All hematopoietic cell types arise from pluripotent stem cells through multiple steps of lineage commitment and subsequent maturation. Cellular decisions regarding proliferation and differentiation are tightly controlled to ensure homeostasis of all circulating blood cells. Disruption of this control can lead to hematological aplasias or malignancies. The efforts of this laboratory are directed towards understanding the development of the erythroid cell lineage by focusing on the transcription factor GATA-1. GATA-1 participates in the regulation of virtually all erythroid-restricted genes including the globin genes. It causes cell cycle arrest and prevents apoptosis of erythroid precursor cells. This project centers around our recent observation that the transcriptional integrator CBP serves as cofactor for GATA-1 and is required for erythroid cell maturation. In addition to GATA-1, CBP regulates various transcription factors involved in cellular differentiation and is associated with translocations found in certain types of leukemias. CBP possesses histone acetylase activity towards all four core histones. Recently, we have discovered that CBP acetylates GATA-1 specifically at functionally important sites.
Under Aim 1 we will determine the precise residues of GATA-1 acetylated in vitro and in vivo. Subsequently, we will monitor acetylation of GATA-1 in vivo during cell cycle progression and cellular differentiation.
In Aim 2 we will analyze the functional consequences of GATA-1 acetylation through biochemical assays and through gene complementation experiments using a unique GATA-1-deficient erythroid cell line. This cell line allows the testing of GATA-1 constructs in the physiological environment of maturing erythroid cells. Studies outlined in Aim 3 are designed to delineate the transcriptional pathways by which CBP controls erythroid maturation. Using various cellular assays, we will determine which domains of CBP are required for erythroid differentiation and GATA-1 activation. Together, these studies should provide new insights into the mechanisms by which cellular differentiation and tissue-specific gene expression is accomplished and might lead to novel approaches for therapy of hematopoietic disorders.
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