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In the filamentous fungus Neurospora crassa, constitutive heterochromatin is marked by tri-methylation of histone H3 lysine 9 (H3K9me3) and DNA methylation. We identified mutations in the Neurospora defective in methylation-1 (dim-1) gene that cause defects in cytosine methylation and implicate a putative AAA-ATPase chromatin remodeler. Although it was well-established that chromatin remodelers can affect transcription by influencing DNA accessibility with nucleosomes, little was known about the role of remodelers on chromatin that is normally not transcribed, including regions of constitutive heterochromatin. We found that dim-1 mutants display both reduced DNA methylation in heterochromatic regions as well as increased DNA methylation and H3K9me3 in some intergenic regions associated with highly expressed genes. Deletion of dim-1 leads to atypically spaced nucleosomes throughout the genome and numerous changes in gene expression. DIM-1 localizes to both heterochromatin and intergenic regions that become hyper-methylated in dim-1 strains. Our findings indicate that DIM-1 normally positions nucleosomes in both heterochromatin and euchromatin and that the standard arrangement and density of nucleosomes is required for the proper function of heterochromatin machinery.

The circadian rhythms found in almost all organisms are driven by molecular oscillators, including transcription/translation feedback loops (TTFLs). However, TTFL-independent oscillators can drive rhythms in both eukaryotes and prokaryotes. The fungus Neurospora crassa is a model organism for studying the molecular mechanism of the circadian clock. Although a circadian TTFL involving the proteins FRQ, WC-1, and WC-2 is well-characterized in N. crassa, rhythms can still be observed in the absence of this feedback loop. These rhythms are said to be driven by 1 or more FRQ-less oscillator(s) (FLOs). The prd-1 mutation lengthens the period in frq wild type and was previously shown to severely disrupt FRQ-less rhythms in frq null mutants under several different conditions; therefore, the prd-1 gene product is a candidate for a component of a FLO. We report here that prd-1 also disrupts free-running rhythms in wc-1 null mutants, confirming its effects on FRQ-less rhythms. We have now mapped and identified the prd-1 gene as NCU07839, a DEAD-box RNA helicase dbp-2. Complementation with the wild-type gene corrects the rhythm defects of the prd-1 mutant in the complete circadian system (when the FRQ-based TTFL is intact) and also the free-running FRQ-less rhythm on low choline. A PRD-1–GFP fusion protein localizes to the nucleus. The prd-1 mutant has a single base pair change in the first base of an intron that results in abnormally spliced transcripts. FRQ-less rhythms on low choline, or entrained to heat pulses, were only marginally affected in strains carrying deletions of 2 other RNA helicases ( prd-6 and msp-8). We conclude that PRD-1 is a member of an RNA helicase family that may be specifically involved in regulating rhythmicity in N. crassa in both the complete circadian system and FLO(s).

Methylation of histone H3 at lysine 36 (H3K36me), a widely-distributed chromatin mark, largely results from association of the lysine methyltransferase (KMT) SET-2 with RNA polymerase II (RNAPII), but most eukaryotes also have additional H3K36me KMTs that act independently of RNAPII. These include the orthologs of ASH1, which are conserved in animals, plants, and fungi but whose function and control are poorly understood. We found that Neurospora crassa has just two H3K36 KMTs, ASH1 and SET-2, and were able to explore the function and distribution of each enzyme independently. While H3K36me deposited by SET-2 marks active genes, inactive genes are modified by ASH1 and its activity is critical for their repression. ASH1-marked chromatin can be further modified by methylation of H3K27, and ASH1 catalytic activity modulates the accumulation of H3K27me2/3 both positively and negatively. These findings provide new insight into ASH1 function, H3K27me2/3 establishment, and repression in facultative heterochromatin., eLife digest Not all genes in a cell’s DNA are active all the time. There are several ways to control this activity. One is by altering how the DNA is packaged into cells. DNA strands are wrapped around proteins called histones to form nucleosomes. Nucleosomes can then be packed together tightly, to restrict access to the DNA at genes that are not active, or loosely to allow access to the DNA of active genes. Chemical marks, such as methyl groups, can be attached to particular sites on histones to influence how they pack together. One important site for such marks is known as position 36 on histone H3, or H3K36 for short. Correctly adding methyl groups to this site is critical for normal development, and when this process goes wrong it can lead to diseases like cancer. An enzyme called SET-2 oversees the methylation of H3K36 in fungi, plants and animals. However, many species have several other enzymes that can also add methyl groups to H3K36, and their roles are less clear. A type of fungus called Neurospora crassa contains just two enzymes that can add methyl groups to H3K36: SET-2, and another enzyme called ASH1. By performing experiments that inactivated SET-2 and ASH1 in this fungus, Bicocca et al. found that each enzyme works on a different set of genes. Genes in regions marked by SET-2 were accessible for the cell to use, while genes marked by ASH1 were inaccessible. ASH1 also affects whether a methyl group is added to another site on histone H3. This mark is important for controlling the activity of genes that are critical for development. ASH1 is found in many other organisms, including humans. The results presented by Bicocca et al. could therefore be built upon to understand the more complicated systems for regulating H3K36 methylation in other species. From there, we can investigate how to intervene when things go wrong during developmental disorders and cancer.

