Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase

Your manuscript titled, “Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase” was reviewed by two experts in the field and by a member of the Board of Reviewing Editors (BRE). After a full discussion of the study and the reviews, I am happy to report that the reviewers and the BRE member found the study of interest to the journal and therefore we are happy to consider a revised manuscript addressing the following issues:

1) An essential control in the ChIP-ChIP studies of Set1 is the use set1 null background in Set1 ChIP-ChIP studies to verify the significance of the low signals observed. Additionally, you and co-authors need to show what percentages of Set1 localize at active and repressed regions, respectively?

2) Further, the result of ChIP-ChIP studies in the paper need to be verified with manual ChIP, and in all ChIP studies proper controls such as untagged strains need to be used and demonstrated.

3) Co-localization studies do not represent co-recruitment. Please assess Set1 occupancy in WT and atf1 null backgrounds and also evaluate H3K4me3 levels genome-wide in the presence and absence of Atf1.

4) In S. pombe, prominent heterochromatin regions include pericentromeres, subtelomeres, rNDA, and the silent mating type locus. Although Figure 1C has tried to classify the roles of Set1 and COMPASS in different classes of genes, it is still difficult to evaluate if any classes of genes are preferentially affected by Set1 only or by COMPASS. The authors need to re-analyze the expression data based on those Set1-occupied genes/regions. Also, the GO term analyses on Figure 2D did not reveal any heterochromatin-related terms. The enrichments of Atf1 in heterochromatin regions are quite clear; however, the color-coding makes the Set1 signals almost invisible in Figure 3A and Figure 3–supplement figure 2.

5) Set1 is proposed to act in parallel pathway to Clr3 to assemble H3K9me heterochromatin. However, the exact nature of defects in clr3set1 double mutant is not discussed. One possibility is that double mutant is defective in production of small RNAs that are critical for RNAi-mediated targeting of heterochromatin. Additional experiments including changes in small RNAs might provide information about exact cause of the described changes in H3K9me. Moreover, the authors show widespread upregulation of genes in the double mutant but the biological significance of these changes has not been addressed. Is the double mutant defective in stress responses or does it show developmental defects (such as untimely meiosis etc.)? Inclusion of such results may help connect changes in gene expression to biological processes.

6) The authors should clarify whether or not this new function of Set1 is truly independent of its catalytic activity or methylation of its normal substrate (H3K4). They can either use catalytically dead Set1 (without altering Set1's stability) or H3K4R mutant (which is preferable).

7) Introduction: in the sentence discussing heterochromatin islands, the authors should cite a paper by Zofall et al., Science, 335:96, 2012, that discusses dynamic heterochromatin domains in different parts of the genome. Similarly, the next sentence discussing RNAi and exosome should include reference to Yamanaka et al., Nature, 493:557, 2013.

1) An essential control in the ChIP-ChIP studies of Set1 is the use set1 null background in Set1 ChIP-ChIP studies to verify the significance of the low signals observed.

As pointed out by one of the reviewers, it is often a challenge to detect localization of enzymatic proteins. However, due to the expected low signals of Set1 at certain loci, we have now performed additional manual ChIP experiments to verify Set1 localization. Because we performed ChIP-chip experiment of Set1 using a FLAG antibody (Sigma, M2) against an epitope tagged Set1, we now include in our manual ChIP verification of Set1 targets a control ChIP experiment against an untagged strain. These results are shown in Figure 2–figure supplement 1. Our results are consistent with our recent findings that Set1 localization at active and repressed loci is generally not dependent of the status of H3K4 methylation (Figure 2 in Mikheyeva et al., PLOS Genetics, 2014).

Additionally, you and co-authors need to show what percentages of Set1 localize at active and repressed regions, respectively?

We have now included analysis of the percentage of Set1 ChIP targets at active and repressed loci. These results are shown in Figure 2–figure supplement 2. Briefly, of the 290 loci whose promoters are bound by Set1 (ChIP fold enrichment ≥ 2 at 3+ adjacent probes), 80% of those loci are considered as actively transcribed genes whereas 20% correspond to repressed genes (RNA level below the mean expression level for logarithmically growing cells; Rhind et al., 2011, RNA-seq data).

2) Further, the result of ChIP-ChIP studies in the paper need to be verified with manual ChIP, and in all ChIP studies proper controls such as untagged strains need to be used and demonstrated.

Please see our reply to question 1. In addition, because the Atf1 ChIP-ChIP experiment was performed using a commercial antibody specific for endogenous Atf1, we now include a negative control ChIP experiment using the same antibody in an atf1 null strain. This result is present in Figure 3–figure supplement 2 which shows that the enriched signals at Atf1 targets is significantly higher in atf1+ compared with atf1Δ, suggesting that the antibody binding is specific to Atf1. Moreover, our Atf1 ChIP-ChIP results are in high agreement with recently published ChIP-ChIP data of Atf1 using antibody against an HA epitope of an Atf1-HA tagged strain (Eshaghi et al., PloS One, 2010).

3) Co-localization studies do not represent co-recruitment. Please assess Set1 occupancy in WT and atf1 null backgrounds and also evaluate H3K4me3 levels genome-wide in the presence and absence of Atf1.

