Epigenetic therapy attenuates oxidative stress in BMSCs during ageing

Oxidative stress, a hallmark of ageing, inhibits the osteogenic differentiation of bone marrow‐derived mesenchymal stem cells in long bone. The dysfunction of the cellular antioxidant defence system is a critical cause of oxidative stress, but the mechanism of the decline of antioxidant defence in senescent stem cells remains elusive. Here, we found that EZH2, an epigenetic regulator of histone methylation, acted as a suppressor of the antioxidative defence system in BMSCs from the femur. The increased EZH2 led to a decrease in the levels of antioxidant enzymes and exaggerated oxidative damage in aged BMSCs, resulting in the defect of bone formation and regeneration. Mechanistically, EZH2 enhanced the modification of H3K27me3 on the promoter of Foxo1 and suppressed its function to activate the downstream genes in antioxidant defence. Moreover, epigenetic therapy targeting EZH2‐mediated H3K27me3 modification largely recovered the antioxidant defence in BMSCs and attenuate oxidative damage, leading to the recovery of the osteogenesis in old BMSCs. Taken together, our findings revealed novel crosstalk between histone epigenetic modification and oxidative stress during stem cell ageing, suggesting a possibility of epigenetic therapy in the recovery of BMSCs senescence and treatment of age‐related bone disease.     

Inhibition of EZH2 Attenuates Sorafenib Resistance by Targeting NOTCH1 Activation-Dependent Liver Cancer Stem Cells via NOTCH1-Related MicroRNAs in Hepatocellular Carcinoma

Acquired resistance and intrinsic to sorafenib therapy represents a major hurdle in improving the management of advanced hepatocellular carcinoma (HCC), which has been recently shown to be associated with the emergence of liver cancer stem cells (CSCs). However, it remains largely unknown whether and how histone posttranslational modifications, especially H3K27me3, are causally linked to the maintenance of self-renewal ability in sorafenib-resistant HCC. Here, we found that NOTCH1 signaling was activated in sorafenib-resistant HCC cells and NOTCH1 activation conferred hepatoma cells sorafenib resistance through enhanced self-renewal and tumorigenecity. Besides, the overexpression of EZH2 was required for the emergence of cancer stem cells following prolonged sorafenib treatment. As such, modulating EZH2 expression or activity suppressed activation of NOTCH1 pathway by elevating the expression of NOTCH1-related microRNAs, hsa-miR-21-5p and has-miR-26a-1-5p, via H3K27me3, and consequently weakened self-renewal ability and tumorigenecity and restored the anti-tumor effects of sorafenib. Overall, our results highlight the role of EZH2/NICD1 axis, and also suggest that EZH2 and NOTCH1 pathway are rational targets for therapeutic intervention in sorafenib-resistant HCC.     

Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells

We mapped Polycomb-associated H3K27 trimethylation (H3K27me3) and Trithorax-associated H3K4 trimethylation (H3K4me3) across the whole genome in human embryonic stem (ES) cells. The vast majority of H3K27me3 colocalized on genes modified with H3K4me3. These commodified genes displayed low expression levels and were enriched in developmental function. Another significant set of genes lacked both modifications and was also expressed at low levels in ES cells but was enriched for gene function in physiological responses rather than development. Commodified genes could change expression levels rapidly during differentiation, but so could a substantial number of genes in other modification categories. SOX2, POU5F1, and NANOG, pluripotency-associated genes, shifted from modification by H3K4me3 alone to colocalization of both modifications as they were repressed during differentiation. Our results demonstrate that H3K27me3 modifications change during early differentiation, both relieving existing repressive domains and imparting new ones, and that colocalization with H3K4me3 is not restricted to pluripotent cells.     

EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.     

Aberrant overexpression of EZH2 and H3K27me3 serves as poor prognostic biomarker for esophageal squamous cell carcinoma patients

It has been reported that the trimethylation of histone 3 on lysine 27 (H3K27me3) is required for enhancer of zeste homology 2 (EZH2)-mediated repression of various genes essential for tumorigenesis and tumor development. Here, we reported the expression of EZH2 and H3K27me3 in esophageal squamous cell carcinoma (ESCC) specimens was higher than the pericarcinoma esophageal specimens. Their expression was positively associated with the poor prognosis of ESCC patients. EZH2 expression, histological grade and distant lymph node metastasis were all independent factors for poor prognosis of ESCC. In addition, enforced expression of EZH2 in esophageal cancer-derived cells could increase the overall H3K27me3 level. Our results suggested the expression of EZH2 and H3K27me3 could serve as biomarkers in the prediction of ESCC patients’ survival and ESCC metastasis.     

Polycomb Repressive Complex 2 Regulates Lineage Fidelity during Embryonic Stem Cell Differentiation

Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, making it difficult to determine the precise roles of PRC2 during lineage commitment. Moreover, while studies suggest that PRC2 prevents DNA methylation, how these two epigenetic regulators coordinate to regulate lineage programs is poorly understood. Using several PRC2 mutant ESC lines that maintain varying levels of H3K27me3, we found that partial maintenance of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). In contrast, genes that function to specify other lineages failed to be repressed in these cells, suggesting that PRC2 is also necessary for lineage fidelity. We also found that loss of H3K27me3 leads to a modest gain in DNA methylation at PRC2 target regions in both ESCs and in SMNs. Our study demonstrates a critical role for PRC2 in safeguarding lineage decisions and in protecting genes against inappropriate DNA methylation.     

Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.