S14 protein is the first tissue-specific component of the nuclear matrix to be purified and to be directly implicated in metabolic regulation by thyroid hormone and dietary constituents. The goal of this project is to define the function of the S14 protein. S14 gene expression is rapidly and markedly increased by thyroid hormone (T3) and dietary carbohydrate only in tissues involved in the synthesis of fat for storage or export (liver, adipose, lactating mammary), and the protein is not similar to other reported sequences. We hypothesized that S14 protein is involved in the tissue-specific regulation of lipid metabolism, and our structural and functional studies both support this concept. Major structural observations included our demonstration that S14 is a nuclear protein by immunohistochemistry, and that it exhibits zonated expression in liver identical to that of acetyl CoA-carboxylase, a rate-determining enzyme of long chain fatty acid synthesis. Western analysis of fractionated nuclei and immunoelectron microscopy both localized S14 protein to the nuclear matrix, a structure that is increasingly recognized as playing a key role in the spatial organization of gene expression. Moreover, ligand blot analysis using S14 protein that we purified by immunoaffinity chromatography revealed the presence of an acceptor protein for S14 within the nuclear matrix. Our functional studies directly implicated S14 protein in metabolic regulation. Antisense-mediated inhibition of S14 expression in hepatocytes specifically impaired the induction of malic enzyme (ME) activity and mRNA, as well as lipogenesis itself, by T3 and glucose. We propose to further define the function of this uniquely regulated component of the nuclear matrix by defining the mechanism whereby the antisense-mediated knockout of S14 protein expression inhibits ME expression and lipogenesis in cultured hepatocytes. Analysis of the relationship of the ME gene with the nuclear matrix during induction by T3 and dietary carbohydrate will be performed as a prelude to study of the potential role of 514 protein in gene-matrix attachment. Adipocyte cell lines stably transfected with an inducible S14 expression construct will be used in complementary studies of the effects of over-expression of the protein on both gene expression and metabolism. S14 protein represents a unique link between tissue-specific nuclear architecture and metabolic regulation, and our preliminary studies provide a firm structural and functional basis for the proposed experiments.
