Genome-wide redistribution of is linked to PNAS PLUS genotoxic stress and defective growth

Evelina Y. Basenkoa,1, Takahiko Sasakia,1, Lexiang Jib, Cameron J. Prybola, Rachel M. Burckhardta, Robert J. Schmitzc, and Zachary A. Lewisa,2

aDepartment of Microbiology, University of Georgia, Athens, GA 30602; bInstitute of Bioinformatics, University of Georgia, Athens, GA 30602; and cDepartment of Genetics, University of Georgia, Athens, GA 30602

Edited by Jay C. Dunlap, Geisel School of Medicine at Dartmouth, Hanover, NH, and approved October 2, 2015 (received for review June 10, 2015) H3K9 directs formation by recruiting silent . Pc-target domains are often referred to as fac- multiple heterochromatin protein 1 (HP1)-containing complexes ultative heterochromatin because these regions are condensed that deacetylate and methylate cytosine bases in DNA. and transcriptionally repressed in some but not all cell types In Neurospora crassa, a single H3K9 complex, (14). Polycomb repressive complex-2(PRC2)methylatesH3K27, called the DIM-5,-7,-9, CUL4, DDB1 Complex (DCDC), is required which in animals can be bound by PRC1 to promote mitotically for normal growth and development. DCDC-deficient mutants are heritable gene silencing (15, 16). However, the mechanisms that hypersensitive to the genotoxic agent methyl methanesulfonate control Pc complexes are not fully understood. In some situations, (MMS), but the molecular basis of genotoxic stress is unclear. We H3K27me3-independent recruitment of PRC1 can occur, leading found that both the MMS sensitivity and growth phenotypes of to subsequent PRC2 recruitment and deposition of H3K27me3 embryonic DCDC-deficient strains are suppressed by mutation of (17, 18). Pc components are absent from the model yeasts Sac- ectoderm development Su-(var)3-9; E(z); Trithorax (set)-7 or , charomyces cerevisiae and Schizosaccharyomyces pombe, whereas encoding components of the H3K27 methyltransferase Polycomb PRC1 appears to be absent from all fungi (19). PRC2 is present repressive complex-2 (PRC2). Trimethylated H3K27 (H3K27me3) in some fungi, however, and H3K27me3 directs transcriptional undergoes genome-wide redistribution to constitutive heterochro- repression of PRC2-target domains in Neurospora, Fusarium matin in DCDC- or HP1-deficient mutants, and introduction of an graminearum, Epichloë festucae, and Cryptococcus neoformans H3K27 missense mutation is sufficient to rescue phenotypes of (19–23). Thus, the application of microbial genetic approaches DCDC-deficient strains. Accumulation of H3K27me3 in heterochro- to study the Pc system in fungi can provide mechanistic insights matin does not compensate for silencing; rather, strains deficient for into this evolutionarily conserved chromatin regulatory system. both DCDC and PRC2 exhibit synthetic sensitivity to the topoisomer- Although the Pc system is best known for its ability to repress ase I inhibitor Camptothecin and accumulate γH2A at heterochro- matin. Together, these data suggest that PRC2 modulates the response developmentally regulated genes, recent studies in higher eukary- to genotoxic stress. otes link H3K27 methylation to DNA replication and repair. In human cells, PRC2 associates more stably with chromatin Polycomb | heterochromatin | | H3K27me3 | genotoxic stress following oxidative or UV-induced DNA damage, and levels of both PRC2 and H3K27me3 are increased at induced double-strand breaks(DSBs)(24–26). Moreover, knockdown of PRC2 increases ovalent modifications of histones and DNA partition ge- Cnomes into discrete functional domains. Heterochromatin refers to highly condensed parts of the genome that are tran- Significance scriptionally inert and rich in repeated DNA sequences (1). Failure to establish or maintain heterochromatin leads to cata- Regulators of chromatin structure play critical roles in DNA- strophic defects in chromosome segregation, DNA replication, based processes. (K) Methyltransferase 1 (KMT1) ho- and DNA repair, highlighting its functional importance (1–3). At mologs perform methylation of H3 lysine-9 and are best known the molecular level, heterochromatin domains are defined by for their essential role in heterochromatin formation and tran- hypoacetylated histones, histone H3K9 methylation (H3K9me), scriptional silencing. Heterochromatin formation is also important and DNA methylation (1). The fungus Neurospora crassa shares for maintenance of genome stability, although the mechanisms these features with higher eukaryotes and is an established model are not well understood. We report that altered activity of Poly- to study the control and function of heterochromatin (4). In comb repressive complex-2 (PRC2), a histone lysine-27 methyl- Neurospora, the H3K9 methyltransferase complex, named the transferase complex, is responsible for genotoxic stress, poor growth, and defective development in KMT1-deficient mutants of defective in methylation (DIM)-5,-7,-9, Cullin 4, DNA damage- Neurospora crassa binding protein 1 (DDB1) Complex (DCDC), initiates hetero- . Mammalian KMT1 and PRC2 are required for chromatin formation at degenerate DNA repeat sequences that development and are frequently mutated in cancer. This work are products of the genome defense system repeat-induced point provides information about the cellular consequences of KMT1 and PRC2 deficiency and provides insights into the regulatory and mutation (RIP) (5–7). DCDC trimethylates H3K9 to create binding functional relationships of these conserved enzymes. sites for multiple heterochromatin protein 1 (HP1)-containing complexes, which in turn direct methylation of cytosine bases in

Author contributions: E.Y.B., T.S., R.J.S., and Z.A.L. designed research; E.Y.B., T.S., L.J., C.J.P., GENETICS DNA and deacetylation of histones (8–10). As in other eukary- and R.M.B. performed research; T.S. and C.J.P. contributed new reagents/analytic tools; E.Y.B., otes, heterochromatin formation is sufficient to silence T.S., L.J., C.J.P., R.M.B., R.J.S., and Z.A.L. analyzed data; and Z.A.L. wrote the paper. in Neurospora. Reporter genes flanked by RIP’d DNA are not The authors declare no conflict of interest. expressed, and spreading of heterochromatin causes aberrant gene This article is a PNAS Direct Submission. silencing in strains that lack DNA methylation modulator-1 Data deposition: The sequence reported in this paper has been deposited in NCBI Short (DMM-1) (11, 12). Together, heterochromatin domains comprise Reads Archive (accession no. SRP058573). ∼20% of the Neurospora genome and include structurally im- 1E.Y.B. and T.S. contributed equally to this work. portant loci such as the (7, 13). 2To whom correspondence should be addressed. Email: [email protected]. In animals, plants, and some fungi, Polycomb (Pc) group pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. teins establish and maintain a second type of transcriptionally 1073/pnas.1511377112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1511377112 PNAS | Published online November 2, 2015 | E6339–E6348 Downloaded by guest on September 30, 2021 sensitivity to ionizing radiation (25). It is possible that Pc proteins strain. We identified a mutation in the gene encoding the PRC2 are recruited to silence transcription in the vicinity of a DNA lesion, component embryonic ectoderm development (EED) in mouse but the precise roles of PRC2 and H3K27me3 in the DNA damage (19, 47). H3K9me3-deficient mutants exhibit redistribution of response are unclear. H3K27me3, leading to induction of PRC2-target genes. We show Similarly, defects in constitutive heterochromatin formation that gain of H3K27me3 in constitutive heterochromatin domains are associated with genome instability. Replication fork stalling is not compensatory for gene silencing but rather leads to growth is observed in S. pombe heterochromatin domains (27), and defects and altered sensitivity to genotoxic agents. Our data Clr4KMT1-deficient mutants, which lack , exhibit ille- suggest that PRC2 modulates the response to genotoxic stress. gitimate mitotic recombination that is exacerbated by mutation of the replication fork protection complex (3, 28). In Drosophila, Results cytological studies revealed that H3K9me-deficient mutants ex- Δdim-5 mutants exhibit hypersensitivity to the DNA-damaging hibit spontaneous DSBs in heterochromatin, and it was proposed agent MMS, suggesting that the DIM-5KMT1 MTase is required for that this damage is due to defective DNA replication (2, 29, 30). normal DNA replication or repair (5). We examined growth of Mice lacking Lysine (K) Methyltransferase 1A (KMT1A) (SUV39H1) heterochromatin-defective mutants in the presence of additional and KMT1B (SUV39H2) exhibit genome instability and high DNA replication and repair inhibitors (Fig. 1A). As controls, rates of lymphoma development (31, 32), and both KMT1 en- we included mutagen-sensitive-9 (mus-9), lacking the Neurospora zymes and HP1 proteins have been implicated in DNA repair in ATM homolog required for the DNA damage response (48), and animals (33–42). These and other studies suggest that hetero- mei-3, lacking the Neurospora homolog of RAD51 required for chromatin components have important roles during both DNA (49). Elimination of DNA methylation replication and DNA repair, but the heterochromatin-dependent did not impact growth on any of the tested genotoxic agents. mechanisms that maintain genome integrity remain poorly defined. Strains lacking the DCDC components DIM-5, DIM-7, or DIM-9 Heterochromatin formation is important for genome mainte- were hypersensitive to MMS compared with wild type, as pre- nance in Neurospora as well. H3K9 methylation is required for viously described (5). These strains were sensitive to other geno- normal vegetative growth and for sexual development (6). DCDC- toxic agents including the topoisomerase II inhibitor etoposide deficient mutants exhibit chromosome segregation defects and hy- (50), the interstrand cross-linking agents cisplatin and Mitomycin C persensitivity to the DNA-damaging agent methylmethanesulfonate (51, 52), and Bleomycin (53), which is thought to cause oxidative (MMS) (5), and mutants lacking the catalytic subunit of DCDC, damage and trigger DSBs. DCDC-deficient cells were efficiently defective in methylation-5/kmt1 (dim-5), exhibit increased rates of killed by low concentrations of MMS (0.025%), whereas other mitotic recombination (43). In addition, during normal replicative agents only led to growth inhibition of Δdim-5, Δdim-7, Δdim-9, growth, dim-5 strains display induction and redistribution of γH2A, and hpo strains; that is, these strains were able to grow after pro- a phosphorylated form of H2A that is induced by DNA damage or longed incubation in the presence of drugs (>4 d), whereas growth DNA replication stress (44–46). These data suggest that hetero- of mus-9 and mei-3 did not improve. In contrast, Δdim-5, Δdim-7, chromatin is critically important in Neurospora, but the cause of Δdim-9,andhpo displayed only limited sensitivity to the topo- genotoxic stress and impaired growth in heterochromatin-defective isomerase I inhibitor Camptothecin (CPT) (50). Mutants lacking mutants is not understood. the DCDC component CUL4 were more sensitive to all genotoxic To understand the molecular consequences of heterochromatin agents tested, which is expected given the established role of CUL4 depletion, we isolated a genetic suppressor of a DCDC-deficient in multiple complexes that regulate DNA replication and repair

