The genes for human (h) growth hormone (GH) and chorionic somatomammotropin (CS) belong to a family that includes those encoding prolactin and proliferin. These homologous genes are essential for normal human growth and development. We have shown that the transfected hGH and hCS genes are efficiently expressed in rat pituitary tumor cells (GC) but not in non-pituitary cells. We have identified several cis-acting elements in the 5'- flanking DNa of these genes that regulate hGH and hCS promoter activity. These include: (i) positively and negatively acting thyroid hormone response elements (TREs); (ii) elements that modulate TRE functions; (iii) tissue-specific response elements (TSREs); (iv) a positive control element similar to one involved in adenovirus major late promoter function; (v) a repressor element that blocks the activity of the adenovirus-related element; and (vi) a basal regulatory element (BRE). These studies demonstrate fundamental differences between the rat (r) GH and hGH genes, that underscores the need for characterization of the human genes. In the proposed studies we will define and study the mechanism of action of each of these and other elements that participate in mediating the tissue-specific and hormone regulated expression of these genes. This will involve functional assays in which various gene constructs will be transfected into GC cells, primary human placental cultures and other cells. The elements will be fused to heterologous promoters or other DNA elements to evaluate their enhancer-like, repressor-like, intrinsic promoter-like, and hormone response activities and spatial/phase relationships required for their respective activities. Hormones and effectors to be studied include thyroid and glucocorticoid hormones, GH releasing hormone (GHRH), cAMP, IGF-I, insulin, phorbol esters, EGF and FGF. To relate the regulated control of the transfected genes with that of the endogenous genes, the effects of these hormones will be examined in primary cultures of human acromegalic pituitary cells that express the hGH gene and placental syncytiotrophoblasts that express the hCS gene. Trans-acting factors that are involved in mediating the specific regulatory functions will be identified by DNA-footprinting and gel migration assays using nuclear proteins derived from GC and other eukaryotic cells and partially purified thyroid hormone receptors. These studies should provide a comprehensive understanding of hGH/hCS gene expression and regulation that will be relevant for understanding the control of their expression in man and for gene expression in general.
Showing the most recent 10 out of 23 publications
