The molecular basis of adipocyte-specific gene expression is not well understood at present. Probing the mechanisms regulating gene transcription in this cell type will serve to improve our understanding of metabolic diseases, including diabetes and obesity, and will also provide new insights into the cell differentiation problem. My lab recently described the first adipocyte-specific enhancer that functions in cultured cells and transgenic mice. Molecular dissection of this enhancer from the adipocyte P2 gene has indicated (in Preliminary Data) that full activity requires the binding of several nuclear factors. The DNA binding of one factor (ARF6) at 2 separate sites in the enhancer stimulates marker gene expression specifically in adipose cells. ARF6 binding activity is observed only in adipose cells, while preadipocytes, muscle cells and fibroblasts lack this activity. Thus, a candidate transcription factor responsible for the activation of adipogenic gene expression is described. We propose a full molecular analysis of ARF6 structure and biological function. We will clone this factor by expression screening of CDNA libraries or using sequence data generated from affinity-purified ARF6 protein. The ability of ARF6 (and some other enhancer binding factors) to activate the aP2 enhancer will be investigated by """"""""transactivation"""""""" experiments, utilizing expression vectors. We will also use transfection studies in fibroblasts and other cell types to analyze the ability of ARF6 expression to activate endogenous adipocyte genes and adipocyte differentiation per se. These transfection assays will also facilitate structure-function analysis of the ARF6 molecule, to determine domains required for transcriptional activity and, if applicable, ability to activate cell determination or differentiation. The expression patterns of ARF6 during adipocyte differentiation and embryological development of the mouse will be investigated to approach its site and timing of action, and also to determine whether biological actions of ARF6 are determined solely by expression of this MRNA and protein. If appearance of ARF6 protein does not correlate with its binding activity or activator function, we will investigate the requirements for accessory factors or protein modifications. ARF6 expression patterns will also be examined in rodent obesity-diabetes syndromes that have an excessive adipocyte number. Finally, we will approach ARF6 function in an in vivo context using techniques of transgenesis. ARF6 will be overexpressed in both adipose tissue and in a non-tissue specific fashion. Adipose cell number, size and gene expression patterns will be monitored in transgenic strains of mice. Conversely, we will mutate ARF6 in mice using homologous recombination- based gene targeting in embryonic stem cells, followed by the creation of germ line chimeras. Ultimately, a full understanding of ARF6 activity in vivo will require the achievement of both ectopic expression and loss-of- function mutations.
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