Sheng Zeng

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.

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).

SUV39H1 mediated SIRT1 trans-repression contributes to cardiac ischemia–reperfusion injury

Ischemic reperfusion (I/R) contributes to deleterious cardiac remodeling and heart failure. The deacetylase SIRT1 has been shown to protect the heart from I/R injury. We examined the mechanism whereby I/R injury represses SIRT1 transcription in the myocardium. There was accumulation of trimethylated histone H3K9 on the proximal SIRT1 promoter in the myocardium in mice following I/R injury and in cultured cardiomyocytes exposed to hypoxia–reoxygenation (H/R). In accordance, the H3K9 trimethyltransferase SUV39H1 bound to the SIRT1 promoter and repressed SIRT1 transcription. SUV39H1 expression was up-regulated in the myocardium in mice following I/R insults and in H/R-treated cardiomyocytes paralleling SIRT1 down-regulation. Silencing SUV39H1 expression or suppression of SUV39H1 activity erased H3K9Me3 from the SIRT1 promoter and normalized SIRT1 levels in cardiomyocytes. Meanwhile, SUV39H1 deficiency or inhibition attenuated I/R-induced infarction and improved heart function in mice likely through influencing ROS levels in a SIRT1-dependent manner. Therefore, our data uncover a novel mechanism for SIRT1 trans-repression during cardiac I/R injury and present SUV39H1 as a druggable target for the development of therapeutic strategies against ischemic heart disease.

Aggf1 attenuates hepatic inflammation and activation of hepatic stellate cells by repressing Ccl2 transcription.

Liver injury represents a continuum of pathophysiological processes involving a complex interplay between hepatocytes, macrophages, and hepatic stellate cells. The mechanism whereby these intercellular interactions contribute to liver injury and fibrosis is not completely understood. We report here that angiogenic factor with G patch and FHA domains 1 (Aggf1) was downregulated in the livers of cirrhotic patients compared to healthy controls and in primary hepatocytes in response to carbon tetrachloride (CCl4) stimulation. Overexpression of Aggf1 attenuated macrophage chemotaxis. Aggf1 interacted with NF-κB to block its binding to the Ccl2 gene promoter and repressed Ccl2 transcription in hepatocytes. Macrophages cultured in the conditioned media collected from Aggf1-overexpressing hepatocytes antagonized HSC activation. Taken together, our data illustrate a novel role for Aggf1 in regulating hepatic inflammation and provide insights on the development of interventional strategies against cirrhosis.Liver injury represents a continuum of pathophysiological processes involving a complex interplay between hepatocytes, macrophages, and hepatic stellate cells. The mechanism whereby these intercellular interactions contribute to liver injury and fibrosis is not completely understood. We report here that angiogenic factor with G patch and FHA domains 1 (Aggf1) was downregulated in the livers of cirrhotic patients compared to healthy controls and in primary hepatocytes in response to carbon tetrachloride (CCl4) stimulation. Overexpression of Aggf1 attenuated macrophage chemotaxis. Aggf1 interacted with NF-κB to block its binding to the Ccl2 gene promoter and repressed Ccl2 transcription in hepatocytes. Macrophages cultured in the conditioned media collected from Aggf1-overexpressing hepatocytes antagonized HSC activation. Taken together, our data illustrate a novel role for Aggf1 in regulating hepatic inflammation and provide insights on the development of interventional strategies against cirrhosis.

HIC1 epigenetically represses CIITA transcription in B lymphocytes

Differentiation of B lymphocytes into isotope-specific plasma cells represents a hallmark event in adaptive immunity. During B cell maturation, expression of the class II transactivator (CIITA) gene is down-regulated although the underlying epigenetic mechanism is not completely defined. Here we report that hypermethylated in cancer 1 (HIC1) was up-regulated in differentiating B lymphocytes paralleling CIITA repression. Over-expression of HIC1 directly repressed endogenous CIITA transcription in B cells. Reporter assay and chromatin immunoprecipitation (ChIP) assay confirmed that HIC1 bound to the proximal CIITA type III promoter (-545/-113); mutation of a conserved HIC1 site within this region abrogated CIITA trans-repression. More important, depletion of HIC1 with small interfering RNA (siRNA) restored CIITA expression in differentiating B cells. Mechanistically, HIC1 preferentially interacted with and recruited DNMT1 and DNMT3b to the CIITA promoter to synergistically repress CIITA transcription. On the contrary, silencing of DNMT1/DNMT3b or inhibition of DNMT activity with 5-aza-dC attenuated CIITA trans-repression. Therefore, our data identify HIC1 as a novel factor involved in B cell differentiation acting as an epigenetic repressor of CIITA transcription.

