""""""""Spot 14"""""""" (S14) is a nuclear protein unique to lipogenic tissues (liver, white and brown adipose, lactating mammary). S14 abundance is rapidly increased by stimuli for enhanced long-chain fatty acid synthesis, such as thyroid hormone and dietary carbohydrate. During the current funding period we showed that S14 acts at the transcriptional level in the induction of lipogenic enzymes in hepatocytes. We found a functional interaction with the proximal 197 bp of the promoter of the gene coding liver-type pyruvate kinase (L-PK), a key regulatory enzyme for provision of substrate for lipogenesis. Protein interaction studies in the yeast two-hybrid system showed that S14 forms homodimers in mammalian cells via a conserved """"""""zipper"""""""" region, and coimmunoprecipitation showed that S14 also interacts directly with a 36 kD hepatic protein (p36). We assigned the human S14 gene to an area known to be amplified in poor- prognosis breast cancers (11q13.5), and found S14 gene amplification and expression in selected breast cancer cell lines. Metabolic relevance was indicated by the findings that S14 was expressed in 67 percent of primary breast cancers and that this was highly concordant with overexpression of lipogenic enzymes by immunohistochemistry. Moreover, S14 was expressed in lactating, but not nonlactating, normal breast. We propose three specific aims. First we will examine S14 interactions with the proximal L-PK promoter in S14 antisense- and control-treated rat hepatocytes. Three complementary approaches will be used, including functional analysis of a series of well characterized promoter mutants using antisense plus reporter transfection, gel retardation studies to identify S14-dependent protein-DNA complexes, and DNAse I footprinting to localize direct protein-DNA interactions. Second we will produce S14-deficient knockout mice and examine lipid metabolism under conditions that elicit increased tissue-specific expression of the lipogenic pathway. These will include acute premature weaning of pups to a low-fat diet, chronic exposure of adult mice to diets of varying fat content, and effects on lactation. Third, we will use newly- available S14-expressing breast cancer cell lines to further elucidate S14 regulation and function. Regulation of S14 and lipogenic enzyme expression by T3, glucose, and progesterone will be defined. Function will then be assessed by stable transfection of breast cancer cell lines that do or do not express S14 with ecdysone-inducible constructs coding for S14 sense or antisense transcripts. These differing but complementary windows on S14 function will define the mechanism of S14 action at the molecular, cellular, and physiological levels.
