Wenfang Tian

Brahma‐related gene 1 bridges epigenetic regulation of proinflammatory cytokine production to steatohepatitis in mice

Chronic inflammation, inflicted by the spillover of proinflammatory mediators, links metabolic dysfunction to nonalcoholic steatohepatitis (NASH). The epigenetic maneuverings that underscore accelerated synthesis of proinflammatory mediators in response to nutritional inputs are not clearly defined. Here we report that the ATP‐dependent chromatin remodeling proteins Brahma‐related gene 1 (Brg1) and Brahma (Brm) were up‐regulated in vitro in cultured hepatocytes treated with free fatty acid or glucose and in vivo in animal models of NASH. Occupancy of Brg1 and Brm on the promoter regions of proinflammatory genes was increased in vitro in cells and ex vivo in liver tissues. Estradiol suppressed the induction and recruitment of Brg1/Brm by palmitate. Recruitment of Brg1 and Brm relied on nuclear factor kappa B/p65; reciprocally, Brg1 and Brm contributed to the stabilization of p65 binding. Importantly, overexpression of Brg1/Brm enhanced, whereas knockdown of Brg1/Brm attenuated, the induction of proinflammatory mediators in hepatocytes challenged with excessive nutrient. Mechanistically, Brg1 and Brm were involved in the maintenance of a chromatin microenvironment marked by active histone modifications and friendly to the access of the general transcriptional machinery. Finally, depletion of Brg1/Brm by short hairpin RNA attenuated the release of proinflammatory mediators in the liver and significantly ameliorated hepatic pathology in NASH mice. Conclusion: Our data illustrate a Brg1‐dependent pathway that connects the epigenetic regulation of proinflammatory genes to the pathogenesis of NASH and point to a potential druggable target in the therapeutic intervention of NASH. (HEPATOLOGY 2013;58:576–588)

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.

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.

Pro-Inflammatory Stimuli Engage Brahma Related Gene 1 (Brg1) and Brahma (Brm) 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 steers an epigenetic complex to activate endothelin-induced pro-inflammatory transcription in vascular smooth muscle cells

Endothelin (ET-1) was initially identified as a potent vasoconstrictor contributing to the maintenance of vascular rhythm. Later studies have implicated ET-1, when aberrantly up-regulated within the vasculature, in a range of human pathologies associated with disruption of vascular homeostasis. ET-1 has been shown to invoke strong pro-inflammatory response in vascular smooth muscle cells (VSMCs); the underlying mechanism, however, remains elusive. Here, we report that the transcriptional modulator MRTF-A mediates the activation of pro-inflammatory mediators by ET-1 in VSMCs. ET-1 increased nuclear enrichment and activity of MRTF-A in cultured VSMCs. MRTF-A silencing attenuated ET-1 induced synthesis and release of pro-inflammatory mediators including IL-6, MCP-1 and IL-1 likely as a result of diminished NF-κB activity. In addition, MRTF-A was indispensible for the accumulation of active histone modifications on the gene promoters. Of intrigue, MRTF-A interacted with and recruited ASH2, a component of the mammalian histone methyltransferase complex, to transactivate pro-inflammatory genes in response to ET-1 treatment. The chromatin remodeling proteins BRG1 and BRM were also required for ET-1-dependent induction of pro-inflammatory mediators by communicating with ASH2, a process dependent on MRTF-A. In conclusion, our data have identified a novel epigenetic complex responsible for vascular inflammation inflicted by ET-1.

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.

MKL1 is an epigenetic modulator of TGF-β induced fibrogenesis.

Transforming growth factor (TGF-β) induced activation of portal fibroblast cells serves as a primary cause for liver fibrosis following cholestatic injury. The underlying epigenetic mechanism is not clear. We studied the role of a transcriptional modulator, megakaryoblastic leukemia 1 (MKL1) in this process. We report here that MKL1 deficiency ameliorated BDL-induced liver fibrosis in mice as assessed by histological stainings and expression levels of pro-fibrogenic genes. MKL1 silencing by small interfering RNA (siRNA) abrogated TGF-β induced transactivation of pro-fibrogenic genes in portal fibroblast cells. TGF-β stimulated the binding of MKL1 on the promoters of pro-fibrogenic genes and promoted the interaction between MKL1 and SMAD3. While SMAD3 was necessary for MKL1 occupancy on the gene promoters, MKL1 depletion impaired SMAD3 binding reciprocally. TGF-β treatment induced the accumulation of trimethylated histone H3K4 on the gene promoters by recruiting a methyltransferase complex. Knockdown of individual members of this complex significantly weakened the binding of SMAD3 and down-regulated the activation of portal fibroblast cells. In conclusion, we have identified an epigenetic pathway that dictates TGF-β induced pro-fibrogenic transcription in portal fibroblast thereby providing novel insights for the development of therapeutic solutions to treat liver fibrosis.

