Epigenetics of human Hepatic Stellate Cells (HSCs) in NASH

NASH 中人肝星状细胞 (HSC) 的表观遗传学

基本信息

批准号：
10680588
负责人：
DAVID A. BRENNER
金额：
$ 62.58万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-15 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10680588
关键词：
ATAC-seq Ablation Acetylation Affect Amino Acid Motifs Apoptosis Area Bar Codes Binding Biological Assay Cell Nucleus Cell Separation Cells Cellular Assay Centrifugation ChIP-seq Characteristics Chromatin Chromatin Structure Chronic Cicatrix Cluster Analysis Collaborations Collagen Collagen Type I DNA DNA Sequence Data Diphtheria Toxin EGR2 gene Enhancers Epigenetic Process Exhibits Experimental Models Extracellular Matrix Fibrosis Gene Expression Gene Expression Profile Genes Genetic Genetic Enhancer Element Genetic Transcription Glass Hepatic Stellate Cell Heterogeneity Human Immunoprecipitation In Vitro Individual Injury Knock-in Knock-out Knockout Mice Laboratories Liver Liver Fibrosis Liver diseases Location LoxP-flanked allele Luciferases Mediating Metabolic Modification Monitor Mus Myofibroblast Nuclear Pathogenesis Patients Phenotype Physiological Play Population Property Regulation Regulatory Element Regulatory Pathway Reporter Risk Factors Role Site Sorting Technology Testing Tissues Transcriptional Activation Transplantation Transposase Vitamin A Xenograft procedure activating transcription factor 3 antifibrotic treatment combinatorial diphtheria toxin receptor epigenome gene repression genetic signature genome wide association study genome-wide genomic locus histone modification human tissue in vivo insight knock-down knockout gene liver injury liver transplantation mouse model non-alcoholic fatty liver non-alcoholic fatty liver disease nonalcoholic steatohepatitis novel strategies overexpression pharmacologic prevent promoter response risk variant sex single nucleus RNA-sequencing single-cell RNA sequencing small hairpin RNA transcription factor transcriptome treatment effect western diet

项目摘要

ABSTRACT: Nonalcoholic fatty liver disease (NAFLD), is a spectrum of liver disease ranging from steatosis (nonalcoholic fatty liver, NAFL) to non-alcoholic steatohepatitis (NASH) with fibrosis. Hepatic Stellate Cells (HSCs) play a critical role in the pathogenesis of NASH. In response to chronic toxic injury, quiescent HSCs (qHSCs) activate into aHSCs/myofibroblasts, that secrete the extracellular matrix to promote liver fibrosis. The mechanism of NASH-mediated activation of human HSCs is not well understood. Phenotypic changes in HSCs occur without a change in the DNA sequence but are regulated on an epigenetic level, e.g. specific modifications in the chromatin structure, which affect DNA accessibility of the regulatory transcription factors (TFs), causing transcriptional activation or repression of their target genes. We will analyze normal, NAFL, and NASH livers that have been declined for liver transplantation. We will compare our observations in human HSCs to the well characterized foz/foz mouse model of NASH. Our proposed study will integrate state-of-the- art single-cell-based technologies, a) Single cell (sc)RNA-Seq on purified human HSCs will identify major human HSC subsets; b) Single nuclei (sn)ATAC-Seq and snRNA-Seq will be performed using whole liver tissue to capture and characterize the areas of open chromatin and matching gene expression of individual HSCs; c) Transcriptional activity of the regulatory promoter/enhancer elements will be further accessed using PLAC-Seq followed by ChIP-Seq with H3K27ac, a mark associated with cellular activation (HiChIP-Seq). The transcriptome (AIM 1) and epigenome (AIM 2) of human HSCs, the genome-wide locations of the regulatory elements and their corresponding TFs that regulate distinct HSC phenotypes and drive NAFL®NASH progression, will be determined. Motif enrichment analysis of regions exhibiting characteristics of active enhancers in combination with gene expression data will enable inference of major classes of transcription factors critical for specific subsets of human HSCs. The factors that drive human HSC activation and thereby promote NAFL progression to NASH will be identified. Selected targets (AIM 3) will be evaluated using the experimental model of NASH in Western-diet (WD)-fed foz/foz mice, using ablation of individual aHSC subsets (via overexpression of Diphtheria toxin receptor (DTR) in Col1a1+ aHSCs in a Cre-loxP-dependent manner), or HSC-specific knockout of the key TFs. Specific factors that prevent or suppress HSC activation (for example, Etv1, E3F3, Egr2, NRF1, Tal1, Atf3) will be pharmacologically targeted, and the in vivo effect of treatment on Co1a1+ aHSC activation will be monitored in live WD-fed reporter LratCol1a1-Fluc foz/foz mice (that upregulate Col- 1a1-driven Luciferase in mouse aHSCs), or humanized patient-specific xenograft Rag2-/-gc-/- mice. Overall, we anticipate identifying new targets for the antifibrotic therapy of NASH.

摘要：非酒精性脂肪肝病（NAFLD），是一系列肝病（非酒精性脂肪肝，NAFL）抗纤维化的非酒精性脂肪性肝炎（NASH）。肝星状细胞（HSC）在NASH的发病机理中起关键作用。响应慢性毒性损伤，静止的HSC （QHSC）激活AHSC/肌纤维细胞，该分泌细胞外基质以促进肝纤维化。这 NASH介导的人HSC激活的机制尚不清楚。 HSC的表型变化发生DNA序列没有变化而发生，但在表观遗传水平上进行调节，例如具体的染色质结构的修饰，会影响调节转录因子的DNA可及性（TF），导致其靶基因的转录激活或表达。我们将分析正常，NAFL和纳什的生命因肝移植而被拒绝。我们将比较人类的观察纳什（NASH）的hscs to the良好的FOZ/FOZ小鼠模型。我们提出的研究将整合 ART单细胞技术，a）纯化的人类HSC上的单细胞（SC）RNA-seq将识别主要人类HSC子集； b）将使用整个肝脏进行单个核（SN）ATAC-SEQ和SNRNA-SEQ 组织以捕获和表征开放染色质和匹配基因表达的区域 HSC； c）使用调节启动子/增强子元素的转录活性将进一步访问 plac-seq，然后是chip-seq，带有H3K27AC，这是与细胞激活相关的标记（Hichip-Seq）。这人类HSC的转录组（AIM 1）和表观基因组（AIM 2），该调节的全基因组位置元素及其相应的TFS调节不同的HSC表型并驱动Nafl®Nash 进展将确定。对活性特征的区域的基序富集分析增强子与基因表达数据结合使用，可以推断主要的转录类别人类HSC特定子集至关重要的因素。驱动人类HSC激活的因素，从而将确定促进NAFL向NASH的发展。选定的目标（AIM 3）将使用使用单个AHSC子集的消融，西方 - 迪埃特（WD）foz/foz小鼠NASH的实验模型（通过以CRE-LOXP依赖性的方式过表达Col1a1+ AHSC中的白喉毒素受体（DTR）或密钥TFS的HSC特定敲除。预防或抑制HSC激活的特定因素（例如， ETV1，E3F3，EGR2，NRF1，TAL1，ATF3将是药理学靶向的，并且在体内对治疗的影响对 CO1A1+ AHSC激活将在实时WD-FED的记者Lratcol1a1-Fluc Foz/Foz小鼠中进行监测（这上调了col- 小鼠AHSC中的1A1驱动的荧光素酶或人源化患者特异性异种移植物RAG2 - / - GC - / - 小鼠。总体而言，我们预计识别纳什抗纤维化疗法的新靶标。