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Title:DNase 1 hypersensitive sites in rabbit cytochrome P-450IIC subfamily genes and binding of liver nuclear proteins
Author(s):Ahn, Jongsook Kim
Doctoral Committee Chair(s):Kemper, Byron W.
Department / Program:Molecular and Integrative Physiology
Discipline:Molecular Physiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Molecular
Biology, Animal Physiology
Abstract:Cytochrome P450 genes are expressed in different tissues and show a heterogenous response upon the treatment with various inducers. Increased levels of mRNAs of the P450IIC genes after phenobarbital treatment is primarily due to an increase in the rate of transcription. The frequency of transcriptional initiation determines the level of transcription and this event is mainly achieved by the interaction of trans-acting regulatory proteins with cis-acting DNA elements of a gene. Therefore, to elucidate the mechanism of gene regulation of the P450IIC genes, identification of cis-acting regulatory elements in the P450IIC genes and the corresponding trans-acting factors is required.
My thesis contains two chapters. Chapter one covers studies of the chromatin structural features of the P450IIC genes and chapter two presents studies of protein binding to DNase 1 hypersensitive regions of the P450IIC genes. In order to find DNA regions that might contain regulatory elements governing differential induction and tissue-specific expression of the P450IIC genes, chromatin structures of the 5$\sp\prime$ flanking regions of the P450IIC genes were studied. A distal hypersensitive site was present about $-$2.2 kb and a proximal site was located about $-$0.2 kb upstream from the RNA initiation site. The distal site, detected in the phenobarbital inducible genes, was not found in the C3 gene which is constitutively expressed regardless of phenobarbital treatment. Comparison of the proximal hypersensitivity between liver and kidney from control and phenobarbital-treated rabbits revealed that open chromatin structures near the promoter region correlate with the expressed states of the P450IIC genes.
Open chromatin structures represented as nuclease hypersensitivity serve as targets for specific interactions with trans-acting regulatory proteins as has been documented in other well studied eukaroytic genes. Therefore, we carried out protein binding studies for the DNase 1 hypersensitive sites detected from the previous work. Since P450IIC genes are liver-specific genes, we expected to see interactions between hepatocyte specific trans-acting factors and their cognate cis-acting elements. Using a gel mobility shift assay and competition experiments, I have demonstrated hepatocyte nuclear factor-1 like protein binding to the distal ($-$2.2 kb) and proximal ($-$0.45 kb) hypersensitive regions of the phenobarbital inducible C1 and C2 genes. In contrast, the C3 gene did not show binding to an hepatocyte nuclear factor-1 although it contains potential binding motifs for the hepatocyte nuclear factor-1.
As a whole, my studies did not show any gross changes in either chromatin structures or protein binding to hypersensitive regions after phenobarbital treatment. However, detection of the major DNase 1 hypersensitive sites and observation of hepatocyte nuclear factor-1 like factor binding to these regions will provide fundamental information for future gene regulatory studies on the phenobarbital inducible P450IIC genes.
Issue Date:1991
Type:Text
Language:English
URI:http://hdl.handle.net/2142/20553
Rights Information:Copyright 1991 Ahn, Jongsook Kim
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9136526
OCLC Identifier:(UMI)AAI9136526


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