Pericentric Heterochromatin Generated by HP1 Protein Interaction-defective Histone Methyltransferase Suv39h1

Pericentric regions form epigenetically organized silent heterochromatin structures that accumulate histone H3 lysine 9 trimethylation (H3K9me3) and HP1. At pericentric regions, Suv39h is the major enzyme that generates H3K9me3. Suv39h also interacts directly with HP1, a methylated H3K9-binding protein. However, it is not well characterized how HP1 interaction is important for Suv39h accumulation and Suv39h-mediated H3K9me3 formation at the pericentromere. To address this, we introduced the HP1 binding-defective N-terminally truncated mouse Suv39h1 (ΔN) into Suv39h-deficient embryonic stem cells. Interestingly, pericentric accumulation of ΔN and ΔN-mediated H3K9me3 was observed to recover, but HP1 accumulation was only marginally restored. ΔN also rescued DNA methyltransferase Dnmt3a and -3b accumulation and DNA methylation of the pericentromere. In contrast, other pericentric heterochromatin features, such as ATRX protein association and H4K20me3, were not recovered. Finally, derepressed major satellite repeats were partially silenced by ΔN expression. These findings clearly showed that the Suv39h-HP1 binding is dispensable for pericentric H3K9me3 and DNA methylation, but this interaction and HP1 recruitment/accumulation seem to be crucial for complete formation of heterochromatin.     

A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells

The Rb/E2F complex represses S-phase genes both in cycling cells and in cells that have permanently exited from the cell cycle and entered a terminal differentiation pathway. Here we show that S-phase gene repression, which involves histone-modifying enzymes, occurs through distinct mechanisms in these two situations. We used chromatin immunoprecipitation to show that methylation of histone H3 lysine 9 (H3K9) occurs at several Rb/E2F target promoters in differentiating cells but not in cycling cells. Furthermore, phenotypic knock-down experiments using siRNAs showed that the histone methyltransferase Suv39h is required for histone H3K9 methylation and subsequent repression of S-phase gene promoters in differentiating cells, but not in cycling cells. These results indicate that the E2F target gene permanent silencing mechanism that is triggered upon terminal differentiation is distinct from the transient repression mechanism in cycling cells. Finally, Suv39h-depleted myoblasts were unable to express early or late muscle differentiation markers. Thus, appropriately timed H3K9 methylation by Suv39h seems to be part of the control switch for exiting the cell cycle and entering differentiation.     

Sirt7 inhibits Sirt1-mediated activation of Suv39h1

Sirtuins regulate a variety of cellular processes through protein deacetylation. The best-known member of mammalian sirtuin family, Sirt1, plays important roles in the maintenance of cellular homeostasis by regulating cell metabolism, differentiation and stress responses, among others. Sirt1 activity requires tight regulation to meet specific cellular requirements, which is achieved at different levels and by specific mechanisms. Recently, a regulatory loop between Sirt1 and another sirtuin, Sirt7, was identified. Sirt7 inhibits Sirt1 autodeacetylation at K230 and activation thereby preventing Sirt1-mediated repression of adipocyte differentiation by inhibition of the PPARγ gene. Here, we extend the regulatory complexity of Sirt7-dependent restriction of Sirt1 activity by demonstrating that Sirt7 reduces activation of a previously described prominent Sirt1 target, the histone methyltransferase Suv39h1. We show that removal of the acetyl-group at K230 in Sirt1 due to the absence of Sirt7 leads to hyperactivation of Sirt1 and thereby to constantly increased activity of Suv39h1.     

