Evolutionarily Ancient BAH–PHD Protein Mediates Polycomb Silencing

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

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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. Downloaded by guest on September 30, 2021 (∼15× coverage) (Fig. 1D). When we scored the percentage of genes that are also up-regulated in strains lacking H3K27 Oak Ridge single-nucleotide polymorphisms (SNPs) across the methylation. genome (28), we found a region on linkage group I that was enriched for Oak Ridge SNPs and included an early stop mutation Δepr-1 and Δset-7 Strains Share a Sexual Development Defect. Since (Q206*, CAG→TAG) in NCU07505 (Fig. 1E). Δepr-1 and Δset-7 strains exhibit similar transcriptional profiles, To verify that the early stop in NCU07505 is the causative we wondered if they also shared vegetative growth and sexual mutation in epr-1UV1, we targeted a wild-type copy of NCU07505 development phenotypes as well. To assess if Δepr-1 strains have to the his-3 locus in the epr-1UV1 mutant background. This ec- an altered vegetative growth rate, we measured linear growth Δ Δ “ ” topic copy of NCU07505 complemented the mutation (i.e., it rates of wild-type, set-7, and epr-1 strains with race tubes Δ restored drug sensitivity) (Fig. 1F). In addition, we found that (29). We confirmed that set-7 strains do not have a linear Δ deletion of NCU07505 resulted in resistance to Hygromycin B, growth defect (22) and found that epr-1 strains also grow at similar to the epr-1UV1 strain (Fig. 1F). We subsequently isolated wild-type rates (SI Appendix, Fig. S2E). Loss of SET-7 has been implicated in promoting sexual development in mutants that are and characterized three additional alleles of epr-1 generated in Δ Δ the mutagenesis, further supporting the notion that mutations in homozygous sterile (30). To determine if set-7 and epr-1 NCU07505 support drug resistance (SI Appendix, Fig. S1). strains aberrantly promote sexual differentiation in the absence of a mating partner, we singly inoculated crossing plates (31) Δ Δ EPR-1 and SET-7 Repress an Overlapping Set of H3K27-Methylated with wild-type, set-7,or epr-1 strains. After 2 wk of unfertil- Genes. Although our selection was designed to isolate mutants ized growth at 25 °C, we observed the development of few false Δ Δ with specific defects in Polycomb repression, we could conceiv- perithecia with wild-type controls, whereas epr-1 and set-7 ∼ ably recover mutants that globally altered transcription or led to developed 10- and 100-fold more false perithecia than wild antibiotic resistance independent of hph or nat-1 up-regulation. type, respectively (Fig. 2C and SI Appendix, Fig. S2F). These data To determine if EPR-1 was specifically required for repression of suggest that EPR-1, and SET-7 to a greater extent, repress pre- H3K27-methylated genes, we performed mRNA sequencing mature sexual development, which is reminiscent of fertilization- independent seed development observed in plant Polycomb (mRNA-seq) on Δepr-1 siblings and compared the gene- mutants (32, 33). expression profile to previously published wild-type and Δset-7 datasets (23): 632 genes were up-regulated and 974 genes were H3K27 Methylation Is Essentially Normal in Δepr-1. As a first step to down-regulated greater than twofold in Δepr-1 strains compared < assess if the transcriptional silencing and sexual development to wild-type strains (P 0.05) (SI Appendix, Fig. S2A). The up- defects shared between Δepr-1 and Δset-7 strains were due to a GENETICS Δ regulated gene set in epr-1 was significantly enriched for common global loss of H3K27 methylation, we performed a χ2 = = × −10 H3K27-methylated genes [ (1, n = 632) 40.8, P 1.684 10 ] Western blot on whole-cell lysates to detect H3K27me3 in wild- (SI Appendix, Fig. S2A), and H3K27-methylated genes up- type, Δset-7, and Δepr-1 strains. The total levels of H3K27me3 in Δ Δ regulated in both epr-1 and set-7 significantly overlapped Δepr-1 strains were comparable to that in wild type (Fig. 3A and −16 (P = 1.436 × 10 ) (Fig. 2A). To verify our mRNA-seq results, SI Appendix, Fig. S3 A–C). However, because only a subset of we performed reverse transcription followed by quantitative H3K27-methylated genes are derepressed in both Δepr-1 and PCR (RT-qPCR) on RNA isolated from biological triplicates of Δset-7 strains, we wanted to know if H3K27 methylation might wild-type, Δset-7, and Δepr-1 strains. Five of six examined be specifically lost at up-regulated genes in Δepr-1 strains. To H3K27-methylated genes found up-regulated in both Δset-7 and examine this possibility, we performed H3K27me2/3 chromatin Δepr-1 strains by mRNA-seq were confirmed by RT-qPCR immunoprecipitation followed by sequencing (ChIP-seq) on two (Fig. 2B and SI Appendix, Fig. S2B). In contrast, only one of Δepr-1 siblings and compared the data to that for wild type (34). six H3K27-methylated genes found exclusively up-regulated in We found that the global distribution of H3K27me2/3 in Δepr-1 Δset-7 or Δepr-1 by mRNA-seq was confirmed by RT-qPCR (SI appeared to mirror that of wild type (Fig. 3B). Comparison of Appendix, Fig. S2 C and D). Thus, these data show that loss of H3K27me2/3 levels associated with individual genes showed EPR-1 derepresses a significant number of H3K27-methylated good agreement between the averaged wild-type and Δepr-1

AB C10,000 Upregulated 75 H3K27-methylated wild type *** 1,000 993 genes by mRNA-seq 50 ∆set-7 ∆epr-1 ∆set-7 25 ∆epr-1 153 15 ** 100