A VMM MMS Etoposide Cisplatin Mitomycin C Bleomycin CPT WT

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cul4 mei-3

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VMM +MMS wt BCdim-9 WT 100 dim-9; sup-24 dim-9 80 dim-9; sup-24 60 40 * % Survival 20 * 0 0 0.005 0.010 0.015 0.020 0.025 % MMS

Fig. 1. The sup-24 mutation suppresses MMS sensitivity of Δdim-9.(A) For the indicated strains, serial dilutions of fungal cells (conidia; 104–101) were spotted on VMM with or without the indicated genotoxic agent: MMS (0.025%), Etoposide (600 μg/mL), Cisplatin (100 μg/mL), Mitomycin C (60 μg/mL), Bleomycin (0.15 μg/mL), and CPT (0.25 μg/mL). (B) Suspensions of conidia (104–102) were spotted on VMM with or without 0.025% MMS. (C) Viability of spores (number of colonies) on the indicated MMS concentration is shown for wild type, Δdim-9, and the Δdim-9; sup-24 strain. Asterisks indicate statistically significant differences between Δdim-9 and the Δdim-9; sup-24 strain (Student’s t test; P < 0.00002).

E6340 | www.pnas.org/cgi/doi/10.1073/pnas.1511377112 Basenko et al. Downloaded by guest on September 30, 2021 (54, 55). The fact that heterochromatin-deficient mutants exhibit A W252 * PNAS PLUS broad sensitivity to DNA-damaging agents suggests that DCDC- EED * deficient strains suffer from genotoxic stress, either due to defects in DNA repair or to failures during DNA replication. This conclusion is supported by our previous results that showed γH2A levels are elevated in the Δdim-5 mutant (46).

Isolation and Identification of a Genetic Suppressor of Δdim-9. To determine why loss of H3K9 methylation causes sensitivity to genotoxic agents, we selected for genetic suppressors of a DCDC- deficient mutant. We mutagenized Δdim-9 cells by exposure to UV light and plated cells on medium supplemented with 0.025% MMS. This concentration of MMS kills DCDC-deficient cells but not wild-type cells. We chose the Δdim-9 strain because it en- codes an adaptor protein that links the DIM-7/DIM-5 subunits to the CUL4/DDB1 ubiquitin ligase module. We reasoned that it might be possible to isolate suppressor mutations that restore activity of DIM-5, which is properly targeted to heterochromatin B in the Δdim-9 strain (5). A cross of one putative suppressor strain dim-9 dim-9; WT sup-24 (suppressor-24) yielded two classes of Δdim-9 progeny in ap- proximately equal numbers: MMS-tolerant and MMS-hypersen- H3K9me3 sitive (Fig. 1B). We quantified colony forming units to compare H3K27me3 the level of MMS sensitivity in wild type, Δdim-9, and Δdim-9; sup-24 strains and found that MMS tolerance of Δdim-9; sup-24 H3 was similar to wild type (Fig. 1C). This suggested that a single mutation led to suppression of the Δdim-9 MMS-hypersensitivity CBB phenotype and that this mutation is unlinked to dim-9. To identify the causative mutation, we performed bulked segre- gant analysis combined with whole genome sequencing (BSA-seq) C wildtype (56). We crossed a single Δdim-9; sup-24 homokaryon, generated in 100 dim-5 the Oak Ridge strain background (OR), to a polymorphic wild-type 80 set-7 Neurospora strain (Mauriceville, MV). Progeny were isolated and dim-5; set-7 genotyped to identify Δdim-9; sup-24 strains. We then isolated and pooled genomic DNA from 14 Δdim-9; sup-24 progeny and se- 60 quencedtheDNAinbulk.Analysisof the sequenced data revealed 40

that regions on Linkage Group II (LGII) and LGIV contained SNPs % Viability that were exclusively from the OR strain background and contained mutations (Fig. S1). The LGII region contained the dim-9 deletion. 20 The region at the right end of LGIV contained a G to A base 0 substitution in the gene encoding the single Neurospora EED ho- 0 0.005 0.010 0.015 0.020 0.025 molog (Fig. 2A). This mutation is predicted to introduce a stop %MMS codon in place of a tryptophan codon at position 252. We hereafter sup-24 refer to this allele as eed . Fig. 2. PRC2 function is required for MMS hypersensitivity of DCDC-deficient Neurospora EED is a component of PRC2 and is required for mutants. (A) The frequency of OR and MV SNPs in progeny from a Δdim-9; methylation of H3K27 (19). We therefore asked if H3K27me3 sup-24 × MV cross are shown for LGIV. An OR-specific region on the right arm levels are altered in the Δdim-9; eedsup-24 double mutant. We iso- of LGIV harbors a nonsense mutation in the Neurospora eed gene. The site of lated total histones from wild type, Δdim-9,andΔdim-9; eedsup-24 the mutation is illustrated in a schematic cartoon above the plot. Gray shaded regions depict WD40 domains. (B) Western blots of acid-extracted histones strains and performed Western blots using antibodies that recognize Δ Δ H3K9me3 and H3K27me3 (Fig. 2B). As loading controls, we per- are shown from wild type, dim-9,and dim-9; sup-24 probed with anti- bodies for H3K9me3 and H3K27me3. A gel stained with Coomassie Blue formed Western blots with antibodies for H3 and stained total and a Western blot probed with antibodies to H3 are shown as loading histones with Coomassie Blue. H3K9me3 was detected in wild type controls. (C) Viability of spores (number of colonies) on the indicated MMS sup-24 but was absent in Δdim-9 and Δdim-9; eed strains, demon- concentrations is shown for wild type, Δdim-5, Δset-7, and the Δdim-5; Δset-7 strating that the suppressor mutation is unable to restore H3K9 double mutant. MTase activity in the Δdim-9 background. H3K27me3 was detected in wild type and Δdim-9 strains but was not detected in the Δdim-9; eedsup-24 double mutant, suggesting that the eedsup-24 allele en- Δdim-5; Δset-7 double-mutant progeny were able to survive in the codes a nonfunctional protein. These data raised the possibility presence of MMS, confirming that elimination of PRC2 leads that PRC2 causes the MMS-sensitivity phenotype observed for to genetic suppression of the MMS-hypersensitivity phenotype of GENETICS DCDC-deficient mutants. DCDC-deficient mutants. To test this, we crossed a Δdim-5 strain to a Δset-7 strain obtained from the Neurospora knockout collection (57). The H3K27 Methylation Is Targeted to Constitutive Heterochromatin in dim-5 and set-7 genes the catalytic subunits of the DCDC H3K9me-Deficient Mutants. Heterochromatin components impact H3K9 MTase (DIM-5KMT1) and the PRC2 H3K27 MTase H3K27me3 localization in plant and animal cells (58–62), and (SET-7KMT6) complex, respectively. We isolated three siblings Jamieson and Selker observed similar results in Neurospora (63). of each genotype and determined the level of MMS sensitivity for We therefore performed ChIP-seq to determine if changes in the each strain by plating spores on increasing concentrations of the distribution of H3K27me3 were correlated with sensitivity of DNA- genotoxic agent (Fig. 2C). Wild type and Δset-7 displayed similar damaging agents in Δdim-5. As previously reported, H3K27me3 MMS tolerance, whereas Δdim-5 was hypersensitive. In contrast, was localized to large chromatin domains of transcriptionally