Angiogenic Factor With G Patch and FHA Domains 1 Is a Novel Regulator of Vascular Injury

Objective— Phenotypic modulation of vascular smooth muscle cells represents a hallmark event in vascular injury. The underlying mechanism is not completely sorted out. We investigated the involvement of angiogenic factor with G patch and FHA domains 1 (Aggf1) in vascular injury focusing on the transcriptional regulation of vascular smooth muscle cell signature genes. Approach and Results— We report here that Aggf1 expression was downregulated in several different cell models of phenotypic modulation in vitro and in the vessels after carotid artery ligation in mice. Adenovirus-mediated Aggf1 overexpression dampened vascular injury and normalized vascular smooth muscle cell signature gene expression. Mechanistically, Aggf1 interacted with myocardin and was imperative for the formation of a serum response factor–myocardin complex on gene promoters. In response to injurious stimuli, kruppel-like factor 4 was recruited to the Aggf1 promoter and enlisted histone deacetylase 11 to repress Aggf1 transcription. In accordance, depletion of kruppel-like factor 4 or histone deacetylase 11 restored Aggf1 expression and abrogated vascular smooth muscle cell phenotypic modulation. Finally, treatment of a histone deacetylase 11 inhibitor attenuated vascular injury in mice. Conclusions— Therefore, we have unveiled a previously unrecognized role for Aggf1 in regulating vascular injury.

Brg1 deficiency in vascular endothelial cells blocks neutrophil recruitment and ameliorates cardiac ischemia-reperfusion injury in mice

BACKGROUND Increased neutrophil infiltration and the ensuing inflammatory response represent a hallmark event in cardiac ischemia-reperfusion injury (IRI). It remains poorly defined how the epigenetic machinery contributes to this process. METHODS AND RESULTS Here we report that mice with endothelial specific deletion of brahma related gene 1 (BRG1), a chromatin remodeling protein, exhibited amelioration when subjected to cardiac ischemia-reperfusion as evidenced by a reduction in infarct size as well as better recovery of heart function. Endothelial BRG1 deficiency also attenuated cardiac fibrosis following IRI when compared to wild type littermates. Interestingly, ablation of BRG1 in the endothelium suppressed neutrophil infiltration and down-regulated the levels of pro-inflammatory mediators in the heart following IRI. Further studies revealed that BRG1 activated the transcription of PODOCALYXIN (PODXL), an L-SELECTIN ligand crucial for neutrophil adhesion, in vascular endothelial cells in response to hypoxia-reoxygenation (HR). BRG1 knockdown by small interfering RNA abrogated HR-induced PODXL expression and blocked the adhesion of neutrophils to endothelial cells. Mechanistically, BRG1 alters the chromatin structure surrounding the PODXL promoter by interacting with JMJD2B, a histone H3K9 demethylase. Depletion of JMJD2B abrogated PODXL induction by HR and inhibited the adhesion of neutrophils to endothelial cells. CONCLUSION Our data suggest that trans-activation of PODXL by the BRG1-JMJD2B complex in endothelial cells may promote neutrophil infiltration and consequently the pathogenesis of cardiac ischemia-reperfusion injury.