Down‐regulation of Suv39h1 attenuates neointima formation after carotid artery injury in diabetic rats

Patients with diabetes have an increased risk of vascular complications. Suv39h1, a histone methyltransferase, plays a protective role against myocardial injury in diabetes. Herein, we intend to explore whether Suv39h1 could affect neointimal formation after vascular injury in diabetic rats and reveal the underlying mechanism. In this study, we generated adenovirus expressing Suv39h1 as well as lentivirus expressing Suv39h1‐targeting shRNA and evaluated the significance of Suv39h1 in vascular smooth muscle cells (VSMCs) under diabetic conditions. In vitro, we examined proliferative and migratory behaviours as well as the underlying signalling mechanisms in VSMCs in response to high glucose treatment. In vivo, we induced diabetes in SD rats with streptozocin and established the common carotid artery balloon injury model. Suv39h1 was found to be both necessary and sufficient to promote VSMC proliferation and migration under high glucose conditions. We observed corresponding changes in intracellular signalling molecules including complement C3 and phosphor‐ERK1/2. However, either up‐regulating or down‐regulating Suv39h1, phosphor‐p38 level was not significantly affected. Consistently, Suv39h1 overexpression led to accelerated neointima formation, while knocking down Suv39h1 reduced it following carotid artery injury in diabetic rats. Using microarray analyses, we showed that altering the Suv39h1 level in vivo dramatically altered the expression of myriad genes mediating different biological processes and molecular function. This study reveals the novel role of Suv39h1 in VSMCs of diabetes and suggests its potential role as a therapeutic target in diabetic vascular injury.     

Altered telomere homeostasis and resistance to skin carcinogenesis in Suv39h1 transgenic mice

The Suv39h1 and Suv39h2 H3K9 histone methyltransferases (HMTs) have a conserved role in the formation of constitutive heterochromatin and gene silencing. Using a transgenic mouse model system we demonstrate that elevated expression of Suv39h1 increases global H3K9me3 levels in vivo. More specifically, Suv39h1 overexpression enhances the imposition of H3K9me3 levels at constitutive heterochromatin at telomeric and major satellite repeats in primary mouse embryonic fibroblasts. Chromatin compaction is paralleled by telomere shortening, indicating that telomere length is controlled by H3K9me3 density at telomeres. We further show that increased Suv39h1 levels result in an impaired clonogenic potential of transgenic epidermal stem cells and Ras/E1A transduced transgenic primary mouse embryonic fibroblasts. Importantly, Suv39h1 overexpression in mice confers resistance to a DMBA/TPA induced skin carcinogenesis protocol that is characterized by the accumulation of activating H-ras mutations. Our results provide genetic evidence that Suv39h1 controls telomere homeostasis and mediates resistance to oncogenic stress in vivo. This identifies Suv39h1 as an interesting target to improve oncogene induced senescence in premalignant lesions.     

An HP1 isoform-specific feedback mechanism regulates Suv39h1 activity under stress conditions

The presence of H3K9me3 and heterochromatin protein 1 (HP1) are hallmarks of heterochromatin conserved in eukaryotes. The spreading and maintenance of H3K9me3 is effected by the functional interplay between the H3K9me3-specific histone methyltransferase Suv39h1 and HP1. This interplay is complex in mammals because the three HP1 isoforms, HP1α, β, and γ, are thought to play a redundant role in Suv39h1-dependent deposition of H3K9me3 in pericentric heterochromatin (PCH). Here, we demonstrate that despite this redundancy, HP1α and, to a lesser extent, HP1γ have a closer functional link to Suv39h1, compared to HP1β. HP1α and γ preferentially interact in vivo with Suv39h1, regulate its dynamics in heterochromatin, and increase Suv39h1 protein stability through an inhibition of MDM2-dependent Suv39h1-K87 polyubiquitination. The reverse is also observed, where Suv39h1 increases HP1α stability compared HP1β and γ. The interplay between Suv39h1 and HP1 isoforms appears to be relevant under genotoxic stress. Specifically, loss of HP1α and γ isoforms inhibits the upregulation of Suv39h1 and H3K9me3 that is observed under stress conditions. Reciprocally, Suv39h1 deficiency abrogates stress-dependent upregulation of HP1α and γ, and enhances HP1β levels. Our work defines a specific role for HP1 isoforms in regulating Suv39h1 function under stress via a feedback mechanism that likely regulates heterochromatin formation.     

