Cell-restricted transcriptional modulators play a critical role in the process of lineage selection during hematopoiesis. In addition, the controls that dictate programming of globin expression during erythroid ontogeny rely on higher-order structures that enable proteins bound at distal (LCR) and proximal (promoter) controlling elements to activate transcription of the selected gene within the context of chromatin. Protein-based interactions between factors bound at these elements must play an important part in establishing and maintaining such a structure. However, the molecular details of how this process is coordinated remains to be established. We have been investigating the molecular and biological function of Erythroid Kruppel-like Factor (EKLF). EKLF is a red cell-restricted transcription factor that is an essential component for completion of the erythroid program. Molecular and genetic data demonstrate that EKLF plays a major role in the developmental switch to adult beta-globin expression. It accomplishes this by means of its strong transcriptional activation function when bound to its cognate CACCC element at the beta- globin promoter, and by its ability to interact with coactivators that generate the proper chromatin configuration at the beta-like globin locus. EKLF is post-translationally modified by both acetylation and phosphorylation, and its subcellular localization may play a role in its function. This proposal is designed to test the hypothesis that these observations are interrelated, and thus that EKLF is an integrator of diverse signals that serve to regulate beta-globin expression. This will be accomplished by: (1) demonstrating that EKLF modification status plays a regulated role in protein-protein interactions, both with its known partners (CBP/p300, BRM1) and with other, yet to be identified, EKLF-associated proteins; (2) testing the functional importance of previous and novel EKLF mutants by means of a biological rescue assay of EKLF-null cells; (3) determining the parameters that control EKLF subcellular localization in primitive and definitive erytbroid cells.
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