Molecular regulation of hepatic cell differentiation and maturation

肝细胞分化和成熟的分子调控

基本信息

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
项目类别：
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

项目摘要

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.

项目概要我们的应用程序建立在已发表的知识之上，即在胚胎发育直至出生后，特定的肝细胞经历分化过程，在此过程中它们采用生理功能并与成人肝脏相关的形态。我们的初步研究删除了含有1的锌指HIT型（Znhit1）在妊娠中期的胚胎成肝细胞中表现出在出生后的特定和重要作用肝脏的生存、正常的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因子。 Zhnit1 作为 Snf2 相关核心亚基的提议角色 CREB 结合蛋白激活蛋白 (SRCAP)-染色质重塑复合物可能启动必要的通过插入替代组蛋白 H2A.Z 来改变染色质结构以影响基因表达。此应用程序的目标是确定 Znhit1 是否允许或中断访问转录因子针对不同的基因靶标和/或增强子，从而提供向肝脏的转变细胞分化。我们假设 Znhit1 作为 SRCAP 复合体的一部分，调节肝细胞分化依赖性转录程序。在目标 1 中，我们将确定 Znhit1 是否对肝脏有影响功能是由于 1) 自主肝母细胞和/或肝细胞缺陷以及 2) 主调节器访问基因靶标和/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肝细胞删除 Znhit1，并确定肝母细胞和/或肝细胞转录程序是否 Hnf4a 和 Foxa2 等主调控因子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肝细胞成体主调节器功能的发育开关。判断是否 Zhnit1 是抑制胎儿和/或激活成人肝细胞程序的开关的一部分，我们将使用 AAV/DJ8-Ttr-Znhit1-GPF 在小鼠模型中强制在特定时间点表达 Zhit1，培养 iPSC 生成的肝细胞样细胞。在目标 3 中，我们将确定 H2A.Z 是否被纳入特定位点以分化依赖性方式受 Znhit1 调节。为了回答这个问题，我们将使用 Znhit1-3xFlag-P2A-Zsgreen 敲入小鼠执行 Znhit1 ChIP-Seq 并与 Znhit1 依赖性进行比较 H2A.Z 结合位点。我们的研究将为肝细胞身份的分子调控提供新的见解和差异化。