Histone 3 modifications and blood pressure in the Beijing Truck Driver Air Pollution Study

Context: Histone modifications regulate gene expression; dysregulation has been linked with cardiovascular diseases. Associations between histone modification levels and blood pressure in humans are unclear.

Objective: We examine the relationship between global histone concentrations and various markers of blood pressure.

Materials and methods: Using the Beijing Truck Driver Air Pollution Study, we investigated global peripheral white blood cell histone modifications (H3K9ac, H3K9me3, H3K27me3, and H3K36me3) associations with pre- and post-work measurements of systolic (SBP) and diastolic (DBP) blood pressure, mean arterial pressure (MAP), and pulse pressure (PP) using multivariable mixed-effect models.

Results: H3K9ac was negatively associated with pre-work SBP and MAP; H3K9me3 was negatively associated with pre-work SBP, DBP, and MAP; and H3K27me3 was negatively associated with pre-work SBP. Among office workers, H3K9me3 was negatively associated with pre-work SBP, DBP, and MAP. Among truck drivers, H3K9ac and H3K27me were negatively associated with pre-work SBP, and H3K27me3 was positively associated with post-work PP.

Discussion and conclusion: Epigenome-wide H3K9ac, H3K9me3, and H3K27me3 were negatively associated with multiple pre-work blood pressure measures. These associations substantially changed during the day, suggesting an influence of daily activities. Blood-based histone modification biomarkers are potential candidates for studies requiring estimations of morning/pre-work blood pressure.     

The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male''s life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitrocell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.     

Histone H3 lysine-trimethylation markers are decreased by recombinant methioninase and increased by methotrexate at concentrations which inhibit methionine-addicted osteosarcoma cell proliferation

Methionine addiction is a fundamental and general hallmark of cancer cells, which require exogenous methionine, despite their ability to synthesize normal amounts of methionine from homocysteine. In contrast, methionine-independent normal cells do not require exogenous methionine in the presence of a methionine precursor. The methionine addiction of cancer cells is due to excess transmethylation reactions. We have previously shown that histone H3 lysine marks are over-methylated in cancer cells and the over-methylation is unstable when the cancer cells are restricted of methionine. In the present study, we show that methionine-addicted osteosarcoma cells are sensitive to both methotrexate (MTX) and recombinant methioninase (rMETase), but they affect histone H3 lysine-methylation in the opposite direction. Concentrations of MTX and rMETase, which inhibit osteosarcoma cells viability to 20%, had opposing effects on the status of histone methylation of H3K9me3 and H3K27me3. rMETase significantly decreased the amount of H3K9me3 and H3K27me3. In contrast, MTX significantly increased the amount of H3K9me and H3K27me3. The results suggest that increase or decrease in these methylated histone lysine marks is associated with proliferation arrest of methionine-addicted osteosarcoma.     

Epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer

Emerging evidence suggests that microRNAs (miRNAs) serve an important role in tumorigenesis and development. Although the low expression of miR‐125a‐5p in gastric cancer has been reported, the underlying mechanism remains unknown. In the current study, the low expression of miR‐125a‐5p in gastric cancer was verified in paired cancer tissues and adjacent non‐tumour tissues. Furthermore, the GC islands in the miR‐125a‐5p region were hypermethylated in the tumour tissues. And the hypermethylation was negatively correlated with the miR‐125a‐5p expression. Target gene screening showed that the histone methyltransferase Suv39H1 was one of the potential target genes. In vitro studies showed that miR‐125a‐5p could directly suppress the Suv39H1 expression and decrease the H3K9me3 levels. On the other hand, the Suv39H1 could induce demethylation of miR‐125a‐5p, resulting in re‐activation of miR‐125a‐5p. What is more, overexpessing miR‐125a‐5p could also self‐activate the silenced miR‐125a‐5p in gastric cancer cells, which suppressed cell migration, invasion and proliferation in vitro and inhibited cancer progression in vivo. Thus, we uncovered here that the epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer, suggesting that miR‐125a‐5p might be not only the potential prognostic value as a tumour biomarker but also potential therapeutic targets in gastric cancer.     

FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells

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