In addition to showing reduced Set1 enrichment at pericentromeric repeats, the rDNA array, and ste11 (Figure 3D), we have included additional statistical analyses of the H3K4me3 microarray data to determine all genomic regions displaying significant changes in H3K4me3 in response to atf1Δ. We found that many loci exhibit reduced levels of H3K4me3 in an atf1Δ strain. These data are provided in the new Figure 3–source data 1. The Results and Methods sections of the manuscript contain additional text summarizing these results and an explanation of the analytical methods, respectively. Because Set1 is generally enriched at active genes, it complicates our ability to assess the effect of Set1 occupancy at Atf1 repressed loci in an atf1 null strain compared with wild-type. However, we expect Atf1-mediated repressed loci to exhibit derepression and hence increased H3K4me3 levels in atf1Δ strain. We now include it in Figure 3–figure supplement 3, which illustrates gene promoters corresponding to Atf1-bound genes (fbp1, srk1) that are known to be upregulated in atf1Δ and display increased H3K4me3 levels. A complete list of all such genomic regions are included in Figure 3–source data 1.

4) In S. pombe, prominent heterochromatin regions include pericentromeres, subtelomeres, rNDA, and the silent mating type locus. Although Figure 1C has tried to classify the roles of Set1 and COMPASS in different classes of genes, it is still difficult to evaluate if any classes of genes are preferentially affected by Set1 only or by COMPASS. The authors need to re-analyze the expression data based on those Set1-occupied genes/regions.

Because most of the upregulated probes are exclusive to individual experiments or groups of experiments (i.e., probes upregulated in spp1Δ and shg1Δ or sdc1Δ and ash2Δ differ almost completely with upregulated probes in the set1Δ and other experiments), it is not possible to derive a common subset of genes likely affected by the COMPASS only. This is probably due to certain COMPASS subunits (i.e., Set1, Ash2 and Sdc1) having roles outside of the COMPASS complex that could antagonize the function of COMPASS. However, in order to facilitate a clearer comparison between the classes of genes affected in the individual set1/COMPASS deletion strains summarized in Figure 1, we have reanalyzed the Gene Ontology enrichment in Figure 1–source data 1 to encapsulate GO terms with significant enrichment (p < 0.01) by experiment. This new result is now shown in Figure 1–source data 2. This analysis replicates the more detailed GO analysis in Figure 1–source data 1 into a format more amenable to comparing the functional effects of the various set1/COMPASS deletion mutants.

Also, the GO term analyses on Figure 2D did not reveal any heterochromatin-related terms.

The most current Gene Ontology mappings provided by pombase.org, the reference genome database for S. pombe, is predominantly limited to annotation for protein coding genes. The set of mostly noncoding transcripts located within heterochromatin regions therefore currently has limited GO annotation. Only two S. pombe chromatin regulatory proteins are mapped to the “heterochromatin” GO term (GO:0000792), vs. transcripts within heterochromatin regions. We have revised the manuscript to clarify this point.

The enrichments of Atf1 in heterochromatin regions are quite clear; however, the color-coding makes the Set1 signals almost invisible in Figure 3A and Figure 3–supplement figure 2.

We have reformatted Figure 3A and Figure 3–supplement figure 2 to provide a better contrast for the enriched ChIP signals of Atf1 and Set1.

5) Set1 is proposed to act in parallel pathway to Clr3 to assemble H3K9me heterochromatin. However, the exact nature of defects in clr3set1 double mutant is not discussed. One possibility is that double mutant is defective in production of small RNAs that are critical for RNAi-mediated targeting of heterochromatin. Additional experiments including changes in small RNAs might provide information about exact cause of the described changes in H3K9me.

We have performed several experiments to further elucidate the nature of functional interactions between set1 and clr3. First, consistent with the drastically reduced H3K9me levels in cells null for both set1 and clr3, the double mutant also exhibits severe depletion of the HP1/Swi6 proteins at pericentromeric heterochromatin accompanied by increased levels of Pol II occupancy. These results are now shown in Figure 4–figure supplement 1. We also assessed the levels of siRNAs in the set1 and clr3 mutants. We found that whereas loss of either set1 or clr3 resulted in increased levels of siRNAs, the siRNA level in the set1Δ clr3Δ double mutant was dramatically diminished relative to wild-type. This is a likely consequence of the failure of the RNAi machinery requiring H3K9me to act in cis to contribute to heterochromatin assembly (i.e., the RITS complex tethering to H3K9me via Chp1 to generate siRNAs; Noma et al., Nature Genetics, 2005). This result is shown in Figure 4F.

Moreover, the authors show widespread upregulation of genes in the double mutant but the biological significance of these changes has not been addressed. Is the double mutant defective in stress responses or does it show developmental defects (such as untimely meiosis etc.)? Inclusion of such results may help connect changes in gene expression to biological processes.

We have performed experiments that reveal defects in mating and sporulation in the set1Δ clr3Δ double mutant. These new results are present in Figure 5–figure supplement 4.

6) The authors should clarify whether or not this new function of Set1 is truly independent of its catalytic activity or methylation of its normal substrate (H3K4). They can either use catalytically dead Set1 (without altering Set1's stability) or H3K4R mutant (which is preferable).

We have performed additional experiments that show Set1 localization at active and repressed loci is not impaired due to the absence of H3K4 methylation (set1F ^H3K4me-) or its catalytic activity (set1-SETΔ). These new results are now shown in Figure 2–figure supplement 1.

7) Introduction: in the sentence discussing heterochromatin islands, the authors should cite a paper by Zofall et al., Science, 335:96, 2012, that discusses dynamic heterochromatin domains in different parts of the genome. Similarly, the next sentence discussing RNAi and exosome should include reference to Yamanaka et al., Nature, 493:557, 2013.

We have now included these references to the indicated sentences in the revised manuscript.