Molecular regulation of hepatic cell differentiation and maturation

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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.