Genome-wide redistribution of H3K27me3 is linked to genotoxic stress and defective growth

Evelina Y. Basenko; Takahiko Sasaki; Lexiang Ji; Cameron J. Prybol; Rachel M. Burckhardt; Robert J. Schmitz; Zachary A. Lewis

doi:10.1073/pnas.1511377112

Edited by Jay C. Dunlap, Geisel School of Medicine at Dartmouth, Hanover, NH, and approved October 2, 2015 (received for review June 10, 2015)

Significance

Regulators of chromatin structure play critical roles in DNA-based processes. Lysine (K) Methyltransferase 1 (KMT1) homologs perform methylation of H3 lysine-9 and are best known for their essential role in heterochromatin formation and transcriptional silencing. Heterochromatin formation is also important for maintenance of genome stability, although the mechanisms are not well understood. We report that altered activity of Polycomb repressive complex-2 (PRC2), a histone lysine-27 methyltransferase complex, is responsible for genotoxic stress, poor growth, and defective development in KMT1-deficient mutants of Neurospora crassa. Mammalian KMT1 and PRC2 are required for development and are frequently mutated in cancer. This work provides information about the cellular consequences of KMT1 and PRC2 deficiency and provides insights into the regulatory and functional relationships of these conserved enzymes.

Abstract

H3K9 methylation directs heterochromatin formation by recruiting multiple heterochromatin protein 1 (HP1)-containing complexes that deacetylate histones and methylate cytosine bases in DNA. In Neurospora crassa, a single H3K9 methyltransferase complex, called the DIM-5,-7,-9, CUL4, DDB1 Complex (DCDC), is required for normal growth and development. DCDC-deficient mutants are hypersensitive to the genotoxic agent methyl methanesulfonate (MMS), but the molecular basis of genotoxic stress is unclear. We found that both the MMS sensitivity and growth phenotypes of DCDC-deficient strains are suppressed by mutation of embryonic ectoderm development or Su-(var)3-9; E(z); Trithorax (set)-7, encoding components of the H3K27 methyltransferase Polycomb repressive complex-2 (PRC2). Trimethylated histone H3K27 (H3K27me3) undergoes genome-wide redistribution to constitutive heterochromatin in DCDC- or HP1-deficient mutants, and introduction of an H3K27 missense mutation is sufficient to rescue phenotypes of DCDC-deficient strains. Accumulation of H3K27me3 in heterochromatin does not compensate for silencing; rather, strains deficient for both DCDC and PRC2 exhibit synthetic sensitivity to the topoisomerase I inhibitor Camptothecin and accumulate γH2A at heterochromatin. Together, these data suggest that PRC2 modulates the response to genotoxic stress.

Covalent modifications of histones and DNA partition genomes into discrete functional domains. Heterochromatin refers to highly condensed parts of the genome that are transcriptionally inert and rich in repeated DNA sequences (1). Failure to establish or maintain heterochromatin leads to catastrophic defects in chromosome segregation, DNA replication, and DNA repair, highlighting its functional importance (1 ⇓–3). At the molecular level, heterochromatin domains are defined by hypoacetylated histones, histone H3K9 methylation (H3K9me), and DNA methylation (1). The fungus Neurospora crassa shares these features with higher eukaryotes and is an established model to study the control and function of heterochromatin (4). In Neurospora, the H3K9 methyltransferase complex, named the defective in methylation (DIM)-5,-7,-9, Cullin 4, DNA damage-binding protein 1 (DDB1) Complex (DCDC), initiates heterochromatin formation at degenerate DNA repeat sequences that are products of the genome defense system repeat-induced point mutation (RIP) (5 ⇓–7). DCDC trimethylates H3K9 to create binding sites for multiple heterochromatin protein 1 (HP1)-containing complexes, which in turn direct methylation of cytosine bases in DNA and deacetylation of histones (8 ⇓–10). As in other eukaryotes, heterochromatin formation is sufficient to silence transcription in Neurospora. Reporter genes flanked by RIP’d DNA are not expressed, and spreading of heterochromatin causes aberrant gene silencing in strains that lack DNA methylation modulator-1 (DMM-1) (11, 12). Together, heterochromatin domains comprise ∼20% of the Neurospora genome and include structurally important loci such as the centromeres (7, 13).

In animals, plants, and some fungi, Polycomb (Pc) group proteins establish and maintain a second type of transcriptionally silent chromatin. Pc-target domains are often referred to as facultative heterochromatin because these regions are condensed and transcriptionally repressed in some but not all cell types (14). Polycomb repressive complex-2 (PRC2) methylates H3K27, which in animals can be bound by PRC1 to promote mitotically heritable gene silencing (15, 16). However, the mechanisms that control Pc complexes are not fully understood. In some situations, H3K27me3-independent recruitment of PRC1 can occur, leading to subsequent PRC2 recruitment and deposition of H3K27me3 (17, 18). Pc components are absent from the model yeasts Saccharomyces cerevisiae and Schizosaccharyomyces pombe, whereas PRC1 appears to be absent from all fungi (19). PRC2 is present in some fungi, however, and H3K27me3 directs transcriptional repression of PRC2-target domains in Neurospora, Fusarium graminearum, Epichloë festucae, and Cryptococcus neoformans (19 ⇓⇓⇓–23). Thus, the application of microbial genetic approaches to study the Pc system in fungi can provide mechanistic insights into this evolutionarily conserved chromatin regulatory system.

Although the Pc system is best known for its ability to repress developmentally regulated genes, recent studies in higher eukaryotes link H3K27 methylation to DNA replication and repair. In human cancer cells, PRC2 associates more stably with chromatin 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 sensitivity to ionizing radiation (25). It is possible that Pc proteins are recruited to silence transcription in the vicinity of a DNA lesion, but the precise roles of PRC2 and H3K27me3 in the DNA damage response are unclear.

