The ultimate objective of this work is to understand how hormones and their receptors interact to regulate transcription of target genes. Within this context, one of the most compelling concepts is that gene regulation involves an interactive network of transcription factors and chromatin-remodeling machines. RUSH proteins, new members of the SWI/SNF family of global gene regulators, are potential intermediates in the cross-talk between progesterone and prolactin signaling pathways. Recent data support a model in which progesterone receptor binding to accessible response elements in uteroglobin and Muc1 genes triggers an ATP-dependent remodeling activity. RUSH proteins are potential candidates that are readily available via a progesterone-dependent alternative splicing mechanism coupled to a prolactin-dependent tyrosine phosphorylation event. Co- immunoprecipitation experiments confirm a physical association of RUSH with Jak 2, the tyrosine kinase that mediates the PRL signaling pathway in the mammary gland. Moreover, tyrosine phosphorylation of RUSH is obligatory for DNA binding. However, as members of the SW1/SNF family of nuclear receptor coactivators, RUSH proteins have been implicated in chromatin remodeling. Thus it is important to determine whether RUSH proteins act as PRL signal transducers, function as chromatin remodeling machines, or act directly as transcription regulators. It is equally important to determine whether RUSH is a member of a multi-component complex. A clear view of transcriptional regulation of RUSH is also necessary. These goals will be achieved through five specific aims.
In aim one, the 5'-flanking region of the RUSH gene will be characterized by mutational analysis and transient transfection assays.
In aim two, a targeted cloning strategy will be used to characterize RUSH binding sites in the promoter of the rabbit Muc-1 gene.
In aim three, site-directed mutagenesis will be used to define which of the tyrosine phosphorylation site(s) is functionally relevant to DNA binding.
In aim four, CASTing will be used to identify proteins that interact with RUSH to enhance DNA binding.
In aim five, the dynamic association of RUSH with active chromatin, matrix associated splicing complexes and/or RNA processing centers will be evaluated by immunogold labeling, immunofluorescence, chromatin fractionation, and Western analysis. Defining the interaction between prolactin and progesterone in the regulation of gene transcription is central to understanding the mechanism of hormone action in the uterus, and may be critical to good reproductive health.
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