Eukaryotic organisms employ a variety of important but poorly understood mechanisms to control the expression of their genes. A full understanding of these mechanisms is an important issue since regulation of gene expression plays an important role in the development and differentiation of functionally distinct cell types in a precise spatial manner. The genomic regulatory network that controls gene expression ultimately determines form and function in each species. In addition, regulation of transcription reflects the ability of cells to respond to extracellular signals and environmental stresses. The operational nature of the regulatory programming specified by the interplay between cis-regulatory DNA sequences, the cognate transcription factors and the coactivators of transcription has been determined for the human IFN-beta gene, whose transcription is activated in response to virus infection. Activation of the IFN-beta gene is a transient phenomenon requiring three distinct sets of transcription factors, which with the help of the HMG I(Y) protein bind to enhancer DNA cooperatively to assemble the enhanceosome. The enhanceosome activates transcription by instructing an ordered recruitment of chromatin modifying activities such as Histone Acetyl Transferases (CBP and GCN5) and ATP-dependent remodeling machines (SWI/SNF), which alter the local chromatin structure in a way that allows subsequent assembly of the basal transcriptional complex and initiation of transcription. In parallel, GCN5 and CBP acetylate HMG I at distinct lysine residues conferring opposite effects on enhanceosome stability. The overall goals of this proposal are to elucidate the mechanisms of enhanceosome assembly/disassembly in vivo and the molecular basis by which histone acetylation acts to form a """"""""histone code"""""""" read by other proteins to bring about chromatin remodeling and transcriptional activation. Our approach will use cells lacking the HMG I(Y) gene. We will transduce several HMG I(Y) derivatives deficient in distinct functions of the protein and we will investigate enhanceosome assembly in vivo by chromatin immunoprecipitation experiments. Furthermore, we will decode the """"""""histone acetylation code"""""""" by identifying the histones and the specific residues acetylated in vivo in response to virus infection and their role in gene activation. Finally, we will carry out in vivo and in vitro experiments to understand the nature of chromatin remodeling at the IFN-beta promoter and how the general transcriptional machinery is instructed by the enhanceosome to assemble on the remodeled IFN-beta promoter.
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