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. 2021 Jan 26;118(4):e2010003118.
doi: 10.1073/pnas.2010003118.

IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains

Masayuki Kamei  1 , Abigail J Ameri  1 , Aileen R Ferraro  1 , Yael Bar-Peled  1 , Fangzhou Zhao  2 , Christina L Ethridge  3 , Kathleen Lail  4 , Mojgan Amirebrahimi  4 , Anna Lipzen  4 , Vivian Ng  4 , Igor V Grigoriev  4   5 , Robert J Schmitz  3 , Yi Liu  2 , Zachary A Lewis  6
Affiliations

IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains

Masayuki Kamei et al. Proc Natl Acad Sci U S A. .

Abstract

Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X chromosome inactivation, and cancer. In animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungus Neurospora crassa, and recent work in this organism has generated insights into PRC2 control and function. To identify components of the facultative heterochromatin pathway, we performed a targeted screen of Neurospora deletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We found the Neurospora homolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and normal H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinct Neurospora ISW-containing complexes. A triple mutant lacking three ISW accessory factors and disrupting multiple ISW complexes led to widespread up-regulation of PRC2 target genes and altered H3K27 methylation patterns, similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway in Neurospora, and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.

Keywords: H3K27me3; IMITATION SWITCH (ISWI); Polycomb Repressive Complex 2 (PRC2); chromatin remodeling; facultative heterochromatin.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
ISW is required for repression of PRC2 target genes. (A) The boxplot depicts the average expression level (TPM) and the interquartile range for PRC2 target genes for each of the indicated strains. (B) The heatmap shows relative expression levels of PRC2 target genes sorted by chromosomal position (rows) in the indicated deletion strain (columns). (C) ISW is required for normal patterns of H3K27me2/3. H3K27me2/3 enrichment determined by ChIP-seq was plotted using the Integrated Genome Viewer for the indicated strains across one chromosome (linkage group VII [LGVII]). The bottom graph shows a magnified view of the left end of LGVII. (D) The heat map depicts H3K27me2/3 across all 309 H3K27me2/3-enriched domains in the Neurospora genome. Each heat map row depicts a 3-kb window centered at the left boundary of an H3K27me2/3-enriched peak identified in WT cells.
Fig. 2.
Fig. 2.
ISW catalytic activity is required for repression and H3K27me2/3. (A) The schematic illustrates the predicted protein domains in the Neurospora ISW sequence. The sequence alignment indicates the amino acid conservation between the ATPase domain of Neurospora ISW, S. cerevisiae ISW-1, and Drosophila melanogaster ISWI. The location of the conserved lysine residue mutated to arginine in this study is indicated. (B) Both WT ISW-3xHA and the ISWK214R-3xHA fusion proteins are expressed at similar levels. Total protein extracts were analyzed by Western blotting using an anti-HA antibody or by staining with Coomassie Brilliant Blue (CBB) as a loading control for the indicated strains. (C) The graph depicts the linear growth rates of the indicated strains measured using “race tubes.” (D) For the indicated strains, serial dilutions of fungal cells (conidia; 106 to 103 from left to right) were spotted on Vogel’s Minimal Medium (VMM) with or without the genotoxic agent methyl methanesulfonate (MMS; 0.025%). (E) The Integrated Genome Viewer genome browser image depicts H3K27me2/3 enrichment determined by ChIP-seq for the indicated strains. The location of four genes analyzed by RT-qPCR and ChIP-qPCR are indicated with dashed lines. (F) Relative gene expression data are shown for the genes NCU08085, NCU08086, NCU08101, and NCU08102 for the indicated strains. Expression is relative to the constitutive vma-2 transcript and normalized to WT. (G) Enrichment of H3K27me2/3 across the four genes shown in E and F was examined by ChIP-qPCR for the indicated strains. ChIP enrichment at the indicated locus was normalized to enrichment at the euchromatic hH4 gene locus.
Fig. 3.
Fig. 3.