Similarly, defects in constitutive heterochromatin formation are associated with genome instability. Replication fork stalling is observed in S. pombe heterochromatin domains (27), and Clr4^KMT1-deficient mutants, which lack H3K9me2, exhibit illegitimate mitotic recombination that is exacerbated by mutation of the replication fork protection complex (3, 28). In Drosophila, cytological studies revealed that H3K9me-deficient mutants exhibit spontaneous DSBs in heterochromatin, and it was proposed that this damage is due to defective DNA replication (2, 29, 30). Mice lacking Lysine (K) Methyltransferase 1A (KMT1A) (SUV39H1) and KMT1B (SUV39H2) exhibit genome instability and high rates of lymphoma development (31, 32), and both KMT1 enzymes and HP1 proteins have been implicated in DNA repair in animals (33 ⇓⇓⇓⇓⇓⇓⇓⇓–42). These and other studies suggest that heterochromatin components have important roles during both DNA replication and DNA repair, but the heterochromatin-dependent mechanisms that maintain genome integrity remain poorly defined.

Heterochromatin formation is important for genome maintenance in Neurospora as well. H3K9 methylation is required for normal vegetative growth and for sexual development (6). DCDC-deficient mutants exhibit chromosome segregation defects and hypersensitivity to the DNA-damaging agent methylmethanesulfonate (MMS) (5), and mutants lacking the catalytic subunit of DCDC, defective in methylation-5/kmt1 (dim-5), exhibit increased rates of mitotic recombination (43). In addition, during normal replicative growth, dim-5 strains display induction and redistribution of γH2A, a phosphorylated form of H2A that is induced by DNA damage or DNA replication stress (44 ⇓–46). These data suggest that heterochromatin is critically important in Neurospora, but the cause of genotoxic stress and impaired growth in heterochromatin-defective mutants is not understood.

To understand the molecular consequences of heterochromatin depletion, we isolated a genetic suppressor of a DCDC-deficient strain. We identified a mutation in the gene encoding the PRC2 component embryonic ectoderm development (EED) in mouse (19, 47). H3K9me3-deficient mutants exhibit redistribution of H3K27me3, leading to induction of PRC2-target genes. We show that gain of H3K27me3 in constitutive heterochromatin domains is not compensatory for gene silencing but rather leads to growth defects and altered sensitivity to genotoxic agents. Our data suggest that PRC2 modulates the response to genotoxic stress.

Results

Δdim-5 mutants exhibit hypersensitivity to the DNA-damaging agent MMS, suggesting that the DIM-5^KMT1 MTase is required for normal DNA replication or repair (5). We examined growth of heterochromatin-defective mutants in the presence of additional DNA replication and repair inhibitors (Fig. 1A). As controls, we included mutagen-sensitive-9 (mus-9), lacking the Neurospora ATM homolog required for the DNA damage response (48), and mei-3, lacking the Neurospora homolog of RAD51 required for homologous recombination (49). Elimination of DNA methylation did not impact growth on any of the tested genotoxic agents. Strains lacking the DCDC components DIM-5, DIM-7, or DIM-9 were hypersensitive to MMS compared with wild type, as previously described (5). These strains were sensitive to other genotoxic agents including the topoisomerase II inhibitor etoposide (50), the interstrand cross-linking agents cisplatin and Mitomycin C (51, 52), and Bleomycin (53), which is thought to cause oxidative damage and trigger DSBs. DCDC-deficient cells were efficiently killed by low concentrations of MMS (0.025%), whereas other agents only led to growth inhibition of Δdim-5, Δdim-7, Δdim-9, and hpo strains; that is, these strains were able to grow after prolonged incubation in the presence of drugs (>4 d), whereas growth of mus-9 and mei-3 did not improve. In contrast, Δdim-5, Δdim-7, Δdim-9, and hpo displayed only limited sensitivity to the topoisomerase I inhibitor Camptothecin (CPT) (50). Mutants lacking the DCDC component CUL4 were more sensitive to all genotoxic agents tested, which is expected given the established role of CUL4 in multiple complexes that regulate DNA replication and repair (54, 55). The fact that heterochromatin-deficient mutants exhibit broad sensitivity to DNA-damaging agents suggests that DCDC-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).

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).

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 encodes 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 in the Δdim-9 strain (5). A cross of one putative suppressor strain (suppressor-24) yielded two classes of Δdim-9 progeny in approximately equal numbers: MMS-tolerant and MMS-hypersensitive (Fig. 1B). We quantified colony forming units to compare 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 was similar to wild type (Fig. 1C). This suggested that a single mutation led to suppression of the Δdim-9 MMS-hypersensitivity phenotype and that this mutation is unlinked to dim-9.

To identify the causative mutation, we performed bulked segregant analysis combined with whole genome sequencing (BSA-seq) (56). We crossed a single Δdim-9; sup-24 homokaryon, generated in the Oak Ridge strain background (OR), to a polymorphic wild-type Neurospora strain (Mauriceville, MV). Progeny were isolated and genotyped to identify Δdim-9; sup-24 strains. We then isolated and pooled genomic DNA from 14 Δdim-9; sup-24 progeny and sequenced the DNA in bulk. Analysis of the sequenced data revealed that regions on Linkage Group II (LGII) and LGIV contained SNPs that were exclusively from the OR strain background and contained mutations (Fig. S1). The LGII region contained the dim-9 deletion. The region at the right end of LGIV contained a G to A base substitution in the gene encoding the single Neurospora EED homolog (Fig. 2A). This mutation is predicted to introduce a stop codon in place of a tryptophan codon at position 252. We hereafter refer to this allele as eed^sup-24.

