Molecular regulation of hepatic cell differentiation and maturation

肝细胞分化和成熟的分子调控

• 批准号: 10224185
• 负责人: Stacey S Huppert
• 金额: $ 40.28万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-23 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224185
• 关键词: Acute Liver Failure; Adopted; Adult; Architecture; Binding; Binding Sites; Birth; CREBBP gene; Cell Differentiation process; Cell Maturation; Cell model; Cell physiology; Cells; ChIP-seq; Childhood; Chromatin; Chromatin Remodeling Factor; Complex; Coupled; DNA; DNA Binding; Defect; Derivation procedure; Development; Drug toxicity; Embryo; Embryonic Development; Endoderm; Enhancers; Environment; Etiology; Failure; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genomics; Goals; HNF4A gene; Hepatic; Hepatocyte; Hepatotoxicity; Histone H2A; Human; In Vitro; Knock-in Mouse; Knowledge; Lead; Liver; Liver Failure; Molecular; Morphology; Mus; Newborn Infant; Organ; Physiological; Play; Pregnancy; Process; Proteins; Publishing; Regulation; Repression; Research; Role; Site; Specific qualifier value; Time; Tissue Engineering; Zinc Fingers; chromatin remodeling; embryonic stem cell; fetal; gene repression; genetic signature; healing; in vivo; induced pluripotent stem cell; insight; liver function; loss of function; mouse model; postnatal; progenitor; programs; response to injury; transcription factor

PROJECT SUMMARY Our application builds on the published knowledge that during embryonic development until after birth, specified hepatocytes undergo a process of differentiation where they adopt the physiological functions and morphology associated with the adult liver. Our preliminary studies deleting Zinc finger HIT-type containing 1 (Znhit1) at mid-gestation in embryonic hepatoblasts demonstrates a specific and essential role in the postnatal liver for survival, normal cellular architecture, and molecular gene signatures without changing the expression of the six key hepatic cell master regulators. Znhit1's proposed role as a core subunit of the Snf2-Related CREB-binding protein Activator Protein (SRCAP)-chromatin remodeling complex may initiate the necessary changes in chromatin architecture through insertion of the alternative histone H2A.Z to influence gene expression. The goal of this application is to determine whether Znhit1 allows or disrupts access of transcription factors to different gene targets and/or enhancers, and thereby provides a switch towards hepatic cell differentiation. We hypothesize that Znhit1, part of the SRCAP complex, regulates the hepatocyte differentiation-dependent transcriptional program. In Aim 1 we will determine whether Znhit1's impact on liver function is due to 1) an autonomous hepatoblast and/or hepatocyte defect and 2) master regulator access to gene targets and/or enhancers. For this we will use in vivo mouse models to perform temporal hepatoblast and hepatocyte deletion of Znhit1, and to determine if the hepatoblast and/or hepatocyte transcriptional program and genomic binding sites of master regulators such as Hnf4a and Foxa2 are impacted. In Aim 2 we will define what regulates the developmental switch for hepatocyte adult master regulator function. To determine whether Znhit1 is part of the switch to enable repression of the fetal and/or activation of the adult hepatocyte program, we will use AAV/DJ8-Ttr-Znhit1-GPF to force expression of Znhit1 at specific time points in mouse models and in culture iPSC-generated hepatocyte-like cells. In Aim 3, we will determine whether H2A.Z is incorporated into specific sites in a differentiation-dependent manner regulated by Znhit1. To answer this question, we will use a Znhit1-3xFlag-P2A-Zsgreen knock-in mouse to perform Znhit1 ChIP-Seq and compare to Znhit1 dependent H2A.Z bound sites. Our research will generate new insight into the molecular regulation of hepatic cell identity and differentiation.

项目摘要 我们的应用建立在已发表的知识基础上，即在胚胎发育期间直到出生后， 特定的肝细胞经历分化过程，在此过程中它们具有生理功能， 与成人肝脏相关的形态学。我们初步研究了删除锌指HIT型含1 （Znhit 1）在妊娠中期胚胎肝母细胞中表现出特定的和重要的作用，在出生后的 肝脏生存、正常细胞结构和分子基因特征而不改变表达 六个关键的肝细胞主调节器。Znhit 1作为Snf 2相关蛋白的核心亚基的作用 CREB结合蛋白激活蛋白（SRCAP）-染色质重塑复合物可以启动必要的 通过插入替代性组蛋白H2A.Z影响基因改变染色质结构 表情此应用程序的目标是确定Znhit 1是否允许或中断访问 转录因子与不同的基因靶标和/或增强子的相互作用，从而提供了向肝靶向转录因子的转变。 细胞分化我们假设Znhit 1是SRCAP复合物的一部分，调节肝细胞 分化依赖的转录程序。在目标1中，我们将确定Znhit 1对肝脏的影响是否 功能是由于1）自主肝母细胞和/或肝细胞缺陷和2）主调节剂进入 基因靶和/或增强子。为此，我们将使用体内小鼠模型进行颞叶肝母细胞和 Znhit 1的肝细胞缺失，并确定成肝细胞和/或肝细胞转录程序是否 并且主调节因子如Hnf 4a和Foxa 2的基因组结合位点受到影响。在目标2中，我们将定义 是什么调节了成人肝细胞主调节器功能的发育开关。以确定是否 Znhit 1是能够抑制胎儿肝细胞程序和/或激活成人肝细胞程序的开关的一部分， 我们将使用AAV/DJ 8-Ttr-Znhit 1-GPF在小鼠模型中的特定时间点强制Znhit 1表达， 在培养iPSC产生肝细胞样细胞中。在目标3中，我们将确定H2A.Z是否并入 特定的网站在一个分化依赖的方式调节Znhit 1。为了回答这个问题，我们将使用 Znhit 1 - 3xFlag-P2 A-Zsgreen敲入小鼠进行Znhit 1 ChIP-Seq并与Znhit 1依赖性 H2A.Z结合位点。我们的研究将对肝细胞特性的分子调控产生新的见解 和差异化。