60 43 64 10 *** ** ** *** ** 10 10.5

*** False perithecia 5 ** * *** **

-16 Normalized fold change ns P = 1.436 x 10 * ns ns 1 0

NCU07152NCU05173NCU09640NCU07624NCU09178NCU08570NCU10038NCU08097 wild type ∆epr-1 ∆set-7

Fig. 2. Δepr-1 and Δset-7 strains share defects in transcriptional silencing and sexual development. (A) Venn diagram depicting H3K27-methylated genes that

appear up-regulated by mRNA-seq in both Δepr-1 and Δset-7 strains, only in Δepr-1 strains, or only in Δset-7 strains, using a significance cutoff of log2(mutant/ wild type) > 1 and P < 0.05 using the Benjamin–Hochberg correction for multiple comparisons. Significance of genes up-regulated in both Δepr-1 and Δset-7 strains was determined using a hypergeometric test. (B) RT-qPCR of H3K27-methylated genes that were replaced with antibiotic resistance genes (NCU07152, NCU05173) and used for initial selection of mutants, and H3K27-methylated genes that appeared up-regulated in both Δepr-1 and Δset-7 strains by mRNA- seq (NCU09640, NCU07624, NCU09178, NCU08570, NCU010038, NCU08097). Each value was normalized to expression of actin gene (act) and presented relative to wild type. Filled bars represent the mean from biological triplicates and error bars show SD. (***P < 0.001, **P < 0.01, *P < 0.05, and ns, not significant; all relative to wild type by two-tailed, unpaired t test). (C) Quantification of false perithecia developed in a Petri dish (85-mm diameter) after 2 wk of unfertilized growth are shown for the indicated strains. Horizontal lines and numbers indicate the mean of two biological replicates (open circles).

Wiles et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 30, 2021 ABr-1 ep wild type∆set-7∆epr-1wild type ∆set-7∆ H3K27me2/3 ChIP-seq LG VI H3K27me3 wild type

∆epr-1 hH3 C D ) 11 1.5 H3K27me2/3 ChIP-qPCR wild type epr-1 ns 10 NCU02856 ∆set-7 ∆epr-1 9 1.0 ns

ns 8 NCU08834 % Input 0.5 7 ns (H3K27me2/3 in ∆ 2

log 2 ns ns * 6 R = 0.9105 0.0 ** ** ** ** ** 67891011 hH4 Tel IL NCU07152 NCU05173 NCU08834 NCU02856

log2(H3K27me2/3 in wild type)

Fig. 3. epr-1 is not required for H3K27 methylation. (A) Western blot showing H3K27me3 and total histone H3 (hH3) in the indicated strains. Biological replicates are shown. The same lysate was run on separate gels, and hH3 was used as a sample processing control. (B) ChIP-seq track showing average levels of H3K27me2/3 from two biological replicates of wild-type and Δepr-1 strains on LG VI. Open circle indicates the middle of the centromere region. Gray bar represents 500 kb. The y axis is 0 to 800 RPKM for wild type and 0 to 1,200 RPKM for Δepr-1. (C) Scatter plot showing the correlation of H3K27me2/3 levels at all genes (black dots) in wild-type and Δepr-1 strains based on biological replicates of ChIP-seq data. Line of best fit displayed in red (R2 = 0.9105). Repre- sentative genes that gained (NCU02856)orlost(NCU08834) H3K27me2/3 in Δepr-1 are indicated. (D) H3K27me2/3 ChIP-qPCR to confirm ChIP-seq data at six regions in wild-type, Δset-7 and Δepr-1 strains: hH4 (negative control), Tel IL (unchanged H3K27me2/3 in Δepr-1), NCU07152 (unchanged H3K27me2/3 in Δepr- 1), NCU05173 (unchanged H3K27me2/3 in Δepr-1), NCU08834 (loss of H3K27me2/3 in Δepr-1), and NCU02856 (gain of H3K27me2/3 in Δepr-1). Filled bars represent the mean of biological triplicates and error bars show SD (**P < 0.01, *P < 0.05, and ns, not significant; all relative to wild type by two-tailed, unpaired t test).