Fig. 2.

PRC2 function is required for MMS hypersensitivity of DCDC-deficient mutants. (A) The frequency of OR and MV SNPs in progeny from a Δdim-9; sup-24 × MV cross are shown for LGIV. An OR-specific region on the right arm of LGIV harbors a nonsense mutation in the Neurospora eed gene. The site of the mutation is illustrated in a schematic cartoon above the plot. Gray shaded regions depict WD40 domains. (B) Western blots of acid-extracted histones are shown from wild type, Δdim-9, and Δdim-9; sup-24 probed with antibodies for H3K9me3 and H3K27me3. A gel stained with Coomassie Blue and a Western blot probed with antibodies to H3 are shown as loading controls. (C) Viability of spores (number of colonies) on the indicated MMS concentrations is shown for wild type, Δdim-5, Δset-7, and the Δdim-5; Δset-7 double mutant.

Fig. S1.

The frequency of OR and MV SNPs in progeny from a dim-9; sup-24 × MV cross are shown for all seven Neurospora LGs. An OR-specific region on the right arm of LGIV harbors a nonsense mutation in the Neurospora eed gene. The OR-specific region on LGII harbors the dim-9 gene. The short OR-specific region on LGI is detected in multiple strains backcrossed to MV. This region does not contain any mutations and may represent a translocation present in the MV strain.

Neurospora EED is a component of PRC2 and is required for methylation of H3K27 (19). We therefore asked if H3K27me3 levels are altered in the Δdim-9; eed^sup-24 double mutant. We isolated total histones from wild type, Δdim-9, and Δdim-9; eed^sup-24 strains and performed Western blots using antibodies that recognize H3K9me3 and H3K27me3 (Fig. 2B). As loading controls, we performed Western blots with antibodies for H3 and stained total histones with Coomassie Blue. H3K9me3 was detected in wild type but was absent in Δdim-9 and Δdim-9; eed^sup-24 strains, demonstrating that the suppressor mutation is unable to restore H3K9 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; eed^sup-24 double mutant, suggesting that the eed^sup-24 allele encodes a nonfunctional protein. These data raised the possibility that PRC2 causes the MMS-sensitivity phenotype observed for 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 dim-5 and set-7 genes encode the catalytic subunits of the DCDC H3K9 MTase (DIM-5^KMT1) and the PRC2 H3K27 MTase (SET-7^KMT6) complex, respectively. We isolated three siblings of each genotype and determined the level of MMS sensitivity for each strain by plating spores on increasing concentrations of the genotoxic agent (Fig. 2C). Wild type and Δset-7 displayed similar MMS tolerance, whereas Δdim-5 was hypersensitive. In contrast, Δdim-5; Δset-7 double-mutant progeny were able to survive in the presence of MMS, confirming that elimination of PRC2 leads to genetic suppression of the MMS-hypersensitivity phenotype of DCDC-deficient mutants.

H3K27 Methylation Is Targeted to Constitutive Heterochromatin in H3K9me-Deficient Mutants.

Heterochromatin components impact H3K27me3 localization in plant and animal cells (58 ⇓⇓⇓–62), and Jamieson and Selker observed similar results in Neurospora (63). We therefore performed ChIP-seq to determine if changes in the distribution of H3K27me3 were correlated with sensitivity of DNA-damaging agents in Δdim-5. As previously reported, H3K27me3 was localized to large chromatin domains of transcriptionally repressed genes (19). In contrast, in the Δdim-5 mutant, H3K27me3 was detected exclusively at A:T-rich regions of the genome that are marked by H3K9 methylation in wild-type cells (Fig. 3A). The enrichment of H3K27 methylation was highly reproducible in replicate experiments. We calculated normalized ChIP enrichment values (NCLS values) for individual genomic features (genes or repeats) and generated scatterplots (Fig. 3B; note that repeats in Neurospora share only <80% identity and completely identical stretches are very short). Replicate experiments for wild type or Δdim-5 yielded Pearson correlation coefficients of 0.992 and 0.993, respectively. In contrast, no correlation was observed when comparing H3K27me3 enrichment values in wild type and Δdim-5 (R = 0.152). This revealed global redistribution of H3K27me3 from genes to repeated sequences in Δdim-5. We next examined global changes in H3K27 methylation by creating heat maps of H3K9me3 and H3K27me3 at constitutive heterochromatin domains and at Pc-target domains (i.e., domains targeted in wild-type cells by DCDC or PRC2, respectively; see Materials and Methods) (Fig. 3 C and D). In the Δdim-5 strain, H3K27me3 enrichment was significantly reduced at all regions that are typically targeted by PRC2. In contrast, H3K27me3 enrichment was gained in most constitutive heterochromatin domains. Compared with the wild-type pattern of H3K9me3, H3K27me3 enrichment in heterochromatin was generally lower and more variable across individual heterochromatin domains (compare Fig. 3 C and D).

Fig. 3.

Genotoxic stress of H3K9me3- and HP1-deficient strains is correlated with global redistribution of H3K27me3. (A) The y 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.