The cullin-4 (CUL4) complex DCDC ( D IM-5/-7/-9/ C UL4/ D DB1 c omplex) is essential for DNA methylation and heterochromatin formation in Neurospora crassa . Cullins form the scaffold of cullin-RING E3 ubiquitin ligases (CRLs) and are modified by the covalent attachment of NEDD8, a ubiquitin-like protein that regulates the stability and activity of CRLs. We report that neddylation is not required for CUL4-dependent DNA methylation or heterochromatin formation but is required for the DNA repair functions. Moreover, the RING domain protein RBX1 and a segment of the CUL4 C terminus that normally interacts with RBX1, the E2 ligase, CAND1, and CSN are dispensable for DNA methylation and heterochromatin formation by DCDC. Our study provides evidence for the noncanonical functions of core CRL components.

The eukaryotic nucleosome, the fundamental unit of chromatin, plays an important role in packaging and organizing the genetic material (1). Each nucleosome consists of 146 bp of DNA wrapped around an octameric core histone complex consisting of two H2A-H2B dimers flanking a (H3-H4)2 tetramer (1, 2). All core histones have a basic N-terminal domain, a globular domain organized by the histone fold and a C-terminal tail (1, 2). The histone N-terminal tails, which extend out from the core particle, are involved in the establishment of chromatin structural states, whereas their histone fold domains mediate histone-histone and histone-DNA interactions (2). Core histones are susceptible to a variety of post-translational modifications (PTMs), which include methylation, acetylation, phosphorylation, ubiquitination, SUMOylation and ADP-ribosylation. These modifications, which occur mainly on the N-terminal tails (3, 4), can affect interactions of nucleosomes with trans-acting factors, and are thought to play a role in the assembly and disassembly of chromatin states, and can control the accessibility of DNA for important cellular processes including transcription, replication, gene silencing and DNA repair (5-9). Distinct modifications of the histone tails can recruit specific chromatin-binding proteins, and modifications on the same or different histone tails may be interdependent and generatevarious combinations on any individual nucleosome (10-12). Mass spectrometry has been widely used for assessing histone PTMs. It provides direct information about the sites and types of modifications, differentiates isobaric modifications (e.g., acetylation vs. tri-methylation) (13), and allows for quantitative analysis (14). PTM information obtained by mass spectrometric analysis facilitates genome-wide functional studies and provides the foundation for studies of modifications in specific regions of genomes using chromatin immunoprecipitation (ChIP) with antibodies recognizing specifically modified histones (15-17). Neurospora crassa is a convenient model eukaryote, showing genomic features lacking in some other simple eukaryotes (e.g. yeast), including DNA methylation and certain histone modifications (e.g. H3K27 methylation). Studies using this organism have contributed significantly to the fundamental understanding of circadian rhythms, DNA methylation, genome defense systems, mitochondrial protein import, post-transcriptional gene silencing, DNA repair and other processes (18). Being a multicellular filamentous fungus, Neurospora also provides a system to study cellular differentiation and development (19). However, no comprehensive investigation of the PTMs of Neurospora core histones has been reported. In the present study, we extracted core histones from N. crassa and systematically mapped histone methylation and acetylation, using a combination of digestion with various proteases and analyses with multiple types of mass spectrometers. Our results allowed for the identification of acetylation and methylation sites of lysine residues that were found previously in Arabidopsis thaliana, Saccharomyces cerevisiae and humans. More importantly, we identified several unique acetylation and methylation sites in core histones of Neurospora. Our analysis on core histone PTMs provides a foundation for examining the regulation of histone modifications and for genome-wide functional studies in this model organism.