Neurospora ISW is present in at least three distinct complexes. (A) Enrichment of ISW-3xFLAG was validated by Western blotting (WB). Total protein extracts were analyzed from a WT control strain and the isw-3xflag strain. ISW-3xFLAG was examined in total extracts, in the unbound fraction following incubation with an M2–anti-FLAG affinity resin (flow-through), in an aliquot of six combined wash fractions, and in the final elution fraction containing purified ISW-3xFLAG. (B–D) Immunoprecipitation experiments were performed using extracts from strains expressing ACF1-3xFLAG, IAF-1-3xFLAG, IAF-2-3xFLAG, and ISW-3xHA, as indicated by (+) or (−). The input fraction, the FLAG immunoprecipitate (IP:FLAG), and the HA immunoprecipitate (IP:HA) were subject to WB and probed with the anti-FLAG or anti-HA antibodies as indicated. The asterisks indicate presumed ACF degradation products. (E) The schematic summarizes the results of protein identification by LC-MS/MS for independent, reciprocal protein purification experiments using ISW-3xFLAG, ACF1-3xFLAG, IAF-1-3xFLAG, and IAF-2-3xFLAG (two replicates each). The arrows extend from the purified protein and point to the captured prey protein. The numbers in parentheses indicate the number of unique peptides corresponding to the identified prey protein for two independent replicate purification and LC-MS/MS experiments.
Fig. 4.
Fig. 4.
ISW complexes partially compensate for one another. (A and D) H3K27me2/3 enrichment determined by ChIP-seq was plotted using the Integrated Genome Viewer for the indicated strains across the left end of linkage group VI. (Scale bar: 100 kb.) (B and E) The heat maps depict H3K27me2/3 across all WT H3K27me2/3-enriched domains in the Neurospora genome for the indicated strains. Each heat map row depicts a 3-kb window centered at the left boundary of an H3K27me2/3-enriched peak identified in WT cells. (C and F) The boxplot depicts the average expression level (transcripts per million) and the interquartile range for 642 PRC2 target genes for the indicated strains. Each dot represents the expression level of a PRC2 target gene colored by statistical significance (red dots, adjusted P < 0.05; gray dots, adjusted P > 0.05).
Fig. 5.
Fig. 5.
ISW controls chromatin structure in PRC2 target domains and is required for normal PRC2 binding. (A) H3K27me2/3 and H3K36me3 enrichment determined by ChIP-seq was plotted using the Integrated Genome Viewer for the indicated strains across linkage group VI (LGVI). The bottom graph shows a magnified view of the left end of LGVI. Two representative regions exhibiting loss of H3K36me3 are highlighted. (B) Heat maps depict H3K36me3 across all PRC2 target genes (Top) or all genes (Bottom) in the WT and ∆isw strains. Each heat map row is centered on the transcriptional start site and depicts enrichment (± 2,000 bp) on either side. (C) Histograms plot the relative frequency of MNase-seq reads containing the indicated fragment sizes for all reads that overlap PRC2 target regions for the WT and ∆isw strains. (D) The soluble (Sol) and chromatin fractions (Chr) were isolated from the indicated strains, and both fractions were analyzed by Western blotting using anti-FLAG and anti-H3 antibodies, as indicated. The asterisk indicates a nonspecific band that cross-reacts with the anti-FLAG antibody; the arrow indicates the position of SUZ12-3xFLAG. (E) The graph indicates the percentage of total SUZ12-3xFLAG detected in the soluble fraction for the indicated strains (± SE; P = 1.1 × 10−9), as determined by image analysis of four replicate experiments.
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References

    1. Schuettengruber B., Bourbon H.-M., Di Croce L., Cavalli G., Genome regulation by polycomb and trithorax: 70 years and counting. Cell 171, 34–57 (2017). - PubMed
    1. Margueron R., Reinberg D., The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011). - PMC - PubMed
    1. Cao R., et al. , Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039–1043 (2002). - PubMed
    1. Czermin B., et al. , Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111, 185–196 (2002). - PubMed
    1. Kuzmichev A., Nishioka K., Erdjument-Bromage H., Tempst P., Reinberg D., Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev. 16, 2893–2905 (2002). - PMC - PubMed

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