These data suggest that H3K9 methylation is required for normal H3K27me3 enrichment patterns. To confirm this, we tested strains in which the single H3 gene had been replaced with an H3^K9R or an H3^K9Q substitution allele to mimic H3 with unmodified lysine-9 or acetylated lysine-9, respectively (46). Normal H3K27 methylation patterns were abolished in these mutants, similar to the case for Δdim-5 strains (Fig. S2). In animal cells, histones harboring H3K9 to M substitutions act as dominant inhibitors of KMT1 enzymes (64). We therefore introduced an H3^K9M substitution allele into the endogenous hH3 locus. Although we were able to generate heterokaryons containing the H3^K9M allele, crosses of transformants did not yield homokaryotic progeny. This indicates that the hH3^K9M allele is lethal, in contrast to hH3^K9Q and hH3^K9R alleles. Heterokaryons of hH3; hH3^K9M produced an intermediate distribution of H3K27me3 and a subtle reduction in DNA methylation (Fig. S2 A–C). Our data show that the H3^K9M protein is a weak dominant inhibitor of H3K9 methylation and that the H3^K9M protein disrupts other chromatin-based processes in Neurospora. Given the partial dominance of the hH3^K9M allele and the apparent pleiotropic effects of the H3^K9M protein in Neurospora, caution should be used when interpreting phenotypes of histone K to M alleles in other organisms.

Fig. S2.

H3K27me3 localization is altered in H3K9me3-deficient mutants. (A) Relative ChIP-seq enrichment across the entire LGVII is shown for H3K9me3 (blue) and H3K27me3 (green) in wild type. The H3K27me3 distribution is shown for the indicated strains. (Scale bar, 0.5 Mb.) (B) H3K27me3 enrichment is shown across a region of LGVII and is shown for the indicated strains. (Scale bar, 50 kb.) (C) For the indicated strains, genomic DNA was digested with methylation-sensitive and -insensitive isoschizomers BfuCI [B] and DpnII [D], and Southern blots were probed for the typically methylated region 8:G3 to determine the level of DNA methylation. Genomic DNA was also digested with HindIII and probed for the hH3 gene to determine the ratio of nuclei containing wild-type hH3 (FghH3) and hH3^K9M. (D) ChIP-seq enrichment across the entire LGVII is shown for H3K9me3 (blue) and H3K27me3 (green) in wild type and Δset-7 grown in minimal medium. Enrichment of H3K27me3 is also shown for wild-type cells (WT) exposed to MMS for the indicated number of hours.

Reduction of cytosine methylation in plant and animal cells 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 dependent on H3K9 methylation (65), and reduced DNA methylation can lead to a concomitant loss of H3K9 methylation in certain situations (60, 66, 67). In Neurospora, the heterochromatin 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). We generated heat maps to ask if H3K27me3 patterns were altered in hpo mutants, which lack HP1 and are sensitive to genotoxic agents, or dim-2 mutants, which lack DNA methylation and are insensitive to genotoxic agents. Consistent with previous data, heat maps revealed that enrichment of H3K9me3 was reduced at the boundaries of heterochromatin domains in the hpo strain (9). Despite the presence of significant H3K9me3 in the hpo mutant, the pattern of H3K27 methylation was similar to the Δdim-5 mutant (Fig. 3C). In contrast, H3K27me3 patterns in dim-2 mutants were more similar to wild type, although a subtle increase of H3K27me3 was observed globally in heterochromatin regions in the dim-2 strain (Fig. 3C). Thus, HP1 is required to prevent redistribution of H3K27me3 to heterochromatin, whereas 5mC has only a minimal role.

In Caenorhabditis elegans, spreading of H3K27me3 into adjacent 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 performed ChIP-seq for H3K27me3 in set-1 and set-2 mutants, which are deficient for H3K4 and H3K36 methylation, respectively (Fig. S2) (70). We observed changes in the relative level of H3K27me3 enrichment in the set-2 strain at some Pc-target domains, but the 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 histone methylation pathways lead to global redistribution of H3K27me3. In addition, we found that H3K27me3 patterns were unaltered when wild-type cells were grown in the presence of MMS (Fig. S2D). Hence, exposure to DNA-damaging agents is not sufficient to trigger redistribution of H3K27me3 to heterochromatin.

It has been proposed that redistribution of H3K27me3 to constitutive heterochromatin domains is a compensatory response for maintenance of heterochromatic gene silencing (62, 71). To test if this is the case in Neurospora, we asked if Δdim-5; Δset-7 double mutants exhibit increased transcription from constitutive heterochromatin domains. We first performed ribosomal RNA subtraction followed by strand-specific total RNA-seq in wild type, Δdim-5, Δset-7, and Δdim-5; Δset-7 double mutants. Expression levels of misregulated genes and their associated gene ontology (GO) annotations are included in Dataset S1. Significantly enriched GO terms are shown in Dataset S2. A significant number of H3K27me3-associated genes were up-regulated in the Δdim-5 mutant, consistent with the observed loss of H3K27me3 from these domains (Fig. 4 A and B). In contrast, we did not detect RNAs originating from heterochromatin domains in any of the strains tested. Box plots of fragments per kilobase per million mapped reads (FPKM) values calculated for repeats and for entire constitutive heterochromatin domains on both plus and minus strands revealed few detectable transcripts in any of the strains examined (Fig. 4 C and D). Although both Δdim-5 and Δset-7 strains showed induction of H3K27me3-associated genes, there was incomplete overlap between the sets of induced genes in the two strains (Fig. S3 A–D). We note that H3K27me3-associated genes had low levels of expression compared with the average gene, even in Δdim-5 and Δset-7 (compare Fig. 4 A and B). Although most of these genes have unknown functions, many genes that were down-regulated in Δdim-5 are associated with metabolism and growth (Dataset S1), which is likely an indirect effect of the poor growth phenotype of Δdim-5.

Fig. 4.