Functionally distinct chromatin domains are delineated by distinct posttranslational modifications of histones, and in some organisms by differences in DNA methylation. Proper establishment and maintenance of chromatin domains is critical but not well understood. We previously demonstrated that heterochromatin in the filamentous fungus Neurospora crassa is marked by cytosine methylation directed by trimethylated Lysine 9 on histone H3 (H3K9me3). H3K9me3 is the product of the DIM-5 Lysine methyltransferase and is recognized by a protein complex containing heterochromatin protein-1 and the DIM-2 DNA methyltransferase. To identify additional components that control the establishment and function of DNA methylation and heterochromatin, we built a strain harboring two selectable reporter genes that are silenced by DNA methylation and employed this strain to select for mutants that are defective in DNA methylation ( dim ). We report a previously unidentified gene ( dim-7 ) that is essential for H3K9me3 and DNA methylation. DIM-7 homologs are found only in fungi and are highly divergent. We found that DIM-7 interacts with DIM-5 in vivo and demonstrated that a conserved domain near the N terminus of DIM-7 is required for its stability. In addition, we found that DIM-7 is essential for recruitment of DIM-5 to form heterochromatin.

Covalent modifications of histones integrate intracellular and extracellular cues to regulate the genome. H3 Lys 9 methylation (H3K9me) can direct heterochromatin formation and DNA methylation, while phosphorylation of H3 Ser 10 (H3S10p) drives gene activation and chromosome condensation. To examine the relationship between H3S10p, H3K9me, and DNA methylation in Neurospora crassa, we built and tested mutants of the putative H3S10 phosphatase, PP1. A PP1-impaired mutant showed increased H3S10p and selective reduction of methylation of H3K9 and DNA. Similarly, amino acid substitutions of H3S10 abolished methylation of H3K9 and DNA. Thus, H3S10 dephosphorylation by PP1 is required for DNA methylation of some loci.

The SET domain is an evolutionarily conserved domain found predominantly in histone methyltransferases (HMTs). The Neurospora crassa genome includes nine SET domain genes ( set-1 through set-9 ) in addition to dim-5 , which encodes a histone H3 lysine 9 HMT required for DNA methylation. We demonstrate that Neurospora set-2 encodes a histone H3 lysine 36 (K36) methyltransferase and that it is essential for normal growth and development. We used repeat induced point mutation to make a set-2 mutant ( set-2 RIP1 ) with multiple nonsense mutations. Western analyses revealed that the mutant lacks SET-2 protein and K36 methylation. An amino-terminal fragment that includes the AWS, SET, and post-SET domains of SET-2 proved sufficient for K36 HMT activity in vitro. Nucleosomes were better substrates than free histones. The set-2 RIP1 mutant grows slowly, conidiates poorly, and is female sterile. Introducing the wild-type gene into the mutant complemented the defects, confirming that they resulted from loss of set-2 function. We replaced the wild-type histone H3 gene ( hH3 ) with an allele producing a Lys to Leu substitution at position 36 and found that this hH3 K36L mutant phenocopied the set-2 RIP1 mutant, confirming that the observed defects in growth and development result from inability to methylate K36 of H3. Finally, we used chromatin immunoprecipitation to demonstrate that actively transcribed genes in Neurospora crassa are enriched for H3 methylated at lysines 4 and 36. Taken together, our results suggest that methylation of K36 in Neurospora crassa is essential for normal growth and development.