Myocardin-related transcription factor A (MRTF-A) plays an essential role in hepatic stellate cell activation by epigenetically modulating TGF-β signaling

Fibrosis following injury is a common adaptive response in the liver, which can lead to irreparable and life-threatening cirrhosis and hepatocellular carcinoma without effectual intervention. The molecular mechanisms underlying fibrogenic response in the liver remains poorly understood. Here we report that mice with deficiency in myocardin-related transcription factor A (MRTF-A) showed resistance to thioacetamide (TAA)-induced liver fibrosis with significantly reduced expression of pro-fibrogenic genes when compared to wild type littermates. Over-expression of MRTF-A enhanced whereas depletion of MRTF-A alleviated pro-fibrogenic transcription induced by TGF-β, a major pro-fibrogenic factor in hepatic stellate cells (HSCs). Mechanistically, MRTF-A silencing in HSCs impacted the chromatin structure by reducing the deposition of methylated histone H3K4 on the promoters of pro-fibrogenic genes. Further analyses revealed that MRTF-A interacted with and recruited several key epigenetic factors involved in H3K4 methylation, including ASH2, WDR5, and SET1, to the promoters of pro-fibrogenic genes in response to TGF-β treatment. Over-expression of ASH2, WDR5, or SET1 enhanced the transactivation of pro-fibrogenic gene promoters by TGF-β in an MRTF-A-dependent manner. In conclusion, MRTF-A regulates liver fibrosis by epigenetically tuning the TGF-β signaling pathway in HSCs.

HDAC4 mediates IFN-γ induced disruption of energy expenditure-related gene expression by repressing SIRT1 transcription in skeletal muscle cells.

Metabolic homeostasis is achieved through balanced energy storage and output. Impairment of energy expenditure is a hallmark event in patients with obesity and type 2 diabetes. Previously we have shown that the pro-inflammatory cytokine interferon gamma (IFN-γ) disrupts energy expenditure in skeletal muscle cells via hypermethylated in cancer 1 (HIC1)-class II transactivator (CIITA) dependent repression of SIRT1 transcription. Here we report that repression of SIRT1 transcription by IFN-γ paralleled loss of histone acetylation on the SIRT1 promoter region with simultaneous recruitment of histone deacetylase 4 (HDAC4). IFN-γ activated HDAC4 in vitro and in vivo by up-regulating its expression and stimulating its nuclear accumulation. HIC1 and CIITA recruited HDAC4 to the SIRT1 promoter and cooperated with HDAC4 to repress SIRT1 transcription. HDAC4 depletion by small interfering RNA or pharmaceutical inhibition normalized histone acetylation on the SIRT1 promoter and restored SIRT1 expression in the presence of IFN-γ. Over-expression of HDAC4 suppressed the transcription of genes involved in energy expenditure in a SIRT1-dependent manner. In contrast, HDAC4 knockdown/inhibition neutralized the effect of IFN-γ on cellular metabolism by normalizing SIRT1 expression. Therefore, our data reveal a role for HDAC4 in regulating cellular energy output and as such provide insights into rationalized design of novel anti-diabetic therapeutics.

Angiogenic factor with G patch and FHA domains 1 (Aggf1) regulates liver fibrosis by modulating TGF-β signaling

Fibrosis is a common pathophysiological process following liver injury and can lead to, if left unattended to, irreversible end-stage liver disease such as cirrhosis. Hepatic stellate cells (HSCs) are a major contributor to liver fibrosis. Here we investigated the involvement of angiogenic factor with G patch and FHA domains 1 (Aggf1) in HSC activation and the underlying mechanisms. Aggf1 expression was down-regulated in the livers in three different mouse models of liver fibrosis following injury. Aggf1 expression was also suppressed in activated HSCs when compared to quiescent HSCs. Over-expression of Aggf1 alleviated liver fibrosis in mice and in cultured HSCs. RNA-sequencing (RNA-seq) analysis performed in HSCs revealed that Aggf1-dependent transcription regulates several key fibrogenic pathways. Mechanistically, Aggf1 regulated liver fibrogenesis by forming a complex with the inhibitor SMAD protein (SMAD7) thereby leading to diminished SMAD3 binding to the pro-fibrogenic gene promoters. On the contrary, SMAD7 knockdown abrogated the effect of Aggf1 and rescued HSC activation. Aggf1 expression was silenced during HSC activation/liver fibrogenesis as a result of DNA methylation. Treatment with a DNA methyltransferase inhibitor (5-Azacytidine) restored Aggf1 expression and repressed liver fibrosis in an Aggf1-dependent manner. In conclusion, our data illustrate a previously unknown role for Aggf1 and shed light on the development of novel therapeutic solutions against liver fibrosis.