Xinyu Weng

A crosstalk between chromatin remodeling and histone H3K4 methyltransferase complexes in endothelial cells regulates angiotensin II-induced cardiac hypertrophy.

Angiotensin II (Ang II) induces cardiac hypertrophy and fibrosis in part by stimulating endothelin (ET-1) transcription. The involvement of the epigenetic machinery in this process is largely undefined. In the present study, we examined the epigenetic maneuvering underlying cardiac hypertrophy and fibrosis following ET-1 transactivation by Ang II. In response to Ang II stimulation, core components of the mammalian chromatin remodeling complex (Brahma-related gene 1, or Brg1, and Brahma or Brm) and histone H3K4 methylation complex (Ash2, absent, small, or homeotic discs 2, or Ash2 and WD domain repeat 5, or Wdr5) were recruited to the ET-1 promoter region in endothelial cells. Over-expression of Brg1/Brm or Ash2/Wdr5 enhanced while depletion of Brg1/Brm or Ash2/Wdr5 attenuated Ang II-induced ET-1 transactivation. Endothelial-specific knockdown of Brg1/Brm or Ash2/Wdr5 ameliorated cardiac hypertrophy both in vitro and in vivo. More important, Brg1/Brm interacted with Ash2/Wdr5 on the ET-1 promoter to catalyze H3K4 methylation. The crosstalk between Brg11/Brm and Ash2/Wdr5 was mediated by myocardin-related transcription factor A (MRTF-A). In conclusion, our data have unveiled an epigenetic complex that links ET-1 transactivation in endothelial cells to Ang II-induced cardiac hypertrophy and fibrosis.

Proinflammatory Stimuli Engage Brahma Related Gene 1 and Brahma in Endothelial Injury

Rationale: Endothelial dysfunction inflicted by inflammation is found in a host of cardiovascular pathologies. One hallmark event in this process is the aggregation and adhesion of leukocyte to the vessel wall mediated by the upregulation of adhesion molecules (CAM) in endothelial cells at the transcriptional level. The epigenetic modulator(s) of CAM transactivation and its underlying pathophysiological relevance remain poorly defined. Objective: Our goal was to determine the involvement of Brahma related gene 1 (Brg1) and Brahma (Brm) in CAM transactivation and its relevance in the pathogenesis of atherosclerosis. Methods and Results: In the present study, we report that proinflammatory stimuli augmented the expression of Brg1 and Brm in vitro in cultured endothelial cells and in vivo in arteries isolated from rodents. Overexpression of Brg1 and Brm promoted while knockdown of Brg1 and Brm abrogated transactivation of adhesion molecules and leukocyte adhesion induced by inflammatory signals. Brg1 and Brm interacted with and were recruited to the CAM promoters by nuclear factor &kgr;B/p65. Conversely, depletion of Brg1 and Brm disrupted the kinetics of p65 binding on CAM promoters and crippled CAM activation. Silencing of Brg1 and Brm also altered key epigenetic changes associated with CAM transactivation. Of intrigue, 17&bgr;-estradiol antagonized both the expression and activity of Brg1/Brm. Most importantly, endothelial-targeted elimination of Brg1/Brm conferred atheroprotective effects to Apoe-/- mice on a Western diet. Conclusions: Our data suggest that Brg1 and Brm integrate various proinflammatory cues into CAM transactivation and endothelial malfunction and, as such, may serve as potential therapeutic targets in treating inflammation-related cardiovascular diseases.

