The interaction of the erythroid transcription factor, GATA-1 with DNA is a major focus of our research. The three dimensional structure of the C-terminal zinc finger of GATA-1 bound to DNA has been solved by NMR. However, GATA-1 has two zinc fingers and we have shown that the N-terminal finger also plays an important role in DNA binding. The two zinc fingers interact with each other in ways that can lead either to enhancement or to inhibition of DNA binding, depending on the sequence of the binding site. We have shown that GATA-1 adopts different conformations that are binding site specific; these variations can be detected by altered migration in electrophoretic mobility shift assays or by differential resistance to proteases. We have also shown that GATA-1 is unable to stimulate transcription when bound to some DNA sites, suggesting allosteric regulation. Several crucial cofactors that interact with the zinc fingers of GATA-1 have been identified, and their ability to bind to GATA-1 may be controlled by the conformation that GATA-1 adopts in response to DNA. Consequently, we are attempting to solve the structure of the two GATA-1 zinc fingers on a number of DNA binding sites by Xray crystallography. Two closely spaced GATA sites are conserved in the GATA-1 promoters of many species and are essential for correct expression of GATA-1 in mice. We have shown that this particular arrangement of binding sites is extremely efficient at supporting transactivation by GATA-1. GATA-2, which is expressed before GATA-1 during erythroid development, shows a distinct mode of interaction with this site, which is dependent on the unique binding properties of N-terminal finger of GATA-2. We are characterizing the interactions of these two proteins with this binding site, in an attempt to understand the basis for the efficiency of this site and the differential interaction of these two GATA factors with it. The chicken folate receptor gene (fr) lies upstream of the ?-globin locus on the opposite side of a chromatin boundary, the chicken HS4 insulator element. Both fr and the ?-globin genes are regulated by GATA factors, although they are expressed at different times during development. A GATA site within the fr promoter is critical to the copy number dependent expression of fr in an erythroid precursor cell line and the basis of this dependence is being investigated. It could be a part of a developmental stage- specific chromatin opening activity.
| Ghirlando, R; Trainor, C D (2000) GATA-1 bends DNA in a site-independent fashion. J Biol Chem 275:28152-6 |
| Trainor, C D; Ghirlando, R; Simpson, M A (2000) GATA zinc finger interactions modulate DNA binding and transactivation. J Biol Chem 275:28157-66 |
