In collaboration with the laboratory of Jim Omichinski we have shown that the p53 transactivation domain interacts in vitro and in vivo with the GATA-1 DNA binding domain. The linker and C-terminal zinc-finger of GATA-1 are required for the interaction. The proteins reciprocally inhibit the transactivation activity of one another in an erythroid precursor cell line, 6C2. GATA-1 may be required to prevent p53 induction during erythropoiesis or megakaryopoesis. In collaboration with Masi Yamamoto, we plan to determine the role of this interaction during hematopoiesis by attempting to rescue GATA-1 null cells and mice with mutants of GATA-1 that do not interact with p53. Screens for GATA-1 mutants of this category are in progress, but have not yielded mutants that do not interfere with p53 independent functions of GATA-1. These efforts continue. EKLF, a transcription factor expressed at a similar time in erythroid development as GATA-1, is also critical to this lineage. Anemias associated with mutations in the EKLF DNA binding domain have been identified in humans. An amino acid in the second zinc finger of EKLF is mutated in patients with congenital dyserythropoietic anemia and in the Nan (Neonatal anemia) mouse model. In collaboration with Jim Bieker we are trying to determine if the anemic phenotype is based on different DNA binding affinities of purified Nan-EKLF relative to wild type, as suggested by earlier studies with nuclear extracts. In those studies it was shown that a T base in the central triplet of the EKLF DNA binding domain resulted in sites that were bound by wild type EKLF, but not by Nan EKLF. That result has been confirmed with purified proteins for two such sites reported in those studies. The magnitude of the affinity differences correlates with the degree of reduction in target gene expression in the Nan mouse. A putative new Nan target gene containing the central triplet T has also been identified.
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