Eukaryotic genomes harbor transposable elements and other repetitive sequences that must be silenced. Small RNA interference pathways play a major role in their repression. Here, we reveal another mechanism for silencing these sequences in Drosophila. Depleting the linker histone H1 in vivo leads to strong activation of these elements. H1-mediated silencing occurs in combination with the heterochromatin-specific histone H3 lysine 9 methyltransferase Su(var)3-9. H1 physically interacts with Su(var)3-9 and recruits it to chromatin in vitro, which promotes H3 methylation. We propose that H1 plays a key role in silencing by tethering Su(var)3-9 to heterochromatin. The tethering function of H1 adds to its established role as a regulator of chromatin compaction and accessibility.

Epigenetic silencing in mammals involves DNA methylation and posttranslational modifications of core histones. Here we show that the H1 linker histone plays a key role in regulating both DNA methylation and histone H3 methylation at the H19 and Gtl2 loci in mouse ES cells. Some, but not all, murine H1 subtypes interact with DNA methyltransferases DNMT1 and DNMT3B. The interactions are direct and require a portion of the H1 C-terminal domain. Expression of an H1 subtype that interacts with DNMT1 and DNMT3B in ES cells leads to their recruitment and DNA methylation of the H19 and Gtl2 imprinting control regions. H1 also interferes with binding of the SET7/9 histone methyltransferase to the imprinting control regions, inhibiting production of an activating methylation mark on histone H3 lysine 4. H1-dependent recruitment of DNMT1 and DNMT3B and interference with the binding of SET7/9 also were observed with chromatin reconstituted in vitro. The data support a model in which H1 plays an active role in helping direct two processes that lead to the formation of epigenetic silencing marks. The data also provide evidence for functional differences among the H1 subtypes expressed in somatic mammalian cells.mouse embryonic stem cell | H1 histone triple knockout T wo major types of epigenetic marks occur in mammalian genomes, DNA methylation and posttranslational modifications of histones (1, 2). The methylation of cytosines in mammalian DNA occurs primarily at CpG dinucleotides and is essential for normal mammalian development (1, 3). Perturbations of DNA methylation patterns are thought to play a role in cancer development (4). There are two classes of mammalian DNA methyltransferases (DNMTs), one that functions primarily to establish DNA methylation de novo (DNMT3A and DNMT3B) and the other to maintain it (DNMT1) (3). DNA methylation can have profound effects on gene expression and is most often associated with transcriptional silencing (1, 2). Accumulating evidence indicates the existence of crosstalk between the DNA methylation and core histone modification systems (1, 2). For example, mouse ES cells deficient for the histone H3 lysine 9 (H3K9) methyltransferases Suv39h1/h2 exhibit hypomethylation of CpGs at a subset of repetitive DNA elements (5). Additionally, several lines of evidence indicate that the presence of unmethylated lysine 4 of histone H3 (H3K4) in chromatin favors de novo methylation of DNA (6-9). Despite these advances, the factors in chromatin that coordinate DNA methylation and histone H3 methylation have not been identified.In this study, we investigated the role of the linker histone H1 in regulating DNA methylation and histone H3 methylation at the H19 and Gtl2 loci in mouse ES cells. H1 is the most distinct of the five histone proteins (H1, H2A, H2B, H3, and H4) in chromatin (10,11). Mammals contain at least eight histone H1 subtypes or variants that differ in amino acid sequences and expression during development (12-14). Functional differences among these subtypes have been difficult to identif...

BackgroundLinker histone H1 is a core chromatin component that binds to nucleosome core particles and the linker DNA between nucleosomes. It has been implicated in chromatin compaction and gene regulation and is anticipated to play a role in higher-order genome structure. Here we have used a combination of genome-wide approaches including DNA methylation, histone modification and DNase I hypersensitivity profiling as well as Hi-C to investigate the impact of reduced cellular levels of histone H1 in embryonic stem cells on chromatin folding and function.ResultsWe find that depletion of histone H1 changes the epigenetic signature of thousands of potential regulatory sites across the genome. Many of them show cooperative loss or gain of multiple chromatin marks. Epigenetic alterations cluster to gene-dense topologically associating domains (TADs) that already showed a high density of corresponding chromatin features. Genome organization at the three-dimensional level is largely intact, but we find changes in the structural segmentation of chromosomes specifically for the epigenetically most modified TADs.ConclusionsOur data show that cells require normal histone H1 levels to expose their proper regulatory landscape. Reducing the levels of histone H1 results in massive epigenetic changes and altered topological organization particularly at the most active chromosomal domains. Changes in TAD configuration coincide with epigenetic landscape changes but not with transcriptional output changes, supporting the emerging concept that transcriptional control and nuclear positioning of TADs are not causally related but independently controlled by the locally associated trans-acting factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0857-0) contains supplementary material, which is available to authorized users.

Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.