Identification of a somatic H3K23me3 methyltransferase SET-19 in C. elegans

Reviewer #1 (Public review):

Summary:

The authors wanted to determine whether the set-19 gene, one of 38 SET-domain containing genes in C elegans, has a clear function in vivo with respect to lysine methylation. The question is not only whether it can modify this histone tail residue, but also what the impact of a loss of this locus is on the inheritance of repressive chromatin states.

Strengths:

The authors clearly achieved their goal, and it is convincingly shown that SET_19 is indeed a somatic cell histone methyltransferase with a striking specificity for H3K23. There is both recombinant protein work, quantitative mapping in vivo, of histone marks and transcriptional changes, and the authors rule out some other hypotheses that have been in the literature. Overall, this provides a compelling argument that SET-19 is indeed the major somatic cell HMT for this residue. Interestingly, the phenotypes are rather minimal, consistent with redundancy in the physiological roles of histone methylation, and redundancy as well in HMT function. For the most part, the data are not over-interpreted. The genetic alleles used, assuming they are confirmed, were revealing and well-documented.

Weaknesses:

The major weaknesses are easily fixed. The major weaknesses mainly reflect a slight overstatement of certain data (claiming insignificance, when it is not clear how that was determined) and claiming a bit too much about SET-32, which was independently claimed to be an H3K23 HMT. Clearly, the two SET domain enzymes are not redundant, nor is the claim that SET-32 has no role in H3K23 methylation completely convincing. Especially in germline or embryonic conditions. Finally, the imaging is not of very high quality, nor are the images fully quantitated. These points can be easily remedied.

Reviewer #2 (Public review):

Summary:

This manuscript identifies SET-19 as a somatic H3K23 methyltransferase in C. elegans, building on previous genetic evidence for a role of set-19 in H3K23me3 regulation. The authors combine quantitative mass spectrometry, western blotting, in vitro methyltransferase assays, ChIP-seq, and RNA-seq to show that loss of set-19 causes a strong reduction of H3K23me3, particularly in somatic tissues, and is associated with derepression of a subset of genes enriched for H3K23me3. They further conclude that SET-19 is dispensable for canonical feeding RNAi and for transgenerational or intergenerational inheritance of RNAi, distinguishing its function from other heterochromatin-associated methyltransferases such as SET-25, SET-32, and the H3K27 HMTs. Overall, the work adds an important piece to the H3K23 methylation pathway and tissue-specific chromatin regulation in C. elegans.

Strengths:

Very strong genetic and biochemical evidence for SET-19 as the major H3K23me3 HMT.

The mass spectrometry and western blot data convincingly demonstrate a strong reduction of H3K23me3 in two independent set-19 alleles and rescue by GFP::SET-19, which is a major strength (Figure 1, including Figure 1f).

The in vitro methyltransferase assays (Figure 2) showing robust H3K23me1/2/3 activity for SET-19 SET+CC and only modest H3K23me activity for SET-32, together with the SAM titration experiment in Figure 2C, are very informative and nicely support the conclusion that SET-19 is a high-activity H3K23 methyltransferase compared to SET-32.

The ChIP-seq analysis is central to the conclusions that H3K23me3 is enriched on chromosome arms, co-localizes with H3K9me3/H3K27me3, and is strongly reduced in set-19 mutants.

Weaknesses:

(1) The global reduction of H3K23me3 in Figure 3b,c and Figure S4c is convincing, but the correlation analysis between H3K23me3 loss and mRNA changes in Figure 3g could be strengthened. Currently, the analysis appears to focus on broad categories; it would be helpful to provide:

Representative genome browser tracks (e.g., exemplary gene coverage plots) for several genes that show clear H3K23me3 peaks in wild type, reduction in set-19, and concomitant upregulation of mRNA levels, and for a few genes that retain H3K23me3 and do not change expression. This would make the link between chromatin changes and transcriptional output more concrete.

(2) In Figure S4C, the authors note a pronounced reduction of H3K23me3 mainly on chromosome arms, but in the current data, it appears that the impact might be arm-specific (i.e., stronger reduction in one arm than the other in a chromosome), with a notable pattern at the X chromosome tip where H3K23me3 seems increased. This is potentially interesting and should be briefly commented on in the Results or Discussion, for example, whether this reflects compensatory activity of another HMT, changes in chromatin organization, or could be a technical artifact.