MRTF-A mediates LPS-induced pro-inflammatory transcription by interacting with the COMPASS complex

ABSTRACT Chronic inflammation underscores the pathogenesis of a range of human diseases. Lipopolysaccharide (LPS) elicits strong pro-inflammatory responses in macrophages through the transcription factor NF-&kgr;B. The epigenetic mechanism underlying LPS-induced pro-inflammatory transcription is not fully understood. Herein, we describe a role for myocardin-related transcription factor A (MRTF-A, also known as MKL1) in this process. MRTF-A overexpression enhanced NF-&kgr;B-dependent pro-inflammatory transcription, whereas MRTF-A silencing inhibited this process. MRTF-A deficiency also reduced the synthesis of pro-inflammatory mediators in a mouse model of colitis. LPS promoted the recruitment of MRTF-A to the promoters of pro-inflammatory genes in an NF-&kgr;B-dependent manner. Reciprocally, MRTF-A influenced the nuclear enrichment and target binding of NF-&kgr;B. Mechanistically, MRTF-A was necessary for the accumulation of active histone modifications on NF-&kgr;B target promoters by communicating with the histone H3K4 methyltransferase complex (COMPASS). Silencing of individual members of COMPASS, including ASH2, WDR5 and SET1 (also known as SETD1A), downregulated the production of pro-inflammatory mediators and impaired the NF-&kgr;B kinetics. In summary, our work has uncovered a previously unknown function for MRTF-A and provided insights into the rationalized development of anti-inflammatory therapeutic strategies.

Myocardin related transcription factor A programs epigenetic activation of hepatic stellate cells

BACKGROUND & AIMS Activation of hepatic stellate cells (HSCs) represents a key process in liver injury and, in the absence of intervention, leads to irreversible cirrhosis contributing significantly to the mortality of patients with liver disease. A missing link in the current understanding of HSC activation is the involvement of the epigenetic machinery. We investigated the role of the myocardin related transcription factor A (MRTF-A) in HSC activation. METHODS Liver fibrosis was induced in wild type (WT) and MRTF-A deficient (KO) mice by CCl4 injection. Expression of mRNA and protein was measured by real-time PCR, Western blotting, and immunohistochemistry. Protein binding to DNA was assayed by chromatin immunoprecipitation (ChIP). Knockdown of endogenous proteins was mediated by either small interfering RNA (siRNA) or short hairpin RNA (shRNA), carried by lentiviral particles. RESULTS KO mice exhibited resistance to CCl4-induced liver fibrosis compared to WT littermates. The expression of activated HSC signature genes was suppressed in the absence of MRTF-A. ChIP assays revealed that MRTF-A deficiency led to the erasure of key histone modifications, associated with transcriptional activation, such as H3K4 di- and tri-methylation, on the promoter regions of fibrogenic genes. Mechanistically, MRTF-A recruited a histone methyltransferase complex (COMPASS) to the promoters of fibrogenic genes to activate transcription. Silencing of individual COMPASS components dampened transactivation of fibrogenic genes in vitro and blocked liver fibrosis in mice. Oestradiol suppressed HSC activation by dampening the expression and binding activity of COMPASS. CONCLUSIONS Our data illustrate a novel mechanism that connects MRTF-A dependent histone H3K4 methylation to HSC activation.

Endothelial MRTF-A mediates angiotensin II induced cardiac hypertrophy

Angiotensin II (Ang II) stimulates endothelin (ET-1) transcription, which contributes to cardiac hypertrophy and fibrosis. We have previously reported that myocardin related transcription factor A (MRTF-A) is indispensable for ET-1 transcription in vascular endothelial cells under hypoxic conditions, indicating that MRTF-A might mediate Ang II-induced pathological hypertrophy. Here we report that Ang II augmented the expression of MRTF-A in cultured endothelial cells and in the lungs of mice with cardiac hypertrophy. Over-expression of MRTF-A enhanced, whereas depletion of MRTF-A attenuated, transcriptional activation of ET-1 gene by Ang II. MRTF-A deficiency ameliorated Ang II induced cardiac hypertrophy and fibrosis in mice paralleling diminished synthesis and release of ET-1. Mechanistically, MRTF-A was recruited to the ET-1 promoter by c-Jun/c-Fos (AP-1) in response to Ang II treatment. Once bound, MRTF-A altered the chromatin structure by modulating histone acetylation and H3K4 methylation on the ET-1 promoter. More importantly, mice with endothelial-specific MRTF-A silencing by lentiviral particles phenocopied mice with systemic MRTF-A deletion in terms of Ang II-induced pathological hypertrophy. In conclusion, we data have unveiled a MRTF-A-containing complex that links ET-1 transactivation in endothelial cells to cardiac hypertrophy and fibrosis by Ang II.