datasets (R2 = 0.9105), and within replicate data for wild-type to a strain lacking eed, a gene encoding a component of PRC2 (R2 = 0.9272) and Δepr-1 (R2 = 0.8941) strains (Fig. 3C and SI necessary for catalytic activity (22). We could not easily in- Appendix, Fig. S3 D and E). We did, however, identify 29 genes troduce a deletion of the H3K27 methyltransferase gene, set-7, with a greater than twofold decrease and nine genes with a due to its tight genetic linkage to epr-1. Strains bearing a deletion greater than twofold increase in H3K27me2/3 levels in Δepr-1 of eed lacked distinct nuclear foci of EPR-1WT and instead dis- compared to wild type. Interestingly, none of these 38 genes with played a diffuse nuclear distribution of EPR-1WT (Fig. 4B). Thus, altered H3K27 methylation were classified among the up- an intact PRC2 complex or H3K27 methylation is required for regulated or down-regulated gene sets in the mRNA-seq analy- proper EPR-1WT subnuclear localization. sis of Δepr-1. To validate the H3K27me2/3 ChIP-seq results, we performed H3K27me2/3 ChIP followed by quantitative PCR An Intact BAH Domain Is Required for Normal Nuclear Distribution of (ChIP-qPCR) on wild-type, Δset-7, and Δepr-1 strains in bi- EPR-1. EPR-1 is predicted (36) to have a BAH domain and PHD ological triplicate. These data confirmed wild-type levels of finger, protein modules implicated in chromatin engagement H3K27me2/3 at a subtelomere (Tel IL) and at the genes (37, 38). To determine if these domains are necessary for the replaced by the antibiotic-resistance markers (NCU07152 and formation of the EPR-1WT foci, we created GFP–EPR-1 con- NCU05173), and also corroborated the loss (NCU08834) and structs in which a previously identified critical tryptophan in ei- gain (NCU02856) of H3K27me2/3 observed in the ChIP-seq of ther the BAH domain (EPR-1BAH) or PHD finger (EPR-1PHD) Δepr-1 strains (Fig. 3D). Altogether, these data show that the was replaced with an alanine (39, 40). We found that EPR-1BAH derepression of H3K27-methylated genes in Δepr-1 strains is not displayed the same diffuse nuclear distribution as EPR-1WT in a due to concomitant loss of H3K27 methylation. Δeed background, consistent with the possibility that the BAH domain mediates interaction with H3K27-methylated chromatin EPR-1 Forms Telomere-Associated Foci Dependent on EED. To local- (Fig. 4C). In contrast, EPR-1PHD still formed nuclear foci that ize EPR-1 in vivo, we used the ccg-1 promoter to drive expres- were equivalent to EPR-1WT in number and proximity to TRF1 sion of wild-type EPR-1 fused with green fluorescent protein foci (Fig. 4D and SI Appendix, Fig. S4D), demonstrating the (GFP) at its N terminus (EPR-1WT) (35) in a strain that had nonessential nature of this conserved tryptophan residue for the fluorescent markers for the nuclear membrane (ISH1), telo- normal nuclear distribution of EPR-1. meres (TRF1), and centromeres (CenH3) (23). We found that EPR-1WT was restricted to the nucleus and formed distinct foci EPR-1 Localizes to H3K27 Methylation Genome-Wide. Since proper that were typically closely associated with TRF1 foci (Fig. 4A). subnuclear localization of EPR-1WT appeared to require H3K27 EPR-1WT foci were significantly closer to telomeres as compared methylation, we performed ChIP-seq to determine if EPR-1WT to centromeres (negative control; P = 0.0403) (SI Appendix, Fig. genomic targets coincided with H3K27me2/3 throughout the S4A), and the number of EPR-1WT and TRF1 foci per nucleus genome (Fig. 5A). Results of EPR-1WT ChIP-seq appeared to were not statistically different (P = 0.7422) (SI Appendix, Fig. match the distribution of H3K27me2/3 in wild type (Fig. 5A) and S4B), although the majority of nuclei examined had more EPR- we found good correlation between EPR-1WT and H3K27me2/3 1WT than TRF1 foci (SI Appendix, Fig. S4C). Considering that relative sequencing coverage over each gene (R2 = 0.8675) (SI H3K27 methylation is predominantly present near chromosome Appendix, Fig. S5A). We also examined the genomic distribution ends in N. crassa (22), it is not unexpected that a putative PcG of EPR-1PHD and EPR-1BAH mutant alleles (Fig. 5A), which protein, such as EPR-1, would colocalize with the telomere were expressed at comparable levels (SI Appendix, Fig. S5 B and marker, TRF1. C). The ChIP-seq coverage of EPR-1PHD was still enriched at To determine if the formation of EPR-1WT foci was dependent H3K27-methylated genes, albeit less so than EPR-1WT, while the on H3K27 methylation, we crossed our fluorescent marker strain EPR-1BAH ChIP-seq did not show enrichment (SI Appendix, Fig.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1918776117 Wiles et al. Downloaded by guest on September 30, 2021 EPR-1 TRF1 CenH3 ISH1 Merge A WT epr-1

B ; ∆eed WT epr-1 C BAH epr-1

D PHD epr-1

Fig. 4. EPR-1 forms telomere-associated foci that are dependent on EED and the BAH domain of EPR-1. Maximum-intensity projection images of fluorescence microscopy z-stacks showing EPR-1 (GFP-EPR-1, green) for epr-1WT (A), epr-1WT; Δeed (B), epr-1BAH (C), and epr-1PHD (D). Telomeres (TRF1-TagRFP-T, red),

centromeres (CenH3-iRFP670, magenta), the nuclear membrane (ISH1-TagBFP2, blue), and merged images are shown for reference. Each image shows a single GENETICS conidium with multiple nuclei. (Scale bars, 2 μm.)

