An H3K14ub-H3K9me3 feedback circuit governs heterochromatin spreading and inheritance in fission yeast

Domain: chemistry
Published: 2026-03-05
Editorial outcome: major_revision

Abstract
Heterochromatin is a bistable chromatin state essential for genome stability and gene regulation. Its spreading and inheritance have long been explained by a “read-write” cycle in which histone methyltransferases bind pre-existing tri-methylation of histone H3 lysine 9 (H3K9me3) and propagate this mark to neighboring nucleosomes. However, the weak affinity and limited catalytic stimulation provided by H3K9me3 alone challenge this model. The fission yeast H3K9 methyltransferase Clr4 functions within the CLRC complex, which also catalyzes histone H3 lysine 14 ubiquitination (H3K14ub). Here we show that H3K14ub and H3K9me3 form a feedback loop: H3K14ub strongly stimulates Clr4 activity on nucleosomes, while both H3K14ub and H3K9me3 stabilize CLRC binding to chromatin. Even subtle perturbations that disrupt this feedback, such as mutating one of the three H3 genes to prevent ubiquitination or methylation, or impairing Clr3-mediated H3K14 deacetylation, compromises heterochromatin spreading and inheritance. Conversely, counteracting activities, such as H3K14 acetylation by Mst2 and H3K9 demethylation by Epe1, synergistically constrains heterochromatin expansion. Thus, rather than relying solely on the weak H3K9me3 “read-write” cycle, heterochromatin is maintained through an integrated circuit of ubiquitination, deacetylation, and methylation, which governs spreading and inheritance.

Introduction
Heterochromatin is a transcriptionally repressive chromatin state that plays a critical role in maintaining genome stability by silencing repetitive elements, suppressing recombination, and regulating gene expression programs 1-3. Dysregulation of heterochromatin has been linked to a wide range of human diseases, while its targeted modulation can enhance cancer immunotherapy by reactivating repeat expression and promoting immune recognition of tumor cells 4,5.

Heterochromatin is characterized by specific chromatin features, including hypoacetylated histones and methylation of histone H3 on lysine 9. Its assembly involves three interconnected phases: initiation, spreading, and inheritance 1,3,6. Initiation occurs at defined nucleation sites, where sequence-specific factors or non-coding RNAs recruit histone-modifying enzymes. Once established, heterochromatin spreads into adjacent regions independent of DNA sequence, resulting in extended repressive domains. These domains can then be stably inherited across cell divisions, even in the absence of the initiating signal. While heterochromatin initiation has been well characterized, the molecular mechanisms underlying its spreading and inheritance remain incompletely understood.

A defining feature of heterochromatin is its bistable behavior, in which genomic regions can switch between active and silent states yet maintain sharp boundaries between them 7,8. Bistability usually arises from the interplay of reinforcing and antagonizing activities that generate switch-like behavior 9. However, the precise molecular circuits that confer bistability to heterochromatin domains remain poorly defined.

The fission yeast Schizosaccharomyces pombe has been widely used to dissect the molecular mechanisms of heterochromatin assembly. In this organism, heterochromatin forms primarily at pericentric repeats, telomeres, and the silent mating-type region, all of which share common repetitive elements 10. At these sites, RNA interference (RNAi) machinery processes repeat-derived transcripts into small interfering RNAs (siRNAs), which guide the H3K9 methyltransferase Clr4 to initiate heterochromatin formation 11-13 (Fig. S1). Heterochromatin then spreads from initiation sites through a “read-write” mechanism in which Clr4 uses its chromodomain to bind existing H3K9me3 and methylates adjacent nucleosomes 14,15. The “read-write” coupling is also critical for heterochromatin inheritance: during DNA replication, parental histones marked with H3K9me3 are distributed to daughter strands 16,17, where they guide Clr4 to restore the methylation pattern on newly synthesized histones 15,18,19 (Fig. S1). However, the binding of Clr4 chromodomain to H3K9me3 and the stimulation of Clr4 activity by H3K9me3 are relatively weak 14,15, and whether they form the sole basis of heterochromatin bistability is unknown.

Central to heterochromatin formation is the Clr4 histone H3K9 methyltransferase 20,21. It also functions as part of the CLRC E3 ubiquitin ligase complex, which includes Clr4, Cul4, Rik1, Raf1, and Raf2 22-26. Like other Cullin-based E3 complexes, Cul4 forms the scaffold, Raf1 is the presumptive substrate recognition subunit, and Rik1 links Raf1 to Cul4 27,28. The function of Raf2 is currently unknown but has been implicated to regulate CLRC integrity 29. In addition to its methyltransferase activity, CLRC catalyzes monoubiquitylation of histone H3 on lysine 14 (H3K14ub), a modification that enhances Clr4 activity in vitro 30-33. However, the genomic distribution and biological function of H3K14ub in vivo have remained unclear due to the lack of a specific detection reagent.

Heterochromatin spreading and inheritance also require the histone deacetylase Clr3, which specifically removes acetyl groups from H3K14 34-36. Clr3-mediated deacetylation has been proposed to stabilize heterochromatin by reducing histone turnover 37-39, but it remains unknown whether this activity also promotes heterochromatin through regulation of H3K14ub, which is mutually exclusive with H3K14 acetylation.

In this study, we define a chromatin-based positive feedback loop between H3K14 ubiquitylation and H3K9 methylation that is essential for heterochromatin spreading and inheritance. Using a H3K14ub-specific antibody 40, we show that H3K14ub co-localizes with H3K9me3 across the genome and depends on CLRC and Clr4 enzymatic activity for deposition. Conversely, H3K14ub promotes H3K9me3 on nucleosomes via stimulation of Clr4, establishing a mutually reinforcing circuit. This loop is modulated by Clr3-mediated H3K14 deacetylation, which controls the extent of spreading and inheritance. In addition, Raf1 overexpression can enhance feedback strength and restore heterochromatin spreading and inheritance when the circuit is compromised. Finally, we show that H3K9 demethylase Epe1 and H3K14 acetyltransferase Mst2 function together to limit feedback activation and prevent unchecked heterochromatin expansion. The balance between reinforcing and antagonizing activities thus establishes a bistable system. These findings reveal a previously unrecognized layer of crosstalk between histone deacetylation, ubiquitylation, and methylation that promotes robust heterochromatin formation and maintenance.