Histone Methyltransferase SET1 Mediates Angiotensin II–Induced Endothelin-1 Transcription and Cardiac Hypertrophy in Mice

Objective— Endothelin-1 is a potent vasoconstrictor derived from vascular endothelium. Elevated endothelin-1 levels are observed in a host of cardiovascular pathologies including cardiomyopathy. The epigenetic mechanism responsible for endothelin-1 induction in these pathological processes remains elusive. Approach and Results— We report here that induction of endothelin-1 expression in endothelial cells by angiotensin II (Ang II) was accompanied by the accumulation of histone H3K4 trimethylation, a preeminent histone modification for transcriptional activation, on the endothelin-1 promoter. In the meantime, Ang II stimulated the expression and the occupancy of Suv, Ez, and Trithorax domain 1 (SET1), a mammalian histone H3K4 trimethyltransferase, on the endothelin-1 promoter, both in vitro and in vivo. SET1 was recruited to the endothelin-1 promoter by activating protein 1 (c-Jun/c-Fos) and synergized with activating protein 1 to activate endothelin-1 transcription in response to Ang II treatment. Knockdown of SET1 in endothelial cells blocked Ang II–induced endothelin-1 synthesis and abrogated hypertrophy of cultured cardiomyocyte. Finally, endothelial-specific depletion of SET1 in mice attenuated Ang II–induced pathological hypertrophy and cardiac fibrosis. Conclusions— Our data suggest that SET1 epigenetically activates endothelin-1 transcription in endothelial cells, thereby contributing to Ang II–induced cardiac hypertrophy. As such, screening of small-molecule compound that inhibits SET1 activity will likely offer a new therapeutic solution to the treatment of cardiomyopathy.

Sin3B mediates collagen type I gene repression by interferon gamma in vascular smooth muscle cells

Collagen type I is the primary component of the extracellular matrix (ECM). Repression of collagen type I gene (COL1A2) transcription by the pro-inflammatory cytokine interferon gamma (IFN-γ) in vascular smooth muscle cells (VSMCs) is a key step during atherogenesis that leads to the destabilization of the atherosclerotic plaque. The epigenetic mechanism underlying IFN-γ induced COL1A2 repression is not clearly appreciated. We show here that Sin3B, a component of the eukaryotic histone deacetylase (HDAC) complex, was recruited to COL1A2 transcription start site in response to IFN-γ treatment in VSMCs paralleling COL1A2 repression. Short hairpin RNA (shRNA) mediated silencing of Sin3B abrogated collagen repression by IFN-γ and blocked the erasure of active histone marks and the accumulation of repressive histone marks on COL1A2 transcription start site as evidenced by chromatin immunoprecipitation (ChIP) assays. Sin3B cooperated with G9a, a histone H3K9 methyltransferase, to induce a repressive chromatin structure surrounding the collagen gene transcription start site in response to IFN-γ stimulation. Sin3B was recruited by regulatory factor for X-box 5 (RFX5) to the collagen site through a mechanism that involved HDAC2 mediated deacetylation of RFX5. Together, our data indicate that a repressor complex that contains RFX5, HDAC2, Sin3B, and G9a is responsible for IFN-γ induced COL1A2 repression in VSMCs. Targeting individual component of this complex will likely yield potential therapeutic solutions against atherosclerosis.