S5D). To validate the ChIP-seq of GFP–EPR-1–expressing strains, resistance gene (Fig. 5C). This suggests that while the PHD we performed ChIP-qPCR for representative regions (Fig. 5B). finger of EPR-1 is not essential for recruitment to H3K27 Consistent with the ChIP-seq data, EPR-1WT—and EPR-1PHD methylated chromatin, it is not entirely dispensable for gene to a lesser degree—were enriched at examined regions bearing silencing. H3K27 methylation. In addition, the ChIP-qPCR confirmed that EPR-1BAH,aswellasEPR-1WT in a Δeed background, lack such EPR-1 Is a Homolog of Plant EBS/SHL and Widely Distributed across enrichment. Eukaryotes. To determine if EPR-1 orthologs exist outside of N. As an orthogonal approach to ChIP, we determined the crassa, we performed sequence similarity searches to identify chromatin targets of EPR-1 by fusing an Escherichia coli DNA homologs, followed by phylogenetic and domain architectural adenine methyltransferase (Dam) (41) to the C terminus of en- analysis of those to identify genuine orthologs. Consequently, we dogenous EPR-1 and assayed adenine-methylated DNA frag- were able to identify orthologs in various fungal species as well as ments by sequencing (DamID-seq) (42) (Fig. 5A). Using DamID- a wide range of other eukaryotes (Fig. 6A). Notably, we de- seq of EPR-1WT–Dam and methyl-sensitive restriction enzyme termined the Arabidopsis thaliana paralogs EBS and SHL as Southern blots, we found that EPR-1WT–Dam localizes to H3K27- orthologs of EPR-1 in our analyses, which have been erroneously methylated genes and this is dependent upon EED (Fig. 5A and SI reported as plant-unique proteins (21, 25, 26). Similar to EPR-1, Appendix, Fig. S5 E and F). Mutation of the PHD finger in the the plant paralogs, EBS and SHL, bind H3K27 methylation and EPR-1–Dam fusion did not abolish its targeting to H3K27- have been implicated in gene repression (17, 18, 21). As one methylated chromatin (Fig. 5A). These results were consistent approach to investigate if other EPR-1 orthologs may have roles with our ChIP-seq findings. We conclude that EPR-1 localizes to independent of H3K27 methylation, we checked if any species H3K27-methylated regions of the genome and that proper re- has an EPR-1 ortholog but lacks a SET-7 (H3K27 methyl- cruitment of EPR-1 to chromatin requires both an intact BAH transferase) ortholog. With the exception of Chytridiomycota domain and the integral PRC2 component, EED. and Fonticula lineages, in which the presence of SET-7 homo- logs was deemed ambiguous due to lack of a definitive pre-SET Both the BAH Domain and PHD Finger of EPR-1 Are Necessary for domain, all examined species with EPR-1 homologs had clear Gene Repression. Our localization studies of EPR-1PHD and SET-7 homologs (Fig. 6A). This result is consistent with EPR-1 EPR-1BAH, while suggestive, did not directly test the role of the orthologs mediating H3K27 methylation-based repression in a PHD finger and BAH domain of EPR-1 in H3K27 methylation- wide variety of species. mediated silencing. We therefore utilized our antibiotic- In contrast to EPR-1, A. thaliana EBS is not entirely restricted resistance reporters, used in the initial selection, to test more to H3K27-methylated genes. Indeed, the majority of EBS-bound directly their possible involvement in gene repression. We tar- genes are devoid of H3K27me3 and instead are associated with geted ectopic copies of epr-1WT, epr-1BAH,orepr-1PHD to the his-3 an “active” chromatin mark, H3K4me3 (43), via the PHD finger locus in an epr-1UV1 strain bearing the antibiotic-resistance genes of EBS (17). To understand this discrepancy, we examined the and scored drug resistance. Whereas epr-1WT restored sensitivity underlying protein sequence of the PHD finger of EPR-1. to Hygromycin B and Nourseothricin, epr-1BAH remained re- Whereas aromatic residues implicated in methylated histone sistant to both drugs (Fig. 5C). In contrast, introduction of epr- recognition in the BAH domain (44) are present in both EPR-1 1PHD apparently resilenced the hph, but not the nat-1, antibiotic- and the plant paralogs, comparable residues in the PHD finger

Wiles et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 30, 2021 A LG III wild type H3K9me3

wild type H3K27me2/3

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GFP ChIP epr-1PHD

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DamID epr-1PHD

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10 B epr-1WT C No drug Hygromycin B Nourseothricin PHD epr-1 wild type 8 epr-1BAH epr-1UV1 WT ) epr-1 ; ∆eed

hH4 6 + epr-1WT

4 ns ** UV1

(relative to ** + epr-1BAH GFP ChIP enrichment ChIP GFP

2 ns ns epr-1 ****** ****** ns *** ***

0 + epr-1PHD Tel IL NCU05173 NCU07152 H3K9me3 promoter promoter region cells: 104 103 102 101 104 103 102 101 104 103 102 101

Fig. 5. BAH domain of EPR-1 required for localization to H3K27-methylated chromatin. (A) ChIP-seq and DamID-seq tracks showing average levels from two biological replicates for the indicated genotypes on LG III. The y axis is 0 to 800 RPKM for H3K9me3 and H3K27me2/3 ChIP-seq, 0 to 500 RPKM for all GFP ChIP- seq, and 0 to 3,500 RPKM for all DamID-seq. Gray bar represents 500 kb. (B) GFP ChIP-qPCR to validate GFP–EPR-1 ChIP-seq data at four genomic regions: Tel IL (H3K27-methylated), NCU05173 promoter (H3K27-methylated), NCU07152 promoter (H3K27-methylated), and H3K9me3 region (negative control, LG VI centromere). All data are normalized to a negative, euchromatic control, hH4. Filled bars represent the mean and error bars show SD from three biological replicates (***P < 0.001, **P < 0.01, and ns, not significant; all relative to wild type by two-tailed, unpaired t test). (C) Serial dilution spot test silencing assay

for the indicated strains. All strains harbor PNCU05173::hph and PNCU07152::nat-1.

(18, 45, 46) are present in EBS and SHL, but lacking in EPR-1 examination revealed that GFP–EPR-1 forms approximately (Fig. 6B, highlighted in red). Furthermore, a single amino acid three to five foci per nucleus. This implies that domains of substitution replacing an aromatic tyrosine residue with an ala- H3K27 methylation within and between the seven N. crassa nine residue in the PHD finger of a plant SHL was sufficient to chromosomes generally self-associate. This is consistent with the diminish in vitro H3K4me3-binding greater than twofold (18). observed intra- and interchromosomal contacts among H3K27- Intriguingly, this residue corresponds to the naturally occurring methylated regions of the genome in Hi-C experiments (49). alanine 275 in EPR-1 (Fig. 6B). This suggests that while many Similar, and perhaps equivalent, higher-order chromatin struc- species have EPR-1 homologs, they may not necessarily be bi- tures, referred to as Polycomb bodies (24), are known to be valent histone readers like plant EBS and SHL. mediated by PRC1 components in both plant and animal cells (50–53). Our group has previously shown that SET-7, the Discussion catalytic component of PRC2, is required for normal three- Deciphering the basic mechanisms of Polycomb repression has dimensional genome organization (23). It would be interesting to been difficult, in part, due to the diversity (47), redundancy (48), learn if EPR-1, the only known effector of H3K27 methyl- as well as interdependence (15) of protein players involved. For ation in N. crassa, is also essential for this wild-type chromatin this reason, the model organism N. crassa, which employs H3K27 organization. methylation catalyzed by PRC2 for gene repression (22) yet Despite the loss of transcriptional silencing observed in epr-1 conspicuously lacks PRC1 components (7), represents an ideal mutants, they do not exhibit appreciably altered H3K27 meth- organism to uncover fundamental aspects of Polycomb silencing. ylation; this is striking for a few reasons. First, it suggests that Here we have identified an effector of H3K27 methylation in H3K27 methylation-mediated silencing is a unidirectional path- fungi, EPR-1. This provides insight into how Polycomb silencing way in N. crassa, in which EPR-1 acts downstream of PRC2. This can function in the absence of PRC1. is in contrast to findings in plants and animals, in which PRC1 Our ChIP and DamID results demonstrated that EPR-1 components can affect the recruitment or activity of PRC2 (21, colocalizes with H3K27 methylation genome-wide and cytological 54, 55), a fact that has hampered the elucidation of the direct

