Molecular regulation of hepatic cell differentiation and maturation

肝细胞分化和成熟的分子调控

• 批准号: 10456054
• 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10456054
• 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.

项目总结 我们的应用程序建立在已公布的知识基础上，即在胚胎发育期间直到出生后， 特定的肝细胞经历一个分化的过程，在这个过程中它们采用了生理功能和 与成人肝脏相关的形态。我们的初步研究删除了含有1的锌指命中型 在胚胎肝母细胞中，妊娠中期的(Znhit1)基因在出生后的发育过程中具有特殊而重要的作用 肝脏用于生存、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要调节因子中。S提出作为SnF2相关核心亚单位的作用 CREB结合蛋白激活蛋白(SRCAP)-染色质重塑复合体可能启动必要的 通过插入替代组蛋白H_2A.Z来影响基因的染色质结构的变化 表情。此应用程序的目标是确定Znhit1是否允许或中断访问 转录因子到不同的基因靶点和/或增强子，从而提供了向肝脏的转换 细胞分化。我们假设，SRCAP复合体的一部分，即Znhit1，调节肝细胞 依赖分化的转录程序。在目标1中，我们将确定锌离子1‘S对肝脏的影响 功能归因于1)自主的肝母细胞和/或肝细胞缺陷和2)主调节器访问 基因靶点和/或增强剂。为此，我们将使用活体小鼠模型进行颞叶肝母细胞和 肝细胞缺失的Znhit1，并确定肝母细胞和/或肝细胞转录程序 HNF4a和Foxa2等主调控子的基因组结合位点受到影响。在目标2中，我们将定义 是什么调控着肝细胞成体发育开关的主要调节功能。以确定是否 Znhit1是允许抑制胎儿和/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在特定时间点强制小鼠模型和 在培养条件下，IPSC可产生肝细胞样细胞。在目标3中，我们将确定是否将H2A.Z并入 在分化依赖的方式下，特定的位置受Znhit1的调节。要回答这个问题，我们将使用 Znhit1-3xFlag-P2A-Zsgreen敲入小鼠进行Znhit1芯片序列测定及与依赖的Znhit1比较 H_2A.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。