Loss of H3K9 trimethylation alters chromosome compaction and transcription factor retention during mitosis.

Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases and polycomb repressor complexes, binds to chromosomes throughout mitosis and their depletion results in increased chromosome size. In the present study, we show that enzymes that catalyze H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly, however, mutants lacking histone 3 lysine 9 trimethylation (H3K9me3) have unusually small and compact mitotic chromosomes associated with increased histone H3 phospho Ser10 (H3S10ph) and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous first protein lysine methyltransferase Suv39h1 or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wild-type versus Suv39h1/Suv39h2 double-null mouse embryonic stem cells revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.

An LKB1-mitochondria axis controls TH17 effector function.

CD4+ T cell differentiation requires metabolic reprogramming to fulfil the bioenergetic demands of proliferation and effector function, and enforce specific transcriptional programmes1-3. Mitochondrial membrane dynamics sustains mitochondrial processes4, including respiration and tricarboxylic acid (TCA) cycle metabolism5, but whether mitochondrial membrane remodelling orchestrates CD4+ T cell differentiation remains unclear. Here we show that unlike other CD4+ T cell subsets, T helper 17 (TH17) cells have fused mitochondria with tight cristae. T cell-specific deletion of optic atrophy 1 (OPA1), which regulates inner mitochondrial membrane fusion and cristae morphology6, revealed that TH17 cells require OPA1 for its control of the TCA cycle, rather than respiration. OPA1 deletion amplifies glutamine oxidation, leading to impaired NADH/NAD+ balance and accumulation of TCA cycle metabolites and 2-hydroxyglutarate-a metabolite that influences the epigenetic landscape5,7. Our multi-omics approach revealed that the serine/threonine kinase liver-associated kinase B1 (LKB1) couples mitochondrial function to cytokine expression in TH17 cells by regulating TCA cycle metabolism and transcriptional remodelling. Mitochondrial membrane disruption activates LKB1, which restrains IL-17 expression. LKB1 deletion restores IL-17 expression in TH17 cells with disrupted mitochondrial membranes, rectifying aberrant TCA cycle glutamine flux, balancing NADH/NAD+ and preventing 2-hydroxyglutarate production from the promiscuous activity of the serine biosynthesis enzyme phosphoglycerate dehydrogenase (PHGDH). These findings identify OPA1 as a major determinant of TH17 cell function, and uncover LKB1 as a sensor linking mitochondrial cues to effector programmes in TH17 cells.

Complete loss of H3K9 methylation dissolves mouse heterochromatin organization.

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.

m6A RNA methylation of major satellite repeat transcripts facilitates chromatin association and RNA:DNA hybrid formation in mouse heterochromatin.

Heterochromatin has essential functions in maintaining chromosome structure, in protecting genome integrity and in stabilizing gene expression programs. Heterochromatin is often nucleated by underlying DNA repeat sequences, such as major satellite repeats (MSR) and long interspersed nuclear elements (LINE). In order to establish heterochromatin, MSR and LINE elements need to be transcriptionally competent and generate non-coding repeat RNA that remain chromatin associated. We explored whether these heterochromatic RNA, similar to DNA and histones, may be methylated, particularly for 5-methylcytosine (5mC) or methyl-6-adenosine (m6A). Our analysis in mouse ES cells identifies only background level of 5mC but significant enrichment for m6A on heterochromatic RNA. Moreover, MSR transcripts are a novel target for m6A RNA modification, and their m6A RNA enrichment is decreased in ES cells that are mutant for Mettl3 or Mettl14, which encode components of a central RNA methyltransferase complex. Importantly, MSR transcripts that are partially deficient in m6A RNA methylation display impaired chromatin association and have a reduced potential to form RNA:DNA hybrids. We propose that m6A modification of MSR RNA will enhance the functions of MSR repeat transcripts to stabilize mouse heterochromatin.

Repetitive Elements Trigger RIG-I-like Receptor Signaling that Regulates the Emergence of Hematopoietic Stem and Progenitor Cells.

Inflammatory signaling is required for hematopoietic stem and progenitor cell (HSPC) development. Here, we studied the involvement of RIG-I-like receptors (RLRs) in HSPC formation. Rig-I or Mda5 deficiency impaired, while Lgp2 deficiency enhanced, HSPC emergence in zebrafish embryos. Rig-I or Mda5 deficiency reduced HSPC numbers by inhibiting inflammatory signals that were in turn enhanced in Lgp2 deficient embryos. Simultaneous reduction of Lgp2 and either Rig-I or Mda5 rescued inflammatory signals and HSPC numbers. Modulating the expression of the signaling mediator Traf6 in RLR deficient embryos restored HSPC numbers. Repetitive element transcripts could be detected in hemogenic endothelial cells and HSPCs, suggesting a role as RLR ligands. Indeed, ectopic expression of repetitive elements enhanced HSPC formation in wild-type, but not in Rig-I or Mda5 deficient embryos. Manipulation of RLR expression in mouse fetal liver HSPCs indicated functional conservation among species. Thus, repetitive elements transcribed during development drive RLR-mediated inflammatory signals that regulate HSPC formation.