(3) Figure 3d suggests that some actively expressed genes can also display relatively high H3K23me3 levels, which complicates a simple model of H3K23me3 as exclusively repressive. If feasible, a limited additional analysis stratifying genes by both H3K23me3 and H3K9me3/H3K27me3 status might clarify whether these highly expressed, H3K23me3‑marked genes differ in other chromatin features.

(4) The authors argue that SET-19 primarily affects H3K23me3 and not other canonical repressive marks, based largely on mass spectrometry. It would significantly strengthen the mechanistic conclusions if the authors could assess H3K9me3 and H3K27me3 profiles in set-19 mutants, ideally by ChIP-seq or at least by focused ChIP-qPCR at a subset of loci that lose H3K23me3 and are derepressed at the RNA level. This would address whether H3K23me3 loss occurs independently of changes in other heterochromatin marks, or whether there is crosstalk.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors wanted to determine whether the set-19 gene, one of 38 SET-domain containing genes in C elegans, has a clear function in vivo with respect to lysine methylation. The question is not only whether it can modify this histone tail residue, but also what the impact of a loss of this locus is on the inheritance of repressive chromatin states.

Strengths:

The authors clearly achieved their goal, and it is convincingly shown that SET_19 is indeed a somatic cell histone methyltransferase with a striking specificity for H3K23. There is both recombinant protein work, quantitative mapping in vivo, of histone marks and transcriptional changes, and the authors rule out some other hypotheses that have been in the literature. Overall, this provides a compelling argument that SET-19 is indeed the major somatic cell HMT for this residue. Interestingly, the phenotypes are rather minimal, consistent with redundancy in the physiological roles of histone methylation, and redundancy as well in HMT function. For the most part, the data are not over-interpreted. The genetic alleles used, assuming they are confirmed, were revealing and well-documented.

Thanks very much for the positive comments on our work.

The alleles used in this study were confirmed by PCR and Sanger sequencing, and the sequence information will be added in the revised manuscript.

Weaknesses:

The major weaknesses are easily fixed. The major weaknesses mainly reflect a slight overstatement of certain data (claiming insignificance, when it is not clear how that was determined) and claiming a bit too much about SET-32, which was independently claimed to be an H3K23 HMT. Clearly, the two SET domain enzymes are not redundant, nor is the claim that SET-32 has no role in H3K23 methylation completely convincing. Especially in germline or embryonic conditions. Finally, the imaging is not of very high quality, nor are the images fully quantitated. These points can be easily remedied.

Thanks very much for the comments.

We agree that some interpretations in the original manuscript were too strong, particularly regarding the negative results and the role of SET-32. Our in vitro assays show that SET-32 exhibits H3K23me1 activity and, at higher SAM concentrations, activity toward H3K23me2/3. These findings indicate that SET-32 does have a role in H3K23 methylation. SET-32 is expressed in germ cells, oocytes, and embryos. It is quite likely that redundancy of H3K23 methyltransferase activity exists in these tissues. In the revised manuscript, we will tone down the interpretations and expand the Discussion section to include this possibility. We will also replace the relevant images with higher-quality versions and provide quantitative analyses for Figures 6a and 6b.

Reviewer #2 (Public review):

Summary:

This manuscript identifies SET-19 as a somatic H3K23 methyltransferase in C. elegans, building on previous genetic evidence for a role of set-19 in H3K23me3 regulation. The authors combine quantitative mass spectrometry, western blotting, in vitro methyltransferase assays, ChIP-seq, and RNA-seq to show that loss of set-19 causes a strong reduction of H3K23me3, particularly in somatic tissues, and is associated with derepression of a subset of genes enriched for H3K23me3. They further conclude that SET-19 is dispensable for canonical feeding RNAi and for transgenerational or intergenerational inheritance of RNAi, distinguishing its function from other heterochromatin-associated methyltransferases such as SET-25, SET-32, and the H3K27 HMTs. Overall, the work adds an important piece to the H3K23 methylation pathway and tissue-specific chromatin regulation in C. elegans.

Strengths:

Very strong genetic and biochemical evidence for SET-19 as the major H3K23me3 HMT.

The mass spectrometry and western blot data convincingly demonstrate a strong reduction of H3K23me3 in two independent set-19 alleles and rescue by GFP::SET-19, which is a major strength (Figure 1, including Figure 1f).

The in vitro methyltransferase assays (Figure 2) showing robust H3K23me1/2/3 activity for SET-19 SET+CC and only modest H3K23me activity for SET-32, together with the SAM titration experiment in Figure 2C, are very informative and nicely support the conclusion that SET-19 is a high-activity H3K23 methyltransferase compared to SET-32.

