formed around the Ide gene) that survived with the most strict stringency and are\nrelevant to the observed phenotypes.\nWhy are only males affected?\nThis is an important question and the subject of the recent, successful 5-year renewal of the\nNIH R01 that supports this work.\nWhy were the DCIs detected in PGCs maintained in the liver but not in other organs?\nThe most stringently detected DCI in PGCs was in the gene encoding Ide, and Ide mRNA was\ndownregulated in liver in two independent experiments in F2-F4 generations. Based on these\nobservations and given the Ide-relevant metabolic disorders we observed (hyperinsulinemia,\nhyperglycemia, hyperleptinemia) we argue that the emphasis on Ide is justified. We do not rule\nout a possible role for other genes, but down-regulation of hepatic Ide can readily explain the\nphenotype observed. It is possible that the DCIs may also be maintained in other organs, but\nour current analysis focused on the liver because it is where the action of insulin degrading\nenzyme, plays the most critical role in insulin clearance.\nAre other genes in the only DCI dysregulated? If so, what are the contributions of those genes?\nExpression of Hhex and Kif11 are up-regulated in males as a consequence of the new DCI in\nthe Ide region. Both of these loci, along with Ide, have been firmly associated with type 2\ndiabetes in human cohorts by many independent studies. However, as of today nothing is\nknown about their function in obesity or diabetes. As noted above, impairment of insulin\nclearance should be sufficient to produce the physiological phenotype we observed and\nreproduced.\nAre those DCIs detected in PGCs maintained in sperm?\nWe did not analyze sperm in this experiment and do not have any remaining samples that could\nbe analyzed.\nThis reviewer understands that these may be topics for future studies. The large gaps between\nDCIs and the observed phenotype leave the study purely correlative and hard to believe.\nIf we can manipulate TAD formation around the Ide locus in live mice, we would be able to\npresent strong evidence of the causation. Such studies – including inducible CRISPRa/CRISPRi\nmanipulations of the Ide-associating TADs and direct regulation of the affected genes – are\nongoing in our laboratories. However, as outcomes of such experiments will be available only\ntwo or three years later, the causative evidence has to be presented as a separate study in the\nfuture – not to be covered in the current manuscript. However, the fact that new contacts were\nintroduced into the Ide gene and detected in this experiment, resulting in down-regulation of\nhepatic Ide expression and impaired insulin clearance, together with the observation that Ide is\nsimilarly downregulated in liver remaining from a previous transgenerational experiment\n(T3)(Extended data figure 6d) provides strong evidence that the finding is more than an\naccidental correlation. We argue that the reproducibility of the Ide down-regulation phenotype in\n2 independent transgenerational experiments, coupled with the consistent change in chromatin\nin and around the Ide gene is powerful validation that our model is likely to be correct .\nSpecifics:\n  Validation of CI formation in the Ide gene was conducted using ChIP-qPCR, which can be biased. Wouldn’t it be better to\nuse ChIP-seq? ChIP-seq would allow for the validation of the Hi-C data as well.\nThe median size of TADs and subTADs in mammalian cells is approximately one megabase and\n100-200 kb, respectively. Due to technical limitations, especially the number of mouse PGCs\ncollectable from E13.5 embryonic gonads, the Hi-C data currently presented in our study has a\nresolution sufficient for detection of (sub)TADs but not lower-scale chromatin contact events. In\ncontrast, ChIP-seq typically detects protein-DNA interactions with less than 1 kb resolution, and\nChIP-PCR can target precise protein binding sequences with less than a 100 bp window. Due to\nthese technical differences in the targeting windows, ChIP-qPCR provides an opportunity to\ndetect weak protein-DNA interactions at specific loci that may not be readily detectable by ChIP-\nseq.\nFollowing this review comment, we performed ChIP-seq experiments for CTCF interactions with\ngenomic DNA in livers from F3 DMSO and TBT group males after F0 dam exposures to TBT or\nDMSO (no additional PGC samples from this experiment were available for submission of this\nrevision). Strikingly, we observed strong differences in CTCF binding sites in the close\nproximities of the differential TAD boundaries that were estimated by using our Hi-C data,\nagreeing with the reviewer’s suggestion that the ChIP-seq data would allow the validation of the\nHi-C data. We appreciate the reviewer for this constructive suggestion and added outcomes of\nthe CTCF ChIP-seq data to this revision as Extended data Fig. 6 and Extended data Fig. 7).\nThe title and the text all mentioned obesity as the phenotype, but the data showed no significant\nchanges in either body weight or composition. The changes detected reflect glucose metabolic\ndisorders.\nFigure 1a,b show that the body composition of HFD challenged male mice changed as a result\nof the ancestral TBT exposure. We have also published this male-specific increase in body fat\nphenotype in two other publications from previous transgenerational experiments (T2,\nChamorro-Garcia et al, 2017) and T3 (Chamorro-Garcia et al, 2021).\nGiven that significant reprogramming occurs during the meiotic phase of spermatogenesis\n(NCB, 2023, 25:1520), some of the CI changes may be reset and thus disappear in sperm. Are\nthose detected DCIs present in sperm as well?\nWe do not have sperm remaining from this experiment. However, each of the F3 animals\nanalyzed in this manuscript is derived from a different F0 male and F0 female from every other\nanimal. That is, pedigree analysis supplied in response to reviewer 4 clearly demonstrates that\nthe 16 F3 males and 16 F3 females in the DMSO and TBT groups were derived from different\nF0, F1 and F2 matings. How environmentally induced epigenetic changes survive the genomic\nreprogramming during gametogenesis is a central question in the field of TEI. Our current study\nhas anchored early (PGCs) and late (adult phenotypes) points through epigenetic changes at a\nsingle specific locus. Future studies will be needed to connect details of these two points,\nincluding genomic analysis of gametes. The current study does not include analyses of gametes\nalthough we did include PGCs from F1, F2, and F3 generations in both sexes.\nReviewer #4 (Remarks to the Author):\nThis manuscript describes experiments to examine whether 3-D genome interactions as\ndetected by HiC could potentially be involved, or associated with multigenerational phenotypes\nobserved in offspring of mice treated with TBT. Overall, the manuscript is very clearly written\nconsidering the challenge often associated with communicating inter/transgenerational\nliterature. The phenotyping data appear well done. Overall, the data are correlative without a\ntest of causality or replication.