Linker histone dH1K27 dimethylation marks Drosophila heterochromatin independently of H3K9 methylation

Post-translational modifications (PTMs) of histones are important epigenetic determinants and specific core histones PTMs correlate with functional chromatin states. However, despite linker histone H1s are heavily post-translationally modified, little is known about the genomic distribution of H1s PTMs and their association with epigenetic chromatin states. 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 cells, it is also detected at intercalary heterochromatin. ChIPseq experiments show that dH1K27me2 enriched regions cluster at both the assembled and unassembled heterochromatin regions of all four Drosophila chromosomes. More than 98% of the dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. We also show that dH1K27me2 is independent of H3K9 methylation, as it is equally detected in flies carrying a H3K9R mutation. Moreover, dH1K27me2 is not affected by depletion of Su(var)3-9, HP1a and Su(var)4-20. Altogether these results suggest that dH1K27me2 is a novel epigenetic mark of Drosophila heterochromatin that acts upstream of the major Su(var)3- 9/HP1a pathway of heterochromatin formation.

The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.