H3K27me3 does not compensate for loss of silencing at heterochromatin regions. (A–D) Box plots depict average expression level (log[FPKM+1]) from strand-specific RNA-seq experiments for (A) all Neurospora genes, (B) H3K27me3-enriched genes, (C) DNA repeats, and (D) heterochromatin domains for the indicated strains. FPKM values are shown for both plus and minus strands for C and D. (E and F) Box plots show the levels of 20–24 nt RNAs (log[FPKM+1]) for (E) DNA repeats and (F) heterochromatin domains for the indicated strains. The notches indicate the 95% confidence interval around the median.

Fig. S3.

H3K27me3-associated genes are induced in dim-5 and in set-7. (A) Venn diagram comparing genes associated with H3K27me3 to genes induced in dim-5 and set-7. H3K27me3-enriched genes had a log[NLCS] value > 4.5. (B) Venn diagram comparing genes associated with H3K27me3 to genes repressed in dim-5 and set-7. (C) The heat map depicts H3K27me3 enrichment (Left) and relative expression levels (Right) for wild type, dim-5, and set-7. FPKM values are normalized by row. Genes are ordered from top to bottom by the level of H3K27me3 enrichment (highest to lowest). (D) The heat map depicts expression levels in wild type, dim-5, and set-7 for the 548 genes with the highest H3K27me3 enrichment levels (based on NLCS value). FPKM values are normalized by row. (E) The percent of total 20–24 nucleotide RNA reads with the indicated base in the 5′ position is shown.

It is possible that RNA surveillance pathways rapidly degrade heterochromatin-derived transcripts (43). We therefore sequenced small RNA libraries and examined the level of 20–24 nucleotide (nt) RNAs originating from heterochromatin domains. We determined the levels of small RNAs originating from repeats and heterochromatin domains. The Δdim-5 and Δdim-5; Δset-7 strains had higher levels of small RNAs originating from individual RIP’d repeats and from entire heterochromatin domains compared with wild type or Δset-7 (Fig. 4 E and F). The levels of small RNAs from heterochromatin domains were slightly higher in the Δdim-5; Δset-7 double mutant than in the Δdim-5 strain alone, although the additional increase was minimal. We examined the first base of heterochromatin-derived small RNAs and found that the majority began with a U (Fig. S3E), suggesting that these RNAs are products of the siRNA pathway in Neurospora (72). These data suggest that loss of H3K9me3 leads to increased siRNA production from heterochromatin domains and that H3K27me3 plays a minimal role in limiting heterochromatin-derived siRNAs.

Aberrant H3K27me3 Is Responsible for Defective Sexual Development and Poor Growth of dim-5 Strains.

The fact that Δset-7 strains grow normally and are not hypersensitive to MMS (Fig. 2D) suggests that loss of H3K27me3 from facultative heterochromatin does not lead to significant phenotypes. Rather, the data suggest that gain of H3K27me3 at constitutive heterochromatin domains is linked to genotoxic stress in heterochromatin-defective mutants. To determine if MMS sensitivity is caused by aberrant localization of H3K27me3, we replaced the wild-type hH3 gene with an hH3^K27R or an hH3^K27Q substitution allele and crossed the hH3^K27 mutant strains to Δdim-5. The resulting Δdim-5; hH3^K27R or Δdim-5; hH3^K27Q double-mutant progeny were resistant to MMS, suggesting that aberrant H3K27me3 is responsible for MMS hypersensitivity of the Δdim-5 strain (Fig. 5).

Fig. 5.

H3K27 methylation is responsible for MMS sensitivity of H3K9-deficient strains. Suspensions of conidia (10⁴–10¹) of the indicated strains were spot-tested on media with or without MMS (0.02%).

In addition to hypersensitivity to genotoxic agents, DCDC-deficient mutants display slow growth and fail to complete sexual development (6). We asked if these defects were also rescued by removal of PRC2. Both vegetative and sexual development defects of Δdim-5 single mutants were rescued by deletion of the Δset-7 gene. The linear growth rate of Δdim-5; Δset-7 double mutants was significantly faster than Δdim-5 single mutants yet slower than wild type (Fig. 6 A and B). Similarly, homozygous crosses of Δdim-5; Δset-7 double mutants were fertile, in contrast to homozygous crosses of Δdim-5 single mutants. Crosses of Δdim-5; Δset-7 gave rise to fruiting body structures filled with spores (Fig. 6C), although spore production was delayed and the number of mature asci observed in squashed perithecia was reduced compared with wild type. These data demonstrate that altered PRC2 targeting is responsible for vegetative and sexual phase growth defects observed in heterochromatin-defective mutants.

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 heterochromatin domains. Domains are arranged from smallest (Top) to largest (Bottom). (F) Suspensions of 10⁴, 10³, or 10² 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).

γH2A is a phosphorylated form of H2A that is induced by DNA damage or DNA replication stress (44, 45). We previously showed that γH2A is enriched in heterochromatin domains in wild-type cells and that γH2A is deposited throughout the genome in the Δdim-5 mutant, leading to loss of region-specific enrichment (46). We performed ChIP-seq for γH2A in wild type, Δdim-5, Δset-7, and Δdim-5; Δset-7 double mutants (Fig. 6D). As observed previously, γH2A was enriched in constitutive heterochromatin domains in wild type, but enrichment was lost in the Δdim-5 strain. The pattern of γH2A enrichment in the Δset-7 mutant was equivalent to wild type. In the Δdim-5; Δset-7 double mutant, γH2A was enriched at constitutive heterochromatin domains with a distribution that appeared similar to wild type. We created heat maps to visualize γH2A enrichment in all constitutive heterochromatin domains (Fig. 6E). Wild type and Δset-7 strains displayed the highest levels of γH2A enrichment at the edges of RIP’d regions, whereas in the Δdim-5; Δset-7 double-mutant strains, the levels of γH2A enrichment at the boundaries of heterochromatin domains were lower and the borders of individual γH2A peaks were not as discrete as in wild type. In S. pombe, it was proposed that H3K9me was required for γH2A deposition in heterochromatin domains (73). However, our data show that γH2A deposition in heterochromatin can occur independently of H3K9me in N. crassa. One possible reason for the accumulation of γH2A in Δdim-5; Δset-7 strains is that stalled replication forks or frequent DSBs occur at repeat-rich regions of the genome in these cells.

