Regional Control of Histone H3 Lysine 27 Methylation in Neurospora

Regional control of histone H3 lysine 27 methylation in Neurospora

Kirsty Jamiesona,1, Michael R. Rountreea,1, Zachary A. Lewisa,2, Jason E. Stajichb, and Eric U. Selkera,3

aDepartment of Biology and Institute of Molecular Biology, University of Oregon, Eugene, OR 97403; and bDepartment of Plant Pathology and Microbiology, University of California, Riverside, CA 92521

Contributed by Eric U. Selker, February 27, 2013 (sent for review February 11, 2013)

Trimethylated lysine 27 on histone H3 (H3K27me3) is present in at the telomeres (24), we discovered that N. crassa also sports Drosophila, Arabidopsis, worms, and mammals, but is absent from H3K27me3, allowing us to exploit this organism to study the yeasts that have been examined. We identified and analyzed control and function of this histone modification. H3K27me3 in the filamentous fungus Neurospora crassa and in Considering the lack of information on H3K27me3 in fungi, other Neurospora species. H3K27me3 covers 6.8% of the N. crassa we analyzed the distribution and function of H3K27me3 in N. crassa Neurospora. genome, encompassing 223 domains, including 774 genes, all of and two other species of Sizable H3K27me3 N. crassa domains were found concentrated near the telomeres on all which are transcriptionally silent. H3K27me3-marked N. crassa genes are less conserved than unmarked genes and only ∼35% of seven linkage groups (LGs) of , and the distribution genes marked by H3K27me3 in N. crassa are also H3K27me3-marked has been partially conserved in the genus. H3K27me3 covers in Neurospora discreta and Neurospora tetrasperma. We found that a substantial number of specialized silent genes. The PRC2 complex, but not the PRC1 complex, is conserved in N. crassa. three components of the Neurospora Polycomb repressive complex — – We found that three members of the PRC2 complex are re- 2 (PRC2) [Su-(var)3 9; E(z); Trithorax] (SET)-7, embryonic ectoderm quired for H3K27me3 [SET-7, EED and SU(Z)12]; the fourth, development (EED), and SU(Z)12 (suppressor of zeste12)—are re- Neurospora Neurospora protein 55 (NPF; a homolog of p55), is required for quired for H3K27me3, whereas the fourth component, H3K27me3 on just a subset of targets. protein 55 (an N. crassa homolog of p55/RbAp48), is critical for H3K27me3 only at subtelomeric domains. Loss of H3K27me3, Results caused by deletion of the gene encoding the catalytic PRC2 sub- Distribution of H3K27me3 in Neurospora. We used ChIP-sequencing set-7 unit, , resulted in up-regulation of 130 genes, including genes (Seq) to generate a high-resolution map of H3K27me3 distri- in both H3K27me3-marked and unmarked regions. bution throughout the genome of N. crassa (Fig. 1A) and identified 223 H3K27me3 domains, ranging from 0.5 to 107 kb epigenetic | epigenomics | facultative heterochromatin | KMT (average 12.5 kb), together occupying 2.8 Mb of the 41-Mb genome (SI Appendix, Table S1; Dataset S1). This fraction of the olycomb group proteins form multimeric complexes to es- genome (6.8%) is similar to the H3K27me3 occupancy found in Ptablish, maintain, and recognize the trimethylation of histone Arabidopsis, Drosophila, and mammals (25–27). Interestingly, the H3K27 (H3K27me3) (1, 2). Polycomb repressive complex 2 H3K27me3 domains of N. crassa are found predominantly near (PRC2), which was first described in Drosophila and consists of telomeres (Fig. 1A). We identified 774 predicted genes that are four core proteins: enhancer of zeste [E(Z)], extra sex combs completely included within these domains and an additional 165 (ESC), suppressor of zeste12 [SU(Z)12], and p55, is directly re- predicted genes partially covered by H3K27me3 (“border genes”) sponsible for methylation of H3K27 (1, 2). The SET [Su(var)3–9; (Dataset S2). The multigene domain arrangement of H3K27me3 E(z); Trithorax] domain protein E(Z) is the catalytic subunit of in N. crassa is reminiscent of animal systems (19, 28) and contrasts the complex (3). SU(Z)12 and p55 each appear to facilitate nu- the situation observed in Arabidopsis, in which this mark is asso- cleosome binding, whereas ESC apparently boosts the enzymatic ciated with individual genes (20). activity of E(Z) and modestly contributes to nucleosome binding We verified the H3K27me3 distribution determined by ChIP-Seq (4). Embryonic