We propose to study the involvement of coactivators in the function of the beta-globin locus control region (LCR), a genetic element that regulates the chromatin structure, transcription, and replication of the beta-globin genes. Multiple hematopoietic and ubiquitous transcription factors are important for transactivation by the LCR. A common theme among transcriptional regulatory proteins is their involvement in protein-protein interactions with coactivators . Coactivators mediate activation by covalent modification of chromatin or engagement in protein-protein interactions that facilitate assembly of RNA polymerase II preinitiation complexes. To date, no coactivators have been identified that mediate LCR function. Because the LCR disrupts chromatin over long distances (greater than 50-kb), we hypothesize that the LCR recruits chromatin modifying enzymes, specifically histone acetylases (HATs), and a ubiquitin ligase, which acts as coactivators to catalyze a chromatin changes necessary for long-range activation.
In Specific Aim 1, we will assess the roles of the HATs, CBP/p300, P/CAF, and GCN5 in long- range activation by the LCR. To test the hypothesis that HATs mediate transactivation of globin genes through interactions with the LCR, the influence of wild-type HATs and acetylase-deficient mutants on LCR function will be studied in transfection assays.
In Specific Aim 2, we will determine whether the ubiquitin ligase WWP1 and related WW domain proteins influence LCR function. Based on physical and functional interactions between WW domains and the LCR binding protein, NF-E2, we will test the hypothesis that such proteins are important for LCR function.
In Specific Aim 3, we will test whether amino acid sequences of NF-E2 important for CBP/p300 and WW domain binding are required for long-range transactivation by the LCR. We have delineated distinct sequences within the activation domain of NF-E2 necessary for CBP/p300 and WW domain binding and will determine whether these interactions are required for NF-E2 mediated transactivation. These studies represent the first phase of our long-term goal to understand the role of coactivators in LCR function, to test whether hematopoietic signaling pathways modulate their activity, and to ascertain their role in hematopoiesis.
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