Basenko et al. PNAS | Published online November 2, 2015 | E6341 Downloaded by guest on September 30, 2021 repressed genes (19). In contrast, in the Δdim-5 mutant, H3K27me3 H3K27 methylation by creating heat maps of H3K9me3 and was detected exclusively at A:T-rich regions of the genome that are H3K27me3 at constitutive heterochromatin domains and at marked by H3K9 methylation in wild-type cells (Fig. 3A). The en- Pc-target domains (i.e., domains targeted in wild-type cells by DCDC richment of H3K27 methylation was highly reproducible in replicate or PRC2, respectively; see Materials and Methods)(Fig.3C and experiments. We calculated normalized ChIP enrichment values D). In the Δdim-5 strain, H3K27me3 enrichment was significantly (NCLS values) for individual genomic features (genes or repeats) reduced at all regions that are typically targeted by PRC2. In and generated scatterplots (Fig. 3B; note that repeats in Neurospora contrast, H3K27me3 enrichment was gained in most constitutive share only <80% identity and completely identical stretches are heterochromatin domains. Compared with the wild-type pattern very short). Replicate experiments for wild type or Δdim-5 yielded of H3K9me3, H3K27me3 enrichment in heterochromatin was Pearson correlation coefficients of 0.992 and 0.993, respectively. In generally lower and more variable across individual heterochro- contrast, no correlation was observed when comparing H3K27me3 matin domains (compare Fig. 3 C and D). enrichment values in wild type and Δdim-5 (R = 0.152). This These data suggest that H3K9 methylation is required for revealed global redistribution of H3K27me3 from genes to re- normal H3K27me3 enrichment patterns. To confirm this, we peated sequences in Δdim-5. We next examined global changes in tested strains in which the single H3 gene had been replaced with

A 0.5 Mb WT K9me3

WT K27me3

Relative Enrichment dim-5 K27me3

50 kb WT K9me3

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Relative Enrichment dim-5 K27me3

B

CDH3K9me3 H3K27me3 -1kb +1kb -1kb +1kb -1kb +1kb -1kb +1kb -1kb +1kb -1kb +1kb -1kb +1kb -1kb +1kb

WT

dim-5

hpo

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Heterochromatin Polycomb Heterochromatin Polycomb domains domains domains domains

Fig. 3. Genotoxic stress of H3K9me3- and HP1-deficient strains is correlated with global redistribution of H3K27me3. (A)They axis shows relative ChIP-seq enrichment across the entire LGVII (x axis; Top) and across a ∼250-kb region (800–1,050 kb; Bottom) for H3K9me3 and H3K27me3 in wild type and for H3K27me3 in the Δdim-5 mutant. [Scale bar, 0.5 Mb (Top) and 50 kb (Bottom).] (B) Scatter plots of H3K27me3 enrichment illustrate reproducibility between wild-type replicates (Left), Δdim-5 replicates (Middle), or wild type and Δdim-5 samples (Right). Each axis corresponds to the NLCS values obtained for each genomic feature. Genes and tRNAs are shown in black, and repeats are shown in gray. (C) Heat maps show H3K9me3 enrichment for the indicated strains. Each heat map row depicts a 2-kb window centered at the left or right boundary of each constitutive heterochromatin domain (Left) or each Pc-target domain (Right). Domains are arranged from smallest (Top) to largest (Bottom). (D) Heat maps show H3K27me3 enrichment for the indicated strains at constitutive heterochromatin domains (Left) and for Pc-target domains (Right), as in B.

E6342 | www.pnas.org/cgi/doi/10.1073/pnas.1511377112 Basenko et al. Downloaded by guest on September 30, 2021 an H3K9R or an H3K9Q substitution allele to mimic H3 with un- Δdim-5, Δset-7,andΔdim-5; Δset-7 double mutants. Expression PNAS PLUS modified lysine-9 or acetylated lysine-9, respectively (46). Normal levels of misregulated genes and their associated gene ontology H3K27 methylation patterns were abolished in these mutants, (GO) annotations are included in Dataset S1.Significantly similar to the case for Δdim-5 strains (Fig. S2). In animal cells, enriched GO terms are shown in Dataset S2. A significant number histones harboring H3K9 to M substitutions act as dominant of H3K27me3-associated genes were up-regulated in the Δdim-5 inhibitors of KMT1 enzymes (64). We therefore introduced an K9M mutant, consistent with the observed loss of H3K27me3 from H3 substitution allele into the endogenous hH3 locus. Al- these domains (Fig. 4 A and B). In contrast, we did not detect though we were able to generate heterokaryons containing the RNAs originating from heterochromatin domains in any of the H3K9M allele, crosses of transformants did not yield homo- strains tested. Box plots of fragments per kilobase per million karyotic progeny. This indicates that the hH3K9M allele is lethal, mapped reads (FPKM) values calculated for repeats and for entire in contrast to hH3K9Q and hH3K9R alleles. Heterokaryons of hH3; hH3K9M produced an intermediate distribution of H3K27me3 and constitutive heterochromatin domains on both plus and minus a subtle reduction in DNA methylation (Fig. S2 A–C). Our data strands revealed few detectable transcripts in any of the strains Δ Δ show that the H3K9M protein is a weak dominant inhibitor of examined (Fig. 4 C and D). Although both dim-5 and set-7 H3K9 methylation and that the H3K9M protein disrupts other strains showed induction of H3K27me3-associated genes, there chromatin-based processes in Neurospora. Given the partial dom- was incomplete overlap between the sets of induced genes in the inance of the hH3K9M allele and the apparent pleiotropic effects two strains (Fig. S3 A–D). We note that H3K27me3-associated of the H3K9M protein in Neurospora, caution should be used genes had low levels of expression compared with the average when interpreting phenotypes of histone K to M alleles in other gene, even in Δdim-5 and Δset-7 (compare Fig. 4 A and B). Al- organisms. though most of these genes have unknown functions, many genes Reduction of cytosine methylation in plant and animal cells that were down-regulated in Δdim-5 are associated with metabolism produced a similar redistribution of H3K27 methylation (59–62). Positive feedback loops between H3K9 methylation and DNA methylation pathways complicate interpretation of these data, however. In plants and animals, DNA methylation is partly de- A B All Genes H3K27me3-enriched Genes pendent on H3K9 methylation (65), and reduced DNA methyl- 30 30 ation can lead to a concomitant loss of H3K9 methylation in 20 20 certain situations (60, 66, 67). In Neurospora, the heterochromatin 10 10 formation pathway is primarily unidirectional. DNA methylation depends on H3K9me3 and HP1, but H3K9me3 patterns are not

substantially altered by loss of HP1 or DNA methylation (7, 10). total RNA log2 (FPKM+1) 0 total RNA log2 (FPKM+1) 0 We generated heat maps to ask if H3K27me3 patterns were altered Wild Type dim5 set7 dim5; Wild Type dim5 set7 dim5; set7 set7 in hpo mutants, which lack HP1 and are sensitive to genotoxic agents, or dim-2 mutants, which lack DNA methylation and are C Repeats (+ Strand) rand) insensitive to genotoxic agents. Consistent with previous data, heat 30 30 maps revealed that enrichment of H3K9me3 was reduced at the 20 20 boundaries of heterochromatin domains in the hpo strain (9). 10 10 Despite the presence of significant H3K9me3 in the hpo mutant, the pattern of H3K27 methylation was similar to the Δdim-5 mu-

tant (Fig. 3C). In contrast, H3K27me3 patterns in dim-2 mutants total RNA log2 (FPKM+1) 0 total RNA log2 (FPKM+1) 0 were more similar to wild type, although a subtle increase of Wild Type dim5 set7 dim5; Wild Type dim5 set7 dim5; H3K27me3 was observed globally in heterochromatin regions in set7 set7 the dim-2 strain (Fig. 3C). Thus, HP1 is required to prevent re- D Heterochromatin Domains Heterochromatin Domains distribution of H3K27me3 to heterochromatin, whereas 5mC has (+ Strand) (+ Strand) 30 30 only a minimal role. 20 20 In Caenorhabditis elegans, spreading of H3K27me3 into adja- 10 10 cent chromatin is prevented by H3K36 methylation (68, 69). We therefore asked if methylation of other H3 tail residues affects the distribution of H3K27 methylation in Neurospora. We per- 0 0 total RNA log2 (FPKM+1) Wild Type dim5 set7 dim5; total RNA log2 (FPKM+1) Wild Type dim5 set7 dim5; formed ChIP-seq for H3K27me3 in set-1 and set-2 mutants, which set7 set7 are deficient for H3K4 and H3K36 methylation, respectively (Fig.