ectoderm development (EED), the mammalian in three ways. First, we carried out ChIP-microarray experiments homolog of ESC, was found to bind to H3K27me3, raising the for LG VII. Equivalent results were obtained with ChIP-Seq and possibility that it plays a role in the propagation of this histone ChIP-microarray methods (SI Appendix,Fig.S1A). Second, we used mark (5). PRC2 has been conserved throughout evolution, with ChIP-Seq to assess the distribution of H3K27me3 in different core subunits present in metazoans, plants, and even protists (6, 7). Neurospora culture media [Vogel’s (29) and Bird media (30)]; vir- In both animals and plants, H3K27me3 is commonly associated tually identical distributions of H3K27me3 (SI Appendix,Fig.S1B; with transcriptionally silenced genes involved in development (8). Datasets S1 and S3) were observed. Third, we used ChIP followed Deletion of a PRC2 subunit increases the expression of some by real-time quantitative PCR to verify H3K27me3 enrichment at H3K27me3 genes (9–15), but the mechanism for gene repression LG I telomeres and at two genes on LG VII (qChIP; Fig. 1C). by H3K27me3 is largely unknown (16, 17). The distribution of Our study on telomere silencing in N. crassa provided early H3K27me3 varies among organisms; both Drosophila and mammals evidence of both H3K9me3 and H3K27me3 in several telomeric typically exhibit broad H3K27me3 domains of up to several hundred kilobases, including both transcribed and regulatory regions (18, 19). In contrast, H3K27me3 regions are rather short in Arabidopsis,with Author contributions: K.J., M.R.R., Z.A.L., J.E.S., and E.U.S. designed research; K.J., M.R.R., most less than 1 kb, and are largely restricted to the transcribed Z.A.L., J.E.S., and E.U.S. performed research; K.J., M.R.R., Z.A.L., J.E.S., and E.U.S. contrib- regions of single genes (20). This difference in H3K27me3 uted new reagents/analytic tools; K.J., M.R.R., Z.A.L., J.E.S., and E.U.S. analyzed data; and distribution suggests the possibility of distinct mechanisms for K.J., M.R.R., and E.U.S. wrote the paper. the control of this modification in metazoans and plants. The authors declare no conflict of interest. H3K27me3, like H3K9me3, appears to be absent from some Data deposition: ChIP-sequencing data has been deposited in NCBI (http://ncbi.nlm.nih. simple model organisms, such as Saccharomyces cerevisiae.Fis- gov/sra) (accession no. SRA0688854). sion yeast Schizosaccharomyces pombe sports H3K9 methyla- 1K.J. and M.R.R. contributed equally to this work. tion but lacks H3K27 methylation (21). In Neurospora crassa, 2Present address: Department of Microbiology, University of Georgia, Athens, GA 30602. 3 H3K9me3 directs DNA methylation and marks centromeric and To whom correspondence should be addressed. E-mail: [email protected]. GENETICS interstitial segments of heterochromatin, which largely comprise This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. inactivated transposons (22, 23). While studying gene silencing 1073/pnas.1303750110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1303750110 PNAS | April 9, 2013 | vol. 110 | no. 15 | 6027–6032 Downloaded by guest on September 24, 2021 Fig. 1. Genome-wide H3K27me3 ChIP-Seq analysis of wild-type, Δset-7, and Δnpf strains. (A) The distribution of H3K27me3 in the WT strain (blue) grown in Bird’s medium, displayed above the predicted genes (green ticks), is represented to scale on the seven LGs of N. crassa. Below the genes, the absence of H3K27me3 enrichment in a Δset-7 strain (gray) and the regionally affected H3K27me3 distribution in a Δnpf strain (inverted black traces) are displayed. LG I is divided at the right end of its centromere into IL and IR. Red arrows indicate the locations of primer sets used for qChIP experiments. (B) Part of the right arm of LG V near the telomere (dotted line) is expanded to detail mutually exclusive H3K27me3 and H3K9me3 domains. H3K9me3 data for the whole genome are presented in SI Appendix, Fig. S2.(C) Relative H3K27me3 enrichment was determined by qChIP at the telomeres of LG I (IL and IR) and at two genic regions sporting H3K27me3 in LG VII for WT and for strains deleted for genes encoding the PRC2 subunits (SI Appendix, Table S5). (D) H3K27me3 ChIP-Seq read densities for WT, Δset-7, and Δnpf for the regions assayed by qChIP (primers indicated by red arrows).