Hypermethylated in cancer 1 (HIC1) mediates high glucose induced ROS accumulation in renal tubular epithelial cells by epigenetically repressing SIRT1 transcription

Reactive oxygen species (ROS) is a key regulator of an array of physiological and pathological processes. While essential for the host defense mechanism, excessive ROS generation and/or deficient clearance is blamed for the pathogenesis of human diseases. In the present study, we investigated the regulatory role of hypermethylated in cancer 1 (HIC1), a transcription factor, in high glucose-induced ROS accumulation in renal tubular epithelial cells (HK-2). Treatment with high glucose (HG) not only markedly up-regulated HIC1 expression but prompted its translocation into the nucleus. HG stimulation promoted HIC1 binding to the promoter of SIRT1, a known HIC1 target with anti-oxidative ability. The recruitment of HIC1 to the SIRT1 promoter was paralleled by the enrichment of trimethylated histone H3K27 and 5?methyl cytosine, two well-characterized markers for trans-repression. HIC1 silencing with small interfering RNA abrogated SIRT1 repression by HG and at the same time weakened ROS accumulation in HK-2 cells. Knockdown or pharmaceutical inhibition of SIRT1 preempted the effect of HIC1 depletion by restoring ROS accumulation and down-regulating the expression of antioxidant genes. Mechanistically, HIC1 interacted with and recruited EZH2, an H3K27 trimethyltransferase, and DNA methyltransferase 1 (DNMT1) to repress SIRT1 transcription in response to HG stimulation. Depletion or inhibition of EZH2 or DNMT1 rescued SIRT1 expression and blocked ROS accumulation in HG-treated HK-2 cells. In conclusion, our data suggest that epigenetic repression of SIRT1 by HIC1 may contribute to HG-induced elevation of ROS levels in renal tubular epithelial cells.

BRG1 regulates NOX gene transcription in endothelial cells and contributes to cardiac ischemia-reperfusion injury

Excessive accumulation of reactive oxygen species (ROS) is considered a major culprit for a host of cardiovascular diseases. In vascular endothelial cells, ROS production is mediated by NAPDH oxidases (NOX). In the present study we investigated the role of the chromatin remodeling protein BRG1 in NOX trans-activation as well as its implication in cardiac ischemia-reperfusion injury. We report that in response to hypoxia-reoxygenation (HR) BRG1 was recruited to the NOX promoter regions in both immortalized endothelial cells and primary microvascular endothelial cells. BRG1 knockdown attenuated the induction of NOX genes by HR stimulation. Suppression of NOX trans-activation by BRG1 silencing was paralleled by the loss of active histone modifications (acetylation of histones H3 and H4) and the re-appearance of repressive histone modification (dimethylation of histone H3K9) surrounding the NOX promoter. Of interest, the H3K9 demethylase KDM3A bound to the NOX promoters with kinetics similar to BRG1 and interacted with BRG1 to activate NOX transcription. KDM3A depletion ameliorated NOX induction and ROS production in endothelial cells exposed to HR. Finally, mice with endothelial-specific deletion of BRG1 were protected from cardiac ischemia-reperfusion injury. In conclusion, our data suggest that BRG1 may link epigenetic activation of NOX transcription in endothelial cells to cardiac ischemia reperfusion injury.

The chromatin remodeling protein BRG1 regulates APAP-induced liver injury by modulating CYP3A11 transcription in hepatocyte

Acetaminophen (APAP) overdose represents the most frequent cause of acute liver failure. The underlying epigenetic mechanism is not fully understood. In the present study we investigated the mechanism whereby the chromatin remodeling protein brahma related gene 1 (Brg1) regulates APAP induced liver injury in mice. We report that hepatocyte-specific deletion of Brg1 attenuated APAP induced liver injury in mice as evidenced by reduced plasma ALT and AST levels, decreased liver necrosis, amelioration of GSH depletion, and prolonged survival. Brg1 regulated APAP-induced liver injury likely by stimulating the transcription of Cyp3a11, a key cytochrome enzyme involved in APAP metabolism. Immunoprecipitation coupled with DNA affinity microarray identified hepatocyte nuclear factor 4 (HNF4) as a novel binding partner for Brg1. HNF4 recruited Brg1 to the Cyp3a11 promoter and formed a complex with Brg1 to trans-activate Cyp3a11. In contrast, BRG1 deficiency attenuated HNF4 binding to the Cyp3a11 promoter and dampened Cyp3a11 transcription. Therefore, our data suggest that Brg1 might play an essential role mediating APAP induced liver injury in vivo.