S2) (70). We observed changes in the relative level of H3K27me3 E Repeats F Heterochromatin Domains enrichment in the set-2 strain at some Pc-target domains, but the 10 10 global pattern of H3K27me3 was similar to wild type in both strains. Together, these data suggest that defects in the H3K9 methylation pathway but not in other path- 5 5 ways lead to global redistribution of H3K27me3. In addition, we

found that H3K27me3 patterns were unaltered when wild-type small RNA log2 (FPKM+1) 0 0 GENETICS small RNA log2 (FPKM+1) Wild Type dim5 set7 dim5; Wild Type dim5 set7 dim5; cells were grown in the presence of MMS (Fig. S2D). Hence, set7 set7 exposure to DNA-damaging agents is not sufficient to trigger redistribution of H3K27me3 to heterochromatin. Fig. 4. H3K27me3 does not compensate for loss of silencing at hetero- It has been proposed that redistribution of H3K27me3 to chromatin regions. (A–D) Box plots depict average expression level (log + constitutive heterochromatin domains is a compensatory response [FPKM 1]) from strand-specific RNA-seq experiments for (A) all Neurospora genes, (B) H3K27me3-enriched genes, (C) DNA repeats, and (D) hetero- for maintenance of heterochromatic gene silencing (62, 71). To Δ Δ chromatin domains for the indicated strains. FPKM values are shown for test if this is the case in Neurospora, we asked if dim-5; set-7 both plus and minus strands for C and D.(E and F) Box plots show the levels double mutants exhibit increased transcription from constitutive of 20–24 nt RNAs (log[FPKM+1]) for (E) DNA repeats and (F) heterochro- heterochromatin domains. We first performed ribosomal RNA matin domains for the indicated strains. The notches indicate the 95% subtraction followed by strand-specific total RNA-seq in wild type, confidence interval around the median.

Basenko et al. PNAS | Published online November 2, 2015 | E6343 Downloaded by guest on September 30, 2021 and growth (Dataset S1), which is likely an indirect effect of the compared with wild type. These data demonstrate that altered PRC2 poor growth phenotype of Δdim-5. targeting is responsible for vegetative and sexual phase growth de- It is possible that RNA surveillance pathways rapidly de- fects observed in heterochromatin-defective mutants. grade heterochromatin-derived transcripts (43). We therefore γH2A is a phosphorylated form of H2A that is induced by sequenced small RNA libraries and examined the level of 20–24 DNA damage or DNA replication stress (44, 45). We previously nucleotide (nt) RNAs originating from heterochromatin domains. showed that γH2A is enriched in heterochromatin domains in We determined the levels of small RNAs originating from repeats wild-type cells and that γH2A is deposited throughout the ge- and heterochromatin domains. The Δdim-5 and Δdim-5; Δset-7 nome in the Δdim-5 mutant, leading to loss of region-specific strains had higher levels of small RNAs originating from individual enrichment (46). We performed ChIP-seq for γH2A in wild type, RIP’d repeats and from entire heterochromatin domains compared Δdim-5, Δset-7, and Δdim-5; Δset-7 double mutants (Fig. 6D). As with wild type or Δset-7 (Fig. 4 E and F). The levels of small RNAs observed previously, γH2A was enriched in constitutive hetero- from heterochromatin domains were slightly higher in the Δdim-5; chromatin domains in wild type, but enrichment was lost in the Δset-7 double mutant than in the Δdim-5 strain alone, although the Δdim-5 strain. The pattern of γH2A enrichment in the Δset-7 Δ Δ additional increase was minimal. We examined the first base of mutant was equivalent to wild type. In the dim-5; set-7 double γ heterochromatin-derived small RNAs and found that the majority mutant, H2A was enriched at constitutive heterochromatin beganwithaU(Fig. S3E), suggesting that these RNAs are prod- domains with a distribution that appeared similar to wild type. γ ucts of the siRNA pathway in Neurospora(72).Thesedatasuggest We created heat maps to visualize H2A enrichment in all that loss of H3K9me3 leads to increased siRNA production from constitutive heterochromatin domains (Fig. 6E). Wild type and Δset-7 strains displayed the highest levels of γH2A enrichment heterochromatin domains and that H3K27me3 plays a minimal ’ Δ Δ role in limiting heterochromatin-derived siRNAs. at the edges of RIP d regions, whereas in the dim-5; set-7 double-mutant strains, the levels of γH2A enrichment at the Aberrant H3K27me3 Is Responsible for Defective Sexual Development boundaries of heterochromatin domains were lower and the γ and Poor Growth of dim-5 Strains. The fact that Δset-7 strains grow borders of individual H2A peaks were not as discrete as in wild normally and are not hypersensitive to MMS (Fig. 2D) suggests type. In S. pombe, it was proposed that H3K9me was required for γH2A deposition in heterochromatin domains (73). How- that loss of H3K27me3 from facultative heterochromatin does γ not lead to significant phenotypes. Rather, the data suggest that ever, our data show that H2A deposition in heterochromatin can occur independently of H3K9me in N. crassa. One possible gain of H3K27me3 at constitutive heterochromatin domains is γ Δ Δ linked to genotoxic stress in heterochromatin-defective mutants. reason for the accumulation of H2A in dim-5; set-7 strains To determine if MMS sensitivity is caused by aberrant localiza- is that stalled replication forks or frequent DSBs occur at repeat- rich regions of the genome in these cells. tion of H3K27me3, we replaced the wild-type hH3 gene with an hH3K27R or an hH3K27Q substitution allele and crossed the Neurospora PRC2 Modulates the Response to Genotoxic Stress. We hH3K27 mutant strains to Δdim-5. The resulting Δdim-5; hH3K27R Δ K27Q next asked if sensitivity to other DNA-damaging agents was res- or dim-5; hH3 double-mutant progeny were resistant to cued in the Δdim-5; Δset-7 double mutant (Fig. 6F). The double MMS, suggesting that aberrant H3K27me3 is responsible for mutant was more tolerant than Δdim-5 to all genotoxic agents, Δ MMS hypersensitivity of the dim-5 strain (Fig. 5). with one exception: Cells that lacked both DCDC and PRC2 In addition to hypersensitivity to genotoxic agents, DCDC- exhibited increased sensitivity to the topoisomerase I inhibitor deficient mutants display slow growth and fail to complete sexual CPT. To confirm that loss of H3K27me3 leads to increased CPT development (6). We asked if these defects were also rescued by sensitivity, we examined strains with mutant H3 genes resulting in removal of PRC2. Both vegetative and sexual development de- an H3K9 substitution, an H3K27 substitution, or substitutions of Δ K9Q fects of dim-5 single mutants were rescued by deletion of the both K9 and K27. hH3 strains were mildly sensitive to MMS Δset-7 gene. The linear growth rate of Δdim-5; Δset-7 double and CPT. In contrast, hH3K9Q/K27Q or hH3K9Q/K27R strains were mutants was significantly faster than Δdim-5 single mutants yet both able to grow in the presence of MMS but were hypersensi- slower than wild type (Fig. 6 A and B). Similarly, homozygous tive to CPT. A Δset-7; hH3K9Q strain was similarly hypersensitive crosses of Δdim-5; Δset-7 double mutants were fertile, in contrast to CPT (Fig. S4). Together, our results suggest that PRC2 and its to homozygous crosses of Δdim-5 single mutants. Crosses of product H3K27me3 modulate the cellular response to genotoxic Δdim-5; Δset-7 gave rise to fruiting body structures filled with stress in Neurospora. spores (Fig. 6C), although spore production was delayed and the number of mature asci observed in squashed perithecia was reduced Discussion Heterochromatin components are required for normal growth and development and are important for maintenance of genome VMM MMS integrity. We found that in Neurospora, loss of H3K9me3 or HP1 results in redistribution of H3K27me3, which in turn leads WT to growth defects and hypersensitivity to MMS. The fact that Δdim-5 redistribution of H3K27me3 is deleterious in Neurospora is Δ somewhat surprising; however, a similar observation may have set-7 been difficult to make in other experimental systems. In plants hH3H3K27Q and animals, loss of heterochromatin components leads to sim- ilar redistribution of H3K27me3 (17, 58–62), but in these sys- hH3H3K27R tems, depletion of H3K27me3 from Pc-target regions is sufficient Δdim-5; Δset-7 to cause poor growth and developmental defects (74, 75). Thus, in many organisms, it would be difficult to determine if the pheno- Δdim-5; hH3H3K27Q typic defects displayed by H3K9me3-deficient strains were caused Δdim-5; hH3H3K27R by loss of H3K27me3 from native sites or by gain of H3K27me3 in heterochromatin domains. In contrast, because loss of H3K27me3 Fig. 5. H3K27 methylation is responsible for MMS sensitivity of H3K9- produces only minor phenotypic defects in Neurospora, it is clear deficient strains. Suspensions of conidia (104–101) of the indicated strains were that gain of H3K27me3 in typically heterochromatic domains has spot-tested on media with or without MMS (0.02%). severely deleterious consequences. It is not known if redistribution