regions (24). The conventional ChIP experiments did not pro- Although most of the H3K27me3-marked genes are unanno- vide information on whether these two marks truly colocalize in tated, the annotated set does contain representatives of a full N. crassa, however. To address this possibility, we performed spectrum of categories (e.g., metabolism, cellular transport; SI ChIP-Seq for H3K9me3 and compared the distributions of these Appendix,Fig.S4). two marks (SI Appendix, Fig. S2). Interestingly, we found that The high level of novelty among genes marked by H3K27me3 H3K27me3 often neighbors H3K9me3, but each mark forms prompted us to investigate their relative conservation. We found distinct domains with little or no overlap (Fig. 1B; SI Appendix, that H3K27me3-marked genes are substantially less conserved Fig. S2). As found in more limited surveys (22, 23), H3K9me3, than genes not marked by this modification. Seventy-nine per- which mirrors the distribution of DNA methylation in N. crassa cent of N. crassa H3K27me3-marked genes have orthologs found (23), was almost exclusively associated with gene-depleted, A:T- only in fungi, compared with 49% for non–H3K27me3-marked rich sequences altered by repeat-induced point mutation (RIP). genes (Fig. 2A). Moreover, unlike N. crassa genes generally, In contrast, H3K27me3 domains include numerous predicted a high proportion of H3K27me3-marked genes are limited to the genes and the base composition of these regions is not skewed in Neurospora genus or to closely related genera in the Sordar- any obvious way. iomycetes class; 30% are Neurospora-specific (compared with 9% for non– H3K27me3-marked genes) and an additional 26% are Neurospora H3K27me3-Marked Genes Are Distinctive. As a first step limited to the Sordariomycetes (compared with 8% for non– to explore the possible function of the H3K27me3 mark in H3K27me3-marked genes) (Fig. 2A). Neurospora, we surveyed the underlying genes for their evolutionary conservation and predicted functions. It became obvious Conservation of H3K27me3 in Neurospora Species. Our observation that the H3K27me3-marked genes are not representative of the that N. crassa H3K27me3-marked genes show a strong fungal- overall genome. The average predicted size of proteins encoded specific bias raised two potentially related questions: (i) How by H3K27me3 genes is smaller than that of genes not marked by conserved are H3K27me3-marked genes within the Neurospora H3K27me3 (373 vs. 513 amino acids; SI Appendix, Fig. S3). genus? and (ii) To what extent is the mark itself conserved? Furthermore, an unusually high fraction of the 774 H3K27me3- To address these questions, we determined the distribution of marked genes have no predicted function (71.4%; compared H3K27me3 in two other Neurospora species, N. tetrasperma and with 38.2% of genes in the genome overall; SI Appendix, Fig. S4). N. discreta. Our ChIP-Seq analyses demonstrated that H3K27me3

6028 | www.pnas.org/cgi/doi/10.1073/pnas.1303750110 Jamieson et al. Downloaded by guest on September 24, 2021 for unmethylated N. crassa genes; Fig. 2B). Thus, N. crassa genes that are not found in one or both of the sister species are marked by H3K27me3 more frequently than those found in all three species (Fig. 2B; SI Appendix, Table S3). In sum, we found partial conservation of the H3K27me3 mark among three closely related species of Neurospora.

