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：非酒精性脂肪性肝病（NAFLD）是一系列肝脏疾病，包括脂肪变性（非酒精性脂肪肝，NAFL）到伴有肝星状细胞纤维化的非酒精性脂肪性肝炎（NASH）。 (HSC) 在 NASH 的发病机制中发挥着关键作用为了应对慢性毒性损伤，静止的 HSCs。 (qHSC) 激活为 aHSC/肌成纤维细胞，分泌细胞外基质以促进肝纤维化。 NASH 介导的人类 HSC 激活机制尚不清楚。 DNA 序列没有发生变化，但在表观遗传水平上受到调节，例如特定的染色质结构的修饰，影响调节转录因子的 DNA 可及性 (TF)，导致其靶基因转录激活或抑制。我们将分析正常、NAFL 和我们将比较我们在人体中的观察结果。我们提出的研究将整合最新的 HSC 到 NASH 的特征明确的 foz/foz 小鼠模型。基于单细胞的技术，a) 对纯化的人 HSC 进行单细胞 (sc)RNA-Seq 将鉴定主要人类 HSC 子集；b) 将使用整个肝脏进行单核 (sn)ATAC-Seq 和 snRNA-Seq 组织来捕获和表征开放染色质区域以及个体的匹配基因表达 HSC；c) 将使用进一步获取调节启动子/增强子元件的转录活性 PLAC-Seq 之后是带有 H3K27ac（与细胞激活相关的标记）的 ChIP-Seq（HiChIP-Seq）。人类 HSC 的转录组 (AIM 1) 和表观基因组 (AIM 2)，即调控因子的全基因组位置调节不同 HSC 表型并驱动 NAFL®NASH 的元件及其相应的 TF 将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相结合将能够推断主要的转录类别对人类 HSC 特定亚群至关重要的因素驱动人类 HSC 激活的因素。将使用以下方法评估促进 NAFL 进展为 NASH 的选定目标 (AIM 3)。使用西方饮食 (WD) 喂养的 foz/foz 小鼠的 NASH 实验模型，使用单个 aHSC 子集的消融（通过 Col1a1+ aHSC 中白喉毒素受体 (DTR) 以 Cre-loxP 依赖性方式过度表达），或 HSC 特异性敲除关键 TF 的特定因素可阻止或抑制 HSC 激活（例如， Etv1、E3F3、Egr2、NRF1、Tal1、Atf3）将成为药理学目标，并且治疗的体内效果 Co1a1+ aHSC 激活将在活体 WD 喂养的记者 LratCol1a1-Fluc foz/foz 小鼠（上调 Col- 1a1 驱动的小鼠 aHSC 中的荧光素酶）或人源化患者特异性异种移植 Rag2-/-gc-/- 小鼠总体而言，我们。预计确定 NASH 抗纤维化治疗的新靶标。