Evolutionarily ancient BAH–PHD protein mediates Polycomb silencing Elizabeth T. Wilesa,1, Kevin J. McNaughta,1, Gurmeet Kaurb, Jeanne M. L. Selkera, Tereza Ormsbya,2, L. Aravindb, and Eric U. Selkera,3 aInstitute of Molecular Biology, University of Oregon, Eugene, OR 97403; and bNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 Contributed by Eric U. Selker, March 18, 2020 (sent for review October 28, 2019; reviewed by Wolfgang Fischle and Steve Jacobsen) Methylation of histone H3 lysine 27 (H3K27) is widely recognized (NCU07505) that we show is critical for H3K27 methylation- as a transcriptionally repressive chromatin modification but the mediated silencing and therefore named it effector of Polycomb re- mechanism of repression remains unclear. We devised and imple- pression 1 (epr-1). It encodes a protein with a bromo-adjacent ho- mented a forward genetic scheme to identify factors required for mology (BAH) domain and plant homeodomain (PHD) finger. H3K27 methylation-mediated silencing in the filamentous fungus Although epr-1 mutants display phenotypic and gene-expression Neurospora crassa and identified a bromo-adjacent homology changes similar to strains lacking PRC2 components, H3K27 meth- (BAH)-plant homeodomain (PHD)-containing protein, EPR-1 (effec- ylation is essentially unaffected. We demonstrate that EPR-1 forms tor of polycomb repression 1; NCU07505). EPR-1 associates with nuclear foci, reminiscent of Polycomb bodies (24), and its genomic H3K27-methylated chromatin, and loss of EPR-1 de-represses H3K27- distribution is limited to, and dependent upon, H3K27-methylated methylated genes without loss of H3K27 methylation. EPR-1 is not chromatin, which may be recognized through its BAH domain. Fi- fungal-specific; orthologs of EPR-1 are present in a diverse array of nally, we discovered that EPR-1 orthologs are widely distributed eukaryotic lineages, suggesting an ancestral EPR-1 was a compo- across eukaryotes, contrary to previous reports (21, 25, 26), suggesting nent of a primitive Polycomb repression pathway. an ancient role of EPR-1 homologs in Polycomb repression that was then lost on multiple occasions in certain lineages. epigenetics | facultative heterochromatin | H3K27 methylation | Polycomb repressive complex | histone reader Results GENETICS Genetic Selection for Factors Necessary for H3K27 Methylation-Mediated he establishment and maintenance of transcriptionally re- Repression. In an effort to identify factors required for H3K27 Tpressive chromatin is critical for the development of multi- methylation-mediated repression, we engineered a strain of N. crassa cellular organisms (1–4). Polycomb group (PcG) proteins, originally discovered in Drosophila melanogaster (5), form mul- Significance tiple complexes that maintain such chromatin repression (6). Al- though the composition of PcG complexes varies, a few core constituents define two major classes of chromatin-modifying Polycomb group (PcG) proteins are employed by a wide variety complexes, namely Polycomb repressive complex 1 (PRC1) and of eukaryotes for the maintenance of gene repression. Poly- PRC2 (7). According to the “classic model,” PcG-mediated gene comb repressive complex 2 (PRC2), a multimeric complex of PcG proteins, catalyzes the methylation of histone H3 lysine 27 silencing is initiated by targeting of PRC2 to chromatin (8), which (H3K27). In the filamentous fungus, Neurospora crassa, H3K27 catalyzes methylation of H3K27 (9). Canonical PRC1, which methylation represses scores of genes, despite the absence of contains a chromodomain protein (e.g., Polycomb in D. mela- canonical H3K27 methylation effectors that are present in nogaster and CBX2/4/6–8 in mammals), recognizes trimethylated plants and animals. We report the identification and charac- H3K27 (10), catalyzes monoubiquitination of neighboring histone terization of an H3K27 methylation effector, EPR-1, in N. crassa H2A lysine 119 by RING1A/B (11), and promotes chromatin and demonstrate its widespread presence and early eukaryotic compaction (12, 13). In reality, this hierarchical recruitment model origins with phylogenetic analyses. These findings indicate is an oversimplification, as PRC1 can be recruited to PcG targets that an ancient EPR-1 may have been part of a nascent Poly- irrespective of PRC2 activity (14), and PRC1 presence is required comb repression system in eukaryotes. for stable PRC2 association at many Polycomb response elements in D. melanogaster (15). Interdependence of these complexes has Author contributions: E.T.W., K.J.M., and E.U.S. designed research; E.T.W., K.J.M., G.K., limited our understanding of their respective roles and the func- J.M.L.S., and T.O. performed research; E.T.W., K.J.M., G.K., J.M.L.S., T.O., L.A., and E.U.S. tion of their associated chromatin “marks” on gene repression. analyzed data; and E.T.W., K.J.M., G.K., J.M.L.S., T.O., L.A., and E.U.S. wrote the paper. While plants and animals utilize distinct sets of accessory