Eukaryotic genomes are partitioned into active and inactive domains called euchromatin and heterochromatin, respectively. In Neurospora crassa, heterochromatin formation requires methylation of histone H3 at lysine 9 (H3K9) by the SET domain protein DIM-5. Heterochromatin protein 1 (HP1) reads this mark and directly recruits the DNA methyltransferase, DIM-2. An ectopic H3 gene carrying a substitution at K9 (hH3K9L or hH3K9R) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3WT). We investigated whether other residues in the N-terminal tail of H3 are important for methylation of DNA and of H3K9. Mutations in the N-terminal tail of H3 were generated and tested for effects in vitro and in vivo, in the presence or absence of the wild-type allele. Substitutions at K4, K9, T11, G12, G13, K14, K27, S28, and K36 were lethal in the absence of a wild-type allele. In contrast, mutants bearing substitutions of R2, A7, R8, S10, A15, P16, R17, K18, and K23 were viable. The effect of substitutions on DNA methylation were variable; some were recessive and others caused a semi-dominant loss of DNA methylation. Substitutions of R2, A7, R8, S10, T11, G12, G13, K14, and P16 caused partial or complete loss of DNA methylation in vivo. Only residues R8-G12 were required for DIM-5 activity in vitro. DIM-5 activity was inhibited by dimethylation of H3K4 and by phosphorylation of H3S10, but not by acetylation of H3K14. We conclude that the H3 tail acts as an integrating platform for signals that influence DNA methylation, in part through methylation of H3K9., Author Summary DNA methylation is a common feature of eukaryotic genomes. Methylation is typically associated with silenced chromosomal domains and is essential for development of plants and animals. Although the control of DNA methylation is not well understood, recent findings with model organisms, including the fungus Neurospora crassa, revealed connections between modifications of histones and DNA. DNA methylation is dispensable in Neurospora, facilitating genetic studies. Isolation of mutants defective in DNA methylation revealed that a histone H3 methyltransferase, DIM-5, is required for DNA methylation. DIM-5 trimethylates H3K9, which is then recognized by Heterochromatin Protein 1 (HP1), which recruits the DNA methyltransferase DIM-2. We investigated the possibility that H3 provides a platform to integrate information relevant to whether the associated DNA should be methylated. Indeed, we found that DIM-5 is sensitive to methylation of H3K4 and phosphorylation of H3S10. Our analyses further revealed that H3K14 is critical in vivo, but not because acetyl-K14 inhibits DIM-5. We also found that H3R2 is essential for DNA methylation in vivo but not important for DIM-5 activity. Interestingly, we found H3 mutants that show recessive defects in DNA methylation and others with dominant effects. We also defined a set of H3 mutations that are lethal.

Methylation of DNA and of Lysine 9 on histone H3 (H3K9) is associated with gene silencing in many animals, plants, and fungi. In Neurospora crassa, methylation of H3K9 by DIM-5 directs cytosine methylation by recruiting a complex containing Heterochromatin Protein-1 (HP1) and the DIM-2 DNA methyltransferase. We report genetic, proteomic, and biochemical investigations into how DIM-5 is controlled. These studies revealed DCDC, a previously unknown protein complex including DIM-5, DIM-7, DIM-9, CUL4, and DDB1. Components of DCDC are required for H3K9me3, proper chromosome segregation, and DNA methylation. DCDC-defective strains, but not HP1-defective strains, are hypersensitive to MMS, revealing an HP1-independent function of H3K9 methylation. In addition to DDB1, DIM-7, and the WD40 domain protein DIM-9, other presumptive DCAFs (DDB1/CUL4 associated factors) co-purified with CUL4, suggesting that CUL4/DDB1 forms multiple complexes with distinct functions. This conclusion was supported by results of drug sensitivity tests. CUL4, DDB1, and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains, indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3. DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9. These data support a two-step mechanism for H3K9 methylation in Neurospora., Author Summary Eukaryotic genomes are composed of distinct structural and functional domains marked by various covalent modifications of histone proteins and, in some organisms, by methylation of cytosine bases in DNA. Gene-rich euchromatin exists in a relatively open conformation, facilitating DNA transactions such as transcription, whereas the gene-poor heterochromatin is more condensed and is a poor substrate for DNA–based transactions. Heterochromatin promotes genome stability by silencing transposons and may be essential for proper centromere function. DNA methylation is a common feature of heterochromatin in eukaryotes, including the filamentous fungus Neurospora crassa, which has served as a model system to elucidate the control of DNA methylation. All DNA methylation in Neurospora depends on histone H3 lysine-9 (H3K9) methylation, which is recognized by a complex of HP1 (Heterochromatin Protein 1) and the DNA methyltransferase, DIM-2. An important open question is what controls the H3K9 methyltransferase, DIM-5. We report the genetic and proteomic identification of a DIM-5 protein complex, DCDC, and demonstrate that it includes five proteins essential for H3K9 methylation, DNA methylation, proper chromosome segregation, and resistance to DNA damaging agents. In addition, we report molecular and genetic analyses revealing a hierarchy of protein interactions within DCDC.

Background Silencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in Neurospora crassa, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized. Results The selectable marker, hph, was inserted at the subtelomere of Linkage Group VR in an nst-1 (neurospora sir two-1) mutant and was silenced when nst-1 function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, bar, tested at two other subtelomeres, was similarly sensitive to nst-1 function. Mutation of three additional SIR2 homologues, nst-2, nst-3 and nst-5, partially relieved silencing. Two genes showed stronger effects: dim-5, which encodes a histone H3 K9 methyltransferase and hpo, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased. Conclusion We demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in de novo DNA methylation.