Determinants of GATA-1 Binding to DNA

Abstract

Mammalian GATA transcription factors are expressed in various tissues in a temporally regulated manner. The prototypic member, GATA-1, is required for normal erythroid, megakaryocytic, and mast cell development. This family of DNA-binding proteins recognizes a consensus (A/T)GATA(A/G) motif and possesses homologous DNA binding domains consisting of two zinc fingers. The C-terminal finger of GATA-1 recognizes the consensus motif with nanomolar affinities, whereas the N-terminal finger shows a binding preference for a GATC motif, albeit with much reduced affinity (K d Ϸ M). The Nterminal finger of GATA-2 also shows a preference for an AGATCT binding site, with an increased affinity attributed to N-and C-terminal flanking basic residues (K d Ϸ nM). To understand the differences in the binding specificities of the N-and C-terminal zinc fingers of GATA-1, we have constructed a series of swapped domain peptides. We show that the specificity for AGATAA over AGATCT arises from the C-terminal non-finger basic domain. Thus, the N-terminal finger binds preferentially to AGATAA once appended to the C-terminal arm of the C-terminal finger. We further show that this specificity arises from the highly conserved QTRNRK residues. The converse is, however, untrue in the case of the C-terminal finger; swapping of QTRNRK with the corresponding LVSKRA does not switch the DNA binding specificity from AGATAA to AGATCT. These results highlight the important role of residues adjacent to the CXXCX 17 CNAC zinc finger motif (i.e. non-finger residues) in the specific recognition of DNA residues.DNA binding zinc fingers of the form CXXCX 17 CNAC characterize the GATA family of transcription factors. GATA-1, the original member of the family possesses two such zinc fingers (1, 2). The C-terminal finger and flanking basic arm are both necessary and sufficient for binding to the GATA recognition sequence, (A/T)GATA(A/G) (3, 4). Even though the single Nterminal finger does not bind DNA independently with high affinity, it does stabilize GATA-1 interactions with some sites crucial for gene expression (3,(5)(6)(7)(8). Recent reports, however, indicate that the N-terminal finger binds DNA with an affinity much lower than that observed for the C-terminal finger with binding occurring preferentially to GATC motifs (9). Despite the similarities between the C-and N-terminal fingers (Fig. 1, peptides CC11 and NN4, respectively), the N-terminal finger does not recognize the AGATAA consensus motif, indicating that the flanking or poorly conserved linker residues may play a key role in DNA recognition. A role for the flanking residues has been pointed out in the case of the N-terminal finger of GATA-2 (10). Like the N-terminal finger of GATA-1, this homologous finger shows a binding preference for GATC motifs. However, because of the additional basic region at the N terminus a higher binding affinity has been reported for this finger.Solution NMR studies of the complex formed between the C-terminal finger of cGATA-1 and the AGATAA DNA sequence highlight t...

Structural Analysis of MED-1 Reveals Unexpected Diversity in the Mechanism of DNA Recognition by GATA-type Zinc Finger Domains

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Mathematical modeling of GATA-switching for regulating the differentiation of hematopoietic stem cell

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Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPε Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein

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