Growth hormone (GH) is a major regulator of somatic growth. It has been known for decades that GH regulates growth mainly through stimulating gene expression in tissues, including the liver, of insulin-like growth factor-i (IGF-I), but the molecular mechanism by which GH stimulates IGF-I gene expression is poorly understood. The key to understanding this mechanism is identification of GH-responsive regions in the IGF-I gene, but direct identification of such cis-regulatory regions has been difficult, due to the lack of appropriate cell systems and the complexity of IGF-I gene structure. Recent studies employing knockout mouse models and in vivo gene transfer technique demonstrated that signal transducer and activator of transcription 5 (STAT5), an established component of the GH signaling pathway, is essential for GH-stimulated IGF-I gene expression in the liver. Based on this information and using a unique approach, we have identified a distal IGF-I 5'-flanking region that can function as a STAT5-responsive enhancer, that contains multiple consensus STAT5 binding sites, and that is evolutionally conserved. Based on these preliminary observations, we hypothesize that this distal enhancer mediates GH-STAT5 activation of IGF-I gene transcription in the liver. We propose to test this hypothesis in the mouse system.
In specific aim 1, we will confirm that the distal IGFI enhancer contains functional STAT5 binding sites using DNase I footprinting, gel-shift and co-transfection assays.
In specific aim 2, we will determine whether GH causes binding of STAT5 to this distal IGF-I enhancer and whether the STAT5 binding occurs ahead of GH-induced IGF-I transcription in the liver of GH deficient lit/lit mice. The in vivo STAT5-DNA binding will be quantified by chromatin immunoprecipitation assays (CHIP) coupled with real-time PCR.
In specific aim 3, we determine the distal will if IGF-I enhancer is sufficient for GH induction of gene expression in transgenic mice. The results of this research should provide a basis for further elucidating the molecular mechanism by which GH regulates IGF-I gene expression.