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1918776117 Wiles et al. Downloaded by guest on September 30, 2021 A EPR-1 SET-7 B Homolog present Laccaria bicolor W184 A275 W163 W292 Homolog absent Basidiomycota Cryptococcus neoformans Y186 V268 537 AA Ambiguous Stereum hirsutum Calocera viscosa EPR-1 Neurospora crassa Fusarium graminearum Ascomycota Saccharomyces cerevisiae Y49 Y72 Y148 Y155 W170 Aspergillus nidulans W70 Mucoromycota Rhizophagus irregularis EBS (A.t.) 234 AA Blastocladiomycota Allomyces macrogynus W163 Chytridiomycota Gonapodya prolifera Y41 W63 Y65 F141 Y148 Spizellomyces punctatus SHL (A.t.) 228 AA Cryptomycota Rozella allomycis Encephalitozoon cuniculi Microsporidia Anncaliia algerae Enterocytozoon bieneusi Apusozoa Thecamonas trahens Fonticula Fonticula alba

Caenorhabditis elegans Gallus gallus Metazoa Xenopus laevis Homo sapiens

Choanoflagellata Salpingoeca rosetta

Entamoeba histolytica

Amoebozoa GENETICS Arabidopsis thaliana Viridiplantae Micromonas commoda

Rhodophyta Chondrus crispus Alveolata Paramecium tetraurelia Oxytricha trifallax

Rhizaria Plasmodiophora brassicae

Phytophthora sojae Stramenopila Thalassiosira oceanica Heterolobosea Naegleria gruberi

Fig. 6. EPR-1 homologs are present in species beyond fungi. (A) The presence and absence of EPR-1 and SET-7 protein homologs across major species divisions is depicted in a representative tree of eukaryotes. The leaves of the tree are labeled with the names of the divisions. Representative species are featured and their associated squares in the EPR-1 and SET-7 columns indicate the presence or absence of homologs, as indicated. (B) Protein domain structure of EPR-1, as well as EBS and SHL from A. thaliana (A.t.). BAH domains are indicated by light blue, the PHD fingers by dark blue, and regions with no known domains are gray. Aromatic amino acid residues involved in methylated histone recognition in the BAH domain and PHD finger are indicated above the protein structure diagram (black text). Red text above the PHD finger in EPR-1 highlights the absence of aromatic residues at these amino acid positions.

role of PRC components in gene repression. Second, since Δepr- Polycomb silencing system. While animals are a notable excep- 1 strains de-repress H3K27-methylated genes without loss of tion, regarding the absence of EPR-1 homologs with a BAH– H3K27 methylation, it suggests that active transcription does not PHD structure, a human BAH domain-containing protein, necessarily preclude PRC2 activity. This was surprising since BAHD1, has been reported to “read” H3K27me3 (39) and transcriptional shut-off is thought to precede PRC2 activity promote gene silencing (61), although apparently not interact during normal animal development (56) and because artificial with known PRC1 components (62). It is therefore conceivable gene repression can be sufficient to recruit PRC2 (57, 58). Fi- that BAHD1 homologs present in animal lineages are actually nally, the presence of H3K27 methylation on de-repressed genes divergent orthologs of EPR-1 that lack a PHD finger. Regardless Δ in epr-1 strains demonstrates that H3K27 methylation per se is of their ancestry, both human BAHD1 and N. crassa EPR-1 not sufficient for effective gene repression, consistent with pre- Δ represent forms of H3K27 methylation-mediated repression that vious reports (59, 60). It is noteworthy, however, that while set- do not rely on PRC1 components. 7 and Δepr-1 strains share transcriptional and sexual defects, the phenotype of Δset-7 strains is generally more pronounced. This Materials and Methods suggests that PRC2 or H3K27 methylation may have additional Strains, Media and Growth Conditions. All N. crassa strains used in this study roles in gene repression that go beyond recruitment of EPR-1 to are listed in SI Appendix, Table S1. Liquid cultures were grown with shaking chromatin. at 32 °C in Vogel’s minimal medium (VMM) with 1.5% sucrose (63). Crosses Our investigation into the phylogenetic distribution of EPR-1 were performed at 25 °C on modified VMM with 0.1% sucrose (31). Spot homologs indicates that an ancestral EPR-1 emerged prior to the tests were performed at 32 °C on VMM with 0.8% sorbose, 0.2% fructose, divergence of plants, animals, and fungi. This ancestral EPR-1 and 0.2% glucose (FGS). When appropriate, plates included 200 μg/mL may have been an integral component of an early eukaryotic Hygromycin B Gold (InvivoGen) or 133 μg/mL Nourseothricin (Gold