PRC2 Complex Is Conserved in N. crassa. Pioneering work in Dro- sophila demonstrated that the PRC2 complex is responsible for methylation of H3K27. This complex consists of four core subunits in Drosophila—E(Z), ESC, SU(Z)12, and p55 —which are highly conserved in plants and animals, although some subunits are duplicated in higher eukaryotes (6). The genome of N. crassa contains one homolog for each of the PRC2 subunits, with their predicted functional domains largely intact (SI Appendix,Figs.S5 and S6). The N. crassa homolog of the gene for the catalytic subunit, E(Z), is set-7 (31); the homolog of the p55 gene, which we named npf, was previously named chromatin assembly complex 3 (cac-3) because it was potentially a component of the putative Neurospora chromatin assembly factor 1 (CAF-1) complex (31). To determine whether the four putative PRC2 subunits form a complex in N. crassa, we fused a 3XFLAG epitope tag to the amino terminus of the EED homolog expressed under the control of the qa-2 promoter. We purified tagged EED using an anti- FLAG affinity gel and identified EED and associated proteins by mass spectrometry. In addition to EED, we found strong cov- erage for the other three putative PRC2 subunits, SET-7, SU(Z) 12, and NPF, implying that, indeed, a PRC2-like complex forms in N. crassa (SI Appendix, Table S4). To explore the function of the N. crassa PRC2 complex, we obtained knockout strains for the corresponding genes (32). In contrast to their essential role in developmental processes of higher eukaryotes (6), we found that none of the four PRC2 homologs is essential in N. crassa. Indeed, strains with knockouts of set-7, eed, or su(z)12 displayednogrowthdefectsunder standard growth conditions (SI Appendix,Fig.S7A). However, deletion of npf resulted in a slow-growth phenotype; its linear extension rate is ∼84%ofthatofWT(SI Appendix,Fig.S7A and B). In other systems, besides its role in PRC2, NPF (called RbAp46/RbAp48 in mouse) has been shown to be a histone- Fig. 2. Conservation of H3K27me3 genes in Neurospora species. (A) The binding protein and a component of ATP-dependent nuclear phylogenetic tree depicts the classification of Neurospora species and their remodeling complexes (33). Considering that growth was not relationship to common model organisms. The pie charts illustrate conser- retarded in knockouts for the other three PRC2 subunits, it vation of N. crassa orthologs in H3K27me3 domains (H3K27me3 genes) or seems likely the slow-growth of the npf strain is due to a role of outside H3K27me3 domains (non-H3K27me3 genes). (B) Conservation and this protein in complexes other than PRC2. We also found that H3K27me3 status of N. crassa genes with or without H3K27me3 relative to crosses homozygous for a deletion of set-7 were fruitful, in- N. tetrasperma and N. discreta. Full species names of the organisms indicated in A are: Aspergillus nidulans, Arabidopsis thaliana, Coprinopsis cinerea, dicating that H3K27me3 is not essential for the sexual cycle. Homo sapiens, Podospora anserina, and Sordaria macrospora. NPF Is Differentially Required for H3K27me3. We initially used both immunoblotting and ChIP to test mutants lacking components N. crassa covers a similar fraction of each of the three genomes and that of the PRC2 complex for H3K27me3, but found that all three species have a similar number of H3K27me3 domains available antibodies were most reliable for ChIP experiments. (SI Appendix,TableS2; Datasets S4 and S5). The N. tetrasperma We used qChIP to access the level of H3K27me3 enrichment and N. discreta genomes are not yet fully assembled, so it is not near both telomeres on LG I and at two genic regions on LG VII in each of the PRC2 knockout strains (Fig. 1C). H3K27me3 certain that the H3K27me3 domains are preferentially near the enrichment was completely lost from the LG I telomeres in all ends of chromosomes as in N. crassa. four PRC2 mutants. Similarly, H3K27me3 was eliminated at Although all three species show comparable fractions of their the two genic regions in the Δset-7, Δeed,andΔsu(z)12 strains. genomes associated with this mark, we found striking evidence Surprisingly, there was only a partial reduction of H3K27me3 at of dynamics. Among N. crassa