Direct interaction between DNA methyltransferase DIM-2 and HP1 is required for DNA methylation in Neurospora crassa

DNA methylation is involved in gene silencing and genomic stability in mammals, plants, and fungi. Genetics studies of Neurospora crassa have revealed that a DNA methyltransferase (DIM-2), a histone H3K9 methyltransferase (DIM-5), and heterochromatin protein 1 (HP1) are required for DNA methylation. We explored the interrelationships of these components of the methylation machinery. A yeast two-hybrid screen revealed that HP1 interacts with DIM-2. We confirmed the interaction in vivo and demonstrated that it involves a pair of PXVXL-related motifs in the N-terminal region of DIM-2 and the chromo shadow domain of HP1. Both regions are essential for proper DNA methylation. We also determined that DIM-2 and HP1 form a stable complex independently of the trimethylation of histone H3K9, although the association of DIM-2 with its substrate sequences depends on trimethyl-H3K9. The DIM-2/HP1 complex does not include DIM-5. We conclude that DNA methylation in Neurospora is largely or exclusively the result of a unidirectional pathway in which DIM-5 methylates histone H3K9 and then the DIM-2/HP1 complex recognizes the resulting trimethyl-H3K9 mark via the chromo domain of HP1.     

DNA methylation and normal chromosome behavior in Neurospora depend on five components of a histone methyltransferase complex, DCDC

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.     

Heterochromatin Controls γH2A Localization in Neurospora crassa

In response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called γH2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of γH2A followed by next-generation sequencing (ChIP-seq). γH2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal γH2A localization. In contrast, γH2A is not required for H3K9 methylation or DNA methylation. Normal γH2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. γH2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair.     

In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases

The functional significance of mono-, di-, and tri-methylation of lysine residues within histone proteins is under investigation. Evidence from several model organisms suggests that different methylated states of H3 Lys(9) (H3K9) are generated by specific histone methyltransferases (MTases) to mark distinct types of silent chromatin. Sequence alignment of all histone lysine MTases with known product specificity suggested that a key residue in the active site determines how many methyl groups they add. We examined this possibility both in vitro and in vivo and found that a Phe at the position equivalent to Phe(281) of Neurospora crassa DIM-5 or Phe(1205) of human G9a allows the enzyme to perform di and tri-methylation, whereas a Tyr at this position is restrictive, inhibiting tri-methylation and thus yielding a mono- or di-MTase. Phe to Tyr mutants of both DIM-5 and G9a restrict product specificity in vitro and in vivo without compromising overall catalysis. These mutants were employed to probe the biological significance of mono-, di-, and tri-methylation of H3K9 in both mouse embryonic stem cells and N. crassa. G9a F1205Y, when expressed in G9a (-/-) embryonic stem cells, rescued only H3K9 mono-methylation, but not di-methylation, to wild-type levels yet silenced Mage-a gene expression. When expressed in dim-5 strains, DIM-5 F281Y generated significant levels of mono- and di-H3K9 methylation (which are not observed in wild type Neurospora) as well as tri-methyl H3K9. The altered DIM-5 rescued the growth defect characteristic of dim-5 N. crassa but did not fully rescue the gross DNA hypomethylation of dim-5 strains.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Substitutions in the amino-terminal tail of neurospora histone H3 have varied effects on DNA methylation

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 (hH3(K9L) or hH3(K9R)) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3(WT)). 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.     

Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9

We screened radiation-sensitive yeast mutants for DNA damage checkpoint defects and identified Dot1, the conserved histone H3 Lys 79 methyltransferase. DOT1 deletion mutants (dot1Delta) are G1 and intra-S phase checkpoint defective after ionizing radiation but remain competent for G2/M arrest. Mutations that affect Dot1 function such as Rad6-Bre1/Paf1 pathway gene deletions or mutation of H2B Lys 123 or H3 Lys 79 share dot1Delta checkpoint defects. Whereas dot1Delta alone confers minimal DNA damage sensitivity, combining dot1Delta with histone methyltransferase mutations set1Delta and set2Delta markedly enhances lethality. Interestingly, set1Delta and set2Delta mutants remain G1 checkpoint competent, but set1Delta displays a mild S phase checkpoint defect. In human cells, H3 Lys 79 methylation by hDOT1L likely mediates recruitment of the signaling protein 53BP1 via its paired tudor domains to double-strand breaks (DSBs). Consistent with this paradigm, loss of Dot1 prevents activation of the yeast 53BP1 ortholog Rad9 or Chk2 homolog Rad53 and decreases binding of Rad9 to DSBs after DNA damage. Mutation of Rad9 to alter tudor domain binding to methylated Lys 79 phenocopies the dot1Delta checkpoint defect and blocks Rad53 phosphorylation. These results indicate a key role for chromatin and methylation of histone H3 Lys 79 in yeast DNA damage signaling.     

