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

Abstract

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.

Figure 1

Aberrant DNA methylation in the cdp-2 mutant. (a) Schematic of CDP-2. (b) Genomic DNA from wild type (WT), dim-2 and cdp-2 was digested with 5mC-sensitive BfuCI (B) or its 5mC-insensitive isoschizomer DpnII (D), gel-fractionated, visualized by ethidium bromide (EtBr) straining and analyzed by Southern hybridizations with indicated probes corresponding to representative methylated regions (8:A6, 8:G3, 8:F10 and 8:F3). An extra high-molecular-mass band for the 8:F3 region in cdp-2 mutants indicates hypermethylated products. (c) DNA methylation profile of wild-type and cdp-2 strains across N. crassa LG VII. The immunoprecipitation per input ratio for each oligo was plotted as a log₂ value (y axis). In plots of microarray data, blue is RIP product index (ratio of number of TpA and ApT dinucleotides) and red is RIP substrate index (ratio of sum of CpA and TpG dinucleotides and sum of ApC and GpT dinucleotides) per (ApC + GpT) ratio). Regions with RIP show product index >1.0 and substrate index <1.0 (refs. 16,31). Black arrows, seven representative methylated regions of LG VII. Predicted genes, gray arrows; regions with moderate RIP, red bars. (d) DNA methylation profile at CenVII in wild-type (light blue) and cdp-2 (orange) strains. (e) Southern hybridizations confirming hypermethylation at LG VII centromere in cdp-2 (probes in d).

Figure 2

The CDP-2 chromodomain preferentially binds to H3K9 methylation and CDP-2 stability depends on HP1. (a,b) Fluorescence polarization binding assays of recombinant MBP–CDP-2 chromodomain (CD) with indicated fluoresceinated histone peptides. Average results from at least three independent measurements are plotted (mean ± s.d.). (c) CDP-2 colocalizes with HP1. Conidia of indicated strains, which carried wild-type genes for CDP-2 and HP1 and genes for indicated chimeric proteins, were examined by microscopic analyses. Differential interference contrast (DIC), fluorescence (HP1-GFP and CDP-2–RFP) and overlay images. (d) CDP-2 localization depends on DIM-5 but not DIM-2. CDP-2–GFP localization was examined in dim⁺ (WT), dim-5 or dim-2 strains in addition to cdp-2 mutants carrying a functional CDP-2–GFP construct driven by the ccg-1 promoter at the his-3 locus. Functionality of the CDP-2–GFP construct was demonstrated in the dim⁺ background by occurrence of normal DNA methylation (data not shown). (e) CDP-2 localization depends on HP1 but not vice versa. (f) CDP-2 stability depends on HP1. Extracts from strains expressing CDP-2–GFP in WT and hpo strains were analyzed by western blotting with antibodies to GFP.

Figure 3

Mutation of cdp-2 causes lower H3K9me3 and HP1 localization at hypomethylated regions but no difference at hypermethylated regions compared with wild type. (a) Relative enrichment of H3K9me3 at indicated regions for wild-type, hpo and cdp-2 strains. Peaks 28a, 28b, 33a and 33b are in Figure 1c or Figure 3c. A euchromatic gene lacking DNA methylation (hH4-1) was used as internal control. Ratios of intensities measured for hH4-1 and indicated probe were normalized to ratios obtained without immunoprecipitation (total input). (b) Relative enrichment of HP1-GFP at indicated regions for wild-type and cdp-2 strains. (c) Distribution of DNA methylation, H3K9 methylation and HP1 at peaks 28 and 33 for indicated strains. Data in a,b are mean and s.d. of independent biological replicates.

Figure 4

CDP-2 is required for DIM-5 recruitment to regions with moderate RIP. DIM-5 accessibility at indicated regions was assayed by DamID. Genomic DNA isolated from indicated strains was incubated with (+) or without (−) restriction enzyme. Southern blots were probed with hypomethylated peak 34 and 8:A6 regions, the slightly affected 8:G3 region and the euchromatic gene pan-1.

Figure 5

Identification of HCHC. (a) Purification and mass spectrometric analyses of proteins associated with CDP-2. Extract from strain expressing CDP-2–HAT-Flag was treated by two-step purification, separated by SDS-PAGE, visualized by Coomassie blue staining and analyzed by mass spectrometry. Positions of CDP-2-associated proteins are shown. Unlabeled bands were found to be background or degraded proteins. (b) HDA-1 associates with CDP-2 in vivo. WB, western blot. (c) CHAP associates with CDP-2 in vivo. (d) CDP-2 associates with HP1 but not DIM-2 in vivo. Coimmunoprecipitation (IP) analyses in b–d were done with antibodies to epitopes on extracts from strains expressing indicated tagged proteins.

Figure 6

HCHC is required for normal histone H3 and H4 acetylation. (a–d) Relative enrichment of H3K9me3 and acetylated H3 and H4 determined by ChIP for wild-type and hda-1 strains at hypomethylated regions peak 33b (a) and 8:A6 (b) and at hypermethylated regions CenVIIR (c) and peak 33a (d).

Figure 7

DIM-2 accessibility at centromere is enhanced in cdp-2 mutant. DIM-2 accessibility at indicated regions was assayed by DamID. Genomic DNA isolated from indicated strains was incubated with (+) or without (−) restriction enzyme. Southern blots were probed with slightly hypomethylated 8:G3 region, hypermethylated CenVIIR with high RIP and CenIVR and the euchromatic gene pan-1.

Figure 8

HCHC is required for centromere silencing independent of DNA methylation. (a) Serial dilutions of conidia from each of the indicated strains harboring a centromeric bar construct were spot-tested on medium with or without basta. (b) Linear growth rates for three wild-type (black), three dim-2 (red), three cdp-2 (blue) and three cdp-2, dim-2 double mutant (brown). (c) Schematic of establishment and maintenance of heterochromatin in N. crassa.