\nWe agree that causality has not been tested, however we note that the down-regulation of Ide in\nTBT group animals has been replicated in two independent experiments. Replication of such a\nspecific phenotype, down-regulation of Ide expression associated with diet-dependent\nhyperinsulinemia, hyperleptinemia and increased fat mass, supports our model.\nThe phenotypes themselves raise no concerns and are in keeping with obesity-prone developmental programming, and\ntherefore are important. The genomic data and experimental design raise a number of key questions that leave the impact of\nthe work unclear. These issues are of high importance given that the entire field currently suffers from the legacy of such\ncritiques. It is therefore important at this level of publication that the experiments, data and\ndesign rise up to a definitive level. The authors are lauded for the work invested. My significant\nconcerns in order they arose:\n- Can the authors please show a pedigree of the experiment including all animals and color\ncode perhaps which animals were used for what experiments?\nThis has been provided as Extended data Fig. 17. Every single F3 male analyzed was the\nproduct of different F0, F1 and F2 matings.\nIt is unclear why so many mice were originally ordered\nThe number of mice used was based on a power analysis aimed at detecting a 10% difference\nin body fat with sufficient statistical power. We also have a stringent selection criterion for litter\nsize, requiring litters of 6-8 pups with at least two of each sex (one for phenotypic analysis and\nanother for breeding the next generation.\nand what the number of biological replicates is for the Hi-C. Is it an n=1 pooled from many?\nThe number of biological replicates for Hi-C is 5. That is, PGCs isolated from embryos of a\nsingle dam were pooled by sex and each pool was n=1. Hi-C was performed from 5 replicates\n(i.e., embryos from 5 different dams) and the data pooled by treatment, i.e., DMSO group\nmales, DMSO group females, TBT group males, TBT group females.\nPaternal variation appears not to have been eliminated by having an equal number of DMSO\nand TBT pregnancies derived from each male. Is this true?\nEach pregnancy was derived from a single male as demonstrated by pedigree analysis\n(Extended data Fig. 17); therefore the reviewer’s concern is addressed by our experimental\ndesign.\nThe fact that paternal intergenerational effects exist and that they have been suggested to\npotentially elicit CTCF-dependent, should be mentioned. Similarly, given that virgin/primiparous\nmothers lactate and behave differently than multi-parous mothers, were these all virgin crosses?\nWe are aware of the previous studies providing evidence that CTCF binds to genomic DNA in\nsperm and forms chromatin interactions therein. Following the reviewer’s suggestion, we\nmentioned it in Discussion with a new citation as follows:\n“There is also evidence that CTCF binds genomic DNA in sperm to form chromatin\ncontacts, which may function as vehicles of paternally transmitted epigenetic inheritance\n(Gold, Jung, Corces 2018 JBC, PMC6130957).”\nAnd\n“Whether the observed, TBT-induced changes in chromatin contacts around the Ide\ngene in primordial germ cells are originated from altered CTCF binding in sperm, or such\nchanges are preserved in the sperm genome, are interesting questions.”\nThese were all virgin crosses which was intended to eliminate any confounding effects of multi-\nparous vs virgin dams.\n- How are animals chosen? Are they the most median-like for their respective group (eg. the\nmost average sex-specific body weight for the litter? Randomized sampling, when overall\nsample number is low, ‘needlessly’ includes outliers at a frequency that ‘randomly’ may impact\none group more then another. This chance is reduced as sample number increases.\nSimilarly, it provides a robust measure of control to experiments for a field that is often criticized for such\ndesign (eg. inter-/transgenerational effects).\nAnimals selected for breeding the next generation were randomly chosen from the entire group\nof animals which passed the litter size criterion (6-8 pups ≥ 2 of each sex per litter) and after\neliminating the smallest and largest remaining animals (outliers) at weaning. Since our\nexperiments used individuals from n=16 litters for each treatment group and outliers were not\nincluded in the pool from which random animals were selected, there should not be any outlier\neffect that the reviewer noted.\n- What is the rationale for the different times of HFD treatment in F2 and F3. Given the early\ntimepoint associated with HFD in F2, does this mean the F3 are derived from F2 that also\nexperienced HFD?\nLittermates of the animals chosen for HFD analysis were used for breeding the next generation\nin every case; therefore, the animals chosen to breed the F2 and F3 generations were never\nexposed to HFD challenge. The choice of different times for the HFD experiment was based on\nan attempt to shorten the length of the experiment. While we typically used 12 weeks (See\nChamorro-Garcia et al, 2021) for the diet challenge, we shortened this to 5 weeks for this\nexperiment based on another study of ours (Chamorro-Garcia et al, 2018) that only examined\nphenotypes in F1, While the diet challenge in the F2 generation was successful at 5 weeks\n(Figure 1a), it was not successful for the F3 generation (Extended data Figure 1); therefore we\nused reserved animals from the same group that had been maintained on a standard chow diet\nand subjected them to diet challenge at 17-weeks. This diet challenge was successful. We have\nstandardized the diet challenge at 12-weeks in our subsequent experiments and this has proven\nto be reproducible.\n- It sounds like the animals in Fig 1b were also exposed to HFD at 5w of age but this is not\nindicated on the graph. HFD is well known to have substantially different effects when applied\nearly vs late, and HFD creates substantial metabolic effects irrespective of weight-gain.\nThe time of treatment with HFD is indicated in all parts of Figure 1. For the F3 animals in Fig.\n1b,d it was 17 weeks as noted. Extended data Fig 1b shows the effect of treating F3 males with\nHFD beginning at 5 weeks and continuing through 11 weeks and is discussed in the text.\n- The metrics used to conclude that the Hi-C profiles are globally similar with exception of these\ndetected DCI’s are insufficiently self-critiquing. There is no evidence of intra-condition variability\nagainst which to gauge inter-condition variability.\nThe intra-condition variability was far smaller than the inter-condition (DMSO vs TBT)\nvariability. However, this observation needs to be interpreted carefully because comparisons between each\nsingle PGC samples have reduced sensitivity due to the smaller numbers of PGCs and deep\nsequencing reads and so less power to detect differential TADs. The almost completely clear\nbackground of the differential TAD detection within chromosome 19 indicates a very low level of\nnoise derived from the intra-condition variability.\n- X and Y chromosome mapping counts across samples should be shown given the pooling\napproach.