H3K27me3-marked genes, only Δnpf ∼ N. tetrasperma N. discreta ∼ these genic regions in the strain. To explore this further, 35% are marked in both and , 12% we analyzed the distribution of H3K27me3 across the whole are unmethylated in both comparative species, and nearly 25% genome by ChIP-Seq in Δset-7 and Δnpf strains. Consistent are methylated in one comparative species but not the other with the qChIP results, we did not observe enrichment for (Fig. 2B; SI Appendix,TableS3). Conversely, homologs of 2.5% H3K27me3 in the Δset-7 strain, implying that the histone of unmethylated N. crassa genes are marked with H3K27me3 in methyltransferase catalytic subunit of the PRC2 complex is N. tetrasperma and/or N. discreta (Fig. 2B; SI Appendix,Table absolutely required (Fig. 1). Consistent with the initial qChIP S3). Moreover, compared with non–H3K27me3-marked genes, results, we found a differential loss of H3K27me3 in the Δnpf N. crassa a high fraction of genes associated with the H3K27me3 strain (Fig. 1). Both the size and number of H3K27me3 GENETICS mark are absent in one or both of the comparative species domains was reduced in the Δnpf strain compared with WT (SI (∼14% and ∼9%, respectively, compared with ∼6% and ∼3% Appendix,TableS1; Datasets S6 and S7). Although H3K27me3

Jamieson et al. PNAS | April 9, 2013 | vol. 110 | no. 15 | 6029 Downloaded by guest on September 24, 2021 enrichment was reduced across most areas of the genome, increase expression of the majority of H3K27me3-marked genes the mark was specifically absent near the telomeres of all of under the conditions of our experiment. the chromosomes. We conclude that SET-7, EED, and SU(Z) 12, but not NFP, are absolutely required for H3K27me3 in Discussion N. crassa. Elements of the Polycomb repression system, originally un- covered in Drosophila, have been found in a variety of higher H3K27me3 Domains Are Transcriptionally Quiescent. To determine animals and plants, but not in yeast species that have been ex- whether H3K27me3 represents a repressive mark in N. crassa,we amined (S. cerevisiae and S. pombe). Previously we found that analyzed gene expression in a WT strain by RNA-Seq (Fig. 3A). H3K27me3, a hallmark of the Polycomb system, is represented As in other model systems, we found few or no transcripts in the model filamentous fungus N. crassa (24, 34). Here, we produced from the H3K27me3-marked genes. To illustrate present a genome-wide analysis of the distribution of this chro- the negative correlation between H3K27me3-marked genes and matin mark, characterize the underlying machinery, and start to expression, we plotted transcript abundance versus H3K27me3 explore its function and evolutionary dynamics. A sizable frac- level across the genome (Fig. 3C). The H3K27me3-marked tion of the N. crassa genome (6.8%) is marked by H3K27me3, genes (blue) and the genes falling on the domain borders (black) covering 774 genes in 223 domains. These domains, some of showed extremely low transcript levels; the vast majority of which are hundreds of kilobases long, are found preferentially transcripts were produced by non-H3K27me3 genes (green). near the ends of the chromosomes (Fig. 1). Unlike the case in We next asked whether the absence of H3K27me3 in a Δset-7 Arabidopsis, in which H3K27me3 covers single genes in domains strain is sufficient to increase expression of H3K27me3-marked of less than 1 kb (20), the broad domains in N. crassa (12.5 kb genes. Using a stringent threshold for the change of expression average) are reminiscent of Drosophila and mammals, which (∼7.5-fold), we found 130 genes with increased expression in average 70 and 43 kb, respectively (18, 35). the Δset-7 strain relative to the WT strain (Dataset S8). De- H3K27me3 and H3K9me3 are both regarded as repressive repression of four H3K27me3-marked genes was confirmed by marks (1, 8, 15, 16, 36) but are distributed differently. In Northern blot analyses of total RNA (Fig. 3 A and B; SI Ap- N. crassa, as in higher eukaryotes, H3K9me3 is a feature of con- pendix, Fig. S8). In addition, five genes showed lower transcript stitutive heterochromatin and is found principally associated levels in a Δset-7 strain (Dataset S9). Overall, the functional with centromeric