Results
H3K14ub stimulates Clr4 enzymatic activity on nucleosome substrates in vitro
Previous studies investigating the effects of H3K14ub on Clr4 enzymatic activity in vitro primarily used histone tail peptides as substrates 30-33. However, whether H3K14ub modulates Clr4 activity in different substrate context has not been compared. To address this question, we purified recombinant Clr4 SET domain (residues 190-490) as well as a mutant version containing mutations in three phenylalanine residues (F256A, F310A, and F427A, hereafter referred to as Clr4-3FA) known to disrupt the interaction between Clr4 and H3K14ub 31 (Fig. 1a, b). We performed in vitro histone methyltransferase assay using 3[H]-S-adenosyl-methionine (SAM) and recombinant substrates, including H3/H4 tetramers, wild type mononucleosomes, and H3K14ub-modified mononucleosomes. Clr4 exhibits robust methyltransferase activity toward H3/H4 tetramers but shows minimal activity towards unmodified mononucleosomes under the conditions tested, suggesting that nucleosome DNA inhibits Clr4 function (Fig. 1c). In contrast, Clr4 displays strong activity towards H3K14ub mononucleosomes, indicating that H3K14ub alleviates the inhibitory effects of nucleosome DNA.

Further in vitro analyses reveal that the Clr4-3FA mutant retains activity on H3/H4 tetramers but fails to respond to stimulation by H3K14ub mononucleosomes (Fig. 1c). These results suggest that the interaction between Clr4 and H3K14ub is critical for stimulating Clr4 enzymatic activity in the context of nucleosomes. Notably, this stimulatory effect is specific to H3K14ub, as Clr4 shows little activity toward recombinant H3K18ub mononucleosomes or H2BK120ub mononucleosomes (Fig. 1d).

H3K14ub is enriched at heterochromatin in vivo
Biochemical studies have shown that the CLRC complex catalyzes H3K14ub (Fig. 2a) 33. However, the genomic distribution of H3K14ub in vivo has not been characterized. To address this question, we generated a highly specific antibody against H3 K14ub 40. Western blot analysis using recombinant nucleosomes with ubiquitin at different lysine residues confirmed the antibody’s specificity: it robustly recognizes H3K14ub but does not cross-react with H3K18ub or H2BK120ub (Fig. 2b).

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) reveals that H3K14ub is highly enriched at heterochromatin domains, including pericentric repeats, telomeres, and the silent mating-type region, with a distribution closely resembling that of H3K9me3 (Fig. 2c). Both marks are undetectable in rik1∆ cells (Fig. 2c), indicating a strict dependence on CLRC for their deposition. Consistent with this, ChIP-qPCR confirms that H3K14ub is enriched at pericentric dh repeats, but is abolished in strains carrying the K14R substitution in all histone H3, as well as in CLRC mutants (raf1∆, raf2∆, and rik1∆) (Fig. 2d, e). All of these mutants also show complete loss of H3K9me3 (Fig. 2d, e). These findings establish the genome-wide map of H3K14ub in fission yeast and demonstrate that CLRC is indispensable for its deposition in vivo, consistent with a recent study 41. The striking co-localization of H3K14ub with H3K9me3 further suggests that the two modifications act in concert to define heterochromatin domains, a feature also conserved in mammals 40. The results are consistent with previous mass spectrometry analyses demonstrating the coexistence of H3K14ub and H3K9me3 33, and extend those findings by providing genome-wide localization at high resolution.

H3K9me is required for H3K14ub in vivo
To further dissect the relationship between these two modifications, we also examined whether H3K9 methylation is required for H3K14ub. ChIP analyses reveal that both H3K14ub and H3K9me3 are lost from pericentric dh repeats in clr4∆ cells (Fig. 3a, b), as well as in the catalytically inactive mutant clr4-Y451N mutant, which abolishes H3K9 methylation without affecting CLRC integrity 42 (Fig. 3a, b). Similarly, the H3K9R substitution in all histone H3 genes abolishes H3K14ub, demonstrating that H3K9me is essential for H3K14 ubiquitylation in vivo (Fig. 3a, b).

Mechanistically, H3K9me3 stabilizes CLRC recruitment to chromatin 15. Indeed, ChIP analysis shows that Rik1, a core subunit of CLRC, is no longer enriched at dh repeats in clr4∆, clr4-Y451N, or H3K9R cells (Fig. 3c). Two distinct mechanisms mediate Clr4 recognition of modified nucleosomes: its chromodomain binds H3K9me3, an interaction disrupted by the W31G mutation 15, and its SET domain binds H3K14ub, an interaction abolished by the 3FA mutation 31,32 (Fig. 3d). ChIP analyses show that both clr4-W31G and clr4-3FA mutations markedly reduce H3K9me3, H3K14ub, and Rik1 levels at dh repeats (Fig. 3e-g). In addition, ChIP analyses show that Clr4 localization to dh repeats is abolished in H3K9R and H3K14R cells (Fig. S2a), and co-immunoprecipitation analysis show that the Clr4-Rik1 interaction is not affected by H3K9R or H3K14R substitutions (Fig. S2b). These results demonstrate that recruitment of the entire CLRC complex is dependent on both H3K9me and H3K14ub.

Together, these results uncover a positive feedback loop between H3K14ub and H3K9me3: H3K14ub stimulates Clr4 activity to promote H3K9me3, while both H3K9me3 and H3K14ub enhances CLRC recruitment to heterochromatin, thereby facilitating further H3K14 ubiquitylation and H3K9 methylation (Fig. 3h).

H3K14ub-H3K9me3 feedback is critical for heterochromatin spreading and inheritance
Heterochromatin assembly proceeds through initiation, spreading, and inheritance (Fig. S1) 1,3. Spreading and inheritance rely on preexisting modifications and therefore are expected to be particularly sensitive to perturbations of the H3K14ub-H3K9me3 feedback loop.

Fission yeast encodes three histone H3 genes (hht1+, htt2+, and hht3+) expressed at similar levels 42,43 (Fig. 4a). If this feedback loop is critical, then single-copy mutations that prevent H3 ubiquitination (H3K14R) or methylation (H3K9R) should impair spreading or inheritance, even in the presence of two wild-type copies (Fig. 4b). To test this, we analyzed hht3-K9R and hht1-K14R mutants, each tagged with C-terminal Flag at their endogenous loci (Fig. 4a). Western blot confirms that mutant H3 proteins are expressed at levels comparable to their wild type counterparts (Fig. 4c).

To monitor heterochromatin function, we used ade6+ reporter genes inserted at distinct chromosomal locations. Reporter silencing yields red colonies on low adenine (YE) medium, whereas expression results in white or pink colonies. At the otr::ade6+ reporter within pericentric repeats, where heterochromatin is initiated by the RNA interference (RNAi) pathway 11,12, single-copy H3 mutations (hht3-K9R and hht1-K14R) cause only modest silencing defects, as indicated by mostly red colonies (Fig. 4d) and slight reductions in H3K14ub and H3K9me3 (Fig. 4e). These results suggest that initiation sites are relatively resistant to mild perturbations of the feedback loop.

