Co-repressors SMRT and NCoR1 regulate UGT1A1 gene expression

描述(由申请人提供)：描述导致人类UGT1A1基因发育控制和早发性新生儿高胆红素血症的调节事件的特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能会导致中枢神经系统毒性，即使已经采取极端措施防止其持续和积累。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生是一个高度协调的事件，涉及组织特异性的UGT1A1基因抑制。在人源化UGT1(HUGT1)小鼠中，由于肝脏和肠道UGT1A1基因的抑制，新生小鼠的血清胆红素水平严重升高。在一系列旨在研究PXR和CAR对UGT1基因编码基因调控的影响的实验中，我们准备了PXR(hUGT1/PXR-/-)和CAR(hUGT1/CAR-/-)缺陷的hUGT1小鼠。研究发现，PXR基因缺失导致肝组织中UGT1A1基因表达下调，从而导致血清胆红素代谢加速，表现为新生儿发育过程中胆红素的降低。由于PXR等核受体是SMRT和NCoR1共抑制物的靶标，我们进行了实验来研究这些抑制蛋白对UGT1A1基因发育表达的作用。我们发现，在原代新生hUGT1肝细胞中，siRNA敲除SMRT导致UGT1A1基因表达诱导，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，该实验将检查肝脏特异性SMRT基因敲除对hUGT1小鼠UGT1A1基因表达的影响。另一方面，NCOR1在发育过程中抑制UGT1A1基因在肠道组织中的表达具有高度的特异性。HUGT1小鼠(hUGT1/NCor1GI？GI小鼠)肠道组织中NCoR1的靶向缺失可显著逆转新生儿高胆红素血症，并显著诱导肠道UGT1A1基因表达。具体目标2中概述的实验将集中于鉴定参与NCoR1抑制肠道UGT1A1基因表达的NR。在具体目标3中，研究重点将集中在肝脏和肠道分别由于SMRT和NCoR1缺失而发生的表观遗传学变化。这些变化将集中在SMRT/NCoR/NR结合的位置以及肝脏和肠道中UGT1基因座染色质的变化，这是CHIP-SEQ和RNA-SEQ分析的特征。我们将描述导致UGT1A1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。

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.