E6344 | www.pnas.org/cgi/doi/10.1073/pnas.1511377112 Basenko et al. Downloaded by guest on September 30, 2021 PNAS PLUS ABWT set-7 E wildtype set-7 H2A -1kb +1kb -1kb +1kb

dim-5; set-7

Distance (cm) WT dim-5

dim-5 dim-5; Time (hours) set-7 set-7 C

dim-5

WT set-7 dim-5 dim-5; set-7 dim-5; D H3K9me3 WT set-7

set-7 Heterochromatin H2A dim-5 domains

set-7; dim-5

F VMM MMS Cisplatin Mitomycin C Bleomycin Etoposide CPT WT set-7 dim-5 set-7; dim-5 mei-3 mus-9

Fig. 6. Elimination of H3K27me3 rescues growth and developmental defects of an H3K9me3-deficient strain, but double mutants accumulate γH2A and are sensitive to CPT. (A) Conidia (103) produced by the indicated strains were inoculated in the center of a Petri plate, and cultures were allowed to grow for 24 h. (B) The linear growth rate was determined for the indicated strains using race tubes. The growth of two isolates is shown for each genotype. (C) Homozygous crosses were carried out for each of the indicated genotypes. Images of dissected fruiting bodies (perithecia) are shown revealing the presence or absence of meiotic products for each cross. (D) Relative ChIP-seq enrichment across LGVII is shown for γH2A in wild type, Δdim-5, Δset-7, and the Δset-7; Δdim-5 double mutant. (E) Heat maps show γH2A enrichment for the indicated strains. Each heat map depicts a 2-kb window centered at the boundaries of all hetero- chromatin domains. Domains are arranged from smallest (Top) to largest (Bottom). (F) Suspensions of 104,103,or102 conidia of the indicated strains were spot-tested on VMM with or without the indicated genotoxic agent: MMS (0.025%), Cisplatin (100 μg/mL), Mitomycin C (60 μg/mL), Bleomycin (0.15 μg/mL), Etoposide (600 μg/mL), and CPT (0.25 μg/mL).

of H3K27me3 is also linked to growth defects and genotoxic stress through independent mechanisms. Hence, it is likely that ele- in higher eukaryotes, but such a scenario is consistent with the fact vated levels of γH2A are a response to genotoxic stress in Δdim-5 that depletion of heterochromatin components is associated with rather than the cause of genotoxic stress. We propose three genome instability in animals (2, 31). possible mechanisms for H3K27me3-dependent genotoxic stress It has been proposed that redistribution of H3K27me3 to con- that should be explored in future work. It is possible that stitutive heterochromatin in animals is a compensatory mechanism H3K27me3 leads to genotoxic stress by interfering with centro- to silence transcription. We did not find evidence to support such a mere formation, as both dim-5 and hp1 mutants exhibit defects in role in N. crassa. Heterochromatin-derived transcripts were not CenH3 deposition (13). Another possibility is that aberrant increased in cells lacking H3K9me3, H3K27me3, or both. We did H3K27me3 may interfere with chromatin dynamics during the S detect an increase in small RNA production from heterochromatin phase. In S. pombe, HP1 is displaced from the chromatin fiber domains in dim-5 mutants, but small RNA levels were not sub- in a cell cycle-specific manner, facilitating transcription of cen- stantially higher when cells lacked both H3K9me3 and H3K27me3. tromeric repeats during the S phase (78). It is possible that Notably, genotoxic stress triggers aberrant RNA synthesis by H3K27me3 assembles condensed chromatin that is not subject to QDE1 and subsequent quelling defective 2-interacting RNA appropriate regulation over the course of the cell cycle. Alter- (qiRNA) generation in Neursopora (76, 77), suggesting that the natively, it is also possible that H3K27me3 accumulates in het- observed increase in small RNAs may result from replication or erochromatin as part of a genotoxic stress response that triggers repair defects in heterochromatin domains rather than loss of cell cycle arrest or programmed cell death. Data from other transcriptional silencing. organisms and from this study suggest that defective hetero- GENETICS It is not clear why redistribution of H3K27me3 leads to poor chromatin formation is sufficient to cause genotoxic stress. For growth and MMS sensitivity in Neurospora. Previous work sug- example, S. pombe lacks H3K27me3, but heterochromatin- gests that abnormal localization of γH2A is not the cause of the defective mutants suffer frequent illegitimate recombination MMS-sensitivity phenotype. Although site-specific enrichment of (3, 28). In the present study, we found that γH2A is enriched γH2A is lost in the Δdim-5 mutant, this is the result of increased in heterochromatin domains in Δset-7; Δdim-5 double mutants, γH2A in euchromatin rather than a decrease in γH2A at het- suggesting that defective replication or repair occurs in repeat- erochromatin (46). Moreover, double mutants of Δdim-5 and rich domains of Neurospora even when both H3K9me3 and hH2AS131A display an additive increase in MMS sensitivity (46), H3K27me3 are absent. PRC2 recruitment could be a response to suggesting that loss of DIM-5 and γH2A lead to MMS sensitivity genotoxic stress at these sites. Indeed, we found that Neurospora

