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. In collaboration with Jim Bieker we are trying to determine if the anemic phenotype in the Nan mouse model 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. Sites with a C at this position bound both wild type and Nan EKLF. We have shown with purified proteins, that the Kds for Nan binding to four of these T containing EKLF binding sites are higher than those for wild type EKLF, and the results are statistically significant. For a 5th site a difference in Kd was observed, but without statistical significance. The magnitude of the affinity differences correlates with the degree of reduction in EKLF target gene expression in the Nan mouse for three of these sites. A T containing site in the Dematin gene has also been found to be consistent with the model. We have identified a putative new Nan regulated gene (ICAM 4), which has a site with a central triplet T that binds poorly to Nan EKLF, but well to wild type EKLF. ICAM 4 is EKLF regulated, but its expression levels have not yet been tested in the Nan mouse. Thus the anemia in the Nan mouse is likely caused by reduced binding affinity of Nan-EKLF for a subset of EKLF binding sites. Other studies have addressed the question of the possible role of triple stranded DNA:RNA structures in regulation of gene expression. There are severe restrictions on what RNA sequences can form a triplex with double stranded DNA. We have searched the human genome for such sequences, and developed methods for identifying triplex formation in vitro and in vivo. We have identified in a human erythroid cell line a sequence from within a transcript of one of the beta globin genes that can target a major regulatory element of the locus, with significant regulatory effects. This suggests a novel pathway for regulation of expression of the individual globin genes.
| Planutis, Antanas; Xue, Li; Trainor, Cecelia D et al. (2017) Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development. Development 144:430-440 |
| Hasegawa, Atsushi; Kaneko, Hiroshi; Ishihara, Daishi et al. (2016) GATA1 Binding Kinetics on Conformation-Specific Binding Sites Elicit Differential Transcriptional Regulation. Mol Cell Biol 36:2151-67 |
| Zhu, Jianqiong; Chin, Kyung; Aerbajinai, Wulin et al. (2011) Recombinant erythroid Kruppel-like factor fused to GATA1 up-regulates delta- and gamma-globin expression in erythroid cells. Blood 117:3045-52 |
| Giles, K E; Gowher, H; Ghirlando, R et al. (2010) Chromatin boundaries, insulators, and long-range interactions in the nucleus. Cold Spring Harb Symp Quant Biol 75:79-85 |
| Trainor, Cecelia D; Mas, Caroline; Archambault, Patrick et al. (2009) GATA-1 associates with and inhibits p53. Blood 114:165-73 |