\nPrior to Hi-C analysis of PGCs, we verified by PCR that each pool was exclusively comprised of\nmale or female embryos. We include a table in the revised manuscript showing X and Y\nchromosome counts, as requested (Extended data Fig. 16). The Y chromosome counts for\nmales were well balanced among all samples, intra- or intra-condition categories. Female\nsamples show very small but non-zero Y chromosome counts due to known strong similarities in\nthe genomic DNA sequences between the X and Y chromosomes; these counts would not affect\ninterpretation of the Hi-C data. The X chromosome counts were approximately twice greater in\nfemales than males, which is expected, and well-balanced intra- and inter-conditions.\n- Embryonic sufficiency and phenotypes co-vary with the position of each embryo relative to\nblood supply, with those animals closest to the arterial supply being larger than those more\ndistal. In animals that undergo natural birth, this information is lost and inaccessible. In animals\nthat are taken at fetal stages however this variable can be controlled for. Was embryonic\nposition accounted for?\nWhile uterine position was recorded for embryos after they were sexed, since the PGCs from\neach litter were pooled by sex, these data were no longer available.\n- One alternate explanation for constellation of results specifically at the Ide locus is a genomic\nstructural rearrangement. Some translocations/inversions, and in particular genomic\nduplications, can trigger the same pattern of HiC results and longevity of phenotypic changes.\nThis reviewer is not an expert in SDquest; is the tool robust enough to rule out that a\nsubset of animals within a pooled replicate contain an SV?\nAlthough SDquest has a historical significance, this 6-year-old software has never been updated\nsince its original version (ver. 0.1). Several newer software tools have improved our\nunderstanding about large events such as translocation, inversion, or duplication, which are\nreadily accessible though publicly accessible databases. We examined these databases and\nhave not found any evidence of large genomic events that would affect interpretation of our\nchromatin interaction analysis. Whereas the dTADs reported in our current study span 36.5-37.5\nMb coordinates in chromosome 19, the Cyp2c cluster (apparently reflecting gene duplication\nevents) is found at 39.0-40.5 Mb. However, the 1.5-Mbp distance between the Ide-Kif11-Hhex\nregion and the apparent nearest gene duplication event at the Cyp2c cluster sufficiently\nsegregates the former from the latter with no evidence of significant physical or functional links\nbetween them.\nEven a minor fraction of such events should yield a robust and reproducible signal in HiC (and\nphenotypes). This could be approached by examining even two independent biological\nreplicates from distinct families/pedigrees (assuming this is not a striking hotspot event).Again,\nare there replicates (from distinct pedigrees) that could help rule this out? alternatively, if the\nauthors still have DNA from other tissues of the specific F2 and F3 animals involved, a\nmethylation array analysis, WES, DNA microarrays could all be used to assess copy number\nacross individual animals to rule this in or out presumably without enormous costs (or even in\ntechnical replicates of the pooled samples).\nWe examined 5 biological replicates, all from separate pedigrees. Therefore, the possibilities\nraised here are ruled out.\n- Do the authors discount potential roles for the many other DCI’s observed that didn’t survive\nthe approach?\nWe cannot not positively exclude possible roles of the DCIs that were eliminated by our\nstringent filtering criteria in the observed transgenerational phenotypes. Such DCIs may be\nexamined in separate studies in the future. However, our data support the prominent role of the\nsingle DCI around the Ide gene (encoding Insulin degrading enzyme) in the transgenerational\ninheritance of the diet-induced, male-specific increases in WAT depot size, hyperinsulinemia,\nhyperglycemia and hyperleptinemia.\n- Given the issues above, the fairly strong implications about causality should be toned down (or\neliminated until causality is tested) and replaced with alternate explanations / discussions of\nmechanisms through which these changes would be expected to be repeatedly induced and\nhow they might survive (or be saved/protected) through epigenome erasure and development.\nDo the authors suggest that these changes are maintained through toti- / pluripotency? Do they\nhave corroborative evidence for instance using FISH that these contacts are indeed increased?\nWe have toned down the discussion about causality, although another explanation for why\nhepatic Ide expression is down-regulated in 2 independent experiments is not obvious. The\nmost parsimonious explanation is that the new contact introduced into Ide results in its down-\nregulation and the phenotypes we have observed here. Our future experiments, supported by a\nnew 5-year R01 award aim to test causality by manipulating the structure of the Ide region.\n- Early post-weaning period is a phase of life still considered development and overlapping with\nsubstantial endocrine fluctuations, final maturation of the gonads, as well as puberty/menarchy.\nHFD appears to have been administered during this time period in one set animals (F2, if I\nunderstand the methods correctly). HFD is well known to impact 5-8 w old mice very differently\nthan adult mice. Also, mice immediately post-weaning have just moved from mixed milk/chow to\nchow-only diet and their metabolic tissues are undergoing a related wave of environmentally\ntriggered terminal maturation and their microbiome is also highly variable between animals. Was\nthere a rationale I am missing to start an intervention given the high degree of unmeasured\nvariability and developmental rearrangement that is happening in this time window? With all\ndue respect, this is truly unfortunate for an experiment focused on variation/plasticity. All\nfigures/text should make clear that F2 underwent 2 interventions. The authors are applauded for\nthe transparency in the report.\nWhile we agree with the reviewer on this point regarding the possibly different effects of HFD\nchallenge between weeks 5-11 vs 17-22, we also note that the physiological effects of HFD\nchallenge are remarkably similar in F2 and F3 animals (Figure 4, Extended data Figures 8-11).\nTherefore, despite the unfortunately different timing of the diet challenge in F2 and F3\ngenerations, the physiological effects are quite similar.\nMinor\n- No DMSO/TBT legend on Ext data 10, unless I missed it.