Human (h) growth hormone (GH), chorionic somatomammotropin (CS) and prolactin (PRL) comprise a family of genes that are essential for normal human growth and development. Altered GH production has been implicated in various pathological states, including osteoporosis and aging. Knowledge of the regulation of these genes is important for our understanding of their physiologic and pathophysiologic functions. The proposed studies extend previous investigations on the hormonal and tissue-specific factors that mediate transcript ion of hGH/hCS genes.
Aim 1 focuses on the functional significance of DNA bending induced by the thyroid hormone receptor (TR), cell-specific factor GHF1 and Sp1. The contributions of intrinsic and transcription factor-induced DNA bending will be established by circular permutation and phasing analyses. The influence of DNA phasing-induced changes on overall promoter conformation will be correlated with promoter activities using transient transfection assays and in vitro transcription systems. TR mutants that exhibit coupled and uncoupled DNA binding and transcriptional activation responses will be examined by bending analyses to determine whether DNA bending is coupled to DNA binding or transactivation functions.
Aim 2 will examine the structure and mechanism of action of a unique, promoter-dependent negative thyroid hormone response element (nTRE) located in the 3'-flanking DNA of the hGH gene. The precise structure of the nTRE will be established utilizing mutagenesis and analysis of functional activity with transfection assays. The hGH promoter elements that are required for nTRE function will be established by mutagenesis of the hGH 5'-flanking region/promoter and functional analysis using transient transfection assays. The mechanism of nTRE action will be examined by a combination of run-on transcription, in vitro transcription and in vitro polyadenylation and/or splicing experiments to establish whether nTRE action is coupled to transcript ion initiation, elongation or termination/3'-end formation. The physiologic relevance of nTRE function on hGH gene expression will be examined by the ongoing studies of human acromegalic tumor cells. These studies will extend our understanding of the factors that regulate the hormonal and tissue-specific regulation of hGH/hCS gene expression.
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