Angiotensin II induced CSF1 transcription is mediated by a crosstalk between different epigenetic factors in vascular endothelial cells

Endothelium-derived colony stimulating factor (CSF1) plays a key role in a range of human pathologies. Angiotensin II (Ang II) has been documented to stimulate CSF1 transcription although the underlying epigenetic mechanism remains unclear. Here we report that induction of CSF1 transcription by Ang II in vascular endothelial cells paralleled alterations of signature histone modifications surrounding the CSF1 promoter. Specifically, ChIP assays indicated that there was a simultaneous up-regulation of both acetylated H3 and trimethylated H3K4, indicative of transcriptional activation, and down-regulation of dimethyl H3K9, implicated in transcriptional repression, surrounding the proximal CSF1 promoter. Further analysis revealed that silencing of brahma related gene 1 (BRG1), a chromatin remodeling protein, abrogated CSF1 induction by Ang II. In the meantime, BRG1 silencing erased H3 acetylation and H3K4 trimethylation and restored H3K9 dimethylation. Mechanistically, BRG1 interacted with and recruited SET1A, a histone H3K4 methyltransferase, and JMJD1A, a histone H3K9 demethylase, to the CSF1 promoter to alter chromatin structure thereby promoting CSF1 trans-activation in response to Ang II stimulation. Knockdown of either SET1A or JMJD1A blocked CSF1 induction by Ang II. Finally, we demonstrate that the crosstalk between BRG1 and histone modifying enzymes was mediated by the transcription factor AP-1. In conclusion, our data unveil a novel epigenetic mechanism whereby a BRG1-centered complex mediates transcriptional activation of CSF1 by Ang II in vascular endothelial cells.

Brg1 trans-activates endothelium-derived colony stimulating factor to promote calcium chloride induced abdominal aortic aneurysm in mice

Endothelial cell derived secretive factors play pivotal roles maintaining the homeostasis by influencing the behaviors of other cells. When dysregulated, these factors may contribute to the disruption of physiological integrity and promote disease genesis in a number of different tissues and organs. In the present study we investigated how targeted deletion of brahma related gene 1 (Brg1), a chromatin remodeling protein, in endothelium might affect the pathogenesis of abdominal aortic aneurysm (AAA) induced by calcium chloride (CaCl2). We report here that compared to the wild type (WT) littermates, endothelial conditional Brg1 knockout (ecKO) mice exhibited an attenuated phenotype of AAA. Immunostaining and quantitative PCR analyses showed that vascular inflammation was suppressed in ecKO mice as opposed to WT mice likely due to diminished recruitment of macrophages. Further examination revealed that Brg1 deficiency led to a reduction in colony stimulating factor 1 (CSF1) levels. In cultured endothelial cells, Brg1 cooperated with histone H3K9 demethylase KDM3A to activate CSF1 transcription and macrophage recruitment thereby perpetuating vascular inflammation. Depletion of BRG1 or KDM3A in endothelial cells dampened CSF1 production and attenuated macrophage chemotaxis. Therefore, our data suggest that epigenetic activation of CSF1 transcription by Brg1 may contribute to AAA pathogenesis.

The histone methyltransferase SETD1A regulates thrombomodulin transcription in vascular endothelial cells

Thrombomodulin (TM, encoded by the THBD gene) expressed in vascular endothelial cells plays pivotal roles maintaining the equilibrium of coagulation and anti-coagulation. TM levels can be regulated at the transcriptional level although the epigenetic mechanism is underexplored. Here we report that transcriptional activation of TM in both immortalized vascular endothelial cells (EAhy926) and primary human aortic endothelial cells (HAEC) by all-trans retinoic acid (RA) paralleled accumulation of trimethylated histone H3K4, a prominent marker for active chromatin, surrounding the THBD promoter. RA treatment up-regulated the expression of SETD1A (SET1), a dedicated H3K4 methyltransferase, and augmented SETD1A occupancies on the THBD promoter. Further analysis revealed that the sequence-specific transcription factor Kruppel-like factor 4 (KLF4) interacted with and recruited SETD1A to the THBD promoter. Interestingly, SETD1A was recruited to the KLF4 promoter by retinoic acid receptor (RAR) and mediated the up-regulation of KLF4 expression by RA stimulation. In summary, our data illustrate a previously unrecognized pathway in which SETD1A contributes to RA-induced TM expression in vascular endothelial cells by modulating the activity and expression of KLF4.