\nWe thank the reviewer for pointing out this omission, which has been corrected in the revised\nfigure legend for what is now Extended data Fig. 9"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my questions.", "reviewer_2": "The revisions did not addressed my concerns. The texts, like sentence 2 and 3 of abstracts, state epigenetic\ntransgenerational inheritance is questioned and no mechanistic data has been provided. This is incorrect after over a 1000\npublications on the topic with all epigenetic compenents known shown to be involved including chromatin structure. So the\nmanuscript needs to be put in perspective with the current literature and not the bias of the investigators. Remove this type of\ntext throughout the manuscript and put it in perspective to the actual extensive literature on the topic. To suggest this is the\nfirst study to provide a mechanism on the phenomenon and no other mechanisms have been shown to be involved is not\naccurate and needs to be revised. A number of studies do already show chromatin modifications and structure are regulated\nby other epigenetic factors and they all work together to promote transgenerational inheritance. So please provide a revision\nthat has a more appropriate perspective and considers the literature on the topic. The data provided are very good, but the\nmanuscript needs to be revised.", "reviewer_3": "The authors have addressed most of my concerns, and this review has no further questions.", "reviewer_4": "Comments to the authors:\nI must apologize for my delay in submitting this response. I had too many competing obligations so timing was poor. I\nappreciate the patience and apologize to the authors and editor. The manuscript remains one of overall high quality, with\npotentially interesting results. Powerful aspects of the work include the very systematic evaluation of changes in PGCs as\nwell as a regular somatic tissue and the authors are commended for their work. My previous concerns are partly addressed.\nClaims of causality relating specifically to Ide remain untested. As written, the potential effects of other loci, and of the other\ngenes within and just outside the regulated TAD are ignored and this does a disservice to the authors and to the readers.\nThese points are expanded in somewhat more detail below:\n1. The authors have addressed my questions on experimental design and in particular my questions about breeding\nschemes, mouse selection, etc. Often their answers to my questions in the rebuttal are clearer than the manuscript methods\nand results so I encourage them to spell out some of the concerns with an almost verbatim copy and paste from their\nresponses.\n2. Regarding Hi-C interpretations. The individual replicate data should be shown, ideally with matched experimental\npartners. This is the only way to allow the reader to appreciate consistency of the response across pools. And this is\nimportant, as the authors must acknowledge that it will strike many readers as too good to be true that one and only one\ndifferentially regulated region was found; the over-argumentation for a very simple ide-dependent causality do not help their\ncause in that context.\n3. On that note of causality (also brought up by other reviewers), the fact that Ide ‘fits’ the narrative well does not rule out\ncorrelation or consequence. The authors seem to argue that the fact that they find the same DCI pulled out several times\nsupports causality, and that it somehow argues against other explanations driving the phenotypes. I don’t understand the\nlogic. Their approach looked for reproducible changes so it can finds reproducible changes. The equally likely explanation\n(other than a false positive which would require a repeat of the entire experiment to rule out – too much) for the locus change\nis that it is the result of the transgenerational metabolic phenotype induced by TBT. Ie. The changes could be the result of\nthe strong glucoregulatory phenotype or any other non-measured phenotype. A recently funded R01 is irrelevant; it does not\nargue for or against Ide’s direct causal involvement. A quick search of Ide in GEO Profiles suggests it changes or is flagged\nas potentially relevant to >10,000 deposited datasets and the vast majority of these (by quick browsing) have little to do with\nthe phenotypes or interventions discussed here. Therefore Ide also changes in response to a very wide range of\ninterventions, data that support the possibility that Ide differences can be secondary. Even if the authors repeated everything\nagain, and saw all the same things, it would still not change that. It would simply mean that the correlation (cause or\nconsequence) is reproducible. The goals to use CRISPRi/a are noble and the data generated will be interesting. It will be\nimportant to manipulate those other genes too in this reviewer’s view. For the purposes of this manuscript, this is simply an\nexercise of reducing the excessive focus on Ide (see below) and rewording.\n4. Along the same lines, it seems inappropriate to treat the much larger changes at Kif11 and Hhex (and possibly at other\nneighboring genes within the TAD) as unlikely mediators. Kif11 deletion is embryonic lethal and mutation in humans triggers\nmulti-system developmental disorder so it carries substantial phenotypic potential. Hhex deletion in mice triggers many\nphenotypes and is necessary for liver development (by conditional deletion) for hepatoblast differentiation and for bile duct\nmorphogenesis. It would be surprising if those changes did NOT influence liver functon(including insulin clearance).\nSimilarly, why are there no measures of potential gene expression changes at Fgfbp3, Btaf1, Cpeb3, and March5? An\nRNAseq experiment of the extent of transcriptional rewiring that exists in these tissues, and the relative effect size of these\nchanges to others would be of substantial value. Perhaps the authors have such data? The genes immediately outside (up\nand downstream of the TAD are also not measure though they can / would be expected to be equally affected. Indeed, it is\nmost likely that the contact rearrangement is inducing silencing on one direction (Ide direction) and activation in the other\n(eg. the Hhex Kif11 direction). And all of these may be eqaully causal to the multiple evidenced phenotypes. March5\nheterozygotes shows evidence of bodyweight phenotypes with an apparent sex effect in males for instance based on data\nbase searches. At a minimum, qPCR of the set of genes above, and the flanking genes should be performed in F2/3 PGCs\nand liver, should sufficiently inform the reader of the extent of the transcriptional changes evoked at this locus.\n5. Reviewer 3 commented that the phenotypes reported are not really obesity, but rather glucoregulatory. I agree completely\nwith this statement. There is very little evidence of outright obesity in the model, rather a <i> susceptibility to </i>DIO that is\nrelatively mild. This is an important and much more accurate nuance. The glucoregulatory phenotype is much stronger and\ndoesn’t require induction. That issue does raise whether this is not a transgenerational phenotype (F2/F3) but rather that\nTBT triggers a repeating F1 intergenerational phenotype and this should be discussed. Having the emphasis on obesity is\nsomewhat misleading given the much stronger glucoregulatory changes. The title is currently incorrect."