Basenko et al. PNAS | Published online November 2, 2015 | E6345 Downloaded by guest on September 30, 2021 Δdim-5; Δset-7 double mutants exhibit synthetic sensitivity to 0.5% (wt/vol) glucose. When relevant, plates included 200 μg/mL hygromycin or CPT, suggesting that H3K27me3 is required for cells to mount a 400 μg/mL basta (84) or DNA-damaging agents at the indicated concentration. proper response to CPT-induced damage in the Δdim-5 back- We note that MMS is an alkylating agent that can react with components in the ground. The idea that H3K27me3 is part of a DNA damage re- medium. Therefore, the effective inhibitory concentration can vary between experiments due to ambient temperature and age of the plates. For these sponse is also supported by reports that PRC2 and other Pc reasons, strains were tested on multiple concentrations of MMS during each components localize to DNA damage sites in mammalian cells experiment. For survival curves, 200 cells were plated on minimal medium and and that EZH2 depletion leads to increased sensitivity to ionizing on plates with increasing concentrations of MMS. The number of colonies was radiation in mammals (24–26, 79). On the other hand, we did not counted for each plate and plotted as a percentage of the no MMS control. observe redistribution of H3K27me3 when wild-type cells were Three independent strain isolates were used for each concentration of MMS, exposed to genotoxic agents. Additional studies are needed to and the average percent viability was plotted. Linear growth rates were de- identify the mechanisms that link H3K27me3 and genotoxic stress. termined using “race tubes” (85). Our finding that loss of PRC2 activity can lead to differential sensitivity of genotoxic agents has possible implications for improved Molecular Analyses. Neurospora transformation (86), DNA isolation (87), protein isolation, histone isolation, and Western blotting (10) were per- cancer treatment. Components of constitutive heterochromatin and formed as previously described. Antibodies to γH2A (cat. no. ab15083, Abcam), Pc system components are frequently mutated or overexpressed in H3K9me3 (cat. no. 39161, Active Motif), H3K27me3 (cat. no. 39537, Active cancer cells, and it was recently reported that EGFR and BRG1 Motif, or cat. no. 9733, Cell Signaling Technologies), and H3 (cat. no. 07–690, mutant tumors exhibit enhanced sensitivity to topoisomerase II in- Millipore) were used as indicated. ChIP-seq was performed as described (46). hibitors when EZH2 is inhibited (80). In contrast, we found here that The hH3K9M strain was constructed by site-directed mutagenesis as described Neurospora strains lacking PRC2 and DCDC become more re- (46) using the following primers: K9M_F CAG ACC GCC CGC ATG TCC ACC sistant to the topoisomerase II inhibitor etoposide as well as to GGT GGC AAG GCC CCC and K9M_R CAC CGG TGG ACA TGC GGG CGG TCT several other DNA-damaging agents but display enhanced sensitivity GCT TAG TGC GGG. to the topoisomerase I inhibitor CPT. These results raise the pos- Illumina Sequencing. For Illumina sequencing, ChIP-seq libraries were pre- sibility that EZH2 inhibition may have significantly different conse- pared using 10 ng of immunoprecipitated DNA following the instructions quences in different genetic backgrounds. supplied with Illumina Tru-seq ChIP-seq kits (Illumina cat. no. FC-121-2002). H3K9me3, HP1, and 5mC have all been implicated in preventing RNA-seq libraries were prepared from 5 μg total RNA. Ribosomal RNAs were H3K27me3 redistribution to heterochromatin in animals, and depleted using the yeast Ribo-zero kit (cat. no. MRZY1324, Epicentre), and reduction of 5mC leads to redistribution of H3K27me3 in plants RNA libraries were generated with the Illumina Stranded RNA-seq kit PCR (17, 58–62, 71). These observations highlight the need for addi- (cat. no. RS-122-2101). For ChIP-seq, amplification was limited to 4–8 cycles tional studies to fully elucidate the complex functional and reg- to reduce PCR bias against A:T-rich DNA sequences (88). Illumina sequencing ulatory relationships between heterochromatin and Pc system was performed using an Illumina NextSeq500 Instrument at the University of components. Even within the fungi, multiple heterochromatin- Georgia Genomics Facility. dependent mechanisms can impact H3K27me3 distribution. We Data Analysis. Illumina sequence reads have been deposited into the National found here that H3K27me3 redistribution occurs in H3K9me3- Center for Biotechnology Information Short Reads Archive (accession no. and HP1-deficient mutants of N. crassa, in agreement with other SRP058573). Short reads were mapped using bowtie2 (89) or TopHat (90) for ChIP- work (63). In C. neoformans, deletion of an H3K27me3-binding seq or RNA-seq experiments, respectively, to the latest Neurospora genome an- protein leads to accumulation of H3K27me3 at centromeres. notation (version 12), available from the Neurospora genome database (91). For However, redistribution of H3K27me3 requires H3K9me2 in this ChIP-seq data, read numbers were determined for 25 base pair bins using igvtools, fungus (23). This may also be the case in E. festucae, where deletion and the read count for each bin was normalized to the total read number in the of the H3K27 MTase ΔezhB does not rescue the growth defects of sample. Relative enrichment data were visualized using the Integrated Genome the H3K9 MTase-deficient mutant ΔclrD (22). In plants and ani- Viewer (92, 93). For histograms showing relative ChIP enrichment at specific chromosomes or genomic loci, enrichment values for each sample are shown mals, cross-talk between different heterochromatin components relative to the maximum enrichment value for the depicted window. and the Pc system has been linked to cell type-specific changes in The Hypergeometric Optimization of Motif EnRichment (HOMER) soft- chromatin architecture, raising the possibility that different het- ware package (94) was used to identify H3K9me3 or H3K27me3 domains in erochromatin-based mechanisms could regulate H3K27me3 in wild type. The coordinates of H3K9me peaks and H3K27me3 peaks are listed different cells even within the same organism (71, 81, 82). in Datasets S3 and S4, respectively. We constructed a custom genome an- Despite apparent mechanistic differences, the fact that Pc com- notation file containing genes, repeats (46), H3K27me3 domains, and ponents can be conditionally recruited to constitutive hetero- H3K9me3 domains and used HOMER to construct heat maps of ChIP en- chromatin domains in plants, animals, and fungi suggests shared richment across H3K9me3 or H3K27me3 domains (using the –ghist option). mechanisms exist. We speculate that these mechanisms involve HOMER-generated matrix files or FPKM files were loaded into GENE-E genotoxic stress. Indeed, mammalian PRC2 and NuRD complexes (www.broadinstitute.org/cancer/software/GENE-E/) to generate heat map images. For scatter plots, NCLS values were calculated using EpiChIP soft- are simultaneously recruited to chromatin when heterochromatin ware, which calculates enrichment values normalized for total read number components are depleted and when DNA damage is introduced and for length of the feature (95). Differential expression was determined (25, 62). Moreover, the NuRD component BEND3 was recently using Cuffdiff (90). Genes were classified as differentially expressed if they showntoberequiredforPRC2recruitment to heterochromatin passed criteria for statistical significance and had a minimum twofold dif- domains in 5mC- or H3K9me3-deficient cells (62). Given that fungi ference in expression between wild type and the mutant. Functional classi- and plants lack BEND3, other proteins must contribute to PRC2 fications for differentially expressed genes were determined using Fungifun2 (96) with the following parameters. Significant enrichment of recruitment to constitute heterochromatin in these organisms. Our ’ study highlights the power of fungal genetics for investigating the overrepresented functional categories was determined using Fisher s exact test (P < 0.05), and P values were adjusted using the Benjamini–Hochberg control and the functions of the Pc system and suggests that future procedure option. studies in Neurospora are likely to provide clues into the relation- We used 1 μg of total RNA for preparation of small RNA sequencing ships between the Pc system, heterochromatin, and genotoxic stress. libraries according to the manufacturer’s protocol in the TruSeq Small RNA Sample Prep Kit from Illumina. Raw files were trimmed for adapters Materials and Methods using cutadapt v1.7.1 (97), and only reads with sizes ranging from 20 to Strains and Growth Media. All Neurospora strains used in this study are listed in 24 nt were kept. Reads were then mapped to the N. crassa rRNA database Table S1. Strains were grown at 32 °C in Vogel’s minimal medium (VMM) + (98) and annotated tRNA genes (based on N. crassa OR74A v12). Aligned 1.5% (wt/vol) sucrose (83). Liquid cultures were shaken at 150 rpm. Crosses were reads were removed, and then all samples were subsampled based on performed on modified synthetic cross-medium (83). For plating assays, Neu- the sample with the lowest number of reads, ensuring the same input rospora conidia were plated on VMM with 2.0% sorbose, 0.5% fructose, and read number for subsequent analyses. Qualified reads were aligned to the

E6346 | www.pnas.org/cgi/doi/10.1073/pnas.1511377112 Basenko et al. Downloaded by guest on September 30, 2021 N. crassa OR74A v12 using bowtie 1.1.0 with the following parameters:– ACKNOWLEDGMENTS. We thank Laura Banken for technical contributions PNAS PLUS phred33-quals–nomaqround–best–strata–chunkmbs 1024 –e1–l20–n0-a–m to the project. This work would not have been possible without materials generated by the Neurospora Functional Genomics Project (NIH Grant 1000 (99). The FPKM method was used to quantify the small RNA distribution P01GM68087) provided by the Fungal Genetics Stock Center. This work in each given region. Regions that did not meet a minimum coverage re- was supported by American Cancer Society Grant RSG-14-184-01-DMC (to quirement (three reads) were not included in the box plot calculation. Z.A.L.) and NIH Grant R00GM100000 (to R.J.S.).

