Science. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. We found that mutations in either acetylation motif partially impaired the ability of GATA-1 to induce differentiation while mutations in both motifs abrogated it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells. Erythrocyte development is among the best-studied model systems used to define the mechanisms responsible for lineage commitment, cellular differentiation, and tissue-specific gene expression. The lineage-restricted transcription factor GATA-1 orchestrates several key aspects of erythroid development. Functionally important binding sites for GATA-1 are found in the regulatory regions of essentially all erythroid-cell-specific genes (30). Mice deficient for GATA-1 succumb to fatal anemia due to an inability of erythroid precursor cells to survive and mature (14, 43, 44). In addition, GATA-1 induces terminal erythroid maturation when introduced into the GATA-1-deficient erythroid cell line G1E (45). Tissue-specific gene expression is the result of a concerted action of tissue-restricted GSK5182 and ubiquitous transcription factors. Our previous studies indicated that Rat monoclonal to CD4.The 4AM15 monoclonal reacts with the mouse CD4 molecule, a 55 kDa cell surface receptor. It is a member of the lg superfamily, primarily expressed on most thymocytes, a subset of T cells, and weakly on macrophages and dendritic cells. It acts as a coreceptor with the TCR during T cell activation and thymic differentiation by binding MHC classII and associating with the protein tyrosine kinase, lck a widely expressed transcriptional cofactor, CREB-binding protein (CBP), is usually a potent coactivator of GATA-1 (4). CBP associates with GATA-1 in vitro and in vivo and markedly stimulates its activity. Expression of the adenovirus oncoprotein E1A, which interferes with the action of CBP, blocks the effects of CBP on GATA-1 activity, inhibits erythroid differentiation, and reduces the expression of several GATA-1-dependent genes (4). Multiple mechanisms have been invoked to account for the activity of CBP and its close relative p300. GSK5182 First, CBP interacts with TFIIB (23), TATA-binding protein (1, 12, 37, 40), and RNA polymerase II (10, 21, 27) and could thus serve as a bridging molecule between DNA-bound transcription factors and the basal transcription machinery. Second, CBP interacts with numerous transcription factors via dedicated domains and could thus provide a scaffold for the assembly of a high-molecular-weight enhanceosome complex (for a review, see reference 38). Such multifunctional interactions may account for the observed synergy between transcription factors that use CBP and p300 as cofactors. Third, CBP and p300 possess intrinsic and associated histone acetyltransferase activity (3, 9, 28, 39, 46). Acetylated histones are associated with an open chromatin configuration, which in turn might facilitate the accessibility of DNA to other transcription factors (for a review, see recommendations 31 and 35). Finally, CBP and p300 were recently shown to acetylate nonhistone proteins such as the tumor suppressor protein p53 (16), the erythroid Kruppel-like GSK5182 factor (EKLF) (48), and the basal transcription factors, TFIIE and TFIIF (19). In the case of p53, acetylation of the regulatory domain name led to a dramatic increase in DNA binding (16) GSK5182 in vitro, whereas the functional consequences of EKLF, TFIIE, and TFIIF acetylation remain to be explored. In this report, we further define the mechanisms of transcriptional activation of GSK5182 GATA-1 by CBP. We found that CBP acetylates GATA-1 at two highly conserved lysine-rich motifs near the zinc finger domains. In contrast to a recent report which appeared while this paper was under review (7), we found that acetylation of GATA-1 did not affect its ability to bind DNA and that substitutions or deletions in these motifs did not affect DNA binding of mammalian-cell-expressed GATA-1. We also found that mutations in the C-terminal acetylation motif, but not in the N-terminal motif, reduce the binding to CBP and diminish the response to CBP in transient-transfection assays. We further demonstrate that CBP stimulates GATA-1 acetylation in vivo and that coexpression of E1A abolishes GATA-1 acetylation. Certain functions of GATA-1 critically depend on an erythroid environment. For example, the N-terminal zinc finger of GATA-1 is required for function in erythroid cells.