In contrast, the mat3::ade6+ reporter, located in the silent mating type region but distant from the cenH initiation element, specifically measures heterochromatin spreading. Both single-copy H3 mutations show strong spreading defects (white colonies, Fig. 4 f), accompanied by loss of H3K14ub and H3K9me3 at the reporter (Fig. 4g).

The K∆::ade6+ reporter, in which cenH is replaced with ade6+, specifically measures heterochromatin inheritance (Fig. 4h). In this context, silencing depends exclusively on parental histone-mediated inheritance 44. Both single-copy H3 mutations cause strong loss of silencing (Fig. 4h), accompanied by loss of H3K14ub and H3K9me3 at the reporter as well (Fig. 4i).

These results suggest that local saturation of both marks is necessary to sustain read-write coupling, and that interference with even one third of total H3 can perturb this balance 45,46.

The acetylation status of H3K14 regulates H3K14ub-H3K9me3 feedback
The H3K14ub-H3K9me3 feedback loop relies on the availability of unmodified H3K14, since acetylation at this residue blocks ubiquitination. We therefore investigated the role of Clr3, a histone H3K14deacetylase 34-36, in regulating this process. ChIP analysis reveals that clr3∆ causes only modest reductions in H3K14ub and H3K9me3 levels at pericentric dh repeats (Fig. S3a, b). It also results in enhanced enrichment of Rik1 and Clr4 at dh repeats (Fig. S3c, d).

Clr3-mediated H3K14 deacetylation is particularly important for heterochromatin spreading at the silent mating-type locus (Fig. 5a) 36. In clr3∆ cells, both H3K14ub and H3K9me3 are modestly reduced at cenH but completely lost at mat2P (Fig. 5a, b), indicating that H3K14ub spreads together with H3K9me3. Both Rik1 and Clr4 show strong enrichment at cenH, but are absent at mat2P (Fig. 5c and Fig. S3e, f).

Previous studies have shown that clr3Δ increases transcription of repetitive DNA, thereby enhancing RNAi-dependent recruitment of CLRC to heterochromatin initiation sites 15,47, consistent with our findings. However, despite this increased recruitment at nucleation sites, CLRC fails to spread to distal regions such as mat2P. This uncoupling of initiation from spreading demonstrates that RNAi-mediated recruitment alone is insufficient to sustain heterochromatin propagation, and that effective spreading of CLRC requires an intact H3K14ub-H3K9me3 feedback loop.

The modest reduction of H3K14ub and enhanced enrichment of CLRC at dh and cenH in clr3∆ cells suggest that elevated CLRC levels may partially compensate for loss of deacetylation by outcompeting histone acetyltransferases for H3K14 (Fig. 5d). To test this idea, we overexpressed Raf1, the presumptive substrate recognition subunit of CLRC, by placing a Flag-raf1+ construct under the control of the ade6 promoter (OE-raf1+) (Fig. 5e).

In clr3∆ cells, the mat3::ade6+ reporter is expressed, giving rise to white colonies on YE medium. In contrast, OE-raf1+ clr3∆ cells form pink colonies (Fig. 5f), and partially restored H3K14ub and H3K9me3 at mat3::ade6+ (Fig. 5g). Strikingly, OE-raf1+ also partially rescued the spreading defects of hht3-K9R or hht1-K14R, as shown by pink colonies on YE medium (Fig. 5f) and restoration of H3K14ub and H3K9me3 at the reporter (Fig. 5g). In contrast, OE-raf1+ does not rescue silencing defects of swi6∆, consistent with Swi6 functions downstream of H3K9me3 (Fig. 5f). It also cannot rescue silencing defects of clr4-W31G or clr4-3FA, suggesting that OE-raf1+ exerts its effects through the H3K14ub-H3K9me3 feedback loop (Fig. S3g, h).

Raf1 overexpression also partially restored heterochromatin inheritance defects in clr3∆, hht3-K9R or hht1-K14R mutants, as well as in mcm2-2A or mrc1∆, which disrupt proper parental histone segregation to the lagging strand during DNA replication 45,48-50 (Fig. 5h). These results establish that H3K14ac modulates the strength of the H3K14ub-H3K9me3 feedback loop to support both spreading and inheritance.

Regulation of the H3K14ub-h3K9me3 feedback loop by Mst2 and Epe1
If positive feedback drives heterochromatin spreading and inheritance, then negative regulatory mechanisms are equally important to prevent uncontrolled heterochromatin expansion. Two major heterochromatin antagonists are the histone acetyltransferase Mst2, which is specific for H3K14, and the JmjC-domain protein Epe1, a putative H3K9 demethylase 18,19,51-56. Although their biochemical activities differ, the H3K14ub-H3K9me3 feedback loop explains their shared phenotypes: Mst2-mediated H3K14ac counteracts Clr3-mediated H3K14 deacetylation, therefore inhibiting H3K14ub, while Epe1 directly erases H3K9me3 (Fig. 6a). Consistent with this logic, either mst2∆ or epe1∆ suppresses the silencing defects of clr3∆ at both mat3::ade6+ and K∆::ade6+ (Fig. 6b, c), indicating that enhancing one arm of the loop can compensate for weakening another.

Loss of both Mst2 and Epe1, however, removes both inhibitory constraints, causing excessive heterochromatin spreading that silences essential genes and slows growth 51,57 (Fig. 6d, e). This stress triggers a compensatory response in which H3K9me accumulates at the clr4+ locus, down regulating Clr4 expression 51,57 and allowing cells to recover from heterochromatic stress (Fig. 6f). Our model predicts that simultaneous loss of Mst2 and Epe1 synergistically activates the H3K14ub-H3K9me3 feedback loop, driving heterochromatin expansion and triggering this adaptive response.

To test this hypothesis, we examined H3K14ub and H3K9me3 levels at a region outside the boundary element of centromere I (IRC1R) 58. Because mst2∆ epe1∆ rapidly generate epigenetically silenced clr4+, we analyzed mst2∆ epe1∆ swi6∆ cells, which tolerate elevated H3K9 methylation without trigging formation of the epigenetic suppressor 51. ChIP analyses show that mst2∆ or epe1∆ alone has no effects on H3K14ub or H3K9me3 outside of IRC1R (Fig. S4a), because the IRC1R boundary effectively blocks heterochromatin spreading. In contrast, in mst2∆ epe1∆ swi6∆ cells, both H3K14ub and H3K9me3 levels dramatically increased outside IRC1R, suggesting that supercharging the H3K14ub-H3K9me3 feedback loop overrides heterochromatin boundary (Fig. S4a).