1. Grewal SI, Jia S (2007) Heterochromatin revisited. Nat Rev Genet 8(1):35–46. 33. Soria G, Almouzni G (2013) Differential contribution of HP1 proteins to DNA end 2. Peng JC, Karpen GH (2008) Epigenetic regulation of heterochromatic DNA stability. resection and homology-directed repair. Cell Cycle 12(3):422–429. Curr Opin Genet Dev 18(2):204–211. 34. Lee YH, Kuo CY, Stark JM, Shih HM, Ann DK (2013) HP1 promotes tumor suppressor 3. Li PC, et al. (2013) Replication fork stability is essential for the maintenance of BRCA1 functions during the DNA damage response. Nucleic Acids Res 41(11):5784–5798. integrity in the absence of heterochromatin. Cell Reports 3(3):638–645. 35. Bolderson E, et al. (2012) Kruppel-associated Box (KRAB)-associated co-repressor 4. Aramayo R, Selker EU (2013) Neurospora crassa, a model system for (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1β (HP1-β) research. Cold Spring Harb Perspect Biol 5(10):a017921. mobilization and DNA repair in heterochromatin. J Biol Chem 287(33):28122–28131. 5. Lewis ZA, et al. (2010) DNA methylation and normal chromosome behavior in 36. Chiolo I, et al. (2011) Double-strand breaks in heterochromatin move outside of a Neurospora depend on five components of a histone methyltransferase complex, dynamic HP1a domain to complete recombinational repair. Cell 144(5):732–744. DCDC. PLoS Genet 6(11):e1001196. 37. Baldeyron C, Soria G, Roche D, Cook AJ, Almouzni G (2011) HP1alpha recruitment to 6. Tamaru H, Selker EU (2001) A methyltransferase controls DNA methyl- DNA damage by p150CAF-1 promotes homologous recombination repair. J Cell Biol ation in Neurospora crassa. Nature 414(6861):277–283. 193(1):81–95. 7. Lewis ZA, et al. (2009) Relics of repeat-induced point mutation direct heterochro- 38. Luijsterburg MS, et al. (2009) Heterochromatin protein 1 is recruited to various types matin formation in Neurospora crassa. Genome Res 19(3):427–437. of DNA damage. J Cell Biol 185(4):577–586. 8. Tamaru H, et al. (2003) Trimethylated lysine 9 of histone H3 is a mark for DNA 39. Dinant C, Luijsterburg MS (2009) The emerging role of HP1 in the DNA damage methylation in Neurospora crassa. Nat Genet 34(1):75–79. response. Mol Cell Biol 29(24):6335–6340. 9. Honda S, et al. (2012) Heterochromatin protein 1 forms distinct complexes to direct 40. Ayoub N, Jeyasekharan AD, Bernal JA, Venkitaraman AR (2008) HP1-beta mobili- histone deacetylation and DNA methylation. Nat Struct Mol Biol 19(5):471–477, S1. zation promotes chromatin changes that initiate the DNA damage response. Nature 10. Honda S, Selker EU (2008) Direct interaction between DNA methyltransferase DIM-2 453(7195):682–686. and HP1 is required for DNA methylation in Neurospora crassa. Mol Cell Biol 28(19): 41. Liu H, et al. (2013) A method for systematic mapping of protein lysine methylation β – 6044–6055. identifies functions for HP1 in DNA damage response. Mol Cell 50(5):723 735. 11. Lewis ZA, Adhvaryu KK, Honda S, Shiver AL, Selker EU (2010) Identification of DIM-7, 42. Ayrapetov MK, Gursoy-Yuzugullu O, Xu C, Xu Y, Price BD (2014) DNA double-strand a protein required to target the DIM-5 H3 methyltransferase to chromatin. Proc Natl breaks promote methylation of histone H3 on lysine 9 and transient formation of – Acad Sci USA 107(18):8310–8315. repressive chromatin. Proc Natl Acad Sci USA 111(25):9169 9174. 12. Honda S, et al. (2010) The DMM complex prevents spreading of DNA methylation 43. Chicas A, Forrest EC, Sepich S, Cogoni C, Macino G (2005) Small interfering RNAs that from transposons to nearby genes in Neurospora crassa. Genes Dev 24(5):443–454. trigger posttranscriptional gene silencing are not required for the histone H3 Lys9 13. Smith KM, Phatale PA, Sullivan CM, Pomraning KR, Freitag M (2011) Heterochro- methylation necessary for transgenic tandem repeat stabilization in Neurospora – matin is required for normal distribution of Neurospora crassa CenH3. Mol Cell Biol crassa. Mol Cell Biol 25(9):3793 3801. 31(12):2528–2542. 44. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded 14. Schwartz YB, Pirrotta V (2007) Polycomb silencing mechanisms and the management breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273(10): – of genomic programmes. Nat Rev Genet 8(1):9–22. 5858 5868. 15. Müller J, et al. (2002) Histone methyltransferase activity of a Drosophila Polycomb 45. Dickey JS, et al. (2009) H2AX: Functional roles and potential applications. Chromosoma 118(6):683–692. group repressor complex. Cell 111(2):197–208. 46. Sasaki T, et al. (2014) Heterochromatin controls γH2A localization in Neurospora 16. Cao R, et al. (2002) Role of histone H3 lysine 27 methylation in Polycomb-group si- crassa. Eukaryot Cell 13(8):990–1000. lencing. Science 298(5595):1039–1043. 47. Sharan SK, et al. (1991) The albino-deletion complex of the mouse: Molecular 17. Cooper S, et al. (2014) Targeting polycomb to pericentric heterochromatin in em- mapping of deletion breakpoints that define regions necessary for development of bryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment. Cell Reports the embryonic and extraembryonic ectoderm. Genetics 129(3):825–832. 7(5):1456–1470. 48. Wakabayashi M, Ishii C, Hatakeyama S, Inoue H, Tanaka S (2010) ATM and ATR 18. Blackledge NP, et al. (2014) Variant PRC1 complex-dependent H2A ubiquitylation homologes of Neurospora crassa are essential for normal cell growth and mainte- drives PRC2 recruitment and polycomb domain formation. Cell 157(6):1445–1459. nance of chromosome integrity. Fungal Genet Biol 47(10):809–817. 19. Jamieson K, Rountree MR, Lewis ZA, Stajich JE, Selker EU (2013) Regional control of 49. Hatakeyama S, Ishii C, Inoue H (1995) Identification and expression of the Neuros- histone H3 lysine 27 methylation in Neurospora. Proc Natl Acad Sci USA 110(15): pora crassa mei-3 gene which encodes a protein homologous to Rad51 of Saccha- 6027–6032. romyces cerevisiae. Mol Gen Genet 249(4):439–446. 20. Smith KM, et al. (2008) The fungus Neurospora crassa displays telomeric silencing 50. Kaufmann SH (1998) Cell death induced by topoisomerase-targeted drugs: More mediated by multiple sirtuins and by methylation of histone H3 lysine 9. Epigenetics questions than answers. Biochim Biophys Acta 1400(1-3):195–211. Chromatin 1(1):5. 51. Tomasz M, et al. (1987) Isolation and structure of a covalent cross-link adduct be- 21. Connolly LR, Smith KM, Freitag M (2013) The Fusarium graminearum histone H3 K27 tween mitomycin C and DNA. Science 235(4793):1204–1208. methyltransferase KMT6 regulates development and expression of secondary me- 52. Eastman A (1987) The formation, isolation and characterization of DNA adducts tabolite gene clusters. PLoS Genet 9(10):e1003916. produced by anticancer platinum complexes. Pharmacol Ther 34(2):155–166. 22. Chujo T, Scott B (2014) Histone H3K9 and H3K27 methylation regulates fungal al- 53. Haidle CW (1971) Fragmentation of deoxyribonucleic acid by bleomycin. Mol kaloid biosynthesis in a fungal endophyte-plant symbiosis. Mol Microbiol 92(2): Pharmacol 7(6):645–652. – 413 434. 54. Adhvaryu KK, et al. (2015) The cullin-4 complex DCDC does not require E3 ubiquitin 23. Dumesic PA, et al. (2015) Product binding enforces the genomic specificity of a yeast ligase elements to control heterochromatin in Neurospora crassa. Eukaryot Cell – polycomb repressive complex. Cell 160(1-2):204 218. 14(1):25–28. ’ 24. O Hagan HM, Mohammad HP, Baylin SB (2008) Double strand breaks can initiate 55. Higa LA, Zhang H (2007) Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous WD40-repeat proteins to destroy. Cell Div 2:5. promoter CpG island. PLoS Genet 4(8):e1000155. 56. Pomraning KR, Smith KM, Freitag M (2011) Bulk segregant analysis followed by 25. Chou DM, et al. (2010) A chromatin localization screen reveals poly (ADP ribose)- high-throughput sequencing reveals the Neurospora cell cycle gene, ndc-1, to be regulated recruitment of the repressive polycomb and NuRD complexes to sites of allelic with the gene for ornithine decarboxylase, spe-1. Eukaryot Cell 10(6):724–733. DNA damage. Proc Natl Acad Sci USA 107(43):18475–18480. 57. Colot HV, et al. (2006) A high-throughput gene knockout procedure for Neurospora 26. O’Hagan HM, et al. (2011) Oxidative damage targets complexes containing DNA reveals functions for multiple transcription factors. Proc Natl Acad Sci USA 103(27): , SIRT1, and polycomb members to promoter CpG islands. Cancer 10352–10357. – Cell 20(5):606 619. 58. Peters AH, et al. (2003) Partitioning and plasticity of repressive histone methylation GENETICS 27. Zaratiegui M, et al. (2011) RNAi promotes heterochromatic silencing through repli- states in mammalian chromatin. Mol Cell 12(6):1577–1589. cation-coupled release of RNA Pol II. Nature 479(7371):135–138. 59. Deleris A, et al. (2012) Loss of the DNA methyltransferase MET1 induces H3K9 hy- 28. Cam HP, et al. (2005) Comprehensive analysis of heterochromatin- and RNAi-medi- permethylation at PcG target genes and redistribution of H3K27 trimethylation to ated epigenetic control of the fission yeast genome. Nat Genet 37(8):809–819. transposons in Arabidopsis thaliana. PLoS Genet 8(11):e1003062. 29. Peng JC, Karpen GH (2007) H3K9 methylation and RNA interference regulate nu- 60. Murphy PJ, et al. (2013) Single-molecule analysis of combinatorial epigenomic states cleolar organization and repeated DNA stability. Nat Cell Biol 9(1):25–35. in normal and tumor cells. Proc Natl Acad Sci USA 110(19):7772–7777. 30. Peng JC, Karpen GH (2009) Heterochromatic genome stability requires regulators of 61. Reddington JP, et al. (2013) Redistribution of H3K27me3 upon DNA hypomethylation histone H3 K9 methylation. PLoS Genet 5(3):e1000435. results in de-repression of Polycomb target genes. Genome Biol 14(3):R25. 31. Peters AH, et al. (2001) Loss of the Suv39h histone methyltransferases impairs 62. Saksouk N, et al. (2014) Redundant mechanisms to form silent chromatin at peri- mammalian heterochromatin and genome stability. Cell 107(3):323–337. centromeric regions rely on BEND3 and DNA methylation. Mol Cell 56(4):580–594. 32. Allis CD, et al. (2007) New nomenclature for chromatin-modifying enzymes. Cell 63. Jamieson K, Selker E (2015) Control of histone H3 lysine 27 trimethylation in Neu- 131(4):633–636. rospora crassa. PhD dissertation. (University of Oregon, Eugene).

