Cell-restricted transcriptional modulators play a critical role in the process of selective gene regulation during hematopoiesis. 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. Although the details of how EKLF integrates these signals remain to be fully deciphered, recent evidence unexpectedly suggests that it can function as a transcriptional repressor and may also play a role outside of the nucleus. Shifts between EKLF repression and activation are influenced by the cellular environment and by its interaction with corepressors. In addition, its modification by acetylation may also affect these decisions. This proposal is designed to expose the details of how these varied processes are controlled by identifying components of EKLF protein complexes and examining the role of post-translational modifications in these interactions. This will be accomplished by: (1) examining (a) in vitro and (b) italic how EKLF protein interactions with its activation partners are coordinated at the beta-globin promoter; (2) using tandem affinity purification to isolate EKLF protein complexes derived from selected cell types and subcellular fractions; (3) illuminating alternate roles for EKLF by (a) identifying EKLF repression targets and analyzing the role of its acetylation in repression and (b) characterizing cytoplasmic EKLF complexes.