The principal objective is to develop the rat liver S14 gene model to correlate the chromatin structure of this gene with its tissue-specific and hormonal regulation. The level of the rat hepatocellular mRNA (mRNA-S14) coding for the cytosolic protein, """"""""S14""""""""; (17,000 Mr; 4.9 pI) is subject to complex regulation involving hormonal, nutritional, circadian, tissue-specific and developmental factors. The predominant association of this sequence with tissues synthesizing mobilizible fat suggests the S14 protein functions in lipid metabolism. Although the function for this protein is unknown, several features make this system particularily attractive to study. They are: 1) The presence of several DNase I hypersensitive sites at the 5' end of the gene and the hypomethylation of the S14 gene domain correlates with the tissue-specific expression of mRNA-S14; 2) In liver, thyroid hormone rapidly activates S14 gene transcription, whereas glucagon rapidly inactivates S14 gene transcription. Although other systems like MuMTV,P-globin and vitellogenin allow for the analysis of tissue-specific and/or hormonal effects on chromatin structure, these systems do not offer the added advantage of rapidly turning the gene on and off by hormonal administration. The S14 system offers the opportunity to correlate rapid hormonally induced changes in S14 gene activity with changes in S14 chromatin structure; and 3) The availability of cDNA and genomic clones to the S14 gene now make it possible to study this system at the molecular level. Accordingly, this applicaton will focus on: 1) DNA sequence analysis of the 5' flanking sequence of the S14 gene to identify the hypersensitive DNA sequences, i.e., putative regulatory sequences; 2) high resolution mapping of the hypersensitive sites proximal to the 5' end of the S14 gene to determine whether changes in DNA-protein interactions at these sites accompany hormonally induced changes in S14 gene activity; 3) develop an in vitro approach to study site-specific DNA-protein interaction with the S14 gene. These studies are prerequisite steps toward the long term goal of identifying and establishing the function of the chromatin constituents which confer tissue-specific and hormonal regulation of S14 gene transcription. These studies should allow us to identify specific molecular events associated with the hormonally-induced changes in S14 gene transcription. It is anticipated that these studies will lead to a better understanding of the multifactorial regulation of S14 gene expression at the molecular level.
