EKLF is a transcription factor expressed somewhat later in erythroid development than GATA-1, and 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 (CDA) type IV and in the Nan (Neonatal anemia) mouse. Using purified EKLF zinc finger GST fusion proteins, in collaboration with Jim Bieker, we have confirmed that the anemic phenotype in the Nan mouse model is partially based on different DNA binding affinities of Nan-EKLF relative to wild type (WT), as suggested by earlier studies with nuclear extracts. In those studies it was shown that sites with a T base in the central triplet of the EKLF DNA binding motif were bound by WT EKLF, but not by Nan EKLF. Both WT and Nan EKLF bind sites with a C at this position. We have shown that the Kds for Nan binding to five of these T containing EKLF binding sites are higher than those for wild type EKLF. Thus one cause of the anemia in the Nan mouse is reduced binding affinity of Nan-EKLF for a subset of EKLF binding sites. Recently a new binding site for Nan-EKLF was discovered among genes that are up-regulated in Nan relative to WT mice. Many of these genes are usually expressed in macrophages, not erythroid cells. For two genes we have shown that the up-regulation is due to high affinity binding of Nan-EKLF to sites with this newly discovered motif to which WT EKLF does not bind. The protein products of these two target genes are secreted, and thus have cell- extrinsic effects that include inhibition of erythroid development. This finding partially explains the observation that while EKLF +/- heterozygous mice are normal, the Nan anemia occurs in heterozygotes in the presence of a normal copy of the EKLF gene. In collaboration with the laboratory of Jim Omichinski we have shown that p53 and the GATA-1 DNA binding domain interact through the transactivation domain of p53 and the linker and C-terminal zinc-finger of GATA-1. In collaboration with Masi Yamamoto, it was demonstrated that GATA1 monovalently binds to a single GATA motif (Single-GATA) while a monomeric GATA1 and a homodimeric GATA1 bivalently bind to two GATA motifs in palindromic (Pal-GATA) and direct-repeat (Tandem-GATA) arrangements, respectively, and form higher stoichiometric complexes on respective elements. A separate investigation has addressed the possible role of DNA:RNA triple helix formation in regulation of gene expression, particularly in the human beta globin gene locus. There are strict rules for the combinations of bases that can form such structures. We searched for loci that obeyed those rules. Most significantly we have found that an intronic sequence within the adult human beta globin primary transcript can form a triple helix with a target site in the beta globin locus control region (LCR), the major positive compound regulatory element of the beta globin genes. The effect of this interaction is to displace transcription factors and down regulate expression of these genes, providing negative feedback. We have found another example of this regulation elsewhere in the genome, suggesting that this may a general regulatory mechanism.
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| 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 |
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| Trainor, Cecelia D; Mas, Caroline; Archambault, Patrick et al. (2009) GATA-1 associates with and inhibits p53. Blood 114:165-73 |