}, "author_response": "We appreciate the reviewer’s insight regarding the broader involvement of TAD-\nembedded genes. In response, we have updated the discussion section of the\nmanuscript to include the following (lines 298-304):\n\" Our findings are consistent with a mechanism involving altered hepatic Ide expression\nand the fact that the observed downregulation of Ide phenocopies the effects of Ide\nloss-of-function supports a central role for Ide in the phenotypes observed. However,\nthese results do not rule out the potential contributions of Cpeb3 or other TAD-\nembedded genes, in other metabolic tissues. Future studies are warranted to\ninvestigate the role of these genes in metabolic processes and their potential\ninteractions with environmental exposures.\"\nThis addition highlights the need for further exploration while remaining within the scope\nof our current dataset.\nResponse:\nWe agree with the reviewer that hyperinsulinemia and reduced insulin clearance are\ncritical, new findings of this study and that they are, in fact, present prior to HFD\nexposure, although, the insulin effects are strongly exacerbated by HFD. However, we\ndisagree that obesity is a “really minimal phenotype” and point out that we are not using\na standard HFD (40-60% Kcal from fat) but rather a diet with modestly increased fat\ncontent (21.6% Kcal from fat compared with 13.1% in the control diet). To better reflect\nthese findings, we have revised the title of the manuscript to:\n“Heritable Changes in Chromatin Contacts Associated with Transgenerational\nSusceptibility to Diet-Induced Insulin Dysregulation and Obesity.”\nThis updated title acknowledges the metabolic phenotypes observed in the study while\nmaintaining a connection to the broader obesity-related context which we and others\nhave found in multiple experimental systems following TBT exposure"}, {"round": 3, "reviewer_comments": {"reviewer_2": "The previous revisions have improved the manuscript and I appreciate the comments provided in the revision comments section. Although I feel the manuscript should be published, the following comments are provided to allow a further revision to improve the manuscript and put it into more accurate and less biased context. I feel this would improve the manuscript and provide a less biased presentation for the reader, but this is simply a suggestion for the author, and should not prevent publication.\n1) Abstract, the controversial comment in the Abstract is accurate, but this is not due to the extensive literature on the topic of transgenerational inheritance, but this is primarily due to the concept of genetic determinism, which is predominant in today’s science. Therefore, a discussion of controversy without mention of this issue is misleading.\n2) New literature on the lack of DNA methylation erasure is not presented in regards to the comment DNA methylation is erased during embryogenesis. Observations now indicate the lower density DNA methylation that constitutes the majority of DNA methylation is not erased in the primordial germ cell, as occur with high CpG density sites. The transgenerational literature on ncRNA is also not referenced. The reviews listed are primarily older, so inclusion of new literature would be useful, lines 50-64.\n3) Stable generational changes for DNA methylation and ncRNA have been shown in the literature, but this literature is ignored and states TADs only evidence, lines 256-264. Literature clearly demonstrates corresponding DNA methylation and histone modifications are required for chromatin structure and TADs to form, but this is not clarified in the Abstract or Discussion sections.\n4) The bias to TADs or chromatin structure needs to be reduced. The clarification all epigenetic mechanisms are integrated and will be involved in epigenetic transgenerational inheritance needs to be clarified. The literature on this topic is not presented.", "reviewer_4": "This reviewer appreciates the author’s response and efforts. They have addressed most of the concerns. There some\nremaining issues below, and with apologies, one issue of high relevance that must be brought up again.\n_Revisiting copy number variation (CNV)_\nYesterday when presenting my own developmental plasticity work, a postdoc in the crowd asked if I was aware of the\nfollowing paper by Watkins-Chow and Pavan - https://genome.cshlp.org/content/18/1/60.full. I was not. In reading it today on\nthe flight I see it shows what appear to be convincing evidence of a dual signal CNV in Jax C57BL6J animals. The dual\nCNV signal precisely overlaps the signal identified in the manuscript under review here (ie. two likely-coupled CNV signals\nthat match exactly the two Hi-C signals at Ide and Fgfbp3, one shorter one over Fgfbp3, and the second, slightly longer one,\ndirectly over Ide). Watkins/Pavan describe the CNV as being present at frequencies consistent with the possibility that they\nunderpin the transgenerational effects described here. Presumably the origin of the problem was that Jax maintains\nextremely large colonies that make maintaining ‘isogenicity’ near impossible when generating and maintaining their\ncolonies. This reviewer is unaware of what Jax has done since to remedy that situation. We will also be reaching out to Jax\nfor clarity. On quick survey, I cannot find evidence that they have remedied the situation, and the postdoc who informed me\nof this indicated that they personally detected the CNV in many mice, ordered from Jax in 2020. These data raise significant\nconcerns of a genetic underpinning for the current manuscript that have to do with the isogenicity/fidelity of C57BL6J line\nitself.\nBecause of the near impossibility of coincidence, particularly the exact overlap of both CNV signal-elements with the Hi-C\nsignals identified here in this work, this begs a serious re-examination of the possibility that CNV variation underpins the\nincomplete penetrance. This reviewer would recommend replicating some or all of the techniques used by Watkins/Pavan to\ntest the issue. This must of course be done on samples from individual mice (not pools, and ideally those used in the study),\nand from tissues that do not contain large variation in polyploidy that may mask the signal (ie. definitely not liver; ideally\nhaploid sperm… I guess?… I am not a CNV expert, yet). The authors should also consider long-read sequencing as well as\nusing a read mapping/alignment strategy that allows for chimeric- read mapping.\nRegardless, given the essentially perfect overlap with the reported CNV signals, documenting these validation results and\nclearly relating them to how/when the C57BL6J colony used here was distinguished from Jax (or relative to what Jax has\ndone, if anything, about the issue) is necessary. As a scientist, I feel this is completely inappropriate for Jax not to highlight\nboldly in their ordering system if it is still happening. We will also begin carefully looking into this in our lab. I feel for the\nauthors and in many ways hate to have to be the one that brings it up.\n_In the case that the above issue turns out not to be an issue, the following points remain:_\n_From last review Point 4 that highlighted potential relevance of the numerous genes within the TAD:_\nThe authors responded: \"To address the reviewer’s comment, we now include qPCR analysis of the expression of FGFbp3,\nBtaf1, Cpeb3 and March 5 (Extended data Figs. 19, 20). With the exception of a slight increase in hepatic expression of\nCpeb3 in males (~1.3 fold) and a small decrease in expression of Btaf1 in gWAT of F3 males, there were no significant\nchanges observed in the expression of the indicated genes. Therefore, the hypothesis that these genes are equally affected\nto Ide or equally causal to the multiple evidenced phenotypes is not supported.