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. EKLF, a transcription factor expressed at a similar time in erythroid development as GATA-1, is also critical to the 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. Preliminary results suggest that this is the case for a subset of EKLF target genes.
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