heterochromatin, which is characterized by classification of the up-regulated genes is similar to that of the H3K9me3, DNA methylation, a paucity of genes, and an abun- total H3K27me3-marked genes, consisting of primarily un- dance of repeats that show evidence of RIP (23, 37). H3K9me3 is annotated genes (SI Appendix, Figs. S4 and S9). Interestingly, of also found in N. crassa associated with numerous small islands of the 130 de-repressed genes, only 21 fell completely within the sequences mutated by RIP and near telomeres (24), adjacent to H3K27me3 domains identified in the WT strain. This result is where we found H3K27me3. Unlike H3K9me3, we show that similar to what has been observed in Arabidopsis (9). Thus, al- H3K27me3 is in gene-rich regions.Notably,theH3K27me3 though loss of H3K27me3 may be necessary, it is not sufficient to and H3K9me3 regions do not appear to overlap (Fig. 1 and SI

Fig. 3. Deletion of set-7 de-represses a subset of Neurospora genes. (A) RNA-Seq read densities for WT (black) and Δset-7 (green) are displayed below the genes (green ticks) for LG V; H3K27me3 enrichment (blue) is included for reference. (B) Two genes (NCU08907 and NCU11087) within an H3K27me3 domain are expanded along with the corresponding RNA-Seq reads. Northern conﬁrmation of increased NCU08907 expression in the Δset-7 mutant; 18S rRNA stained with methylene blue is shown as a loading control. (C) Transcript abundance, expressed as fragments per kilobase of exon per million fragments mapped (FPKM), plotted vs. H3K27me3 level (reads) for genes contained within H3K27me3 domains (blue circles), genes partially contained in H3K27me3 domains (H3K27me3 “border” genes; black diamonds), and for genes outside of H3K27me3 domains (non-H3K27me3 genes; green crosses). The Δset-7 up-regulated genes that were veriﬁed by Northern blots are indicated by red dots.

6030 | www.pnas.org/cgi/doi/10.1073/pnas.1303750110 Jamieson et al. Downloaded by guest on September 24, 2021 Appendix, Fig. S2). This is consistent with reports from plant and this organism (20). Lack of gene annotation often reflects a lack animal systems that describe mostly mutually exclusive H3K27me3 of characterized orthologs in other species. Indeed, we found that and H3K9me3 distributions (18, 26–28, 38–40). It will be inter- N. crassa H3K27me3-marked genes show a striking lack of con- esting to learn whether the machinery responsible for methylating servation; most are confined to the fungal kingdom, with the H3K9 and H3K27 are inherently incompatible. largest fractions confined to the class Sordariomycete and genus As a step to investigate the mechanism of H3K27me3 in N. crassa, Neurospora (Fig. 2A). Thus, H3K27me3 seems to preferentially we identified and tested homologs of PRC2 components identified mark poorly conserved or “new” genes. Conceivably, as new genes in other organisms (SI Appendix,Fig.S5). We found that H3K27me3 are incorporated into a genome, H3K27me3 could serve as a absolutely depends on three of the PRC2 components: SET-7 “safety” mechanism by silencing them. (equivalent to EZH2), EED, and SU(Z)12. Thus, unlike the situa- To investigate the evolutionary dynamics of H3K27me3 and of tion in Drosophila and other animals, H3K27me3 is not essential in the associated genes, we analyzed the distribution of H3K27me3 N. crassa. Interestingly, we found that the fourth component of the in two closely related Neurospora species. We found that presumptive N. crassa PRC2 complex, NPF (Neurospora homolog H3K27me3 is present in both N. tetrasperma and N. discreta and of Drosophila P55 and mammalian P48), is not required for all that the number and size of the H3K27me3 domains in both H3K27me3. In particular, NPF is only required for H3K27me3 species is similar to N. crassa (SI Appendix, Table S2). Although domains near telomeres; domains farther from telomeres are these closely related species predominantly share a common set somewhat affected, shrinking in the absence of NPF. We conclude of genes, we found that those that were not shared (i.e., those that NPF is not required for the methyltransferase activity of PRC2, that are unique to N. crassa genes, or those only found in only unlike SET-7, EED, and SUZ12. Perhaps NPF and its homologs