Either mst2∆ or epe1∆ results in increase in H3K9me at facultative heterochromatin islands, such as mei4+ 51,59. Although we did not detect significant increase in H3K14ub and only a modest increase in H3K9me3 in the single mutants, we observed a strong increase in both H3K14ub and H3K9me3 at mei4+ in mst2∆ epe1∆ swi6∆ cells (Fig. S4b). These results indicate that Mst2 and Epe1 act synergistically to restrain the H3K14ub-H3K9me3 feedback loop.

We next hypothesize that compromising the H3K14ub-H3K9me3 feedback loop would prevent activation of the adaptative response. To test this, we combined mst2∆ and epe1∆ cells with mutations that weaken the feedback loop (clr4-W31G, clr4-3FA, hht3-K9R, and hht1-K14R). In all cases, the resulting triple mutants no longer produce small colonies (Fig. 6e) and fail to induce adaptive accumulation of H3K9me2 at clr4+ (Fig. 6f). Together, these findings demonstrate that Mst2 and Epe1 function as independent brakes that restrain the H3K14ub-H3K9me3 feedback loop and prevent uncontrolled heterochromatin expansion.

Methods
Fission yeast strains
Yeast strains overexpressing Flag-raf1+ were generated by using Flag-raf1+ to replace ade6+ coding region through CRISPR-mediated recombination 77, allowing it to be driven by the ade6 promoter. All other strains were generated by genetic crosses. A list of fission yeast strains used in this study is provided in Supplementary Data 1.

Protein expression and purification
The SET domain of recombinant Clr4 (residues 190-490) and the 3FA mutant were cloned into a pET28a vector. The expression plasmids were transformed into Rosetta cells and protein expression was induced using 0.15 mM isopropyl-1-thio-D-galactopyranoside. After incubation overnight at 16 ̊C, the cells were harvested and resuspended in lysis buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7.0), supplemented with 2 mM β-mercaptoethanol and lysed with ultrasonication. The lysate was incubated with Talon metal affinity resin (Takara Bio USA, 635501) and then washed with lysis buffer containing 15 mM imidazole. Bound protein was eluted with lysis buffer containing 150 mM imidazole.

In vitro histone methyltransferase assays
Histone methyltransferase assays were performed with recombinant Clr4 SET domain, histone H3/H4 tetramer or nucleosomes in histone methyltransferase buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 1 mM DTT, 2 μM SAM) containing 3H-SAM (400 nM) for 30 minutes at 25°C. Wild type (16-0006), H3K14ub (16-0398), H3K18ub (16-0401), and H2BK120ub (16-0396) nucleosomes were purchased from Epicypher. The samples were resolved by SDS-PAGE and subjected to Coomassie staining to visualize the proteins and then treated with EN3HANCE (Perkin Elmer) to visualize labeled substrates.

Serial dilution analyses
For serial dilution plating assays, ten-fold dilutions of a mid log-phase culture were plated on the indicated medium and grown for 3 days at 30C.

Chromatin immunoprecipitation (ChIP)
Log phase yeast cultures were crosslinked with 1% formaldehyde for 20 minutes at room temperature, followed by the addition of 125 mM glycine for 5 minutes. Cells were then harvested and washed with cold PBS and resuspended in ChIP lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% Deoxycholate, 1 mM PMSF). Cold glass beads were added, and the mixture was vigorously shaken in a MiniBeadBeater (Biospec Products). The lysates were collected and sonicated with Bioruptor® Pico (Diagenode) for 12 cycles (30 seconds on/30 seconds off). After centrifugation at 16,000g for 15 minutes to clarify the lysates, released chromatin was immunoprecipitated overnight at 4°C with antibodies: H3K14ub, H3K9me3 (Active Motif 39161), and myc (Sigma C3956). Protein G Agarose beads (Sigma 11243233001) were added for an additional 2 hours at 4°C. The beads were washed twice with ChIP lysis buffer, once each with ChIP lysis buffer containing 0.5 M NaCl, Wash buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA), and TE buffer (50 mM Tris pH 8.0, 1 mM EDTA). Beads-bounded chromatin was eluted with TES buffer (50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS) at 65°C and then incubated overnight at 65°C to reverse crosslinking. The DNA-protein mixtures were treated with Proteinase K (Invitrogen 10005393), and DNA was purified by phenol: chloroform extraction, followed by ethanol precipitation.

Quantitative PCR (qPCR) was performed using Luna Universal qPCR Master Mix (NEB M3003S) on a StepOne Plus Real-Time PCR System (Applied Biosystems). DNA serial dilutions were used as templates to generate a standard curve of amplification for each pair of primers, and the relative concentration of the target sequence was calculated accordingly. The value from wild-type cells was arbitrarily set to 1 and served as a reference for other samples. A list of DNA oligos used is provided in Supplementary Data 2.

ChIP-seq
Sequencing reads were trimmed using Trimmomatic (v0.38) to remove Illumina adapters and low-quality sequences. Filtered reads were aligned to the Schizosaccharomyces pombe reference genome (ASM294v3) using BWA (v0.7.17). Alignment files were processed with SAMtools (v1.9) to filter mapped reads. Picard (v2.19.0) was used for sorting, PCR duplicate removal, and indexing. Local realignment around indels were performed with GATK3 (v3.6-6). Peak calling was performed with MACS2 (v2.2.7.1). Genome coverage was normalized to counts per million mapped reads (CPM) using bedtools (v2.27.1) to generate standardized BedGraph files. The BedGraph files were visualized using IGV. ChIP-seq data is available in the NCBI database under accession number PRJNA1300844.

Western blot analysis
Cell lysates were prepared by lysing yeast cells with a beadbeater in ChIP lysis buffer. The lysates were sonicated for a total of 10 minutes to fragment chromatin. The cleared lysates were mixed with 2xSDS loading dye and resolved by SDS-PAGE. Western blot analyses were performed with H3K14ub and Flag (Sigma F7425 and Genscript A00187) antibodies.

Protein purification and co-immunoprecipitation
Log phase yeast cells were harvested and washed with 2xHC buffer (300 mM HEPES-KOH at pH 7.6, 2 mM EDTA, 100 mM KCl, 20% glycerol, 2 mM DTT, 1mM PMSF) and frozen in liquid nitrogen. Crude cell extracts were prepared by vigorously blending frozen yeast cells with dry ice using a household blender, followed by incubation with 1xHC buffer containing 250 mM KCl for 30 minutes. The lysate was cleared by centrifugation at 15,000g for 1 hour. The supernatants were incubated with Flag-agarose (Sigma A2220) overnight, and washed eight times with 1xHC containing 250 mM KCl. For co-immunoprecipitation analysis, bound proteins were resolved by SDS-PAGE followed by Western blot analyses with Myc (Sigma C3956), or Flag (Sigma F7425) antibodies.

