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Co-repressors SMRT and NCoR1 regulate UGT1A1 gene expression

共阻遏物 SMRT 和 NCoR1 调节 UGT1A1 基因表达

基本信息

批准号：
8898831
负责人：
Robert H Tukey
金额：
$ 33.79万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8898831
关键词：
Animal Model Benign Bilirubin Birth ChIP-seq Chromatin Complex Development Environmental Risk Factor Epigenetic Process Event Gastrointestinal tract structure Gene Expression Gene Expression Profile Gene Expression Regulation Gene Targeting Genes Genetic Health Hepatic Hepatic Tissue Hepatocyte Human Hyperbilirubinemia In Vitro Interruption Intestines Knock-out Lead Life Link Liver Location Measures Metabolic Metabolism Modification Mus Mutation NCOR1 gene Neonatal Neonatal Jaundice Neuraxis Neurologic Dysfunctions Newborn Infant Nuclear Receptors Pharmaceutical Preparations Play Proteins Publishing Receptor Inhibition Recovery Regulation Repression Repressor Proteins Role Series Serum Small Interfering RNA Syndrome Testing Therapeutic Agents Tissues Toxic effect Transcriptional Regulation UDP-Glucuronosyltransferase 1A1 UGT1A1 gene Vision base design drug metabolism early onset gastrointestinal gene repression human NCOR1 protein in vivo Model model design neonatal death novel pregnane X receptor prevent receptor binding research study transcriptome sequencing whole blood lead

项目摘要

DESCRIPTION (provided by applicant): The characterization of the regulatory events leading to developmental control of the human UGT1A1 gene and the early onset of neonatal hyperbilirubinemia is the central focus of this application. While generally benign, the occurrence of hyperbilirubinemia can lead to central nervous system toxicity, even when extreme measures to prevent its duration and accumulation have been implemented. The underlying mechanisms associated with this well-known syndrome are currently unknown. We will demonstrate in preliminary studies using novel animal models that the onset of neonatal hyperbilirubinemia is a highly coordinated event that involves tissue specific repression of the UGT1A1 gene. In humanized UGT1 (hUGT1) mice, neonatal mice develop severe levels of serum bilirubin, resulting from repression of the liver and intestinal UGT1A1 gene. In a series of experiments designed to examine the impact of PXR and CAR on regulation of the genes encoded by the UGT1 locus, we prepared hUGT1 mice that were deficient in PXR (hUGT1/Pxr-/-) and CAR (hUGT1/Car-/-). From these studies, it was discovered that the deletion of PXR resulted in de-repression of UGT1A1 gene expression in liver tissue, a result that led to accelerated metabolism of serum bilirubin as shown by reduction of bilirubin during neonatal development. Since the nuclear receptors (NRs) such as PXR are targets for co- repressors SMRT and NCoR1, we undertook experiments to examine the role of these repressor proteins on developmental expression of the UGT1A1 gene. We have discovered that in primary neonatal hUGT1 hepatocytes, knockout of SMRT by siRNA leads to induction of UGT1A1 gene expression, potentially linking SMRT repression with the actions of PXR in liver. These preliminary findings will be the basis for experiments performed in Specific Aim 1 which will examine the impact of liver specific knockout of SMRT on UGT1A1 gene expression in hUGT1 mice. NCoR1, on the other hand, is highly specific for repressing UGT1A1 gene expression in intestinal tissue during development. Targeted deletion of NCoR1 in intestinal tissue of hUGT1 mice (hUGT1/NCor1GI�GI mice) leads remarkably to the complete reversal of neonatal hyperbilirubinemia, with dramatic induction of intestinal UGT1A1 gene expression. Experiments outlined in Specific Aim 2 will focus on the identification of the NR involved in NCoR1 repression of intestinal UGT1A1 gene expression. In Specific Aim 3, studies will focus on the epigenetic changes that occur in liver and intestine as a result of SMRT and NCoR1 deletion in these tissues, respectively. These changes will focus on the location of SMRT/NCoR/NR binding and the chromatin changes across the UGT1 locus in liver and intestine as characterized by ChIP-seq and RNA-seq analysis. We will be describing novel mechanisms leading to developmental control of the UGT1A1 gene and its implications toward neonatal hyperbilirubinemia.

描述（由申请方提供）：导致人UGT 1A 1基因发育控制和新生儿高胆红素血症早期发作的调控事件的表征是本申请的中心焦点。虽然通常是良性的，但高胆红素血症的发生可导致中枢神经系统毒性，即使已采取极端措施防止其持续时间和蓄积。与这种众所周知的综合征相关的潜在机制目前尚不清楚。我们将在使用新的动物模型的初步研究中证明，新生儿高胆红素血症的发作是一个高度协调的事件，涉及UGT 1A 1基因的组织特异性抑制。在人源化UGT 1（hUGT 1）小鼠中，新生小鼠由于肝脏和肠道UGT 1A 1基因的抑制而产生严重的血清胆红素水平。在一系列旨在检查PXR和CAR对UGT 1基因座编码的基因调控的影响的实验中，我们制备了PXR（hUGT 1/Pxr-/-）和CAR（hUGT 1/Car-/-）缺陷的hUGT 1小鼠。从这些研究中发现，PXR的缺失导致肝组织中UGT 1A 1基因表达的去抑制，这一结果导致血清胆红素代谢加速，如新生儿发育期间胆红素减少所示。由于核受体（NR）如PXR是共阻遏物SMRT和NCoR 1的靶标，我们进行了实验以检查这些阻遏蛋白对UGT 1A 1基因发育表达的作用。我们已经发现，在原代新生儿hUGT 1肝细胞中，通过siRNA敲除SMRT导致UGT 1A 1基因表达的诱导，可能将SMRT抑制与肝脏中PXR的作用联系起来。这些初步发现将是在特定目标1中进行的实验的基础，该实验将检查SMRT的肝脏特异性敲除对hUGT 1小鼠中UGT 1A 1基因表达的影响。另一方面，NCoR 1在发育过程中对抑制肠组织中UGT 1A 1基因表达具有高度特异性。hUGT 1小鼠（hUGT 1/NCor 1GI？GI小鼠）肠道组织中NCoR 1的靶向缺失可显着完全逆转新生儿高胆红素血症，并显着诱导肠道UGT 1A 1基因表达。具体目标2中概述的实验将重点鉴定参与NCoR 1抑制肠道UGT 1A 1基因表达的NR。在特定目标3中，研究将分别关注由于SMRT和NCoR 1缺失而在肝脏和肠道中发生的表观遗传变化。这些变化将集中在SMRT/NCoR/NR结合的位置以及通过ChIP-seq和RNA-seq分析表征的肝脏和肠道中UGT 1基因座的染色质变化。我们将描述导致UGT 1A 1基因发育控制的新机制及其对新生儿高胆红素血症的影响。