Wiles et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 30, 2021 Biotechnology). Linear growth rates were determined as previously de- was reversed by incubation at 65 °C for 16 h and then samples were treated scribed except 25-mL serological pipettes were used in place of glass tubes with proteinase K for 2 h at 50 °C. DNA was purified using Minelute columns (29). Genomic DNA was isolated as previously described (64). Nutritional (Qiagen) and subsequently used for qPCR with the PerfeCTa SYBR Green supplements required for auxotrophic strains were included in all growth FastMix (QuantBio, 95071-012) on a Step One Plus Real Time PCR System media when necessary. (Life Technologies) using primer pairs in SI Appendix, Table S3, or prepared for sequencing using the NEBNext DNA Library Prep Master Mix Set for Selection for Mutants Defective in H3K27 Methylation-Mediated Silencing. Ten- Illumina (New England BioLabs). ChIP-seq data are available in the NCBI thousand conidia of strain N6279 (created with primers in SI Appendix, Table Gene Expression Omnibus (GEO) database (accession no. GSE128317). S2) were plated on VMM supplemented with FGS and 500 μg/mL histidine and subjected to 0, 3, 6, or 9 s of UV light (Spectrolinker XL-1500 UV ChIP-Seq Mapping and Analysis. The suite of tools available on the open- Crosslinker, Spectronics Corporation) in a dark room and plates were source platform Galaxy (70) was used to map ChIP-seq reads (71) against wrapped in aluminum foil to prevent photoreactivation (65). Plates were the corrected N. crassa OR74A (NC12) genome (49) and to create bigWig incubated at 32 °C for 16 h before being overlaid with 1% top agar con- coverage files normalized to reads per kilobase per million mapped reads taining VMM, FGS, 500 μg/mL histidine, Hygromycin B, and Nourseothricin. (RPKM) (72). ChIP-seq tracks were visualized with the Integrative Genomics Drug-resistant colonies were picked after an additional 48 to 72 h at 32 °C. Viewer (73). MAnorm was used to compare H3K27me2/3 ChIP-seq coverage Initial mutant strains were crossed to a Sad-1 mutant (66) strain (N3756) and on all genes (designated as “peaks”) between samples and data were visu- resultant progeny were germinated on Hygromycin B- and Nourseothricin- alized with Matplotlib (67). Genes were scored by their normalized containing medium to obtain homokaryotic mutants. H3K27me2/3 ChIP-seq coverage in wild type and the top-ranking genes (873) were designated as H3K27-methylated. Whole-Genome Sequencing, Mapping, and Identification of epr-1 Alleles. Homokaryotic mutants resistant to Hygromycin B and Nourseothricin were RNA Isolation, mRNA-seq Library Prep, and RT-qPCR. RNA was extracted from crossed to the genetically polymorphic Mauriceville strain (FGSC 2225) (27) germinated conidia grown for 16 to 18 h with a 1:1:1 glass beads, NETS and resultant progeny were germinated on medium containing Hygromycin (300 mM NaCl, 1 mM EDTA, 10 mM Tris·HCl, 0.2% SDS), acid phenol:chloroform B and/or Nourseothricin to select for strains bearing the causative mutation. mixture (5:1; [pH 4.5]) using a bead beater and ethanol precipitated. RNA was ∼ Genomic DNA from 15 to 20 progeny per mutant were pooled and se- treated with DNase I (Amplification grade; Thermo Fisher Scientific). DNase quencing libraries were prepared with a Nextera kit (Illumina, FC-121-1030). I-treated RNA was used for RNA-seq library preparation as previously de- All whole-genome sequencing data are available in National Center for scribed (23) or cDNA was synthesized using the SuperScript III First Strand- Biotechnology Information (NCBI) Sequence Read Archive (SRA) (accession Synthesis System (Thermo Fisher Scientific) with poly-dT primers. cDNA was no. PRJNA526508). To map the approximate location of a particular causa- used for qPCR using the PerfeCTa SYBR Green FastMix (QuantBio) on a Step tive mutation, the fraction of Oak Ridge (versus Mauriceville) SNPs across the One Plus Real Time PCR System (Life Technologies) using primer pairs in SI N. crassa genome was determined as previously described (28) and visualized Appendix, Table S4. mRNA-seq data are available in the NCBI GEO database = = as a moving average (window size 10 SNPs, step size 1 SNP) with Mat- (accession no. GSE128317). plotlib (67). We utilized FreeBayes and VCFtools to identify genetic variants present in our pooled mutant genomic DNA but absent in the original mRNA-seq Mapping and Analysis. Tools available on Galaxy (70) were used to mutagenized strain (N6279) and the Mauriceville strain (68, 69). Only genetic map mRNA-sequencing reads (intron size < 1 kb) (74) against the corrected variants of high probability that were consistent with the mapping data N. crassa OR74A (NC12) genome (49), to count the number of reads per gene were considered further. (74) and to identify differentially expressed genes (75). P values reported are adjusted for false-discovery rates (75). The χ2 test was used to determine if Western Blotting. N. crassa tissue from a 16-h liquid culture of germinated up-regulated genes in Δepr-1 strains were enriched for genes marked with conidia was collected by filtration, washed with 1× phosphate-buffered sa- H3K27 methylation. Significance of gene set intersections were calculated line (PBS) (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.5), and sus- with a hypergeometric distribution (http://nemates.org/MA/progs/overlap_ pended in 500 μL of ice-cold lysis buffer (50 mM Hepes [pH 7.5], 150 mM stats_prog.html). NaCl, 10% glycerol, 0.02–0.2% Nonidet P-40, 1 mM ethylenediaminetetra- acetic acid [EDTA]) supplemented with 1× Halt Protease Inhibitor Cocktail DamID Southern Hybridizations and Sequencing. Southern hybridizations were (Thermo Scientific). Tissue was sonicated (Branson Sonifier-450) for three carried out as previously described (76), except probes were made with PCR sets of 10 pulses (output = 2, duty cycle = 80), keeping the sample on ice products amplified from wild-type N. crassa genomic DNA (NCU05173,Tel between sets. Insoluble material was pelleted by centrifugation at VIIL) or plasmid pBM61 (his-3) using primer pairs from SI Appendix, Table S5. 14,000 rpm at 4 °C for 10 min and the supernatant used as the Western Preparation of N6-methyladenine-containing DNA for sequencing was per- sample. Anti-H3K27me3 (Cell Signaling Technology, 9733) and anti-hH3 (Abcam, ab1791) primary antibodies were used with IRDye 680RD goat formed using a previously reported procedure (42) using primers in SI Ap- μ anti-rabbit secondary (LI-COR, 926-68071). Anti-GFP (Thermo Fisher, A10262) pendix, Table S6 with the following modifications: 5 g of genomic DNA μ primary antibody was used with goat anti-chicken horseradish peroxidase from N. crassa strains expressing a Dam fusion was digested with 1 Lof μ (HRP)-conjugated secondary antibody (Abcam, 6877). Images were acquired DpnI (New England Biolabs, 20 units/ L); ligation to primer 5050 was carried with an Odyssey Fc Imaging System (LI-COR) and analyzed with Image Studio out overnight at 16 °C; amplification reactions of ligated DNA with primer μ software (LI-COR). 5051 were performed in triplicate with 5 L dNTPs, and an additional PCR cycle was added to the second and third phase of the PCR protocol (4 and 18 cycles, respectively); 3 μg of pooled, amplified DNA was sheared using a ChIP. Liquid cultures were grown for ∼18 h with shaking at 32 °C. Tissue samples for H3K27me2/3 ChIP were cross-linked with 0.5% formaldehyde for Bioruptor (Diagenode) twice on high for 10 min (30 s on/off) at 4 °C; bio- μ 10 min and GFP ChIP samples were cross-linked with 1% formaldehyde for tinylated DNA was purified using 250 L slurry of streptavidin-agarose beads 10 min in 1× PBS. Cross-linking was quenched with 125 mM glycine, tissue (Sigma). DNA was cleaved from the beads with DpnII and libraries were was washed with 1× PBS and collected. Cells were lysed in ChIP lysis buffer prepared for sequencing using the NEBNext DNA Library Prep Master Mix (50 mM Hepes [pH 7.5], 90 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Set for Illumina (New England Biolabs). Deoxycholate) supplemented with 1× Halt Protease Inhibitor Cocktail (Thermo Scientific) using a Branson Sonifier 450. Chromatin was sheared False Perithecia Assay and Image Analysis. To assay of the development of using a Bioruptor (Diagenode) and 2 μL of appropriate antibody (H3K27me2/ false perithecia, strains were grown on modified VMM (31) as described 3, Active Motif 39536, or GFP, MBL 598) was added and incubated with above without a fertilizing strain. Images of plates were acquired after 2 wk rotation at 4 °C overnight. Protein A/G agarose (40 μL; Santa Cruz Biotech- of growth at 25 °C and false perithecia were detected and quantified using nologies) was added to H3K27me2/3 ChIP samples and Protein A agarose (40 the Laplacian of Gaussian blob detection algorithm from scikit-image (77). μL; Sigma) was added to GFP ChIP samples and incubated for 3 h, rotating at 4 °C. Beads were washed twice with ChIP lysis buffer supplemented with Microscopy Image Acquisition and Analysis. Live conidia were suspended in 140 mM NaCl, once with ChIP lysis buffer with 0.5 M NaCl, once with LiCl water and placed on a poly-L-lysine (Sigma) coated coverslip (No. 1.5; VWR) wash buffer (10 mM Tris·HCl [pH 8.0], 250 mM LiCl, 0.5% Nonidet P-40, 0.5% and mounted on a glass slide. Single-plane images for distance measure- Deoxycholate, 1 mM EDTA), and once with TE (10 mM Tris·HCl, 1 mM EDTA), ments were captured with the ELYRA S.1 system (Zeiss) mounted on an AXIO all rotating at 4 °C for 10 min each. DNA was eluted from beads by in- Observer Z1 inverted microscope stand (Zeiss) equipped with a 63×,1.4NA cubation in TES (10 mM Tris·HCl, 1 mM EDTA, 1% SDS) at 65 °C. Cross-linking Plan-Apochromat oil-immersion lens (Zeiss) and analyzed using Imaris