Quantification and Statistical Analysis
For ChIP-qPCR, data are presented as mean ± sd of three biological replicates. Statistical significance was assessed using two-tailed unpaired student’s t-tests for pairwise comparisons.

Data Availability
The ChIP-seq data generated in this study have been deposited in the NCBI database under accession code PRJNA1300844 [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1300844/]. Source data are provided with this manuscript.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Toda et al. propose a novel mechanism to explain the long-standing question of how the vulnerable\nH3K9me3 read-write cycle alone can maintain epigenetic inheritance. The authors identify a H3K14ub–H3K9me3 circuit\nthat forms a mutually reinforcing feedback loop. This work builds upon previous findings that H3K14ub is required for\nH3K9me3 (PMIDs: 40446033, 34524082, 31468675, 34010645).\nThe authors provide convincing results to support several new findings:\n1) H3K9me3 is also necessary for H3K14ub.\n2) Both H3K14ub and H3K9me3 stimulate Clr4 activity to promote H3K9me3, and also contribute to recruiting and\nstabilizing CLRC at heterochromatin.\n3) Any partial disruption of this circuit causes heterochromatin defects.\n4) Impairment of the activities that antagonize H3K14Ub or H3K9me3 alleviates the defects, supporting that both positive\nand negative feedback activities synergistically regulate the propagation and maintenance of heterochromatin.\nAdditionally, this study is the first to map the genome-wide distribution and biological function of H3K14ub in fission yeast.\nThis work is original and significant, with data that support the conclusions, making it of interest to a broad audience in the\nfield of heterochromatin.\nMinor comments for the authors to consider:\nFigure 1c: Please clarify what the X and Y axes represent.\nFigure 1c Legend: Add \"tel1L,\" \"cen1,\" and \"tel2R\" to the legend.\nMating Type Locus: Does H3K14ub colocalize with H3K9me3 at the mating type locus, one of the three major\nheterochromatic regions? This locus was not shown in Figure 1c, although it was used for further analysis in Figures 3-5.\nLine 40: Rea et al. (#12) should be cited with references 6–8, not 9–12.\nLine 50 and 237: \"Histone deacetylation, ubiquitination, and methylation\" might be a more appropriate description than\n\"histone ubiquitination, acetylation, and methylation.\"\nLine 103–104: Data for H3K14R and deletion of raf1 and raf2 are not shown in the main or supplemental figures.\nLine 132: What evidence supports the notion that H3K14ub makes a stronger contribution? The results shown in Figure 2f–\n2g appear to support this.\nThe methods section includes a quantification and statistical analysis section; however, no statistical analysis is presented\nin the ChIP-qPCR figures. It is also unclear what statistical methods the authors would use for comparison.\nFigure 3a: Should the hht3-K9R data look different from hht1-K14R (symbol + a bar)? Does the blue bar indicate the Flag\ntag?\nFigure 3b: Should the mutants on the H3 tail be more visible in the hht mutant?", "reviewer_2": "Methylation of H3K9 (H3K9me), which is catalyzed by the histone methyltransferase Clr4/Suv39, is a conserved mark that\nassembles a higher-order chromatin structure called heterochromatin. In fission yeast, Clr4 forms a complex called CLRC\nthat provides H3K14 ubiquitylation (H3K14ub). Previous studies have shown that H3K14ub promotes Clr4’s H3K9\nmethyltransferase activity in vitro. In this manuscript, the authors generated a specific antibody against H3K14ub. Using this\nantibody, the authors analyzed the genome-wide localization of H3K14ub and demonstrated that it clearly overlaps with\nH3K9me3. The authors also examined the relationship between the factors involved in H3K9me and H3K14ub, showing\nthat H3K9me and H3K14ub form a feedback loop. Finally, the authors demonstrated that this feedback loop is negatively\nregulated by the histone acetyltransferase Mst2 and the H3K9 demethylase Epe1.\nOverall comments:\nAlthough accumulating evidence indicates that H3K14ub plays a pivotal role in depositing H3K9me in heterochromatic\nregions, its genome-wide localization and dynamics are not fully understood. This study used an H3K14ub antibody and\nprovides strong evidence that H3K14ub is closely associated with H3K9me. In addition, it is important to note that this\nfeedback loop is negatively regulated by counteracting histone-modifying enzymes. The experiments were carefully\ndesigned, and most of the conclusions are supported by the presented data. Having said that, the novelty of the findings\nappears limited, as most of the conclusions are predictable from previous genetic studies, and the authors merely verified\nthem by using the H3K14ub antibody. Below, I raise several points that the authors should address to improve the\nmanuscript.\nMajor points:\n1. (Figs. 1 and 2):\nWhile the authors demonstrated that Rik1 (CLRC) recruitment depends on H3K9me and H3K14ub, they did’t show whether\nClr4 recruitment depends on these modifications. To demonstrate interdependence, the authors should show that Clr4\nlocalization is affected by H3K9R or H3K14R amino acid substitutions, or CLRC complex formation.\n2. (Fig 4c, Fig S3c):\nThe finding that Rik1 levels are elevated at cenH in clr3∆ strains is intriguing. While the authors suggest that CLRC\naccumulates at cenH because it cannot spread, does Clr4 localization also increase similarly at cenH? Furthermore, cenH\nfunctions as an H3K9me initiation site by recruiting the RITS complex (doi: 10.1038/ng1452). It would be helpful to discuss\nwhether the RNAi pathway is involved in the increased Rik1 (CLRC) localization at cenH observed in clr3∆ cells.\n3. (Fig. 5):\nThe authors noted that negative regulators, such as Mst2 and Epe1, restrict the H3K14ub-H3K9me3 feedback loop, thereby\npreventing the uncontrolled expansion of heterochromatin. However, they demonstrated heterochromatin expansion only at\nmarker genes inserted in heterochromatic regions. To clarify the roles of Mst2 and Epe1 against the H3K14ub-H3K9me3\nfeedback loop under physiological conditions, the authors could perform ChIP-seq analyses to test whether H3K14ub and\nH3K9me3 expand beyond well-defined heterochromatic regions in mst2Δ and epe1Δ mutants, as shown in Fig. 1c.\n4. (lines 48 and 204):\nThe authors describe the actions of Mst2 and Epe1 as negative feedback. While these factors negatively regulate the\nH3K9me–H3K14ub feedback loop, this is not sufficient to describe it as negative feedback. To do so, the authors need to\ndemonstrate a relationship in which H3K14 acetylation by Mst2 promotes H3K9 demethylation, and, conversely, H3K9\ndemethylation promotes H3K14 acetylation by Mst2.\nMinor points:\n1. (Abstract, lines 39-40):\nReferences #11 and #12 do not include any results regarding H3K14 ubiquitylation activity of CLRC.