Growth (GH) and insulin-like growth factor (IGF-I) are both critically important for growth and differentiation in vertebrates. GH is the principal regulator of IGF-I production and acts primarily by activating IGF-I gene transcription. Characterizing the mechanisms responsible for this transcriptional activation should provide new molecular targets for drug design and ultimately lead to better treatment of metabolic and growth disorders. Our laboratory has identified a chromatin region in the rat IGF-I gene termed HS7 that undergoes GH-regulated structural alteration in parallel with hormone-induced transcriptional regulation. The goals of this project are to characterize the molecular mechanisms that mediate activation of IGF-I gene by GH by evaluating the role of HS7 in transcriptional control. The first set of experiments will test the hypothesis that HS7 mediates GH-induced stimulation of IGF-I gene transcription. Several cell lines known to express IGF-I will be transfected with GH receptor and various reporter genes fused to the IGF-I or heterologous promoters with or without the HS7 site. A parallel set of experiments will characterize the nuclear proteins that bind to the IGF-I at the HS7 site. In vitro gel shift assays and foot printing experiments will be used in conjunction with site-specific mutagenesis to identify the specific nucleotides that are necessary and sufficient for high affinity protein binding. Several experimental approaches will be employed to identify and clone the nuclear proteins that interact with HS7. A series of experiments will test the hypothesis that the cloned proteins are specific mediators of GH-regulated IGF-I gene transcription.