\"\nThe authors are commended for their work. Because it is beyond the scope of this work to rule out involvement of the TAD-\nembedded candidate genes across all relevant metabolic tissues and cell-types, something like the following idea should\nsimply be added to the results and discussion: ‘Our findings are consistent with a mechanism that involves altered haptic Ide\nand Cebp3. The data do not rule out involvement of other genes within the TAD in other metabolic tissues, for instance\nthrough central (eg. hypothalamic) or immune regulation. The Watkins/Pavan paper actually highlight the earlier point, brain\nand spleen appear to be relevant sites of altered expression in those CNV varying mice.\n_From last review Point 5:_\nThe authors responded “In response to this comment, we amended the title to “Heritable changes in chromatin contacts\nlinked to transgenerational susceptibility to diet-induced obesity”. The major change observed is elevation of plasma insulin\nas a result of decreased insulin clearance, particularly in response to increased dietary fat. Therefore, we believe that the\nrevised title is descriptive of what the manuscript presents.”\nThe authors state themselves that the major change is hyperinsulinemia and reduced insulin clearance, so why not put that\nin the title (“susceptibility to diet-induced insulin dysregulation”)?. Those are the most substantial phenotypes; they ARE\nrelevant to metabolic syndrome; they ARE metabolic sequelae of HFD; and they are NOT obesity (which as indicated has a\nreally minimal phenotype). This reviewer would still recommend a more objective/accurate title."}, "author_response": "In response to this comment, we added the following sentences to the third paragraph\nin Discussion:\n“The existence of TEI in mammals remains controversial primary due to the currently\npredominant notion that changes in DNA nucleotide sequence are the exclusive basis of\ninheritance of potentially acquired traits. Another source of the controversy is the wide\nvariety of epigenetic mechanisms that have been proposed to explain mammalian TEI\nwithout describing how more than a single type of molecular epigenetic events can be\ninvolved.”\n2) New literature on the lack of DNA methylation erasure is not presented in regards to\nthe comment DNA methylation is erased during embryogenesis. Observations now\nindicate the lower density DNA methylation that constitutes the majority of\nDNA methylation is not erased in the primordial germ cell, as occur with high CpG\ndensity sites. The transgenerational literature on ncRNA is also not referenced. The\nreviews listed are primarily older, so inclusion of new literature would be useful, lines 50-64.\nWe appreciate the reviewer’s suggestion and have added a discussion on recent\nfindings regarding DNA methylation erasure in embryogenesis. Recent studies (e.g.,\nBen Maamar et al, 2023) indicated that while high-density CpG DNA methylation is\nerased, lower-density methylation is retained, potentially contributing to\ntransgenerational epigenetic inheritance. Additionally, accumulating evidence supports\nthe role of small non-coding RNAs (ncRNAs) in carrying epigenetic information across\ngenerations. We have added the following to the manuscript at Line 54:\n“DNA methylation is erased genome-wide twice each generation in mammals13;\nalthough emerging evidence suggests that lower-density DNA methylation may persist\nin primordial germ cells, contributing to transgenerational inheritance14.”\nIn response to the ncRNA point, we added the following to the manuscript from line 62\n(new text from line 64-66):\n“It remains uncertain how expression of non-coding RNAs, such as those that were\nreported in F1 sperm and seminal fluid16 could be transmitted to multiple future\ngenerations in mammals, although, small non-coding RNAs (ncRNAs) have been linked\nwith epigenetic transgenerational inheritance17.”\n3) Stable generational changes for DNA methylation and ncRNA have been shown in the literature, but this literature is\nignored and states TADs only evidence, lines 256-264. Literature clearly demonstrates corresponding DNA methylation and\nhistone modifications are required for chromatin structure and TADs to form, but this is not clarified in the Abstract or\nDiscussion sections.\nWe addressed this concern as noted above.\n4) The bias to TADs or chromatin structure needs to be reduced. The clarification all epigenetic mechanisms are integrated\nand will be involved in epigenetic transgenerational inheritance needs to be clarified. The literature on this topic is not\npresented.\nWe recognize the need to present a more integrated view of epigenetic mechanisms in\ntransgenerational inheritance. It is our model that DNA methylation, histone modification\nand ncRNA expression may be effectors of transgenerational inheritance that are\ndownstream of changes in higher-order chromatin structure that carry the phenotype.\nThis is discussed from lines 268-277.\nResponse:\nWe thank the reviewer for introducing us to the study of Watkins-Chow and Pavan\n(WC/P), which was published in 2008 to describe the two CNVs around the Ide gene in\nchromosome 19 of the C57BL/6J mice. However, to the extent we investigated, the\nexistence of these CNVs has not been supported by subsequent studies despite the\ncritical importance of this particular strain, which represents the GRCm mouse\nreference genome. The negative reports include a comprehensive study from the\nJackson Laboratory scientists on the genome of C57BL/6J “Eve,” which mentioned the\nWC/P study but showed no evidence of the peri-Ide CNVs in either the founder mouse\nof this strain or its contemporary offspring [Sarsani et al, 2019 PMC6553538]. Although\nwe noticed a few studies on the peri-Ide CNVs published after the WC/P paper, in our\nopinion none of them convincingly reproduced the WC/P CNVs. Therefore, rather than a\nwell-established fact, whether or not a CNV exists near Ide is controversial and certainly\nbears further study.\nBecause of the near impossibility of coincidence, particularly the exact overlap of both\nCNV signal-elements with the Hi-C signals identified here in this work, this begs a\nserious re-examination of the possibility that CNV variation underpins the\nincomplete penetrance.