in one of the other Neurospora species examined) are approxi- other organisms, which have been reported to bind histone H4 (41, mately threefold more frequently marked with H3K27me3 than 42), help tether the PRC2 complex to nucleosomes via its six WD40 are genes in the overall genome (SI Appendix, Fig. S10). In- domains. This is consistent with the observation that a trimeric Esc-E terestingly, among orthologs common to the three species, 35% (z)-Su(z)12 complex trimethylates H3K27 in vitro, but is unable to of the N. crassa H3K27me3-marked genes are also marked by bind nucleosomes (4). It is interesting that various genomic regions H3K27me3 in both N. tetrasperma and N. discreta. A sizable are differentially dependent on NPF. Perhaps regions that do not number of genes that are H3K27me3-marked in N. crassa are not lose H3K27me3 in the Δnpf strain rely on another WD40 domain– marked in at least one of the other species (24.6% “MU” in Fig. containing protein. We are unaware of direct evidence from other 2B) or do not currently have the gene in one (14.1% “M−” in organisms of a comparable, genome-wide influence of NPF homo- Fig. 2B) or both (9.0% “–” in Fig. 2B) other species. Thus, al- logs on H3K27me3 or other histone modifications, but there are though the fraction of the genome that is marked by H3K27me3 clues that the effect is not limited to N. crassa;thecombinedfindings in the three genomes is equivalent, the distribution of the mark of two studies with Arabidopsis revealed that H3K27me3 marks and the associated genes are not highly conserved (Fig. 2B). approximately half of the genes that become de-repressed in a mu- The presence of H3K27me3 and PRC2 components in N. crassa tant for the NPF homolog (20, 43). Although Arabidopsis does not and some other lower eukaryotes (e.g., Chlamydomonas reinhardtii contain the broad H3K27me3 domains observed in N. crassa, Dro- (7)) and absence of this mark and the associated machinery from sophila, and mammals, selective reduction of H3K27me3 re- some other lower eukaryotes [e.g., S. cerevisiae and S. pombe (21, sulting from loss of msi1 could be responsible for the de-repression 24)] suggests that this system has not been retained throughout of a subset of H3K27me3-marked genes and their presumptive evolution (7), and is consistent with its nonessential role in N. indirect targets (20, 43). crassa. Previous studies suggested PRC2 arose before PRC1 and As in other organisms, we found that N. crassa genes marked showed that it is the more conserved of the two Polycomb com- by H3K27me3 produce little or no transcripts. The silence of plexes (2). N. crassa appears to lack PRC1 homologs, raising the these genes is not a simple consequence of this mark, however, question of how the H3K27me3 mark is “read” in this organism. because elimination of H3K27me3 by mutation of set-7 did not derepress the bulk of the genes. The Δset-7 strain showed up- Materials and Methods regulation of 130 genes, but only 21 of these fell within Neurospora Strains and Methods. Neurospora strains used in this study (SI H3K27me3 domains, representing 2.7% of the 774 genes marked Appendix, Table S1) were grown and crossed following standard procedures by H3K27me3 in the WT strain. The 109 genes that showed (48). The Δset-7, Δeed, Δsuz12, and Δnpf strains were generated by the increased expression but are located outside of H3K27me3 Neurospora gene knockout project (32) and obtained from the Fungal Ge- domains represent 1.1% of the total genes not marked by netic Stock Center (FGSC; www.fgsc.net). RNA isolation and Northern blot- H3K27me3. Thus, the increased gene expression observed in the ting was done as described (49), except the mycelium, grown 16 h at 32 °C, set-7 mutant is modestly skewed toward H3K27me3-marked was disrupted using a minibead beater (Biospec). genes. That only a small subset of genes within H3K27me3 domains was up-regulated suggests that, in addition to loss of the ChIP. ChIP was performed as previously described (37) using anti-H3K27me3 repressive mark, activating signals may be required to express (Active Motif 39535; ChIP-Seq and qChIP), anti-H3K27me3 (Upstate 07-449; genes in H3K27me3 domains. There are also indications in hu- ChIP-chip), anti-H3K9me3 (Active Motif 39161; ChIP-Seq), and anti-H3K4me2 man, Drosophila, and Arabidopsis that depletion of Polycomb (Active Motif 39141; qChIP). ChIP-chip procedures, including microarray de- group genes is only sufficient to activate a subset of H3K27me3- sign, sample labeling, microarray hybridization, and data analysis, were marked genes, leading researchers to postulate a secondary layer conducted as described (23). For qChIP, real-time PCR experiments were of regulation (9, 11, 14, 44–47). In Arabidopsis, the up-regulation performed three times using FAST SYBR Green master mix (Kapa) with the of genes not marked by H3K27me3 in an H3K27me3-deficient listed primers (Table S2) and analyzed using a Step One Plus Real Time PCR background is thought to result from induction of transcriptional System (Life Technologies). Relative enrichment of H3K27me3 at representative telomeres and genic regions was determined versus input and then regulators (9). Because the majority of H3K27me3-marked genes standardized to relative enrichment of H3K4me2 at hH4. in N. crassa are unannotated, it is not yet clear if this is the case in Neurospora; it will be interesting to learn whether the up-regulated – Sequencing. Neurospora strains used for ChIP-Seq and RNA-Seq were grown non H3K27me3-marked genes are controlled by any of the up- in liquid media as described in the figures. A detailed description of cDNA regulated H3K27me3-marked genes. preparation, preparation of ChIP-enriched DNA and double-stranded cDNA Although H3K27me3 preferentially marks developmental genes for sequencing, and description of sequence analysis is available in SI Appendix, in animals, it is not yet clear if this is the case in N. crassa, SI Materials and Methods. Sequencing reads can be downloaded from NCBI because the vast majority of the marked genes have not been (accession no. SRA0688854). ChIP-Seq reads and ChIP-chip data were mapped

characterized. Similarly, although some developmental genes are to the N. crassa OR74A reference genome (50) (www.broadinstitute.org/ GENETICS marked by H3K27me3 in Arabidopsis, a high proportion of annotation/genome/neurospora/MultiDownloads.html), N. crassa OR74A H3K27me3-marked genes are also functionally unknown genes in v10genomeassembly,N. tetrasperma FGSC 2508 mat A v2.0 reference

Jamieson et al. PNAS | April 9, 2013 | vol. 110 | no. 15 | 6031 Downloaded by guest on September 24, 2021 genome (51), or the N. discreta FGSC 8579 mat A (US Department of Energy searchCatfirstFun.html). Domain prediction used RSEG software (http:// Joint Genome Institute). smithlab.usc.edu/histone/rseg/) with a bin-size of 500 bp. The RSEG difference program was run to determine domain differences between Bioinformatic Analysis. Orthologs were identified by aligning the three H3K27me3 domains predicted from independent ChIP-Seq experiments. Neurospora genomes with Mercator (52) and identifying orthologs as genes found in the same position between the genomes. Phylogenetic clades were ACKNOWLEDGMENTS. We thank Larry L. David (Oregon Health and Science identified by clustering genes into orthologous groups using OrthoMCL (53), University) for carrying out mass spectrometry on EED-associated proteins which first links genes by similarity with the BLASTP program followed and Doug Turnbull (University of Oregon) for help with high-throughput by the MCL graph algorithm, which identifies groups through a Markov sequencing. We gratefully acknowledge the Neurospora Genome Project Clustering procedure (54). Comparison of orthology relationships and and the Fungal Genetic Stock Center for materials. This work was supported H3K27me3-marked and -unmarked genes was completed with custom Perl by US Public Health Service Grants GM03569, GM093061, and GM068087 (to scripts (https://github.com/hyphaltip/H3K27) written with BioPerl (55). E.U.S.); the University of California Riverside College of Natural and Agricul- Functional classification of genes/proteins was conducted using MIPS FunCat tural Sciences; and grants from the Burroughs Wellcome Fund and the (http://mips.helmholtz-muenchen.de/genre/proj/ncrassa/Search/Catalogs/ Alfred P. Sloan Foundation (to J.E.S.).

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