Basenko et al. PNAS | Published online November 2, 2015 | E6347 Downloaded by guest on September 30, 2021 64. Lewis PW, et al. (2013) Inhibition of PRC2 activity by a gain-of-function H3 mutation 82. Puschendorf M, et al. (2008) PRC1 and Suv39h specify parental asymmetry at con- found in pediatric glioblastoma. Science 340(6134):857–861. stitutive heterochromatin in early mouse embryos. Nat Genet 40(4):411–420. 65. Lehnertz B, et al. (2003) Suv39h-mediated histone H3 lysine 9 methylation directs 83. Davis RH (2000) Neurospora: Contributions of a (Oxford Univ Press, DNA methylation to major satellite repeats at pericentric heterochromatin. Curr Biol Oxford). 13(14):1192–1200. 84. Pall ML (1993) The use of Ignite (Basta;glufosinate;phosphinothricin) to select 66. Smallwood A, Estève PO, Pradhan S, Carey M (2007) Functional cooperation between transformants of bar-containing plasmids in Neurospora crassa. Fungal Genet Newsl HP1 and DNMT1 mediates gene silencing. Genes Dev 21(10):1169–1178. 40(1):58. 67. Estève PO, et al. (2006) Direct interaction between DNMT1 and G9a coordinates DNA 85. Davis RH, de Serres FJ (1970) [4] Genetic and microbiological research techniques for – and histone methylation during replication. Genes Dev 20(22):3089 3103. Neurospora crassa. Methods Enzymol 17(1):79–143. 68. Gaydos LJ, Rechtsteiner A, Egelhofer TA, Carroll CR, Strome S (2012) Antagonism 86. Margolin BS, Freitag M, Selker EU (1997) Improved plasmids for gene targeting at between MES-4 and Polycomb repressive complex 2 promotes appropriate gene the his-3 locus of Neurospora crassa by electroporation. Fungal Genet Newsl 44(1): – expression in C. elegans germ cells. Cell Reports 2(5):1169 1177. 34–36. 69. Patel T, Tursun B, Rahe DP, Hobert O (2012) Removal of Polycomb repressive com- 87. Pomraning KR, Smith KM, Freitag M (2009) Genome-wide high throughput analysis plex 2 makes C. elegans germ cells susceptible to direct conversion into specific so- of DNA methylation in eukaryotes. Methods 47(3):142–150. – matic cell types. Cell Reports 2(5):1178 1186. 88. Ji L, et al. (2014) Methylated DNA is over-represented in whole-genome bisulfite 70. Adhvaryu KK, Morris SA, Strahl BD, Selker EU (2005) Methylation of histone H3 lysine sequencing data. Front Genet 5:341. 36 is required for normal development in Neurospora crassa. Eukaryot Cell 4(8): 89. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat 1455–1464. Methods 9(4):357–359. 71. Tardat M, et al. (2015) Cbx2 targets PRC1 to constitutive heterochromatin in mouse 90. Trapnell C, et al. (2012) Differential gene and transcript expression analysis of RNA- zygotes in a parent-of-origin-dependent manner. Mol Cell 58(1):157–171. seq experiments with TopHat and Cufflinks. Nat Protoc 7(3):562–578. 72. Lee HC, et al. (2010) Diverse pathways generate microRNA-like RNAs and Dicer-independent 91. Galagan JE, et al. (2003) The genome sequence of the filamentous fungus Neuros- small interfering RNAs in fungi. Mol Cell 38(6):803–814. pora crassa. Nature 422(6934):859–868. 73. Rozenzhak S, et al. (2010) Rad3 decorates critical chromosomal domains with 92. Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): gammaH2A to protect genome integrity during S-Phase in fission yeast. PLoS Genet High-performance genomics data visualization and exploration. Brief Bioinform 6(7):e1001032. 14(2):178–192. 74. Goodrich J, et al. (1997) A Polycomb-group gene regulates homeotic gene expres- 93. Robinson JT, et al. (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24–26. sion in Arabidopsis. Nature 386(6620):44–51. 94. Heinz S, et al. (2010) Simple combinations of lineage-determining transcription 75. Lewis EB (1978) A gene complex controlling segmentation in Drosophila. Nature 276(5688):565–570. factors prime cis-regulatory elements required for macrophage and B cell identities. – 76. Lee HC, et al. (2010) The DNA/RNA-dependent RNA polymerase QDE-1 generates Mol Cell 38(4):576 589. aberrant RNA and dsRNA for RNAi in a process requiring replication protein A and a 95. Hebenstreit D, et al. (2011) EpiChIP: Gene-by-gene quantification of epigenetic DNA helicase. PLoS Biol 8(10):e1000496. modification levels. Nucleic Acids Res 39(5):e27. 77. Lee HC, et al. (2009) qiRNA is a new type of small interfering RNA induced by DNA 96. Priebe S, Kreisel C, Horn F, Guthke R, Linde J (2015) FungiFun2: A comprehensive damage. Nature 459(7244):274–277. online resource for systematic analysis of gene lists from fungal species. Bioinformatics 78. Chen ES, et al. (2008) Cell cycle control of centromeric repeat transcription and 31(3):445–446. heterochromatin assembly. Nature 451(7179):734–737. 97. Martin M (2011) Cutadapt removes adapter sequences from high-throughput se- 79. Vissers JH, van Lohuizen M, Citterio E (2012) The emerging role of Polycomb re- quencing reads. EMBnet Journal 17(1):10–12. pressors in the response to DNA damage. J Cell Sci 125(Pt 17):3939–3948. 98. Quast C, et al. (2013) The SILVA ribosomal RNA gene database project: Improved data 80. Fillmore CM, et al. (2015) EZH2 inhibition sensitizes BRG1 and EGFR mutant lung processing and web-based tools. Nucleic Acids Res 41(Database issue):D590–D596. tumours to TopoII inhibitors. Nature 520(7546):239–242. 99. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient 81. Weinhofer I, Hehenberger E, Roszak P, Hennig L, Köhler C (2010) H3K27me3 pro- alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25. filing of the endosperm implies exclusion of polycomb group protein targeting by 100. Freitag M, Hickey PC, Khlafallah TK, Read ND, Selker EU (2004) HP1 is essential for DNA methylation. PLoS Genet 6(10):e1001152. DNA methylation in neurospora. Mol Cell 13(3):427–434.

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