Shinji Honda

Direct Interaction between DNA Methyltransferase DIM-2 and HP1 Is Required for DNA Methylation in Neurospora crassa

ABSTRACT 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.

Tools for Fungal Proteomics: Multifunctional Neurospora Vectors for Gene Replacement, Protein Expression and Protein Purification

The completion of genome-sequencing projects for a number of fungi set the stage for detailed investigations of proteins. We report the generation of versatile expression vectors for detection and isolation of proteins and protein complexes in the filamentous fungus Neurospora crassa. The vectors, which can be adapted for other fungi, contain C- or N-terminal FLAG, HA, Myc, GFP, or HAT–FLAG epitope tags with a flexible poly-glycine linker and include sequences for targeting to the his-3 locus in Neurospora. To introduce mutations at native loci, we also made a series of knock-in vectors containing epitope tags followed by the selectable marker hph (resulting in hygromycin resistance) flanked by two loxP sites. We adapted the Cre/loxP system for Neurospora, allowing the selectable marker hph to be excised by introduction of Cre recombinase into a strain containing a knock-in cassette. Additionally, a protein purification method was developed on the basis of the HAT–FLAG tandem affinity tag system, which was used to purify HETEROCHROMATIN PROTEIN 1 (HP1) and associated proteins from Neurospora. As expected on the basis of yeast two-hybrid and co-immunoprecipitation (Co-IP) experiments, the Neurospora DNA methyltransferase DIM-2 was found in a complex with HP1. Features of the new vectors allowed for verification of an interaction between HP1 and DIM-2 in vivo by Co-IP assays on proteins expressed either from their native loci or from the his-3 locus.

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.

The DMM complex prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa

Transposable elements are common in genomes and must be controlled. Many organisms use DNA methylation to silence such selfish DNA, but the mechanisms that restrict the methylation to appropriate regions are largely unknown. We identified a JmjC domain protein in Neurospora, DNA METHYLATION MODULATOR-1 (DMM-1), that prevents aberrant spreading of DNA and histone H3K9 methylation from inactivated transposons into nearby genes. Mutation of a conserved residue within the JmjC Fe(II)-binding site abolished dmm-1 function, as did mutations in conserved cysteine-rich domains. Mutants defective only in dmm-1 mutants grow poorly, but growth is restored by reduction or elimination of DNA methylation using the drug 5-azacytosine or by mutation of the DNA methyltransferase gene dim-2. DMM-1 relies on an associated protein, DMM-2, which bears a DNA-binding motif, for localization and proper function. HP1 is required to recruit the DMM complex to the edges of methylated regions.

Heterochromatin protein 1 forms distinct complexes to direct histone deacetylation and DNA methylation

DNA methylation, methylation of histone H3 at Lys9 (H3K9me3) and hypoacetylated histones are common molecular features of heterochromatin. Important details of their functions and inter-relationships remain unclear, however. In Neurospora crassa, H3K9me3 directs DNA methylation through a complex containing heterochromatin protein 1 (HP1) and the DNA methyltransferase DIM-2. We identified a distinct HP1 complex, HP1, CDP-2, HDA-1 and CHAP (HCHC), and found that it is responsible for silencing independently of DNA methylation. HCHC defects cause hyperacetylation of centromeric histones, greater accessibility of DIM-2 and hypermethylation of centromeric DNA. Loss of HCHC also causes mislocalization of the DIM-5 H3K9 methyltransferase at a subset of interstitial methylated regions, leading to selective DNA hypomethylation. We demonstrate that HP1 forms distinct DNA methylation and histone deacetylation complexes that work in parallel to assemble silent chromatin in N. crassa.

Identification of DIM-7, a protein required to target the DIM-5 H3 methyltransferase to chromatin

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.

Neurospora chromosomes are organized by blocks of importin alpha-dependent heterochromatin that are largely independent of H3K9me3

