Epigenetics of human Hepatic Stellate Cells (HSCs) in NASH

NASH 中人肝星状细胞 (HSC) 的表观遗传学

• 批准号: 10367096
• 负责人: Tatiana Kisseleva
• 金额: $ 62.58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 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/10367096
• 关键词: ATAC-seq; Ablation; Acetylation; Affect; Amino Acid Motifs; Apoptosis; Area; Bar Codes; Binding; Biological Assay; Cell Nucleus; 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 Activation; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genomics; Glass; Hepatic Stellate Cell; Heterogeneity; Human; Immunoprecipitation; In Vitro; Individual; Injury; Knock-in; Knock-out; Knockout Mice; Laboratories; Liver; Liver Fibrosis; Liver diseases; Location; Luciferases; Mediating; Metabolic; Modification; Monitor; Mus; Myofibroblast; Nuclear; Pathogenesis; Patients; Pharmacology; Phenotype; Physiological; Play; Population; Property; Regulation; Regulatory Element; Regulatory Pathway; Reporter; Risk Factors; Role; Site; Small Nuclear RNA; Technology; Testing; Tissues; Transcriptional Activation; Transplantation; Transposase; Vitamin A; Xenograft procedure; activating transcription factor 3; alpha Toxin; antifibrotic treatment; base; combinatorial; diphtheria toxin receptor; epigenome; gene repression; genetic signature; genome wide association study; genome-wide; 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; prevent; promoter; response; risk variant; sex; single cell analysis; single-cell RNA sequencing; small hairpin RNA; transcription factor; transcriptome; transcriptome sequencing; treatment effect; western diet

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：非酒精性脂肪性肝病（NAFLD）是一系列肝脏疾病， 非酒精性脂肪肝（NAFL）至非酒精性脂肪性肝炎（NASH）伴纤维化。肝星状细胞 肝星状细胞（HSC）在NASH的发病机制中起关键作用。在对慢性毒性损伤的反应中，静止的HSC 在一些实施方案中，肝纤维化细胞（qHSC）活化成aHSC/肌成纤维细胞，其分泌细胞外基质以促进肝纤维化。的 NASH介导的人HSC活化的机制还不清楚。HSC的表型变化 在DNA序列没有变化的情况下发生，但在表观遗传水平上受到调节，例如特异性 染色质结构中的修饰，其影响调节转录因子的DNA可及性 (TFs)，导致其靶基因的转录激活或抑制。我们将分析正常，NAFL， 已拒绝接受肝移植的NASH肝脏。我们将比较我们对人类的观察， 将HSC移植到充分表征的foz/foz NASH小鼠模型中。我们提议的研究将结合国家的- a）对纯化的人HSC的单细胞（sc）RNA-Seq将鉴定主要的 人HSC亚群; B）使用全肝进行单核（sn）ATAC-Seq和snRNA-Seq 组织来捕获和表征个体的开放染色质和匹配基因表达的区域。 c）调控启动子/增强子元件的转录活性将使用 PLAC-Seq，然后用H3 K27 ac进行ChIP-Seq，H3 K27 ac是与细胞活化相关的标记（HiChIP-Seq）。的 转录组（AIM 1）和表观基因组（AIM 2）的人HSC，调控基因的全基因组位置， 调节不同HSC表型并驱动NAFL®NASH的元件及其相应TF 进展，将被确定。表现出活性特征的区域的基序富集分析 增强子与基因表达数据的组合将能够推断转录的主要类别 对人类HSC的特定亚群至关重要的因子。驱动人类HSC活化的因素， 促进NAFL进展为NASH。选定的目标（AIM 3）将使用 使用个体aHSC亚群消融的西方饮食（WD）喂养的foz/foz小鼠中NASH的实验模型 (via白喉毒素受体（DTR）在Col 1a 1 + aHSC中以Cre-loxP依赖性方式过表达），或 HSC特异性敲除关键TF。阻止或抑制HSC活化的特定因素（例如， Etv 1、E3 F3、Egr 2、NRF 1、Tal 1、Atf 3）将被靶向，并且治疗对E3 F3、Egr 2、NRF 1、Tal 1、Atf 3的体内作用将被评估。 将在活的WD喂养的报告基因LratCol 1a 1-Fluc foz/foz小鼠（上调Col-1 α 1表达）中监测Co 1a 1 + aHSC活化。 Ial驱动的荧光素酶）或人源化患者特异性异种移植物Rag 2-/-gc-/-小鼠。总的来说，我们 预期确定NASH抗纤维化治疗的新靶点。