The transcription factor Ets-1 plays an important role in hematopoietic cell function, but how it activates transcription is unclear. A number of transcription factors use the transcription coactivators CREB binding protein (CBP) and the related p300 to mediate transactivation of TNA polymerase II (Pol II). How CBP/p300 mediates transactivation is unknown, but it binds basal transcription factors and has associated histone acetyltransferase (HAT) and protein kinase activities that may play a role in activating Pol II. The importance of CBP associated HAT activity in hematopoiesis is suggested by the t(8;16)(p11;p13) translocation found in a subset of acute myeloid leukemias where a putative HAT is fused to CBP. The long term goals of this proposal are to define the mechanisms by which hematopoietic transcription factors stimulate gene expression by interacting with CBP/p300 and how perturbation of these interactions results in a disease state. This proposal is focused on the hypothesis that Ets-1 regulates Pol II transcription by binding specific regions of CBP/p300. Ets-1 activity can be affected by modulating the Ets-1:CBP/p300 interaction, or by regulating factors and enzymatic activities associated with CBP/p300.
Specific Aim I will test if interaction of Ets-1 with specific CBP/p300 domains is required for Ets-1 transcription activity. The applicant will define CBP/p300 sequences necessary and sufficient for Ets-1 binding in vitro and in vivo and will correlate this with transactivation function in vivo.
Specific Aim II : Deletion and point mutagenesis will be used to functionally define the Ets-1 sequences required to interact in vivo and in vitro with CBP/p300. It will be determined whether these Ets-1 sequences are necessary and sufficient for transactivation function in vivo.
Specific Aim III will determine if Ets-1 activity correlates with CBP/p300 associated factors and enzymatic activities. The investigators will measure HAT, protein kinase and Pol II activities and general transcription factors present in Ets-1:CBP/p300 complexes and correlate this with Ets-1 transactivation potential.
