Orientation-Dependent Regulation of RUNX1 and GATA-1 Transcriptional Function by the p300/CBP Coactivators.

The transcription factors RUNX1 and GATA-1, as well as the coactivators p300/CBP, have been implicated in the regulation of primary megakaryopoiesis through studies of knockout mice. In particular, p300/CBP has previously been shown to serve as a coactivator for both RUNX1 and GATA-1 in the transactivation of hematopoietic target genes. Coactivator orientation within transactivating complexes is generally not considered to influence the degree of transcriptional activity, reflecting an inherent flexibility in the spatial requirements for coactivator function. Experiments to further explore this issue showed coexpression of p300 to enhance cooperative transcriptional activation by wild type RUNX1 and GATA-1. Enforced recruitment of p300/CBP to GATA-1 through fusion of GATA-1 with the p300/CBP docking module of adenoviral E1A, E1A(1–89), enhanced GATA-1 activity alone, regardless of whether the fusion was to the amino or carboxy terminal of GATA-1. However, enforced recruitment of p300/CBP to the amino terminus of GATA-1 completely eliminated cooperation with RUNX1, and enforced recruitment of p300/CBP to the carboxy terminus of GATA-1 diminished cooperation with RUNX1. Both GATA-1 fusions retained physical interaction with RUNX1. Similarly, fusion of E1A(1–89) directly to the amino terminus of RUNX1 completely eliminated its transcriptional activity, while fusion to the carboxy terminus diminished RUNX1 transcriptional activity. For both the GATA-1 and the RUNX1 fusions, these repressive effects were attributable to the ectopic recruitment of p300/CBP because a mutation within E1A(1–89) known to specifically diminish p300/CBP recruitment, R2G, rescued the transcriptional activities. Addressing the mechanism of repression by ectopic p300/CBP, we found that E1A(1–89)-GATA-1 caused diminished serine phosphorylation within RUNX1, an effect opposite to that of wild type GATA-1 which enhanced RUNX1 phosphorylation. Similarly, E1A(1–89)-RUNX1 showed complete loss of phosphorylation on cdk target sites, as compared with wild type RUNX1. RUNX1-E1A(1–89) showed diminished phosphorylation on cdk target sites, as compared with wild type RUNX1. From these results, we conclude that p300/CBP may function as a coactivator for the RUNX1-GATA-1 complex when recruited to endogenous, wild type domains. By contrast, ectopic recruitment of p300/CBP to RUNX1, particularly to the amino terminus, targets RUNX1 for repression through inhibition or reversal of phosphorylation. Our results thus offer a novel paradigm in which the function of p300/CBP, coactivator versus repressor, may be determined by its mode of recruitment to certain transcriptional complexes. Notably, some transcription factors, such as GATA-1, have relaxed requirements for the topology of coactivator recruitment, whereas other transcription factors, such as RUNX1, have stringent requirements in this regard.