Lysine 27 dimethylation of Drosophila linker histone dH1 contributes to heterochromatin organization independently of H3K9 methylation.

Post-translational modifications (PTMs) of core histones are important epigenetic determinants that correlate with functional chromatin states. However, despite multiple linker histone H1s PTMs have been identified, little is known about their genomic distribution and contribution to the epigenetic regulation of chromatin. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase, it is also detected at intercalary heterochromatin. ChIPseq experiments show that >98% of dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. Moreover, expression of a mutated dH1K27A form, which impairs dH1K27me2, alters heterochromatin organization, upregulates expression of heterochromatic transposable elements and results in the accumulation of RNA:DNA hybrids (R-loops) in heterochromatin, without affecting H3K9 methylation and HP1a binding. The pattern of dH1K27me2 is H3K9 methylation independent, as it is equally detected in flies carrying a H3K9R mutation, and is not affected by depletion of Su(var)3-9, HP1a or Su(var)4-20. Altogether these results suggest that dH1K27me2 contributes to heterochromatin organization independently of H3K9 methylation.

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1.

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.

Drosophila melanogaster linker histone dH1 is required for transposon silencing and to preserve genome integrity.

Histone H1 is an intrinsic component of chromatin, whose important contribution to chromatin structure is well-established in vitro. Little is known, however, about its functional roles in vivo. Here, we have addressed this question in Drosophila, a model system offering many advantages since it contains a single dH1 variant. For this purpose, RNAi was used to efficiently deplete dH1 in flies. Expression-profiling shows that dH1 depletion affects expression of a relatively small number of genes in a regional manner. Furthermore, depletion up-regulates inactive genes, preferentially those located in heterochromatin, while active euchromatic genes are down-regulated, suggesting that the contribution of dH1 to transcription regulation is mainly structural, organizing chromatin for proper gene-expression regulation. Up-regulated genes are remarkably enriched in transposons. In particular, R1/R2 retrotransposons, which specifically integrate in the rDNA locus, are strongly up-regulated. Actually, depletion increases expression of transposon-inserted rDNA copies, resulting in synthesis of aberrant rRNAs and enlarged nucleolus. Concomitantly, dH1-depleted cells accumulate extra-chromosomal rDNA, show increased γH2Av content, stop proliferation and activate apoptosis, indicating that depletion causes genome instability and affects proliferation. Finally, the contributions to maintenance of genome integrity and cell proliferation appear conserved in human hH1s, as their expression rescues proliferation of dH1-depleted cells.