\n2. (Fig. 1c):\nAlthough the authors clearly showed the genome-wide localization of H3K14ub by ChIP-seq analysis using H3K14ub\nantibody, its heterochromatic localization and coexistence with H3K9me have been previously demonstrated by ChIP-\nMS/MS analysis (doi: 10.15252/embr.201948111). The authors should refer to the published study in the text.\n3. (Fig. 1c):\nH3K14ub and H3K9me3 are not detected at the mat region of chromosome 2. If this is the case, the author should accurately\ndescribe it and provide possible reasons, such as the strain used.\n4. (ChIP-qPCR):\nTo determine significant or non-significant differences, it would be better to apply statistical analyses to ChIP-qPCR data.\n5. (Fig. S2):\nUbiquitylated nucleosomes were used as the substrate for in vitro histone methyltransferase assays. However, the Methods\nsection does not include the description how the ubiquitylated nucleosomes were reconstituted.\n6. (Reference #34):\nThe authors should provide the correct citation.", "reviewer_3": "Toda et al. present an analysis of the role of the histone modification H3K14ub in heterochromatin formation in fission yeast.\nThis is a very neat genetic analysis and reveals that H3K14ub and H3K9me2/3 depend on each other in a finely balanced\nsystem that involves the H3K14ac and H3K9me.\nThis analysis brings long-awaited clarity about the role of H3K14ub in the heterochromatin and gene silencing system of\nfission yeast. Due to its highly conserved machinery, these results are likely to be relevant for many other systems, including\nhuman cells, where the significance of H3K14ub in heterochromatin has now been well established. The characterization of\nthe feedback loop using highly targeted mutants reveals novel dependencies, showing that H3K9me is critical for H3K14ub\nand vice versa.\nThe data is of high quality and clarity and is presented in a consistent and clear manuscript. Provided the concerns below\nare addressed, this manuscript is highly adequate for publication in Nature Communications.\nIssues:\n- Statistics for the ChIP-qPCR data are specified as mean +/- SD of 2 technical replicates. This is inadequate and should be\nbased on at least 3 biological replicates.\n- The antibody is custom-made and there is no information on how the authors are planning to share it. Given China's strict\nexport rules, there should be a provision for distribution and some information on how the antibody can be obtained by the\ninternational research community."}, "author_response": "We thank the reviewers for their time and constructive comments on our manuscript. In response, we have performed\nadditional experiments and revised the text to address their concerns. The manuscript has also been reformatted\nto conform to the style of Nature Communications, with expanded Introduction and Discussion sections, and the\nconversion of Figure S1 to Figure 1. Below, we provide a detailed, point-by-point response to the reviewers’\ncomments.\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Toda et al. propose a novel mechanism to explain the long-standing question of how the\nvulnerable H3K9me3 read-write cycle alone can maintain epigenetic inheritance. The authors identify a H3K14ub–\nH3K9me3 circuit that forms a mutually reinforcing feedback loop. This work builds upon previous findings that\nH3K14ub is required for H3K9me3 (PMIDs: 40446033, 34524082, 31468675, 34010645).\nThe authors provide convincing results to support several new findings:\n1) H3K9me3 is also necessary for H3K14ub.\n2) Both H3K14ub and H3K9me3 stimulate Clr4 activity to promote H3K9me3, and also contribute to recruiting and\nstabilizing CLRC at heterochromatin.\n3) Any partial disruption of this circuit causes heterochromatin defects.\n4) Impairment of the activities that antagonize H3K14Ub or H3K9me3 alleviates the defects, supporting that both\npositive and negative feedback activities synergistically regulate the propagation and maintenance of\nheterochromatin.\nAdditionally, this study is the first to map the genome-wide distribution and biological function of H3K14ub in fission\nyeast. This work is original and significant, with data that support the conclusions, making it of interest to a broad\naudience in the field of heterochromatin.\nWe thank the reviewer for this insightful and highly positive assessment of our work. We appreciate their recognition\nof the novelty, significance, and broad relevance of our findings.\nMinor comments for the authors to consider:\nFigure 1c: Please clarify what the X and Y axes represent.\nWe have clarified the identities of X (genome coordinates) and Y (reads per million mapped reads) axes in the\nrevised legend (now Figure 2c).\nFigure 1c Legend: Add \"tel1L,\" \"cen1,\" and \"tel2R\" to the legend.\nWe have added description of \"tel1L\" and \"cen1\", and “mat” (which replaces tel2R) in the revised legend (now Figure\n2c).\nMating Type Locus: Does H3K14ub colocalize with H3K9me3 at the mating type locus, one of the three major\nheterochromatic regions? This locus was not shown in Figure 1c, although it was used for further analysis in Figures\n3-5.\nThe silent mating-type was not included in the genome assembly because commonly used laboratory strains often\ncontain rearrangements at this locus. The strain used in our study carries the mat1-Msmt0 configuration, which\nretains an intact silent mat region. We have now incorporated this region into the analysis and find that both H3K14ub\nand H3K9me3 are detected at this locus (new Figure 2c).\nLine 40: Rea et al. (#12) should be cited with references 6–8, not 9–12.\nReferences have been removed from the abstract in the revised manuscript. Therefore, this comment no longer\napplies.\nLine 50 and 237: \"Histone deacetylation, ubiquitination, and methylation\" might be a more appropriate description\nthan \"histone ubiquitination, acetylation, and methylation.\"\nWe changed the text accordingly to reflect the reviewer’s suggestion.\nLine 103–104: Data for H3K14R and deletion of raf1 and raf2 are not shown in the main or supplemental figures.\nWe now explicitly refer to Fig. 2d, e, where the data for H3K14R and the raf1∆ and raf2∆ mutants are shown.\nLine 132: What evidence supports the notion that H3K14ub makes a stronger contribution? The results shown in\nFigure 2f–2g appear to support this.\nBecause statistical analysis did not reveal a significant difference between the clr4-3FA and clr4-W31G mutants, we\nhave removed this statement from the revised text.\nThe methods section includes a quantification and statistical analysis section; however, no statistical analysis is\npresented in the ChIP-qPCR figures. It is also unclear what statistical methods the authors would use for comparison.\nWe have now performed three biological replicates and statistical analyses for the ChIP-qPCR data and assessed\nsignificance using two-tailed unpaired Student’s t-tests. The statistical annotations have been added to corresponding\nfigures.