The ChIP-seq analysis is central to the conclusions that H3K23me3 is enriched on chromosome arms, co-localizes with H3K9me3/H3K27me3, and is strongly reduced in set-19 mutants.

Thanks very much for the positive comments on our work.

Weaknesses:

(1) The global reduction of H3K23me3 in Figure 3b,c and Figure S4c is convincing, but the correlation analysis between H3K23me3 loss and mRNA changes in Figure 3g could be strengthened. Currently, the analysis appears to focus on broad categories; it would be helpful to provide:

Representative genome browser tracks (e.g., exemplary gene coverage plots) for several genes that show clear H3K23me3 peaks in wild type, reduction in set-19, and concomitant upregulation of mRNA levels, and for a few genes that retain H3K23me3 and do not change expression. This would make the link between chromatin changes and transcriptional output more concrete.

Thanks very much for the suggestion.

To address this point, we will include representative genome browser tracks for selected genes in the revised manuscript. These examples will help better illustrate the relationship between H3K23me3 loss and mRNA expression changes.

(2) In Figure S4C, the authors note a pronounced reduction of H3K23me3 mainly on chromosome arms, but in the current data, it appears that the impact might be arm-specific (i.e., stronger reduction in one arm than the other in a chromosome), with a notable pattern at the X chromosome tip where H3K23me3 seems increased. This is potentially interesting and should be briefly commented on in the Results or Discussion, for example, whether this reflects compensatory activity of another HMT, changes in chromatin organization, or could be a technical artifact.

Thanks very much for bringing up this point.

As shown in Figure S4C, the overall chromosomal distribution pattern of H3K23me3 is broadly similar between wild type and set-19 mutants, with pronounced enrichment over one chromosomal arm, whereas the center and the opposite arm show relatively lower signal. In set-19 mutants, this asymmetry becomes more pronounced, with a larger difference between the highly enriched arm and the lower-signal regions. This pattern is particularly evident on chromosomes I, II, V, and X. These observations suggest that the effect of set-19 loss on H3K23me3 is not uniform across chromosomal regions.

Substantial H3K23me3 signal remains in specific regions in set-19 mutants, suggesting that additional enzyme(s) also contribute to H3K23me3 methylation. For example, SET-19 appears to function predominantly in somatic tissues, yet the ChIP-seq assays were performed using whole animals, including the germline. Alternatively, there might be compensatory activity of another HMT. In the revised manuscript, we will state these points more explicitly in the Results section and discuss the residual and locally increased H3K23me3 signals.

(3) Figure 3d suggests that some actively expressed genes can also display relatively high H3K23me3 levels, which complicates a simple model of H3K23me3 as exclusively repressive. If feasible, a limited additional analysis stratifying genes by both H3K23me3 and H3K9me3/H3K27me3 status might clarify whether these highly expressed, H3K23me3 marked genes differ in other chromatin features.

Thanks very much for the suggestion.

To address this point, we will perform additional stratified analyses of H3K23me3-marked genes according to their H3K9me3 and/or H3K27me3 status. We will also compare highly and weakly expressed H3K23me3-marked genes to examine whether they differ in other chromatin features, including H3K9me3, H3K27me3, and, if feasible, H3K4me3 and H3K36me3.

(4) The authors argue that SET-19 primarily affects H3K23me3 and not other canonical repressive marks, based largely on mass spectrometry. It would significantly strengthen the mechanistic conclusions if the authors could assess H3K9me3 and H3K27me3 profiles in set-19 mutants, ideally by ChIP-seq or at least by focused ChIP-qPCR at a subset of loci that lose H3K23me3 and are derepressed at the RNA level. This would address whether H3K23me3 loss occurs independently of changes in other heterochromatin marks, or whether there is crosstalk.

Thanks very much for the suggestions.

As suggested, H3K9me3 and H3K27me3 ChIP-seq in wild-type and set-19 mutants will be performed. We will compare their genome-wide distributions and identify loci with significantly altered H3K9me3 and/or H3K27me3 enrichment. These analyses should help clarify whether H3K23me3 loss occurs largely independently of H3K9me3/H3K27me3 changes or reflects potential crosstalk among these repressive chromatin marks. In addition, we will examine H3K9me3 and H3K27me3 enrichment at genes showing both H3K23me3 loss and increased mRNA expression in set-19 mutants to assess whether derepression at these loci is accompanied by changes in other canonical repressive marks.