The proposal's long-term goal is to understand how transcription factors (TFs) that control blood cell development and function regulate gene expression by interacting with transcriptional coactivators. CREB-binding protein (CBP) and the highly related p300, are large multi-domain nuclear phosphoprotein coactivators that functionally and physically interact with many nuclear proteins. In humans, chromosomal translocations involving CBP are associated with leukemia, and CBI haplo-insufficiency leads to Rubinstein-Taybi Syndrome (RTS is characterized by mental retardation, craniofacial defects and broad big toes and thumbs, and an abnormal incidence of neoplasms, including leukemia). A crucial role for CBP and p300 has been demonstrated in mice where knockouts of either gene result in death at embryonic days 9 to 11.5. CBP, bu not p300, null heterozygotes also exhibit severe problems in hematopoiesis with marked B-cell and, to some degree, T-cell deficiencies, and a predisposition for developing cancers of hematopoietic origin, including lymphocytic leukemia. However the mechanistic defect(s) that affects transcription in vivo and leads to such severe phenotypes is largely unknown. The proposal focuses on one of the four distinct TF-binding domains in CBP/p300, the evolutionarily conserved Cysteine Histidine-rich domain 1 (CH 1), because it interacts with two factors critical for normal hematopoietic cell function, Ets-1. and STAT5. Using Ets-1 and STAT5 as model TFs, it is proposed to employ CBP and p300 conditional null alleles ii mouse and mouse-cell model systems to elucidate CBP- and p300-dependent functions in normal and malignant hematopoietic transcription.
Aim 1 is to define the molecular rules governing the Ets-1 and STAT5 interaction with the CH domain of CBP and p300 by site-directed mutagenesis, and to correlate this with transactivation function.
Aim 2 is to determine the in vivo role of CBP and p300 in B- and T-cells by using CBP- and p300-conditional knockout mice. The CBI and p300 mutant mice generated in this study will be valuable models for examining the roles of these coactivators in other physiological processes relevant to human health, including memory, reproduction, growth, aging, and metabolism.