Structural basis for the product specificity of histone lysine methyltransferases

DIM-5 is a SUV39-type histone H3 Lys9 methyltransferase that is essential for DNA methylation in N. crassa. We report the structure of a ternary complex including DIM-5, S-adenosyl-L-homocysteine, and a substrate H3 peptide. The histone tail inserts as a parallel strand between two DIM-5 strands, completing a hybrid sheet. Three post-SET cysteines coordinate a zinc atom together with Cys242 from the SET signature motif (NHXCXPN) near the active site. Consequently, a narrow channel is formed to accommodate the target Lys9 side chain. The sulfur atom of S-adenosyl-L-homocysteine, where the transferable methyl group is to be attached in S-adenosyl-L-methionine, lies at the opposite end of the channel, approximately 4 A away from the target Lys9 nitrogen. Structural comparison of the active sites of DIM-5, an H3 Lys9 trimethyltransferase, and SET7/9, an H3 Lys4 monomethyltransferase, allowed us to design substitutions in both enzymes that profoundly alter their product specificities without affecting their catalytic activities.     

DCAF26, an Adaptor Protein of Cul4-Based E3, Is Essential for DNA Methylation in Neurospora crassa

DNA methylation is involved in gene silencing and genome stability in organisms from fungi to mammals. Genetic studies in Neurospora crassa previously showed that the CUL4-DDB1 E3 ubiquitin ligase regulates DNA methylation via histone H3K9 trimethylation. However, the substrate-specific adaptors of this ligase that are involved in the process were not known. Here, we show that, among the 16 DDB1- and Cul4-associated factors (DCAFs) encoded in the N. crassa genome, three interacted strongly with CUL4-DDB1 complexes. DNA methylation analyses of dcaf knockout mutants revealed that dcaf26 was required for all of the DNA methylation that we observed. In addition, histone H3K9 trimethylation was also eliminated in dcaf26^KO mutants. Based on the finding that DCAF26 associates with DDB1 and the histone methyltransferase DIM-5, we propose that DCAF26 protein is the major adaptor subunit of the Cul4-DDB1-DCAF26 complex, which recruits DIM-5 to DNA regions to initiate H3K9 trimethylation and DNA methylation in N. crassa.     

Heterochromatin is required for normal distribution of Neurospora crassa CenH3

Centromeres serve as platforms for the assembly of kinetochores and are essential for nuclear division. Here we identified Neurospora crassa centromeric DNA by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) of DNA associated with tagged versions of the centromere foundation proteins CenH3 (CENP-A) and CEN-C (CENP-C) and the kinetochore protein CEN-T (CENP-T). On each chromosome we found an ～150- to 300-kbp region of enrichment for all three proteins. These regions correspond to intervals predicted to be centromeric DNA by genetic mapping and DNA sequence analyses. By ChIP-seq we found extensive colocalization of CenH3, CEN-C, CEN-T, and histone H3K9 trimethylation (H3K9me3). In contrast, H3K4me2, which has been found at the cores of plant, fission yeast, Drosophila, and mammalian centromeres, was not enriched in Neurospora centromeric DNA. DNA methylation was most pronounced at the periphery of centromeric DNA. Mutation of dim-5, which encodes an H3K9 methyltransferase responsible for nearly all H3K9me3, resulted in altered distribution of CenH3-green fluorescent protein (GFP). Similarly, CenH3-GFP distribution was altered in the absence of HP1, the chromodomain protein that binds to H3K9me3. We conclude that eukaryotes with regional centromeres make use of different strategies for maintenance of CenH3 at centromeres, and we suggest a model in which centromere proteins nucleate at the core but require DIM-5 and HP1 for spreading.     

Methylation of histone H3 lysine 36 is required for normal development in Neurospora crassa

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.     

Connections between epigenetic gene silencing and human disease

Alterations in epigenetic gene regulation are associated with human disease. Here, we discuss connections between DNA methylation and histone methylation, providing examples in which defects in these processes are linked with disease. Mutations in genes encoding DNA methyltransferases and proteins that bind methylated cytosine residues cause changes in gene expression and alterations in the patterns of DNA methylation. These changes are associated with cancer and congenital diseases due to defects in imprinting. Gene expression is also controlled through histone methylation. Altered levels of methyltransferases that modify lysine 27 of histone H3 (K27H3) and lysine 9 of histone H3 (K9H3) correlate with changes in Rb signaling and disruption of the cell cycle in cancer cells. The K27H3 mark recruits a Polycomb complex involved in regulating stem cell pluripotency, silencing of developmentally regulated genes, and controlling cancer progression. The K9H3 methyl mark recruits HP1, a structural protein that plays a role in heterochromatin formation, gene silencing, and viral latency. Cells exhibiting altered levels of HP1 are predicted to show a loss of silencing at genes regulating cancer progression. Gene silencing through K27H3 and K9H3 can involve histone deacetylation and DNA methylation, suggesting cross talk between epigenetic silencing systems through direct interactions among the various players. The reversible nature of these epigenetic modifications offers therapeutic possibilities for a wide spectrum of disease.     

Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome

To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.     

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6dim-3 allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6^dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.