Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

Post‐translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We identified a bivalent combination, a dually marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent sequential ChIP‐Seq identified dually marked single nucleosome regions, with reduced number of sites and decreased signal in old livers, confirming mass spectrometry results. We detected H3K9me3 and H3K14ac bulk ChIP‐Seq signal in reChIP nucleosome regions, suggesting a correlation between H3K9me3/H3K14ac bulk bivalent genomic regions and dually marked single nucleosomes. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied both bulk and single nucleosome bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.     

Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

Nucleosomes are the basic units of chromatin and critical for storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks, in which post-translational modifications of histones play a key role. However, the mode of action and the structural implications at the single-molecule level of nucleosomes is still poorly understood. Here we apply a high-throughput atomic force microscopy imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants three additional methyl groups at lysine 36 of histone H3 (H3K36me3), phosphorylation of H3 histones at serine 10 (H3S10phos), and acetylation of H4 histones at lysines 5, 8, 12, and 16 (H4K5/8/12/16ac). Our data set of more than 25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5-bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild-type nucleosomes and additionally unwrap stochastically from both sides, similar to centromere protein A (CENP-A) nucleosomes and in contrast to the highly anticooperative unwrapping of wild-type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild-type nucleosomes and also retain the same level of anticooperativity. Our findings help to put the mode of action of these modifications into context. Although H3K36me3 likely acts partially by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.     

N-terminal phosphorylation of HP1α increases its nucleosome-binding specificity

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin. Although HP1 phosphorylation has been described in several organisms, the biological implications of this modification remain largely elusive. Here we show that HP1''s phosphorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation assays and conventional chromatography, we demonstrated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the N-terminus of human HP1α. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1α bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1α showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1α''s intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1''s recognition of H3K9me-marked nucleosomes.     

H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non‐coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2‐dependent H3K27me3 and SETD8‐dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3‐specific intracellular antibody or H3K27me3‐mintbody. By combining live‐cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP‐seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.     

Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1β with the Nucleosome

Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1β to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1β with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1β-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.     

Divergence and Selectivity of Expression-Coupled Histone Modifications in Budding Yeasts

Various histone modifications are widely associated with gene expression, but their functional selectivity at individual genes remains to be characterized. Here, we identify widespread differences between genome-wide patterns of two prominent marks, H3K9ac and H3K4me3, in budding yeasts. As well as characteristic gene profiles, relative modification levels vary significantly amongst genes, irrespective of expression. Interestingly, we show that these differences couple to contrasting features: higher methylation to essential, periodically expressed, ‘DPN’ (Depleted Proximal Nucleosome) genes, and higher acetylation to non-essential, responsive, ‘OPN’ (Occupied Proximal Nucleosome) genes. Thus, H3K4me3 may generally associate with expression stability, and H3K9ac, with variability. To evaluate this notion, we examine their association with expression divergence between the closely related species, S. cerevisiae and S. paradoxus. Although individually well conserved at orthologous genes, changes between modifications are mostly uncorrelated, indicating largely non-overlapping regulatory mechanisms. Notably, we find that inter-species differences in methylation, but not acetylation, are well correlated with expression changes, thereby proposing H3K4me3 as a candidate regulator of expression divergence. Taken together, our results suggest distinct evolutionary roles for expression-linked modifications, wherein H3K4me3 may contribute to stabilize average expression, whilst H3K9ac associates with more indirect aspects such as responsiveness.     

The DNA dioxygenase Tet1 regulates H3K27 modification and embryonic stem cell biology independent of its catalytic activity

Tet enzymes (Tet1/2/3) oxidize 5-methylcytosine to promote DNA demethylation and partner with chromatin modifiers to regulate gene expression. Tet1 is highly expressed in embryonic stem cells (ESCs), but its enzymatic and non-enzymatic roles in gene regulation are not dissected. We have generated Tet1 catalytically inactive (Tet1^m/m) and knockout (Tet1^−/−) ESCs and mice to study these functions. Loss of Tet1, but not loss of its catalytic activity, caused aberrant upregulation of bivalent (H3K4me3⁺; H3K27me3⁺) developmental genes, leading to defects in differentiation. Wild-type and catalytic-mutant Tet1 occupied similar genomic loci which overlapped with H3K27 tri-methyltransferase PRC2 and the deacetylase complex Sin3a at promoters of bivalent genes and with the helicase Chd4 at active genes. Loss of Tet1, but not loss of its catalytic activity, impaired enrichment of PRC2 and Sin3a at bivalent promoters leading to reduced H3K27 trimethylation and deacetylation, respectively, in absence of any changes in DNA methylation. Tet1^−/−, but not Tet1^m/m, embryos expressed higher levels of Gata6 and were developmentally delayed. Thus, the critical functions of Tet1 in ESCs and early development are mediated through its non-catalytic roles in regulating H3K27 modifications to silence developmental genes, and are more important than its catalytic functions in DNA demethylation.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.