The ACF chromatin-remodeling complex is essential for Polycomb repression
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Evaluation Summary:
The chromatin mark, H3K27me, is deposited by the Polycomb complex PRC2 and is associated with repressed genes. There are two important findings in this paper: 1) that the promoters of some H3K27me-repressed genes are regulated by nucleosome positioning and 2) the H3K27me repressed genes are a diverse group that can be derepressed by different mechanisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Establishing and maintaining appropriate gene repression is critical for the health and development of multicellular organisms. Histone H3 lysine 27 (H3K27) methylation is a chromatin modification associated with repressed facultative heterochromatin, but the mechanism of this repression remains unclear. We used a forward genetic approach to identify genes involved in transcriptional silencing of H3K27-methylated chromatin in the filamentous fungus Neurospora crassa . We found that the N. crassa homologs of ISWI (NCU03875) and ACF1 (NCU00164) are required for repression of a subset of H3K27-methylated genes and that they form an ACF chromatin-remodeling complex. This ACF complex interacts with chromatin throughout the genome, yet association with facultative heterochromatin is specifically promoted by the H3K27 methyltransferase, SET-7. H3K27-methylated genes that are upregulated when iswi or acf1 are deleted show a downstream shift of the +1 nucleosome, suggesting that proper nucleosome positioning is critical for repression of facultative heterochromatin. Our findings support a direct role of the ACF complex in Polycomb repression.
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Author Response
Reviewer #2 (Public Review):
In this paper Wiles et al. show that mutations in the iswi and acf genes, which encode components of a nucleosome remodeling complex, lead to expression of a subset of H3K27me-repressed genes. The strengths of the paper include the detailed genomic analysis supporting the statements that Iswi and Acf regulate a subset of H3K27me3-repressed genes. Data showing that the +1 nucleosome shifts 50bp in H3K27me-genes upregulated in the iswi mutant is also very strong. There is strong data documenting the proteins that Iswi interacts with in N. crassa. The data showing the nucleosome shift in the acf mutant is not as strong. The summary figure is highly speculative because there is no data for discrete localization of Acf. Another piece of data that is lacking is what happens to H3K36me in iswi …
Author Response
Reviewer #2 (Public Review):
In this paper Wiles et al. show that mutations in the iswi and acf genes, which encode components of a nucleosome remodeling complex, lead to expression of a subset of H3K27me-repressed genes. The strengths of the paper include the detailed genomic analysis supporting the statements that Iswi and Acf regulate a subset of H3K27me3-repressed genes. Data showing that the +1 nucleosome shifts 50bp in H3K27me-genes upregulated in the iswi mutant is also very strong. There is strong data documenting the proteins that Iswi interacts with in N. crassa. The data showing the nucleosome shift in the acf mutant is not as strong. The summary figure is highly speculative because there is no data for discrete localization of Acf. Another piece of data that is lacking is what happens to H3K36me in iswi and acf mutants. Knowing this is important because a similar set of genes seem to be derepressed in an ash1 mutant as in the acf and iswi mutants, although the level of depression in ash1 is not as great as in iswi mutants. The summary diagram shows loss of H3K36me as a separate mechanism than loss of the ACF complex. We don't know that since there was no analysis of H3K36me in iswi or acf mutants. Still, the major findings of the paper are important.
We feel that the data showing the nucleosome shift in ∆acf1 are quite strong (Figure 5I). We agree that the model is somewhat speculative but we feel it is useful and have addressed concerns e.g. by performing H3K36me ChIP-seq (more below). Although we were unable to ChIP ACF, we find the combination of the ACF1-DamID and nucleosome shifts specifically at the K27me upregulated genes in ∆acf1 provides good evidence that ACF is acting at and localizing to these genomic locations. We have added H3K36me ChIP data in ∆acf1 and ∆iswi.
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Evaluation Summary:
The chromatin mark, H3K27me, is deposited by the Polycomb complex PRC2 and is associated with repressed genes. There are two important findings in this paper: 1) that the promoters of some H3K27me-repressed genes are regulated by nucleosome positioning and 2) the H3K27me repressed genes are a diverse group that can be derepressed by different mechanisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #3 (Public Review):
This manuscript clearly shows that, in Neurospora, ACF chromatin remodeler represses some of H3K27-methylated genes in an H3K27me-dependent fashion. This is the first report to demonstrate ISWI class remodeler functions through H3K27me. The main weakness of the manuscript is the modest impact on mechanistic understanding of how ACF or H3K27me functions. Also, the authors' model needs additional support to firmly establish the causality between ACF-dependent nucleosome repositioning and transcriptional repression.
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Reviewer #2 (Public Review):
In this paper Wiles et al. show that mutations in the iswi and acf genes, which encode components of a nucleosome remodeling complex, lead to expression of a subset of H3K27me-repressed genes. The strengths of the paper include the detailed genomic analysis supporting the statements that Iswi and Acf regulate a subset of H3K27me3-repressed genes. Data showing that the +1 nucleosome shifts 50bp in H3K27me-genes upregulated in the iswi mutant is also very strong. There is strong data documenting the proteins that Iswi interacts with in N. crassa. The data showing the nucleosome shift in the acf mutant is not as strong. The summary figure is highly speculative because there is no data for discrete localization of Acf. Another piece of data that is lacking is what happens to H3K36me in iswi and acf mutants. …
Reviewer #2 (Public Review):
In this paper Wiles et al. show that mutations in the iswi and acf genes, which encode components of a nucleosome remodeling complex, lead to expression of a subset of H3K27me-repressed genes. The strengths of the paper include the detailed genomic analysis supporting the statements that Iswi and Acf regulate a subset of H3K27me3-repressed genes. Data showing that the +1 nucleosome shifts 50bp in H3K27me-genes upregulated in the iswi mutant is also very strong. There is strong data documenting the proteins that Iswi interacts with in N. crassa. The data showing the nucleosome shift in the acf mutant is not as strong. The summary figure is highly speculative because there is no data for discrete localization of Acf. Another piece of data that is lacking is what happens to H3K36me in iswi and acf mutants. Knowing this is important because a similar set of genes seem to be derepressed in an ash1 mutant as in the acf and iswi mutants, although the level of depression in ash1 is not as great as in iswi mutants. The summary diagram shows loss of H3K36me as a separate mechanism than loss of the ACF complex. We don't know that since there was no analysis of H3K36me in iswi or acf mutants. Still, the major findings of the paper are important.
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Reviewer #1 (Public Review):
Polycomb repression of heterochromatic genes differs in different organisms but has been most widely studied in Drosophila. Neurospora lacks components of Drosophila polycomb repression complexes.
Using a powerful forward genetic screen the authors found that components of the ACF complex were required to maintain repression of H3K27 methylated heterochromatic genes in Neurospora.
ACF binds widely to chromatin across the genome and is not restricted to heterochromatic genes. This indicates that it also functions outside of heterochromatin. Its interaction with heterochromatin is affected somewhat by the loss of H3K27 methylation.
ACF appears to be necessary to position the +1 nucleosome over the promoter of H3K27 methylated heterochromatic genes.
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