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

Abstract

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.

Fig. 1.

DNA methylation-dependent silencing of hph^m and bar^m reporter genes. (A) Suspensions of 10⁴, 10³, or 10² conidia of strain N2977, grown in the absence (host) or presence of 5-azacytidine (host+5AC), and a dim-2 knock-down strain (dim-2 kd; strain N3854) were spot-tested on media with or without basta or hygromycin. (B) Genomic DNA from the parental strain (host) and a dim-2 knockdown (dim-2 kd) was digested with the methyl-cytosine-sensitive restriction enzyme Sau3AI or its insensitive isoschizomer, DpnII, and used for Southern hybridizations with a probe corresponding to the bar reporter gene. The arrow indicates loss of DNA methylation in the dim-2 kd strain.

Fig. 2.

The dim-7^UV64 strain exhibits reduced DNA methylation. (A) Suspensions of 10⁴, 10³, or 10² conidia from strains N2977 (host) and N3312 (dim-7^UV64) were spot-tested on media with or without basta or hygromycin, as indicated. (B) Genomic DNA from strain N2977 (host), N1851 (Δdim-2), and N3312 (dim-7^UV64) was digested with Sau3AI or DpnII and used for Southern hybridizations with probes corresponding to the methylated 8:A6, 8:G3, or 8:F10 regions (3).

Fig. 3.

H3K9me3 levels are globally reduced in the dim-7^UV64 mutant. (A) Histones were extracted from WT (N150), Δdim-2 (N1851), Δdim-5 (N3074), and dim-7^UV64 (N3312) strains and subjected to Western blotting using antibodies to H3K9me3 or H3K4me2. (B) For WT and the dim-7^UV64 strain, radio-labeled products obtained by multiplex PCR using using whole-cell extracts (input) or the indicated immunoprecipitate fraction as a template are shown. PCR products corresponding to heterochromatin (8:A6, 9:E1, and 8:G3) (3) or control euchromatin regions (H4 and pcna) are indicated at the right of the panel. (C) The average enrichment of each PCR product relative to the indicated euchromatin internal control is shown graphically for three independent experiments. (D) The distribution of H3K9me3 on LGVII in WT and dim-7^UV64. Enrichment values (IP/input) for ChIP-chip experiments using antibodies to H3K9me3 are shown as log₂ values. (E) The distribution of HP1-GFP is shown in multinucleate conidia for WT and the dim-7^UV64 strain (DIC, differential interference contrast).

Fig. 4.

DIM-7 is essential for DNA and H3K9 methylation and interacts with DIM-5. (A) Genomic DNA from WT (N150), Δdim-2 (N1851), dim-7^UV64 (N3312), dim-7^UV64;::dim-7⁺(N3868), and Δdim-7 (N3855) was digested using Sau3AI or DpnII and used for Southern hybridizations with probes corresponding to the methylated 8:A6 or 8:F10 regions (3). The blots were also probed with mtr, an unmethylated gene, to confirm that the digests were complete. (B) Histones were extracted from WT (N150), Δdim-5 (N3074), and Δdim-7 (N3855) and subjected to Western blotting using antibodies to H3K9me3 or H3K4me2. (C) Immunoprecipiation experiments were performed using extracts from strains expressing DIM-7–3XFLAG, DIM-5–3XHA, or both (indicated by + or −). The input fraction, the α-HA immunoprecipitate fraction (IP:αHA), and the α-FLAG immunoprecipitate fraction (IP:αFLAG) were subjected to Western blotting and probed with the α-FLAG or α-HA antibodies as indicated (WB).

Fig. 5.

DIM-7 is required for recruitment of DIM-5 to heterochromatin domains. Genomic DNA from WT (N150), which does not express DIM-5-Dam, as well as dim+ (N3864) and Δdim-7 (N3865) strains expressing DIM-5-Dam were incubated with or without DpnI, which cuts GATC only when the adenine is methylated. As an indicator of completely digested DNA, genomic DNA from the WT strain was incubated with the cytosine methylation-insensitive enzyme DpnII. Digested DNA was used for Southern hybridizations with probes corresponding to the heterochromatic 8:A6 and 8:G3 regions and the euchromatic mtr and sms-2 genes.