Reviewers: W.F., Max Planck Institute for Biophysical Chemistry; and S.J., University of proteins to recognize methylated H3K27 (10, 16–18), they are California, Los Angeles. generally thought to mediate repression in the context of a ca- The authors declare no competing interest. nonical PRC1 complex (7, 19–21). In fungal lineages that employ Published under the PNAS license. H3K27 methylation as a repressive chromatin mark, however, Data deposition: All ChIP-seq, DamID-seq, and mRNA-seq data have been deposited in the core PRC1 components are notably absent (7). This raises the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession question of how H3K27 methylation mediates repression in the no. GSE128317). All whole-genome sequencing data have been deposited in the NCBI Sequence Read Archive (SRA), https://www.ncbi.nlm.nih.gov/sra/ (accession no. absence of PRC1. It suggests that either 1) H3K27 methylation PRJNA526508). “ ” per se may be repressive, or 2) there is a reader of H3K27 1E.T.W. and K.J.M. contributed equally to this work. methylation that functions outside the context of canonical PRC1. 2Present address: The Laboratory of Proteases of Human Pathogens, Institute of Organic To elucidate the repressive mechanism of H3K27 methylation Chemistry and Biochemistry, Czech Academy of Sciences, 166 10 Prague 6, in fungi, we developed and employed a forward genetics ap- Czech Republic. proach to identify effectors of Polycomb repression using Neu- 3To whom correspondence may be addressed. Email: [email protected]. rospora crassa. H3K27 methylation covers ∼7% of the N. crassa This article contains supporting information online at https://www.pnas.org/lookup/suppl/ genome and is responsible for the repression of scores of genes doi:10.1073/pnas.1918776117/-/DCSupplemental. (22, 23). We found four mutant alleles of an undescribed gene www.pnas.org/cgi/doi/10.1073/pnas.1918776117 PNAS Latest Articles | 1of10 Downloaded by guest on September 30, 2021 in which we replaced the open reading frames (ORFs) of two PRC2- (Fig. 1B). One mutant isolated in this manner and characterized here repressed genes (23), NCU05173 and NCU07152, with the antibiotic- is epr-1 (Fig. 1C). resistance genes hph and nat-1, respectively (Fig. 1A). Strains that bear these gene replacements and lack the H3K27 methyltransferase Mapping and Identification of epr-1 as NCU07505. In order to map (SET-7) are resistant to Hygromycin B and Nourseothricin, whereas and identify the causative mutation in the epr-1UV1 mutant, we a wild-type strain with these gene replacementsissensitivetothese crossed epr-1UV1, which is in an Oak Ridge genetic background, drugs (Fig. 1C). We subjected conidia collected from such an to a highly polymorphic wild-type strain named “Mauriceville” antibiotic-sensitive, otherwise wild-type strain to UV mutagenesis and (27). We then pooled the genomic DNA from Hygromycin selected for mutants that derepressed both the hph and nat-1 genes B-resistant progeny and subjected it to whole-genome sequencing A B Hygromycin B mRNA-seq + 175 wild type No drug Nourseothricin 150 ∆set-7 UV 125 100 75 50 OFF ON PNCU05173::hph PNCU05173::hph 25 P ::nat-1OFF P ::nat-1ON Average normalized counts Average 0 NCU07152 NCU07152 NCU05173 NCU07152 C No drug Hygromycin B Nourseothricin wild type ∆set-7 epr-1UV1 cells: 104 103 102 101 104 103 102 101 104 103 102 101 D E Q206* 537 AA epr-1UV1 wild type (Oak Ridge) X (Mauriceville) * NCU07505 100 LG I Pool genomic DNA 80 of antibiotic-resistant progeny 60 40 Next-generation sequencing 20 % Oak Ridge SNPs 0 0246810 Bioinformatic analysis Position (Mb) F No drug Hygromycin B wild type ∆set-7 epr-1UV1 epr-1UV1 + NCU07505WT ∆NCU07505 cells: 104 103 102 101 104 103 102 101 Fig. 1. Forward genetics identifies a gene, epr-1, required for H3K27 methylation-mediated repression. (A) mRNA-seq results for two genes repressed by the N. crassa H3K27 methyltransferase, encoded by set-7.(B) Selection scheme, utilizing reporter genes illustrated in A, to identify factors required for H3K27 methylation-mediated silencing. (C) Serial dilution spot test silencing assay for the indicated strains plated on the indicated media. All strains harbor UV1 UV1 PNCU05173::hph and PNCU07152::nat-1.(D) Scheme for genetic mapping of critical mutation in epr-1 .(E) Whole-genome sequencing of pooled epr-1 mutant genomic DNA identified a region on the left arm of linkage group (LG) I that is enriched for Oak Ridge SNPs and contains a premature stop codon in the BAH domain of NCU07505 (BAH domain, light blue; PHD finger [split], dark blue; no annotated domains, gray). Each translucent point represents a running average of SNPs (window size = 10 SNPs, step size = 1 SNP). (F) Serial dilution spot test silencing assay for the indicated strains. epr-1UV1 + WT NCU07505 has a wild-type copy of NCU07505 at the his-3 locus. All strains harbor PNCU05173::hph. 2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1918776117 Wiles et al.