The human growth hormone (GH) gene cluster is regulated by a locus control region (LCR) that contains 5 DNasel hypersensitive sites (HS), located between -14kb and -32kb 5' of the gene cluster. When this region is present in a transgene construct, transgenic mouse lines reproducibly express the hGH gene in the pituitary and the hCS gene in the placenta at copy-number-dependent levels and in a site-of- integration independent fashion. The overall goal of this proposal is to understand the dominant mechanism by which the hGH LCR regulates expression from the hGH locus in these two tissues. The hypothesis states that the hGH LCR functions primarily through chromatin structure and epigenetic modifications.
Aim I is to structural and functionally characterize the LCR determinants involved in activation of the hGH gene cluster in the placenta and pituitary. Specifically, the structure of the chromatin domain encompassing the hGH gene cluster in expressing tissues will be established, the positions of each HS in somatotrope and placental nuclei will be fine-mapped, correlations between histone acetylation at critical control determinants within the LCR and gene expression from the hGH cluster will be established, as will the role of nucleosomal organization in mediating LCR functions.
Aim II will determine whether the LCR is conserved in the mouse. A large genomic clone containing the mGH gene will be mapped and any HS homologues identified will be tested for LCR function in the mouse pituitary.
Aim III will analyze the role of HSI and HSII in somatotrope restriction. The HSI,II cis elements will be functionally mapped, footprinted, and trans-acting factors sought. Whether the homologous hGH promoter is required for HSI,II activity will be determined. The question of whether HSI,II activity is required to maintain the hGH gene in an active state will be studied using developmentally-timed, recombination- activated gene expression in mouse models. Finally, Aim IV will identify determinants of placental- and somatotrope-restriction of hCS and hGH expression my mutation of individual HS in the context of the intact hGH locus followed by analysis of gene expression in vivo. The significance of these investigation will be a detailed understanding of the mechanism of expression of the hGH gene cluster and its regulation as a unit by the LCR. This will provide insight into the function of other LCRs and serve as a foundation for a sophisticated understanding of the pathophysiology of human growth and development, that may lead in turn to the design of new means of medical intervention.
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