The interaction of the erythroid transcription factor, GATA-1 with DNA is a major focus of our research. Vertebrate GATA factors have two zinc fingers that comprise the DNA binding domain. The C-terminal finger is the main DNA binding finger, and the three dimensional structure of this finger of GATA-1 bound to DNA has been solved by NMR. The structure reveals a finger and helix that bind to the major groove of DNA and an adjacent basic arm that binds in the minor groove. However, the GATA-1 N-terminal finger also plays an important role in DNA binding, especially at genomic locations that contain multiple closely spaced GATA sites. The N-finger is also essential for interaction with the critical cofactor FOG. Mutations in the N-finger of GATA-1 that interfere with its ability to bind to DNA, but do not affect its interaction with FOG are associated with thrombocytopenia and thalassemia in humans and anemia in transgenic mice. More severe forms of similar disorders are associated with GATA-1 mutations that disrupt FOG binding without altering DNA binding. Wild-type GATA-1 can rescue GATA-1 knockdown mice from lethal anemia, but a GATA-1 molecule lacking the N-terminal zinc finger cannot. Some GATA-1 point mutations that disrupt FOG binding have less severe erythroid phenotypes than the mutant lacking the N-finger, again suggesting that the N-finger has a FOG independent role in erythropoiesis in addition to its FOG dependent role. We are interested in understanding the mechanism of the FOG independent contribution of the N-finger. We are currently investigating three new complex GATA sites that occur in the GATA-2 promoter and the P13 kinase promoter. Two sites in the GATA-2 promoter appear to be involved in the activation and shut off of the gene. We have shown that GATA-1 can adopt different conformations that are DNA binding site specific, and these variations can be detected by altered migration in electrophoretic mobility shift assays or by differential resistance to proteases. These variations are not due to DNA bending since we have established that GATA-1 bends DNA slightly in a binding site independent manner. We have also demonstrated that GATA-1 is unable to stimulate transcription when bound to some DNA sites. In addition to FOG, there are several other cofactors that interact with the zinc fingers of GATA-1, and their ability to bind to GATA-1 may be influenced by the conformation that GATA-1 adopts in response to DNA. Consequently, we are attempting to solve the structure of the linked GATA-1 zinc fingers on a number of double DNA binding sites by X-ray crystallography. Meanwhile we have taken a biochemical approach to study the N-terminal finger interactions with DNA. We have shown that the binding specificity of the N- and C-terminal zinc fingers is distinct. Binding site selection experiments using the GATA-1 C-finger or the GATA-1 N-finger fused to the basic arm of the C-finger show that both of these peptides prefer GATA containing binding sites. However, the N-finger of GATA-2 prefers sites containing GATC. Recently, it has been shown that the N-finger of GATA-1 also binds to DNA independently, but with such low affinity that a selection experiment could not be performed. However, conventional binding studies show that this finger also prefers the GATC site, unlike the fusion protein containing the N-finger and C-arm mentioned above. These observations lead to the conclusion that the C-terminal basic arm can change the specificity of the GATA-1 N-finger. Fusing this same basic arm to the GATA-2 N-finger also changes its specificity from GATC to GATA, suggesting that the GATA-1 basic arm controls the specificity at the last base of the four base core binding site. A cluster of amino acids, QTNRK, within this arm of the C-terminal finger, is largely responsible for this preference. These amino acids can convert the specificity of the N-finger of GATA-1 from GATC to GATA when they replace five analogously positioned N-finger amino acids. Because some biologically important GATA binding sites contain a combination of a canonical and non-canonical sequence, the mode of DNA recognition at these sites is significant and the N-finger of the GATA proteins may be particularly important here. DNA recognition by the N-finger of GATA-3 is also important for the regulation of some genes. Three GATA recognition sequences in the IL13 gene promoter form a high affinity-binding site for two molecules of GATA-3 and two of the sites have GATG as their core sequence. All three sites are necessary for full activity of this promoter at limiting GATA-3 concentrations and the N-finger is involved in binding to these sites. The IL 5 gene also contains some palindromic GATA binding sites that may be important for gene expression and require the N-finger of GATA-3. The new site in the P13 kinase promoter is a target of GATA-2 and/or GATA-3. We continue to examine the regulation of the chicken folate receptor gene that is adjacent to a 16 KB region of condensed chromatin. The gene is GATA regulated, but is expressed at a different stage in development than the beta-globin genes that reside on the other side of the 16 KB region and are also GATA regulated. We have shown that in addition to GATA factors, the folate receptor gene is regulated by AML-1, as the AML-1-ETO fusion protein inhibits its transcription. We do not yet know whether AML-1 contributes to the formation of the boundary between the gene and the condensed chromatin.