Heterochromatin establishment during early mammalian development is regulated by pericentromeric RNA and characterized by non-repressive H3K9me3.

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.

H1 linker histones silence repetitive elements by promoting both histone H3K9 methylation and chromatin compaction.

Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.

Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis.

Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. Our study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.

Acetate Promotes T Cell Effector Function during Glucose Restriction.

Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8+ T cells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Ex vivo acetate treatment increases IFN-γ production by exhausted T cells, whereas reducing ACSS expression in T cells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote T cell function during cancer.

H3K9me3-heterochromatin loss at protein-coding genes enables developmental lineage specification.

Gene silencing by chromatin compaction is integral to establishing and maintaining cell fates. Trimethylated histone 3 lysine 9 (H3K9me3)-marked heterochromatin is reduced in embryonic stem cells compared to differentiated cells. However, the establishment and dynamics of closed regions of chromatin at protein-coding genes, in embryologic development, remain elusive. We developed an antibody-independent method to isolate and map compacted heterochromatin from low-cell number samples. We discovered high levels of compacted heterochromatin, H3K9me3-decorated, at protein-coding genes in early, uncommitted cells at the germ-layer stage, undergoing profound rearrangements and reduction upon differentiation, concomitant with cell type-specific gene expression. Perturbation of the three H3K9me3-related methyltransferases revealed a pivotal role for H3K9me3 heterochromatin during lineage commitment at the onset of organogenesis and for lineage fidelity maintenance.

Interplay among H3K9-editing enzymes SUV39H1, JMJD2C and SRC-1 drives p66Shc transcription and vascular oxidative stress in obesity.

Aims: Accumulation of reactive oxygen species (ROS) promotes vascular disease in obesity, but the underlying molecular mechanisms remain poorly understood. The adaptor p66Shc is emerging as a key molecule responsible for ROS generation and vascular damage. This study investigates whether epigenetic regulation of p66Shc contributes to obesity-related vascular disease. Methods and results: ROS-driven endothelial dysfunction was observed in visceral fat arteries (VFAs) isolated from obese subjects when compared with normal weight controls. Gene profiling of chromatin-modifying enzymes in VFA revealed a significant dysregulation of methyltransferase SUV39H1 (fold change, -6.9, P < 0.01), demethylase JMJD2C (fold change, 3.2, P < 0.01), and acetyltransferase SRC-1 (fold change, 5.8, P < 0.01) in obese vs. control VFA. These changes were associated with reduced di-(H3K9me2) and trimethylation (H3K9me3) as well as acetylation (H3K9ac) of histone 3 lysine 9 (H3K9) on p66Shc promoter. Reprogramming SUV39H1, JMJD2C, and SRC-1 in isolated endothelial cells as well as in aortas from obese mice (LepOb/Ob) suppressed p66Shc-derived ROS, restored nitric oxide levels, and rescued endothelial dysfunction. Consistently, in vivo editing of chromatin remodellers blunted obesity-related vascular p66Shc expression. We show that SUV39H1 is the upstream effector orchestrating JMJD2C/SRC-1 recruitment to p66Shc promoter. Indeed, SUV39H1 overexpression in obese mice erased H3K9-related changes on p66Shc promoter, while SUV39H1 genetic deletion in lean mice recapitulated obesity-induced H3K9 remodelling and p66Shc transcription. Conclusion: These results uncover a novel epigenetic mechanism underlying endothelial dysfunction in obesity. Targeting SUV39H1 may attenuate oxidative transcriptional programmes and thus prevent vascular disease in obese individuals.

SIRT6-dependent cysteine monoubiquitination in the PRE-SET domain of Suv39h1 regulates the NF-κB pathway.

Sirtuins are NAD+-dependent deacetylases that facilitate cellular stress response. They include SirT6, which protects genome stability and regulates metabolic homeostasis through gene silencing, and whose loss induces an accelerated aging phenotype directly linked to hyperactivation of the NF-κB pathway. Here we show that SirT6 binds to the H3K9me3-specific histone methyltransferase Suv39h1 and induces monoubiquitination of conserved cysteines in the PRE-SET domain of Suv39h1. Following activation of NF-κB signaling Suv39h1 is released from the IκBα locus, subsequently repressing the NF-κB pathway. We propose that SirT6 attenuates the NF-κB pathway through IκBα upregulation via cysteine monoubiquitination and chromatin eviction of Suv39h1. We suggest a mechanism based on SirT6-mediated enhancement of a negative feedback loop that restricts the NF-κB pathway.

Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation.

