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.
The human UDP-glucuronosyltransferase 1A1 (UGT1A1) gene is developmentally regulated with UGT1A1 playing a key role in the metabolism of total serum bilirubin, thus serving as the key rate-limiting metabolic step in the onset of neonatal hyperbilirubinemia. To date, an understanding of the regulatory events that control expression of the UGT1A1 gene during development have been elusive. Having developed novel humanized UGT1 mice, we will exploit key new discoveries that nuclear receptors along with co-repressor proteins SMRT and NCoR1 repress the UGT1A1 gene in liver and intestinal tissue, leading to delayed bilirubin glucuronidation by UGT1A1.
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