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 我们的应用基于公开的知识，即在胚胎开发期间直到出生后， 指定的肝细胞经历了一个分化过程，它们采用生理功能和 与成年肝脏相关的形态。我们的初步研究删除了包含1的锌指命中型hit型 （Znhit1）在胚胎肝细胞中妊娠中期，在产后表现出特定而重要的作用 肝脏生存，正常的细胞结构和分子基因特征，而不改变表达 在六个关键的肝细胞主调节器中。 Znhit1提出的角色是SNF2相关的核心亚基 CREB结合蛋白活化剂蛋白（SRCAP） - 染色质重塑络合物可能会启动必要的 通过插入替代组蛋白H2A.Z来影响基因，染色质结构的变化 表达。此应用程序的目的是确定ZNHIT1是否允许或破坏访问 到不同基因靶标和/或增强子的转录因子，从而提供了肝的转换 细胞分化。我们假设SRCAP复合物的一部分Znhit1调节肝细胞 与分化有关的转录程序。在AIM 1中，我们将确定Znhit1是否对肝脏的影响 功能是由于1）自主肝细胞和/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，我们将使用体内鼠标模型执行颞肝细胞和 Znhit1的肝细胞缺失，并确定肝细胞和/或肝细胞转录程序是否 HNF4A和FOXA2等主要调节剂的基因组结合位点受到影响。在AIM 2中，我们将定义 是为了调节肝细胞成人主调节函数的发育开关。确定是否 ZNHIT1是使胎儿抑制和/或成人肝细胞计划的抑制作用的一部分， 我们将使用AAV/DJ8-TTR-ZNHIT1-GPF在鼠标模型和 在培养中，IPSC生成的肝细胞样细胞。在AIM 3中，我们将确定是否将H2A.Z纳入 以Znhit1调节的分化依赖性方式的特定位点。要回答这个问题，我们将使用 Znhit1-3xflag-p2a-Zsgreen敲入鼠标以执行Znhit1芯片seq并与Znhit1相比 H2A.Z绑定站点。我们的研究将对肝细胞身份的分子调节产生新的见解 和分化。