\nFigure 3a: Should the hht3-K9R data look different from hht1-K14R (symbol + a bar)? Does the blue bar indicate the\nFlag tag?\nIn the revised Figure 4a, we used red star to indicate K14R, green start to indicate K9R, and blue bar to indicate the\nFlag tag. We have updated the figure legend to clarify these details.\nFigure 3b: Should the mutants on the H3 tail be more visible in the hht mutant?\nWe have adjusted the figure to make the H3 tail mutants more clearly visible.\nReviewer #2 (Remarks to the Author):\nMethylation of H3K9 (H3K9me), which is catalyzed by the histone methyltransferase Clr4/Suv39, is a conserved mark\nthat assembles a higher-order chromatin structure called heterochromatin. In fission yeast, Clr4 forms a complex\ncalled CLRC that provides H3K14 ubiquitylation (H3K14ub). Previous studies have shown that H3K14ub promotes\nClr4’s H3K9 methyltransferase activity in vitro. In this manuscript, the authors generated a specific antibody against\nH3K14ub. Using this antibody, the authors analyzed the genome-wide localization of H3K14ub and demonstrated\nthat it clearly overlaps with H3K9me3. The authors also examined the relationship between the factors involved in\nH3K9me and H3K14ub, showing that H3K9me and H3K14ub form a feedback loop. Finally, the authors\ndemonstrated that this feedback loop is negatively regulated by the histone acetyltransferase Mst2 and the H3K9\ndemethylase Epe1.\nOverall comments:\nAlthough accumulating evidence indicates that H3K14ub plays a pivotal role in depositing H3K9me in\nheterochromatic regions, its genome-wide localization and dynamics are not fully understood. This study used an\nH3K14ub antibody and provides strong evidence that H3K14ub is closely associated with H3K9me. In addition, it is\nimportant to note that this feedback loop is negatively regulated by counteracting histone-modifying enzymes. The\nexperiments were carefully designed, and most of the conclusions are supported by the presented data. Having said\nthat, the novelty of the findings appears limited, as most of the conclusions are predictable from previous genetic\nstudies, and the authors merely verified them by using the H3K14ub antibody. Below, I raise several points that the\nauthors should address to improve the manuscript.\nThe reviewer notes that “the experiments were carefully designed and most of the conclusions are supported by the\npresented data,” and we thank the reviewer for this positive assessment. We respectfully disagree, however, with the\nconcern that “the novelty of the findings appears limited.” While prior genetic studies suggested that H3K14ub\ndepends on the Cul4-Rik1-Raf1-Raf2 E3 ubiquitin ligase, our study reveals an unexpected and previously\nunrecognized requirement for Clr4 enzymatic activity in establishing H3K14ub. This finding could not have been\ninferred from existing genetic data and directly supports our central conclusion that H3K9me3 and H3K14ub form a\nself-reinforcing feedback loop. Importantly, the use of an H3K14ub-specific antibody enables direct genome-wide and\nlocus-specific interrogation of this modification, moving beyond genetic inference and previous mass spectrometry\nstudies. This allows us to define the spatial coupling and regulatory logic of H3K14ub and H3K9me3 in vivo, providing\nmechanistic insight into how heterochromatin integrity is maintained. We therefore believe that the work offers a\nsubstantive advance rather than a simple confirmation of prior genetic models.\nMajor points:\n1. (Figs. 1 and 2):\nWhile the authors demonstrated that Rik1 (CLRC) recruitment depends on H3K9me and H3K14ub, they didn’t show\nwhether Clr4 recruitment depends on these modifications. To demonstrate interdependence, the authors should show\nthat Clr4 localization is affected by H3K9R or H3K14R amino acid substitutions, or CLRC complex formation.\nWe performed ChIP analysis of Clr4, and the new data (new Figure S2a) show that Clr4 localization at dh repeats is\nabolished by both H3K9R and H3K14R mutations. To assess whether these mutants affect CLRC assembly, we also\nperformed co-immunoprecipitation analyses of Clr4 and Rik1 and found that their interaction is not affected by either\nthe H3K9R or H3K14R mutations (new Figure S2b).\n2. (Fig 4c, Fig S3c):\nThe finding that Rik1 levels are elevated at cenH in clr3∆ strains is intriguing. While the authors suggest that CLRC\naccumulates at cenH because it cannot spread, does Clr4 localization also increase similarly at cenH? Furthermore,\ncenH functions as an H3K9me initiation site by recruiting the RITS complex (doi: 10.1038/ng1452). It would be helpful\nto discuss whether the RNAi pathway is involved in the increased Rik1 (CLRC) localization at cenH observed in clr3∆\ncells.\nWe performed ChIP analysis of Clr4, and the new data (new Figure S3d-f) show that Clr4 localization at dh and cenH\nis increased inclr3Δcells, similar to what we observe for Rik1. As the reviewer points out, the Rik1 at cenH\ninclr3Δcells may result from increased transcription at this locus. At present, we cannot distinguish whether CLRC\naccumulation arises primarily from impaired spreading away from initiation sites or from increased transcription-\ndependent recruitment via the RNAi pathway. Importantly, however, this ambiguity does not affect our conclusion that\nCLRC fails to spread to distal regions at the mating-type locus in clr3∆ cells. We have now cited the referred study in\nthe revised manuscript.\n3. (Fig. 5):\nThe authors noted that negative regulators, such as Mst2 and Epe1, restrict the H3K14ub-H3K9me3 feedback loop,\nthereby preventing the uncontrolled expansion of heterochromatin. However, they demonstrated heterochromatin\nexpansion only at marker genes inserted in heterochromatic regions. To clarify the roles of Mst2 and Epe1 against the\nH3K14ub-H3K9me3 feedback loop under physiological conditions, the authors could perform ChIP-seq analyses to\ntest whether H3K14ub and H3K9me3 expand beyond well-defined heterochromatic regions in mst2Δ and epe1Δ\nmutants, as shown in Fig. 1c.\nWe thank the reviewer for this thoughtful suggestion regarding the assessment of H3K14ub and H3K9me3 spreading\nunder physiological conditions. We have previously performed genome-wide analyses of H3K9 methylation\nin mst2Δ and epe1Δ single-mutant cells, which showed only modest effects on heterochromatin spreading (Wang et\nal., 2015). In contrast, robust and readily detectable heterochromatin expansion was observed in mst2Δ epe1Δ\nswi6Δ cells. The mst2Δ epe1Δ swi6Δ background was used in place of a clean mst2Δ epe1Δ background\nbecause mst2Δ epe1Δ cells rapidly acquire a compensatory epigenetic suppressor that silences clr4+, allowing cells\nto revert toward a wild-type like chromatin state (Wang et al., 2015). Deletion of swi6+ prevents this adaptive\nresponse and permits stable analysis of heterochromatin expansion.