Neurospora PRC2 Modulates the Response to Genotoxic Stress.

We next asked if sensitivity to other DNA-damaging agents was rescued in the Δdim-5; Δset-7 double mutant (Fig. 6F). The double mutant was more tolerant than Δdim-5 to all genotoxic agents, with one exception: Cells that lacked both DCDC and PRC2 exhibited increased sensitivity to the topoisomerase I inhibitor CPT. To confirm that loss of H3K27me3 leads to increased CPT sensitivity, we examined strains with mutant H3 genes resulting in an H3K9 substitution, an H3K27 substitution, or substitutions of both K9 and K27. hH3^K9Q strains were mildly sensitive to MMS and CPT. In contrast, hH3^K9Q/K27Q or hH3^K9Q/K27R strains were both able to grow in the presence of MMS but were hypersensitive to CPT. A Δset-7; hH3^K9Q strain was similarly hypersensitive to CPT (Fig. S4). Together, our results suggest that PRC2 and its product H3K27me3 modulate the cellular response to genotoxic stress in Neurospora.

Fig. S4.

(A) Loss of H3K27me3 and H3K9me3 leads to MMS tolerance and synthetic sensitivity to CPT.

Discussion

Heterochromatin components are required for normal growth and development and are important for maintenance of genome integrity. We found that in Neurospora, loss of H3K9me3 or HP1 results in redistribution of H3K27me3, which in turn leads to growth defects and hypersensitivity to MMS. The fact that redistribution of H3K27me3 is deleterious in Neurospora is somewhat surprising; however, a similar observation may have been difficult to make in other experimental systems. In plants and animals, loss of heterochromatin components leads to similar redistribution of H3K27me3 (17, 58 ⇓⇓⇓–62), but in these systems, depletion of H3K27me3 from Pc-target regions is sufficient to cause poor growth and developmental defects (74, 75). Thus, in many organisms, it would be difficult to determine if the phenotypic defects displayed by H3K9me3-deficient strains were caused by loss of H3K27me3 from native sites or by gain of H3K27me3 in heterochromatin domains. In contrast, because loss of H3K27me3 produces only minor phenotypic defects in Neurospora, it is clear that gain of H3K27me3 in typically heterochromatic domains has severely deleterious consequences. It is not known if redistribution of H3K27me3 is also linked to growth defects and genotoxic stress in higher eukaryotes, but such a scenario is consistent with the fact that depletion of heterochromatin components is associated with genome instability in animals (2, 31).

It has been proposed that redistribution of H3K27me3 to constitutive heterochromatin in animals is a compensatory mechanism to silence transcription. We did not find evidence to support such a role in N. crassa. Heterochromatin-derived transcripts were not increased in cells lacking H3K9me3, H3K27me3, or both. We did detect an increase in small RNA production from heterochromatin domains in dim-5 mutants, but small RNA levels were not substantially higher when cells lacked both H3K9me3 and H3K27me3. Notably, genotoxic stress triggers aberrant RNA synthesis by QDE1 and subsequent quelling defective 2-interacting RNA (qiRNA) generation in Neursopora (76, 77), suggesting that the observed increase in small RNAs may result from replication or repair defects in heterochromatin domains rather than loss of transcriptional silencing.

It is not clear why redistribution of H3K27me3 leads to poor growth and MMS sensitivity in Neurospora. Previous work suggests that abnormal localization of γH2A is not the cause of the MMS-sensitivity phenotype. Although site-specific enrichment of γH2A is lost in the Δdim-5 mutant, this is the result of increased γH2A in euchromatin rather than a decrease in γH2A at heterochromatin (46). Moreover, double mutants of Δdim-5 and hH2A^S131A display an additive increase in MMS sensitivity (46), suggesting that loss of DIM-5 and γH2A lead to MMS sensitivity through independent mechanisms. Hence, it is likely that elevated levels of γH2A are a response to genotoxic stress in Δdim-5 rather than the cause of genotoxic stress. We propose three possible mechanisms for H3K27me3-dependent genotoxic stress that should be explored in future work. It is possible that H3K27me3 leads to genotoxic stress by interfering with centromere formation, as both dim-5 and hp1 mutants exhibit defects in CenH3 deposition (13). Another possibility is that aberrant H3K27me3 may interfere with chromatin dynamics during the S phase. In S. pombe, HP1 is displaced from the chromatin fiber in a cell cycle-specific manner, facilitating transcription of centromeric repeats during the S phase (78). It is possible that H3K27me3 assembles condensed chromatin that is not subject to appropriate regulation over the course of the cell cycle. Alternatively, it is also possible that H3K27me3 accumulates in heterochromatin as part of a genotoxic stress response that triggers cell cycle arrest or programmed cell death. Data from other organisms and from this study suggest that defective heterochromatin formation is sufficient to cause genotoxic stress. For example, S. pombe lacks H3K27me3, but heterochromatin-defective mutants suffer frequent illegitimate recombination (3, 28). In the present study, we found that γH2A is enriched in heterochromatin domains in Δset-7; Δdim-5 double mutants, suggesting that defective replication or repair occurs in repeat-rich domains of Neurospora even when both H3K9me3 and H3K27me3 are absent. PRC2 recruitment could be a response to genotoxic stress at these sites. Indeed, we found that Neurospora Δdim-5; Δset-7 double mutants exhibit synthetic sensitivity to CPT, suggesting that H3K27me3 is required for cells to mount a proper response to CPT-induced damage in the Δdim-5 background. The idea that H3K27me3 is part of a DNA damage response is also supported by reports that PRC2 and other Pc components localize to DNA damage sites in mammalian cells and that EZH2 depletion leads to increased sensitivity to ionizing radiation in mammals (24 ⇓–26, 79). On the other hand, we did not observe redistribution of H3K27me3 when wild-type cells were exposed to genotoxic agents. Additional studies are needed to identify the mechanisms that link H3K27me3 and genotoxic stress.