Eukaryotic genomes are organized into chromatin domains with three-dimensional arrangements that presumably result from interactions between the chromatin constituents-proteins, DNA, and RNA-within the physical constraints of the nucleus. We used chromosome conformation capture (3C) followed by high-throughput sequencing (Hi-C) with wild-type and mutant strains of Neurospora crassa to gain insight into the role of heterochromatin in the organization and function of the genome. We tested the role of three proteins thought to be important for establishment of heterochromatin, namely, the histone H3 lysine 9 methyltransferase DIM-5, Heterochromatin Protein 1 (HP1), which specifically binds to the product of DIM-5 (trimethylated H3 lysine 9 [H3K9me3]), and DIM-3 (importin alpha), which is involved in DIM-5 localization. The average genome configuration of the wild-type strain revealed strong intra- and inter-chromosomal associations between both constitutive and facultative heterochromatic domains, with the strongest interactions among the centromeres, subtelomeres, and interspersed heterochromatin. Surprisingly, loss of either H3K9me3 or HP1 had only mild effects on heterochromatin compaction, whereas dim-3 caused more drastic changes, specifically decreasing interactions between constitutive heterochromatic domains. Thus, associations between heterochromatic regions are a major component of the chromosome conformation in Neurospora, but two widely studied key heterochromatin proteins are not necessary, implying that undefined protein factors play key roles in maintaining overall chromosome organization.

Normal chromosome conformation depends on subtelomeric facultative heterochromatin in Neurospora crassa

Significance Two forms of heterochromatin, constitutive and facultative, cause gene silencing in eukaryotes. In Neurospora crassa, H3K27me2/3-marked facultative heterochromatin reversibly represses scores of specialized genes, whereas H3K9me3-marked constitutive heterochromatin permanently silences repetitive DNA. Interactions between heterochromatin provide a structural framework for the genome, and this is thought to be functionally important. Histone marks underlying constitutive and facultative heterochromatin are nonessential in N. crassa, permitting tests of their roles in genome organization and gene expression. Although linkages between regions of constitutive heterochromatin are the most prominent feature of the 3D structure of the genome, loss of the facultative mark has a much greater effect on genome architecture than does loss of key features of constitutive heterochromatin, i.e., H3K9me3 and Heterochromatin Protein 1. High-throughput chromosome conformation capture (Hi-C) analyses revealed that the 3D structure of the Neurospora crassa genome is dominated by intra- and interchromosomal links between regions of heterochromatin, especially constitutive heterochromatin. Elimination of trimethylation of lysine 9 on histone H3 (H3K9me3) or its binding partner Heterochromatin Protein 1 (HP1)—both prominent features of constitutive heterochromatin—have little effect on the Hi-C pattern. It remained possible that di- or trimethylation of lysine 27 on histone H3 (H3K27me2/3), which becomes localized in regions of constitutive heterochromatin when H3K9me3 or HP1 are lost, plays a critical role in the 3D structure of the genome. We found that H3K27me2/3, catalyzed by the Polycomb Repressive Complex 2 (PRC2) member SET-7 (SET domain protein-7), does indeed play a prominent role in the Hi-C pattern of WT, but that its presence in regions normally occupied by H3K9me3 is not responsible for maintenance of the genome architecture when H3K9me3 is lost. The Hi-C pattern of a mutant defective in the PRC2 member N. crassa p55 (NPF), which is predominantly required for subtelomeric H3K27me2/3, was equivalent to that of the set-7 deletion strain, suggesting that subtelomeric facultative heterochromatin is paramount for normal chromosome conformation. Both PRC2 mutants showed decreased heterochromatin–heterochromatin contacts and increased euchromatin–heterochromatin contacts. Cytological observations suggested elimination of H3K27me2/3 leads to partial displacement of telomere clusters from the nuclear periphery. Transcriptional profiling of Δdim-5, Δset-7, Δset-7; Δdim-5, and Δnpf strains detailed anticipated changes in gene expression but did not support the idea that global changes in genome architecture, per se, led to altered transcription.

Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

Significance Modifications of chromatin proteins (e.g. histones) and DNA play vital roles in genome function. Both hypo- and hypermethylation of DNA are associated with human diseases, including cancers, but the underlying mechanisms are not well understood. Using the filamentous fungus Neurospora crassa, one of the simplest eukaryotes with DNA methylation, we report a DNA methylation pathway that depends partially on the histone deacetylase complex HCHC [heterochromatin protein 1 (HP1)–chromodomain protein 2 (CDP-2)–histone deacetylase 1 (HDA-1)– CDP-2/HDA-1–associated protein (CHAP)]. Genome-wide DNA methylation analyses revealed both hypo- and hyper-DNA methylation in strains with defective HCHC components. We show the interrelationship of HCHC components and genetically dissect the proteins to define domains critical for proper DNA methylation and centromeric silencing. This work provides insights into the crosstalk between DNA methylation and histone modifications. DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation.

The Cullin-4 Complex DCDC Does Not Require E3 Ubiquitin Ligase Elements To Control Heterochromatin in Neurospora crassa

ABSTRACT The cullin-4 (CUL4) complex DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) 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.