\nRather than repeating genome-wide profiling, we therefore focused on targeted ChIP-qPCR at representative loci that\nsensitively report heterochromatin expansion: a site outside the IRC1R boundary and the facultative heterochromatin\nisland mei4+. We show that combined loss of Mst2 and Epe1 leads to marked expansion of both H3K14ub and\nH3K9me3 in mst2Δ epe1Δ swi6Δcells (new Figure S4a,b), supporting the conclusion that Mst2 and Epe1 cooperate\nto restrain the H3K14ub-H3K9me3 regulatory circuit.\n4. (lines 48 and 204):\nThe authors describe the actions of Mst2 and Epe1 as negative feedback. While these factors negatively regulate the\nH3K9me–H3K14ub feedback loop, this is not sufficient to describe it as negative feedback. To do so, the authors\nneed to demonstrate a relationship in which H3K14 acetylation by Mst2 promotes H3K9 demethylation, and,\nconversely, H3K9 demethylation promotes H3K14 acetylation by Mst2.\nWe thank the reviewer for this comment. We agree that while our data support a role for Mst2 and Epe1 as negative\nregulators of heterochromatin, they do not meet the strict criteria for a negative feedback loop. This might be\nconfused with Mst2 and Epe1 together elicit the formation of ectopic heterochromatin at clr4+, which forms negative\nfeedback. To avoid any confusion, we have therefore revised the manuscript to describe the actions of Mst2 and\nEpe1 as antagonistic or counteracting activities that negatively regulate heterochromatin spreading and maintenance,\nwithout any referencing negative feedback loop.\nMinor points:\n1. (Abstract, lines 39-40):\nReferences #11 and #12 do not include any results regarding H3K14 ubiquitylation activity of CLRC.\nReferences have been removed from the abstract in the revised manuscript. Therefore, this comment no longer\napplies.\n2. (Fig. 1c):\nAlthough the authors clearly showed the genome-wide localization of H3K14ub by ChIP-seq analysis using H3K14ub\nantibody, its heterochromatic localization and coexistence with H3K9me have been previously demonstrated by ChIP-\nMS/MS analysis (doi: 10.15252/embr.201948111). The authors should refer to the published study in the text.\nWe thank the review for pointing this out. We agree with the reviewer and have now cited this study (doi:\n10.15252/embr.201948111) in the section describing our ChIP-seq analysis.\n3. (Fig. 1c):\nH3K14ub and H3K9me3 are not detected at the mat region of chromosome 2. If this is the case, the author should\naccurately describe it and provide possible reasons, such as the strain used.\nThe silent mating-type was not included in the genome assembly because commonly used laboratory strains often\ncontain rearrangements at this locus. The strain used in our study carries the mat1-Msmt0 configuration, which\nretains an intact silent mat region. We have now incorporated this region into the analysis and find that both H3K14ub\nand H3K9me3 are detected at this locus (new Figure 2c).\n4. (ChIP-qPCR):\nTo determine significant or non-significant differences, it would be better to apply statistical analyses to ChIP-qPCR\ndata.\nWe have now performed three biological replicates and statistical analyses for the ChIP-qPCR data and assessed\nsignificance using two-tailed unpaired Student’s t-tests. The statistical annotations have been added to corresponding\nfigures.\n5. (Fig. S2):\nUbiquitylated nucleosomes were used as the substrate for in vitro histone methyltransferase assays. However, the\nMethods section does not include the description how the ubiquitylated nucleosomes were reconstituted.\nThe ubiquitylated nucleosomes were commercially obtained from Epicypher. We have now added this information to\nthe method section of the revised manuscript.\n6. (Reference #34):\nThe authors should provide the correct citation.\nWe have updated the citation to reference 34, which is now published.\nReviewer #3 (Remarks to the Author):\nToda et al. present an analysis of the role of the histone modification H3K14ub in heterochromatin formation in fission\nyeast. This is a very neat genetic analysis and reveals that H3K14ub and H3K9me2/3 depend on each other in a\nfinely balanced system that involves the H3K14ac and H3K9me.\nThis analysis brings long-awaited clarity about the role of H3K14ub in the heterochromatin and gene silencing system\nof fission yeast. Due to its highly conserved machinery, these results are likely to be relevant for many other systems,\nincluding human cells, where the significance of H3K14ub in heterochromatin has now been well established. The\ncharacterization of the feedback loop using highly targeted mutants reveals novel dependencies, showing that\nH3K9me is critical for H3K14ub and vice versa.\nThe data is of high quality and clarity and is presented in a consistent and clear manuscript. Provided the concerns\nbelow are addressed, this manuscript is highly adequate for publication in Nature Communications.\nWe thank the reviewer for this thoughtful and positive evaluation of our work. We appreciate the reviewer’s\nrecognition of the clarity, quality, and significance of our genetic analysis, as well as the identification of the mutually\ndependent relationship between H3K14ub and H3K9me in heterochromatin formation. We are encouraged that the\nreviewer finds this study provides long-awaited mechanistic insight into the role of H3K14ub in fission yeast and notes\nits broader relevance to conserved heterochromatin systems.\nIssues:\n- Statistics for the ChIP-qPCR data are specified as mean +/- SD of 2 technical replicates. This is inadequate and\nshould be based on at least 3 biological replicates.\nWe have now performed three biological replicates and statistical analyses for the ChIP-qPCR data and assessed\nsignificance using two-tailed unpaired Student’s t-tests. The statistical annotations have been added to corresponding\nfigures.\n- The antibody is custom-made and there is no information on how the authors are planning to share it. Given China's\nstrict export rules, there should be a provision for distribution and some information on how the antibody can be\nobtained by the international research community.\nWe appreciate the reviewer’s concern regarding access to the H3K14ub antibody. The antibody was custom\ngenerated and is available in limited quantities. We are committed to facilitating its use by the research community to\nthe extent possible and will make reasonable efforts to share the reagent with interested investigators upon request."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised version of the manuscript has completely addressed my concerns. I have no further suggestions for the authors.", "reviewer_2": "The authors thoroughly addressed all of my concerns, and their rebuttal statements convinced me of the novelty of this work.\nI therefore recommend publishing this article in its current form in Nature Communications.", "reviewer_3": "My concerns have been addressed."}, "author_response": ""}]

Decision letter