Proopiomelanocortin (POMC) is expressed abundantly in the pituitary gland and arcuate nucleus of the hypothalamus where it undergoes tissue-specific posttranslational processing into multiple biologically active peptides, including adrenocorticotrophic hormone (ACTH), beta-endorphin, beta- and gamma 7 lipotropins, and alpha-,beta-, and gamma-melanocyte stimulating hormones (MSH). The objectives of this research are to understand the molecular mechanisms regulating POMC gene transcription and to determine the physiological function of beta-endorphin in brain and pituitary development. Pituitary cell lines characteristic of melanotrophs will be derived from transgenic mice harboring intermediate lobe tumors induced by the expression of a POMC-SV40 large T antigen fusion gene. The cell lines will be used for studies of POMC gene regulation. The regulatory elements in the POMC gene responsible for corticotroph and melanotroph expression will be identified by a combination of DNAse I and chemical interference footprinting assays of POMC sequences, nuclear protein extracts, functional expression studies using AtT20 corticotroph cells, melanotroph cell lines, and transgenic mice pituitaries. The role of an Spl-like transcription factor in mediating pituitary-specific expression of the POMC gene will be investigated. Novel transcription factors binding to three previously identified elements in the POMC gene promoter will be affinity purified, or cloned, to deduce their structure and characterize their physiological function. A targeted gene mutation that specifically blocks the production of beta-endorphin will be introduced into mice by homologous recombination in embryonic stem cells.
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