Our finding that loss of PRC2 activity can lead to differential sensitivity of genotoxic agents has possible implications for improved cancer treatment. Components of constitutive heterochromatin and Pc system components are frequently mutated or overexpressed in cancer cells, and it was recently reported that EGFR and BRG1 mutant tumors exhibit enhanced sensitivity to topoisomerase II inhibitors when EZH2 is inhibited (80). In contrast, we found here that Neurospora strains lacking PRC2 and DCDC become more resistant to the topoisomerase II inhibitor etoposide as well as to several other DNA-damaging agents but display enhanced sensitivity to the topoisomerase I inhibitor CPT. These results raise the possibility that EZH2 inhibition may have significantly different consequences in different genetic backgrounds.

H3K9me3, HP1, and 5mC have all been implicated in preventing H3K27me3 redistribution to heterochromatin in animals, and reduction of 5mC leads to redistribution of H3K27me3 in plants (17, 58 ⇓⇓⇓–62, 71). These observations highlight the need for additional studies to fully elucidate the complex functional and regulatory relationships between heterochromatin and Pc system components. Even within the fungi, multiple heterochromatin-dependent mechanisms can impact H3K27me3 distribution. We found here that H3K27me3 redistribution occurs in H3K9me3- and HP1-deficient mutants of N. crassa, in agreement with other work (63). In C. neoformans, deletion of an H3K27me3-binding protein leads to accumulation of H3K27me3 at centromeres. However, redistribution of H3K27me3 requires H3K9me2 in this fungus (23). This may also be the case in E. festucae, where deletion of the H3K27 MTase ΔezhB does not rescue the growth defects of the H3K9 MTase-deficient mutant ΔclrD (22). In plants and animals, cross-talk between different heterochromatin components and the Pc system has been linked to cell type-specific changes in chromatin architecture, raising the possibility that different heterochromatin-based mechanisms could regulate H3K27me3 in different cells even within the same organism (71, 81, 82).

Despite apparent mechanistic differences, the fact that Pc components can be conditionally recruited to constitutive heterochromatin domains in plants, animals, and fungi suggests shared mechanisms exist. We speculate that these mechanisms involve genotoxic stress. Indeed, mammalian PRC2 and NuRD complexes are simultaneously recruited to chromatin when heterochromatin components are depleted and when DNA damage is introduced (25, 62). Moreover, the NuRD component BEND3 was recently shown to be required for PRC2 recruitment to heterochromatin domains in 5mC- or H3K9me3-deficient cells (62). Given that fungi and plants lack BEND3, other proteins must contribute to PRC2 recruitment to constitute heterochromatin in these organisms. Our study highlights the power of fungal genetics for investigating the control and the functions of the Pc system and suggests that future studies in Neurospora are likely to provide clues into the relationships between the Pc system, heterochromatin, and genotoxic stress.

Materials and Methods

Strains and Growth Media.

All Neurospora strains used in this study are listed in Table S1. Strains were grown at 32 °C in Vogel’s minimal medium (VMM) + 1.5% (wt/vol) sucrose (83). Liquid cultures were shaken at 150 rpm. Crosses were performed on modified synthetic cross-medium (83). For plating assays, Neurospora conidia were plated on VMM with 2.0% sorbose, 0.5% fructose, and 0.5% (wt/vol) glucose. When relevant, plates included 200 µg/mL hygromycin or 400 µg/mL basta (84) or DNA-damaging agents at the indicated concentration. We note that MMS is an alkylating agent that can react with components in the medium. Therefore, the effective inhibitory concentration can vary between experiments due to ambient temperature and age of the plates. For these reasons, strains were tested on multiple concentrations of MMS during each experiment. For survival curves, 200 cells were plated on minimal medium and on plates with increasing concentrations of MMS. The number of colonies was counted for each plate and plotted as a percentage of the no MMS control. Three independent strain isolates were used for each concentration of MMS, and the average percent viability was plotted. Linear growth rates were determined using “race tubes” (85).

View this table:

Table S1.

Strains used in this study

Molecular Analyses.

Neurospora transformation (86), DNA isolation (87), protein isolation, histone isolation, and Western blotting (10) were performed as previously described. Antibodies to γH2A (cat. no. ab15083, Abcam), H3K9me3 (cat. no. 39161, Active Motif), H3K27me3 (cat. no. 39537, Active Motif, or cat. no. 9733, Cell Signaling Technologies), and H3 (cat. no. 07–690, Millipore) were used as indicated. ChIP-seq was performed as described (46). The hH3^K9M strain was constructed by site-directed mutagenesis as described (46) using the following primers: K9M_F CAG ACC GCC CGC ATG TCC ACC GGT GGC AAG GCC CCC and K9M_R CAC CGG TGG ACA TGC GGG CGG TCT GCT TAG TGC GGG.

Illumina Sequencing.

