Chromatin structure and methylation state of a thyroid hormone-responsive gene in rat liver

The gene for S14 in the rat codes for an mRNA which in lipogenic tissues (liver, fat, mammary gland) responds both to L-triiodothyronine and a high-carbohydrate, fat-free diet. In an effort to understand the molecular basis for the tissue-specific regulation of mRNA-S14 expression, we have examined the organization of this gene in chromatin. Specifically, we examined the distribution of DNase I-hypersensitive sites and DNA methylation sites associated with a 25-kilobase chromosomal domain containing the S14 gene. Our results show that DNase I preferentially digests four regions of the DNA flanking the 5' end of the hepatic S14 gene which have characteristics of DNase I-hypersensitive sites. The sites, identified as HS-1, HS-2, HS-3, and HS-4, are located at or adjacent to the site of transcription initiation and at 1.2, 7, and 8 kilobases upstream from this site, respectively. In lactating mammary gland where the S14 gene is also highly expressed and regulated by L-triiodothyronine, sites HS-1, HS-2, and HS-4 are present, but HS-3 is absent. No hypersensitive sites were detected either within the gene or flanking the 3' end of the gene. In brain, kidney, and spleen, tissues in which mRNA-S14 is expressed at levels less than or equal to 10% of that found in euthyroid liver, sites HS-1 and HS-3 were absent. Despite the marked effect of thyroid state on the abundance of hepatic mRNA-S14, no significant alterations were observed in the DNase I sensitivity of hepatic chromatin containing the S14 gene. Analysis of the DNA methylation pattern at HpaII and HhaI sites showed a positive correlation of hypomethylation of the gene and the contiguous flanking regions with S14 gene expression. All HpaII/MspI sites and most HhaI sites either within or flanking the S14 gene were undermethylated in liver and lactating mammary gland. Although thyroid status had no generalized effect on the site-specific DNA methylation state of the hepatic S14 chromosomal domain, one site (H3) situated in the second exon close to the 3' terminus of the S14 gene appeared to undergo demethylation in the transition from hypo- to euthyroidism. In essence, our results show that the 5' DNA flanking region of the S14 gene contains a tissue-specific DNase I-hypersensitive site which, although not influenced by thyroid status, appears essential for the expression of S14 and its regulation by L-triiodothyronine.(ABSTRACT TRUNCATED AT 400 WORDS)