The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity for major satellite repeat RNA. To analyze an RNA-dependent interaction with chromatin, we purified native nucleosomes from mouse ES cells and detect that Suv39h1 and Suv39h2 exclusively associate with poly-nucleosomes. This association was attenuated upon RNaseH incubation and entirely lost upon RNaseA digestion of native chromatin. Major satellite repeat transcripts remain chromatin-associated and have a secondary structure that favors RNA:DNA hybrid formation. Together, these data reveal an RNA-mediated mechanism for the stable chromatin interaction of the Suv39h KMT and suggest a function for major satellite non-coding RNA in the organization of an RNA-nucleosome scaffold as the underlying structure of mouse heterochromatin.

The chromatin remodelling factor ATRX suppresses R-loops in transcribed telomeric repeats.

ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG)n ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function.

The histone H3K9 methyltransferase SUV39H links SIRT1 repression to myocardial infarction.

Myocardial infarction (MI) dampens heart function and poses a great health risk. The class III deacetylase sirtuin 1 (SIRT1) is known to confer cardioprotection. SIRT1 expression is downregulated in the heart by a number of stress stimuli that collectively drive the pathogenesis of MI, although the underlying mechanism remains largely obscure. Here we show that in primary rat neonatal ventricular myocytes (NRVMs), ischaemic or oxidative stress leads to a rapid upregulation of SUV39H, the mammalian histone H3K9 methyltransferase, paralleling SIRT1 downregulation. Compared to wild-type littermates, SUV39H knockout mice are protected from MI. Likewise, suppression of SUV39H activity with chaetocin attenuates cardiac injury following MI. Mechanistically, SUV39H cooperates with heterochromatin protein 1 gamma (HP1γ) to catalyse H3K9 trimethylation on the SIRT1 promoter and represses SIRT1 transcription. SUV39H augments intracellular ROS levels in a SIRT1-dependent manner. Our data identify a previously unrecognized role for SUV39H linking SIRT1 trans-repression to myocardial infarction.

The histone methyltransferase Suv39h2 contributes to nonalcoholic steatohepatitis in mice.

Uncontrolled inflammatory response highlights the central theme of nonalcoholic steatohepatitis (NASH), a growing global pandemic. Hepatocytes and macrophages represent two major sources of hepatic inflammation during NASH pathogenesis, contributing to excessive synthesis of proinflammatory mediators. The epigenetic mechanism that accounts for the activation of hepatocytes and macrophages in this process remains obscure. Here, we report that compared to wild-type littermates, mice with a deficiency in the histone H3K9 methyltransferase suppressor of variegation 39 homolog 2 (Suv39h2, knockout) exhibited a less severe form of NASH induced by feeding with a high-fat, high-carbohydrate diet. Pro-NASH stimuli increased Suv39h2 expression in cell culture, in mice, and in human livers. In hepatocytes, Suv39h2 bound to the Sirt1 gene promoter and repressed Sirt1 transcription. Suv39h2 deficiency normalized Sirt1 expression, allowing nuclear factor kappa B/p65 to become hypoacetylated and thus dampening nuclear factor kappa B-dependent transcription of proinflammatory mediators. In macrophages, Suv39h2-mediated repression of peroxisome proliferator-activated receptor gamma transcription favored a proinflammatory M1 phenotype over an anti-inflammatory M2 phenotype, thereby elevating hepatic inflammation. CONCLUSION: Suv39h2 plays a pivotal role in the regulation of inflammatory response in hepatocytes and macrophages, contributing to NASH pathogenesis. (Hepatology 2017;65:1904-1919).

The molecular hallmarks of epigenetic control.

Over the past 20 years, breakthrough discoveries of chromatin-modifying enzymes and associated mechanisms that alter chromatin in response to physiological or pathological signals have transformed our knowledge of epigenetics from a collection of curious biological phenomena to a functionally dissected research field. Here, we provide a personal perspective on the development of epigenetics, from its historical origins to what we define as 'the modern era of epigenetic research'. We primarily highlight key molecular mechanisms of and conceptual advances in epigenetic control that have changed our understanding of normal and perturbed development.

Dynamic and flexible H3K9me3 bridging via HP1ß dimerization establishes a plastic state of condensed chromatin.

Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1ß is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1ß bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1ß genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin.

Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program.

DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.

Trim28 Haploinsufficiency Triggers Bi-stable Epigenetic Obesity.

More than one-half billion people are obese, and despite progress in genetic research, much of the heritability of obesity remains enigmatic. Here, we identify a Trim28-dependent network capable of triggering obesity in a non-Mendelian, "on/off" manner. Trim28(+/D9) mutant mice exhibit a bi-modal body-weight distribution, with isogenic animals randomly emerging as either normal or obese and few intermediates. We find that the obese-"on" state is characterized by reduced expression of an imprinted gene network including Nnat, Peg3, Cdkn1c, and Plagl1 and that independent targeting of these alleles recapitulates the stochastic bi-stable disease phenotype. Adipose tissue transcriptome analyses in children indicate that humans too cluster into distinct sub-populations, stratifying according to Trim28 expression, transcriptome organization, and obesity-associated imprinted gene dysregulation. These data provide evidence of discrete polyphenism in mouse and man and thus carry important implications for complex trait genetics, evolution, and medicine.