\nResponse:\nPerhaps we were not clear enough in our description of the phenotypes. The\ntransgenerational predisposition to diet-induced obesity, decreased Ide expression and\nelevated insulin levels is 100% reproducible across all batches of male offspring of TBT-\ntreated pregnant F0 dams and 100% reproducible (absent) across all of female\noffspring. Moreover, we have reproduced this male-specific predisposition to diet-\ninduced obesity in 5 independent transgenerational studies using C57BL/6J conducted\nat UCI [Chamorro-Garcia et al, 2017, Chamorro-Garcia et al, 2019, Chang et al (current\nmanuscript), Chang et al (under review at Endocrinology, Chang et al, in preparation]\nand another conducted at MGH using OG2 mice (Tg(Pou5f1-EGFP)2Mnn) (Shioda et\nal, 2022). The probability of an incompletely penetrant CNV in Ide/Fgfbp3 underlying the\nmale-specific phenotype (and not in their female littermates) in 5 independent\nexperiments is vanishingly small, particularly since Sarsani et al did not observe this\nCNV in any C57BL/6J mice in the Jackson Labs colony. See also the discussion below.\nThis reviewer would recommend replicating some or all of the techniques used by\nWatkins/Pavan to test the issue. This must of course be done on samples from\nindividual mice (not pools, and ideally those used in the study), and from tissues that do\nnot contain large variation in polyploidy that may mask the signal (i.e., definitely not\nliver; ideally haploid sperm… I guess?… I am not a CNV expert, yet). The authors\nshould also consider long-read sequencing as well as using a read mapping/alignment\nstrategy that allows for chimeric-read mapping.\nRegardless, given the essentially perfect overlap with the reported CNV signals,\ndocumenting these validation results and\nclearly relating them to how/when the C57BL6J colony used here was distinguished from Jax (or relative to what Jax has\ndone, if anything, about the issue) is necessary. As a scientist, I feel this is completely\ninappropriate for Jax not to highlight boldly in their ordering system if it is still happening. We will also begin carefully looking into this in our lab. I feel for the authors and in many ways hate to have to be the one that brings it up.\nResponse:\nWe agree with the reviewer that the regions of the peri-Ide CNVs detected by array\nCGH in the WC/P paper and the differential chromatin contacts detected in our current\nstudy by Hi-C are practically identical. Array CGH is not fee of artifacts, which can be\ncaused by wet lab procedures or computational analytic algorithms. Unfortunately, in\nthe WC/P study the wet lab procedures were outsourced to a commercial service\nprovider (Nimblegen Systems). In the absence of information on the wet procedures, we\nare unable to replicate the WC/P study. For the dry analysis part, the WC/P study did\nnot include spatial normalization, which is an important step that effectively removes\nfalse positive artifacts. Therefore, the array CGH data presented in the WC/P paper is\nnot necessarily the gold standard. The chromatin contacts around the Ide gene could\nhave been caused by anomalous DNA labeling, biased DNA amplification, or\ninadequate data normalization in the array CGH experiments, resulting in the false\ndetention of CNV at the same location of the chromatin contacts.\nWe also agree that the presence of the CNVs described by Watkins-Chow and Pavan\ncould influence the Hi-C outcomes we reported since our Hi-C study lacks the resolution\nto readily distinguish between new contacts present in the Ide region and increased\ncopy number of the affected region. Although, it seems impossible that our phenotypes\ncould be caused by the Watkins-Chow and Pavan-reported CNV, we recognizing the\nsignificance of this issue and attempted multiple approaches to assess whether CNVs\ncould play a role in our findings.\nTo investigate potential CNV contributions, we:\n1. Attempted to remove possible effects of CNV in our Hi-C data. To the best of\nour knowledge, the most widely accepted method to remove the effects of CNVs\nin Hi-C data is CAIC normalization (Servant et al, 2018, PMC6127900). We\nattempted to use this method to reduce any possible effects of CNVs in our Hi-C\ndata. Unfortunately, this method broadly suppressed TAD detection throughout\nthe genome in all samples, demonstrating that it was not suitable for the task by\nrendering the differential TAD (dTAD) analysis unreliable.\n2. Reviewed existing literature, including the WC/P study, and considered their\nfindings in the context of our results. While WC/P reported the presence of two\nsmall CNVs near the Ide locus, as noted above, a recent comprehensive study of\nthe C57BL/6J genome by Sarsani et al, 2019 PMC6553538 found no evidence\nfor any CNVs on chromosome 19 other than a small duplication between 4.816-\n4.817 MB, which is nowhere near to Ide (37.27-37.33 Mb), Therefore, whether or\nnot a CNV exists near Ide is controversial.\n3. Considered our findings in light of WC/P who reported that the CNV near to Ide\nled to ~1.5 fold elevation of Ide expression in the tissues they evaluated (spleen\nand brain) and a 1.5-fold elevation of Fgfbp3 in spleen but no changes in brain.\na) In contrast to what would be expected by the additional copy(ies) of Ide\nwere they present in our mice, we found ~1.5 fold decreased expression\nof Ide in liver and no changes in other tissues analyzed in the paper\n(muscle and WAT) only in male animals from the TBT group. No changes\nwere observed in females. We assessed Ide mRNA levels in brain and\nspleen to facilitate direct comparisons between our paper and WC/P and\nfound no changes in Ide expression (Extended data Figure 21). Therefore,\nit is unlikely that our results could be explained by the CNVs described by\nWC/P, even if they existed in the 5 groups of animals we obtained from\nJackson Labs over the past 10 years.\nb) We note that the CTCF ChIP-qPCR shown in Fig 3 and Extended Data\nFig 5 showed enhanced CTCF binding at some (B, C, F, G), but not all (no\nincreased binding to A, D, E) of the known and predicted CTCF binding\nsites in the Ide gene from male TBT group mice. No increased CTCF\nbinding was noted at any site in female liver DNA. This is inconsistent with\nwhat would be expected if the putative dTAD was the result of a CNV\nrather than being a bona fide dTAD.\nc) We randomized all mice obtained from Jackson labs prior to starting the\nexperiment and treated groups of 74 F0 females with either DMSO vehicle\nor TBT. Breeding of subsequent generations used groups of at least 61\n(F1) and 69 (F2) females per treatment. While it might be remotely\npossible that only the TBT group females accidentally received mice with\nCNVs, it is inconceivable that only the male offspring of these mice in\nevery generation received this autosomal CNV compared with their female\nlittermates which showed no evidence of the dTADs or increased CTCF\nbinding. Moreover, as noted above, the male-specific predisposition to\nincreased fat mass and Ide down-regulation has been reproduced in\nmultiple, independent experiments. Therefore, we cannot envision a\nscenario in which genetic inheritance of a CNV that is randomly distributed\nin the population drives this highly reproducible phenotype.\nUltimately, while our data do not absolutely rule out the potential involvement of CNVs\nat the Ide and Fgfbp3 loci in the observed transgenerational phenotype, the data\nprovide strong evidence that it is unlikely that the CNVs described by WC/P, if they exist\nin our animals, could be driving the changes in Ide expression and other metabolic\nphenotypes that are found exclusively in male descendants of TBT-treated F0 pregnant\ndams. Given these constraints, we have expanded the discussion in the manuscript\n(lines 325-351) regarding this issue and highlight it as an area for future research.