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: 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
• 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

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)是一种从脂肪变性到脂肪变性的肝脏疾病 非酒精性脂肪肝(NAFL)到伴有纤维化的非酒精性脂肪性肝炎(NASH)。肝星状细胞 (HSCs)在NASH的发病机制中起关键作用。对慢性毒性损伤的反应，静止的HSCs (QHSCs)激活为AHSCs/肌成纤维细胞，分泌细胞外基质促进肝纤维化。这个 NASH介导的人HSCs激活机制尚不清楚。造血干细胞的表型变化 在不改变DNA序列的情况下发生，但在表观遗传水平上进行调节，例如 染色质结构的改变，影响调节转录因子的DNA可及性 (TFS)，导致其目标基因的转录激活或抑制。我们将分析NORMAL、NAFL和 已拒绝接受肝移植的NASH肝脏。我们将在人体内比较我们的观察结果 HSCs移植到特征良好的Foz/Foz小鼠NASH模型。我们提议的研究将综合最新情况 ART基于单细胞的技术，a)纯化的人类HSC上的单细胞(Sc)RNA-Seq将识别主要的 人HSC亚群；b)单核(SN)ATAC-Seq和SnRNA-Seq将使用全肝进行 组织捕捉和表征个体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进一步使用 Plac-Seq和ChIP-Seq与H3K27ac相关，这是一个与细胞激活相关的标记(HiChIP-Seq)。这个 人类造血干细胞的转录组(AIM 1)和表观基因组(AIM 2)，调控基因的全基因组位置 调节不同HSC表型并驱动NAFL®NASH的元件及其对应的TF 进展，将被确定。显示活动特征的区域的基序富集度分析 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 对特定的人类造血干细胞亚群至关重要的因素。驱动人类HSC激活的因素，从而 将确定将NAFL晋级为NASH。选定的目标(AIM 3)将使用 西方饮食(WD)喂养的FOZ/FOZ小鼠单个AHSC亚群消融的NASH模型 (通过Cre-loxP依赖的方式在Col1a1+AHSCs中过表达白喉毒素受体(DTR))，或 HSC特定的关键TF基因敲除。阻止或抑制HSC激活的特定因素(例如， ETV1、E3F3、Egr2、NRF1、TAL1、ATF3)将成为药理靶向，治疗的体内效应 在WD喂养的LratCol1a1-Fluc Foz/Foz小鼠中，将监测Co1a1+AHSC的激活(上调Col- 小鼠AHSCs中的A1驱动的荧光素酶)，或人源化的患者特异性异种移植Rag2-/-GC-/-小鼠。总体而言，我们 期待为NASH的抗纤维化治疗寻找新的靶点。