Obesity and associated metabolic disorders including diabetes have become epidemic worldwide and threaten the health of millions. A recent confluence of data from human population genetics studies, mouse transgenic experiments, and neuropeptide pharmacology has implicated the proopiomelanocortin (POMC) gene, its encoded peptides, and their cognate G-protein coupled receptors as essential components of the neural circuits that regulate food intake and energy expenditure. Evolution has resulted in functionally and topographically distinct DNA regulatory elements that control POMC transcription in either neurons or pituitary endocrine cells, and which are highly conserved across mammalian species. These distinctions in cell-type specific POMC promoter and enhancer elements are mirrored by unique complements of transcription factors that differentially regulate POMC gene expression in the brain versus pituitary gland. POMC expression in hypothalamic neurons is quantitatively associated with adipose mass over the lifetime of a mouse and we hypothesize that in humans the analogous control of neuronal POMC gene expression is a key regulatory switch determining the ultimate balance of fat storage or utilization.
The first aim of this project is to further characterize the neuronal-specific enhancer elements of the POMC gene using a combination of in vivo transgenic and in vitro primary neuronal culture expression systems, guided by additional cross-species comparative sequencing.
A second aim will specifically determine the interaction of POMC gene regulatory elements with components of the leptin receptor-signaling pathway to result in activation of neuronal POMC gene transcription by the adipocyte-derived hormone. Third, the quantitative and qualitative effects of selective neural enhancer mutations in the context of the endogenous mouse POMC gene will be rigorously tested by genetic knock-in strategies. Fourth, the mRNA """"""""transcriptome"""""""" of POMC neurons isolated by lasercapture microscopy will be defined, and the cognate DNA-binding transcription factors responsible for neuron-specific POMC gene expression will be identified and cloned using both computational genomics and screening of yeast one-hybrid cDNA libraries constructed from amplified POMC neuron-specific RNA. Throughout these studies we will collaborate with our clinical colleagues, who are involved in human genome-wide screens and index case-based gene sequencing projects, in a translational effort to identify and functionally characterize additional polymorphic alleles of either the POMC gene itself or candidate neuronal POMC gene transcription factors underlying altered weight regulation. ? ?
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