For Illumina sequencing, ChIP-seq libraries were prepared using 10 ng of immunoprecipitated DNA following the instructions supplied with Illumina Tru-seq ChIP-seq kits (Illumina cat. no. FC-121-2002). RNA-seq libraries were prepared from 5 μg total RNA. Ribosomal RNAs were depleted using the yeast Ribo-zero kit (cat. no. MRZY1324, Epicentre), and RNA libraries were generated with the Illumina Stranded RNA-seq kit PCR (cat. no. RS-122-2101). For ChIP-seq, amplification was limited to 4–8 cycles to reduce PCR bias against A:T-rich DNA sequences (88). Illumina sequencing was performed using an Illumina NextSeq500 Instrument at the University of Georgia Genomics Facility.

Data Analysis.

Illumina sequence reads have been deposited into the National Center for Biotechnology Information Short Reads Archive (accession no. SRP058573). Short reads were mapped using bowtie2 (89) or TopHat (90) for ChIP-seq or RNA-seq experiments, respectively, to the latest Neurospora genome annotation (version 12), available from the Neurospora genome database (91). For ChIP-seq data, read numbers were determined for 25 base pair bins using igvtools, and the read count for each bin was normalized to the total read number in the sample. Relative enrichment data were visualized using the Integrated Genome Viewer (92, 93). For histograms showing relative ChIP enrichment at specific chromosomes or genomic loci, enrichment values for each sample are shown relative to the maximum enrichment value for the depicted window.

The Hypergeometric Optimization of Motif EnRichment (HOMER) software package (94) was used to identify H3K9me3 or H3K27me3 domains in wild type. The coordinates of H3K9me peaks and H3K27me3 peaks are listed in Datasets S3 and S4, respectively. We constructed a custom genome annotation file containing genes, repeats (46), H3K27me3 domains, and H3K9me3 domains and used HOMER to construct heat maps of ChIP enrichment across H3K9me3 or H3K27me3 domains (using the –ghist option). HOMER-generated matrix files or FPKM files were loaded into GENE-E (www.broadinstitute.org/cancer/software/GENE-E/) to generate heat map images. For scatter plots, NCLS values were calculated using EpiChIP software, which calculates enrichment values normalized for total read number and for length of the feature (95). Differential expression was determined using Cuffdiff (90). Genes were classified as differentially expressed if they passed criteria for statistical significance and had a minimum twofold difference in expression between wild type and the mutant. Functional classifications for differentially expressed genes were determined using Fungifun2 (96) with the following parameters. Significant enrichment of overrepresented functional categories was determined using Fisher’s exact test (P < 0.05), and P values were adjusted using the Benjamini–Hochberg procedure option.

We used 1 μg of total RNA for preparation of small RNA sequencing libraries according to the manufacturer’s protocol in the TruSeq Small RNA Sample Prep Kit from Illumina. Raw files were trimmed for adapters using cutadapt v1.7.1 (97), and only reads with sizes ranging from 20 to 24 nt were kept. Reads were then mapped to the N. crassa rRNA database (98) and annotated tRNA genes (based on N. crassa OR74A v12). Aligned reads were removed, and then all samples were subsampled based on the sample with the lowest number of reads, ensuring the same input read number for subsequent analyses. Qualified reads were aligned to the N. crassa OR74A v12 using bowtie 1.1.0 with the following parameters:–phred33-quals–nomaqround–best–strata–chunkmbs 1024 –e 1 –l 20 –n 0 -a –m 1000 (99). The FPKM method was used to quantify the small RNA distribution in each given region. Regions that did not meet a minimum coverage requirement (three reads) were not included in the box plot calculation.

Acknowledgments

We thank Laura Banken for technical contributions to the project. This work would not have been possible without materials generated by the Neurospora Functional Genomics Project (NIH Grant P01GM68087) provided by the Fungal Genetics Stock Center. This work was supported by American Cancer Society Grant RSG-14-184-01-DMC (to Z.A.L.) and NIH Grant R00GM100000 (to R.J.S.).

Footnotes

↵¹E.Y.B. and T.S. contributed equally to this work.
↵²To whom correspondence should be addressed. Email: zlewis{at}uga.edu.

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., and R.M.B. performed research; T.S. and C.J.P. contributed new reagents/analytic tools; E.Y.B., 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.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequence reported in this paper has been deposited in NCBI Short Reads Archive (accession no. SRP058573).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1511377112/-/DCSupplemental.

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Genome-wide redistribution of H3K27me3 is linked to genotoxic stress and defective growth

Significance

Abstract

Results

Isolation and Identification of a Genetic Suppressor of Δdim-9.

H3K27 Methylation Is Targeted to Constitutive Heterochromatin in H3K9me-Deficient Mutants.

Aberrant H3K27me3 Is Responsible for Defective Sexual Development and Poor Growth of dim-5 Strains.

Neurospora PRC2 Modulates the Response to Genotoxic Stress.

Discussion

Materials and Methods

Strains and Growth Media.

Molecular Analyses.

Illumina Sequencing.

Data Analysis.

Acknowledgments

Footnotes

References

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Search

Significance

Abstract

Results

Isolation and Identification of a Genetic Suppressor of Δdim-9.

H3K27 Methylation Is Targeted to Constitutive Heterochromatin in H3K9me-Deficient Mutants.

Aberrant H3K27me3 Is Responsible for Defective Sexual Development and Poor Growth of dim-5 Strains.

Neurospora PRC2 Modulates the Response to Genotoxic Stress.

Discussion

Materials and Methods

Strains and Growth Media.

Molecular Analyses.

Illumina Sequencing.

Data Analysis.

Acknowledgments

Footnotes

References

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Article Classifications

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