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1918776117 Wiles et al. Downloaded by guest on September 30, 2021 (v9.2.1). Images for volume renderings and max projections were collected using PacI and XbaI restriction sites. For the BAH point mutant, epr-1W184A, with the DeltaVision Ultra microscope system (GE) equipped with a 100×,1.4 two PCR products amplified from wild-type genomic DNA with primers pairs NA UPlanSApo objective (Olympus). Three-dimensional z-stack wide-field 6416 and 6368, and 6367 and 6417 were PCR-stitched together with primers fluorescent (eGFP, TagRFP, iRFP, and TagBFP) images were captured with 6416 and 6417, and similarly cloned into plasmid 2406. For the PHD point an sCMOS camera controlled with Acquire Ultra software. Images were mutant, epr-1W292A, two PCR products amplified from wild-type genomic processed using 10 cycles of enhanced ratio deconvolution and max pro- DNA with primer pairs 6416 and 6400, and 6399 and 6417 were PCR-stitched jections were made using softWoRx (GE, v7.1.0). Imaris (v9.2.1) was used to together with primers 6416 and 6417, and cloned into plasmid 2406. Plas- make volume renderings and TFR1 and EPR1 foci were counted by hand. mids were linearized with NdeI and targeted to his-3 in either N7451 (for complementation) or N7567 (for microscopy), as previously described (82). Bioinformatic Identification of EPR-1 and SET-7 Homologs. PSI-BLAST (78) was Primary transformants were then crossed to N7549 or N7552, respectively. used to initiate sequence similarity searches with representative sequences of EPR-1 (accession no. XP_965052.2) and SET-7 (accession no. XP_965043.2, Replacement of epr-1 with trpC::nat-1. The 5′ and 3′ flanks of epr-1 were PCR- residues 577 to 833) from N. crassa against the NCBI nonredundant (NR) amplified from wild-type genomic DNA with primer pairs 6401 and 6402, database and locally maintained databases of proteins from representative and 6350 and 6351, respectively (SI Appendix, Table S9). The 5′ and 3′ flanks species from different branches of the tree of life. HHpred (79) was used to were separately PCR-stitched with plasmid 3237 (source of nat-1) using perform profile-profile comparisons against the Protein Data Bank (PDB), primer pairs 6401 and 4883, and 4882 and 6351, respectively. These two Pfam, and locally maintained protein sequence profiles. Sequences were “split-marker” PCR products were transformed into strain N7537 and epr-1 clustered using BLASTCLUST (ftp://ftp.ncbi.nih.gov/blast/documents/blastclust. replacements were selected on Nourseothricin-containing medium. Primary html). Multiple sequence alignments were generated using Kalign (80) and transformants were crossed to N3752 (to generate N7567) and N6234 (to then adjusted manually. FastTree (81) was used to assess phylogenetic re- generate N7576). lationships among the proteins retrieved after sequence similarity searches. Customized PERL scripts were used for the analysis of domain architec- Endogenous C-terminal–tagging of EPR-1 with 10xGly::Dam. The regions im- tures and other contextual information about protein sequences. Stringent mediately upstream (5′) and downstream (3′)ofepr-1’s stop codon were criteria of domain-architectural concordance were used to retrieve only PCR-amplified from wild-type genomic DNA with primer pairs 6348 orthologs of all proteins under study along with grouping in phylogenetic and 6349, and 6350 and 6351, respectively (SI Appendix, Table S10). The 5′ tree analysis. In the case of SET-7, only those protein sequences that have a and 3′ regions were separately PCR-stitched with plasmid 3131 (source of complete pre-SET domain followed by a SET domain were considered for 10xGly::Dam::trpC::nat-1) using primer pairs 6348 and 4883, and 4882 and analysis. 6351, respectively. These two split-marker PCR products were transformed into N2718 and knockins were selected on Nourseothricin-containing me- Replacement of NCU05173 and NCU07152 ORFs with hph and nat-1. To delete dium. Primary transformants were crossed to N3752 to obtain homokaryons. NCU07152,the5′ and 3′ regions flanking the ORF were amplified from wild- To make epr-1W292A fusions with Dam, the same approach was taken except type genomic DNA with primers 6385 to 6388 (SI Appendix, Table S2). The the 5′ region was a PCR product stitched from two PCR products amplified GENETICS nat-1 gene was amplified from plasmid 3237 with primers 6269 and 6270. from wild-type genomic DNA with primer pairs 6346 and 6400, and 6399 The resulting three pieces of DNA were stitched by overlap extension using and 6349. primers 6385 and 6388 and the final product was transformed into N4840. Primary transformants were selected on Nourseothricin-containing medium Data Availability. All ChIP-seq, DamID-seq, and mRNA-seq data have been Δ Δ and crossed to a wild-type strain (N3753) to remove set-7 and mus-52 from submitted to the NCBI GEO database (accession no. GSE128317). All whole- ′ the genetic background, resulting in strain N5808. To delete NCU05173,the5 genome sequencing data have been submitted to the NCBI SRA (accession ′ and 3 regions flanking the ORF were amplified from wild-type genomic no. PRJNA526508). DNA with primers 6605 to 6608. The hph gene was amplified from plasmid ′ ′ 2283. The 5 flank was stitched to the 5 portion of hph by overlap extension ACKNOWLEDGMENTS. We thank S. De Silva and C. Musselman (University of ′ ′ using primers 6605 and 2955, and the 3 flank was stitched to the 3 portion Colorado Anschutz Medical Campus), and C. Petell and B. Strahl (University of hph using primers 6608 and 2954. The resulting two pieces of “split of North Carolina School of Medicine) for their significant efforts to marker” DNA were transformed into strain N4840. Primary transformants characterize the binding specificity of EPR-1 in vitro; S. Honda (University were selected on Hygromycin B-containing medium and crossed to N5808 to of Fukui) for providing Neurospora crassa strains utilized in fluorescence generate a homokaryotic strain with both marker genes (N6233). N6233 was microscopy experiments; J. Lyle, A. Leiferman, and N. Meyers for help map- crossed to N623 to introduce his-3, resulting in the strain used for the mu- ping the UV-generated mutants; A. Harvey for help with preliminary micros- tant hunt (N6279). copy experiments; and A. Zemper for chicken anti-GFP and goat anti-chicken HRP antibodies. This work was funded by National Institutes of Health Grants GM127142 and GM093061 (to E.U.S.) and HD007348 (for partial sup- + Creation and Targeting of his-3 ::pCCG::N-GFP::EPR-1 Plasmids. The ORF and 3′ port of K.J.M.); and the American Heart Association Grant 14POST20450071 UTR of epr-1 were PCR-amplified from wild-type genomic DNA with primers (for partial support of E.T.W.). G.K. and L.A. were supported by the Intra- 6416 and 6417 (SI Appendix, Table S8) and cloned into plasmid 2406 (35) mural Research Program of the National Library of Medicine.

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