\n_In the case that the above issue turns out not to be an issue, the following points\nremain:_\nFrom last review Point 4 that highlighted potential relevance of the numerous genes\nwithin the TAD:\nThe authors responded: \"To address the reviewer’s comment, we now include qPCR\nanalysis of the expression of Fgfbp3, Btaf1, Cpeb3, and March5 (Extended data Figs.\n19, 20). With the exception of a slight increase in hepatic expression of Cpeb3 in males\n(~1.3-fold) and a small decrease in expression of Btaf1 in gWAT of F3 males, there\nwere no significant changes observed in the expression of the indicated genes.\nTherefore, the hypothesis that these genes are equally affected as Ide or equally causal\nto the multiple evidenced phenotypes is not supported.\"\nThe authors are commended for their work. Because it is beyond the scope of this work\nto rule out involvement of the TAD-embedded candidate genes across all relevant\nmetabolic tissues and cell types, something like the following idea should\nsimply be added to the results and discussion:\n‘Our findings are consistent with a mechanism that involves altered haptic Ide\nand Cebp3. The data do not rule out involvement of other genes within the TAD in other\nmetabolic tissues, for instance through central (eg. hypothalamic) or immune\nregulation. The Watkins/Pavan paper actually highlight the earlier point; brain and\nspleen appear to be relevant sites of altered expression in those CNV-varying mice.’\nResponse:\nWe appreciate the reviewer’s insight regarding the broader involvement of TAD-\nembedded genes. In response, we have updated the discussion section of the\nmanuscript to include the following (lines 298-304):\n\" Our findings are consistent with a mechanism involving altered hepatic Ide expression\nand the fact that the observed downregulation of Ide phenocopies the effects of Ide\nloss-of-function supports a central role for Ide in the phenotypes observed. However,\nthese results do not rule out the potential contributions of Cpeb3 or other TAD-\nembedded genes, in other metabolic tissues. Future studies are warranted to\ninvestigate the role of these genes in metabolic processes and their potential\ninteractions with environmental exposures.\"\nThis addition highlights the need for further exploration while remaining within the scope\nof our current dataset.\nFrom last review Point 5:\nThe authors responded: “In response to this comment, we amended the title to\n‘Heritable changes in chromatin contacts linked to transgenerational susceptibility to\ndiet-induced obesity.’ The major change observed is elevation of plasma insulin as a\nresult of decreased insulin clearance, particularly in response to increased dietary fat.\nTherefore, we believe that the revised title is descriptive of what the manuscript\npresents.”\nThe authors state themselves that the major change is hyperinsulinemia and reduced\ninsulin clearance, so why not put that in the title (“susceptibility to diet-induced insulin\ndysregulation”)? Those are the most substantial phenotypes; they ARE\nrelevant to metabolic syndrome; they ARE metabolic sequelae of HFD; and they are NOT obesity\n(which as indicated has a really minimal phenotype). This reviewer would still\nrecommend a more objective/accurate title.\nResponse:\nWe agree with the reviewer that hyperinsulinemia and reduced insulin clearance are\ncritical, new findings of this study and that they are, in fact, present prior to HFD\nexposure, although, the insulin effects are strongly exacerbated by HFD. However, we\ndisagree that obesity is a “really minimal phenotype” and point out that we are not using\na standard HFD (40-60% Kcal from fat) but rather a diet with modestly increased fat\ncontent (21.6% Kcal from fat compared with 13.1% in the control diet). To better reflect\nthese findings, we have revised the title of the manuscript to:\n“Heritable Changes in Chromatin Contacts Associated with Transgenerational\nSusceptibility to Diet-Induced Insulin Dysregulation and Obesity.”\nThis updated title acknowledges the metabolic phenotypes observed in the study while\nmaintaining a connection to the broader obesity-related context which we and others\nhave found in multiple experimental systems following TBT exposure"}, {"round": 4, "reviewer_comments": {"reviewer_4": "Well done to the authors. I commend them for their stamina and patience with the outstanding issues and for their\nprofessionality in pursuing as clear as possible a view on the potential of genetic variation to be a confounder. I have no\nfurther comments other than recommending that they remove the superlatives (strongly, highly) from their new CNV\nassociated text, and let the data speak for themselves."}, "author_response": "We thank the reviewer for making the effort to evaluate their own mouse stocks and to\ncreate reagents that can distinguish mice as to ‘reference genotype’ or ‘extra copy’,\nthat is, Ide over-expressors as noted in the highlighted text above. This is a great service\nto the field and we appreciate it very much.\nAs we stated in our comments re CNV variation above, it is extraordinarily unlikely that\nany CNV in Ide can underlie the phenotypes we reported in our manuscript. As\nspecifically noted in item #3 of our response above, we reiterate that our phenotype is\nIde down-regulation, rather than the over-expression reported by WC/P, that our\nphenotype is male-specific (no phenotype or increased chromatin contacts in females),\nand that only some, but not all CTCF binding sites in Ide show increased binding in the\nmale (but not female) TBT group. Moreover, due to the breeding scheme and large\nnumber of animals used, we cannot envision a scenario in which randomly selected\nfemales and males (from a population randomly containing ‘over-expressors’) gives rise\nto Ide CNVs (on the autosome chr 19) only in males of the TBT group, but not their\nfemale littermates, or either sex in vehicle controls AND that this group of Ide ‘over-\nexpressors’ could be driving the observed phenotype. We have reproduced the dTAD in\nIde in an additional experiment and shown that hepatic Ide expression is down-\nregulated in four (4) independent transgenerational experiments over the past 8 years\n(two shown in the current manuscript, one recently published\nhttps://doi.org/10.1210/endocr/bqaf063, and an unpublished experiment for which we\ncould provide the data upon request).\nWe would be open to testing the reagents provided by reviewer 4 in our animals and\nalso to participate in a manuscript discussing the impact of the ‘extra copy’ mice\nsourced from JAX since we would be very interested in knowing which of our mice, if\nany, are Ide ‘over-expressors’. However, we strongly believe these future analyses on\nJAX mice should not delay publication of our current manuscript, which reports novel\nand independently validated findings. We trust this is not Reviewer #4's intent or the\neditor's expectation."}]

Decision letter