DNA-binding specificity of GATA family transcription factors.

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

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On exploring effects of coevolving residues on DNA binding specificity of transcription factors

10.1101/2021.05.20.445059 ◽

2021 ◽

Author(s):

Yizhao Luan ◽

Zhi Xie

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Specificity ◽

Binding Activity ◽

Dna Interactions ◽

Dna Binding Domains ◽

Binding Domains ◽

Dna Binding Activity ◽

Dna Binding Specificity ◽

The Stability

Transcription factors (TFs) regulate gene expression by specifically binding to DNA targets. Many factors have been revealed to influence TF-DNA binding specificity. Coevolution of residues in proteins occurs due to a common evolutionary history. However, it is unclear how coevolving residues in TFs contribute to DNA binding specificity. Here, we systematically analyzed TF-DNA interactions from high-throughput experiments for seven TF families, including Homeobox, HLH, bZIP_1, Ets, HMG_box, zf-C4 and Zn_clus TFs. Based on TF-DNA interactions, we detected TF subclass determining sites (TSDSs) defining the heterogeneity of DNA binding preference for each TF family. We showed that the TSDSs were more likely to be coevolving with TSDSs than with non-TSDSs, particularly for Homeobox, HLH, Ets, bZIP_1 and HMG_box TF families. Mutation of the highly coevolving residues could significantly reduce the stability of TF-DNA complex. The distant residues from the DNA interface also contributed to TF-DNA binding activity. Overall, our study gave evidence of the functional importance of coevolved residues in refining transcriptional regulation and provided clues to the application of engineered DNA-binding domains and protein.

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DNA-binding specificity of the cut repeats from the human cut-like protein.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.129 ◽

1995 ◽

Vol 15 (1) ◽

pp. 129-140 ◽

Cited By ~ 76

Author(s):

R Harada ◽

G Bérubé ◽

O J Tamplin ◽

C Denis-Larose ◽

A Nepveu

Keyword(s):

Dna Binding ◽

Electrophoretic Mobility Shift Assay ◽

Fusion Proteins ◽

Glutathione S Transferase ◽

Mobility Shift ◽

Dna Binding Domains ◽

Binding Domains ◽

Dna Binding Specificity ◽

Mobility Shift Assay ◽

A Site

The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

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DNA-binding specificity and molecular functions of NAC transcription factors

Plant Science ◽

10.1016/j.plantsci.2005.05.035 ◽

2005 ◽

Vol 169 (4) ◽

pp. 785-797 ◽

Cited By ~ 104

Author(s):

Addie N. Olsen ◽

Heidi A. Ernst ◽

Leila Lo Leggio ◽

Karen Skriver

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Specificity ◽

Nac Transcription Factors ◽

Dna Binding Specificity ◽

Molecular Functions

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DNA binding by MADS-box transcription factors: a molecular mechanism for differential DNA bending.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2876 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2876-2887 ◽

Cited By ~ 59

Author(s):

A G West ◽

P Shore ◽

A D Sharrocks

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Serum Response Factor ◽

Dna Bending ◽

Binding Specificity ◽

Single Amino Acid ◽

Specific Gene ◽

Mads Box ◽

Dna Binding Specificity ◽

Single Amino Acid Residue

The serum response factor (SRF) and myocyte enhancer factor 2A (MEF2A) represent two human members of the MADS-box transcription factor family. Each protein has a distinct biological function which is reflected by the distinct specificities of the proteins for coregulatory protein partners and DNA-binding sites. In this study, we have investigated the mechanism of DNA binding utilized by these two related transcription factors. Although SRF and MEF2A belong to the same family and contain related DNA-binding domains, their DNA-binding mechanisms differ in several key aspects. In contrast to the dramatic DNA bending induced by SRF, MEF2A induces minimal DNA distortion. A combination of loss- and gain-of-function mutagenesis identified a single amino acid residue located at the N terminus of the recognition helices as the critical mediator of this differential DNA bending. This residue is also involved in determining DNA-binding specificity, thus indicating a link between DNA bending and DNA-binding specificity determination. Furthermore, different basic residues within the putative recognition alpha-helices are critical for DNA binding, and the role of the C-terminal extensions to the MADS box in dimerization between SRF and MEF2A also differs. These important differences in the molecular interactions of SRF and MEF2A are likely to contribute to their differing roles in the regulation of specific gene transcription.

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Correlated Evolutionary Pressure at Interacting Transcription Factors and DNA Response Elements Can Guide the Rational Engineering of DNA Binding Specificity

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.04.054 ◽

2005 ◽

Vol 350 (3) ◽

pp. 402-415 ◽

Cited By ~ 21

Author(s):

Michele Raviscioni ◽

Peili Gu ◽

Minawar Sattar ◽

Austin J. Cooney ◽

Olivier Lichtarge

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Specificity ◽

Response Elements ◽

Rational Engineering ◽

Dna Binding Specificity

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Structural determinants of DNA recognition by plant MADS-domain transcription factors

Nucleic Acids Research ◽

10.1093/nar/gkt1172 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2138-2146 ◽

Cited By ~ 21

Author(s):

Jose M. Muiño ◽

Cezary Smaczniak ◽

Gerco C. Angenent ◽

Kerstin Kaufmann ◽

Aalt D.J. van Dijk

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Site ◽

Structural Characteristics ◽

Binding Specificity ◽

Structural Motif ◽

Dna Binding Specificity ◽

Dna Binding Site ◽

Carg Box

Abstract Plant MADS-domain transcription factors act as key regulators of many developmental processes. Despite the wealth of information that exists about these factors, the mechanisms by which they recognize their cognate DNA-binding site, called CArG-box (consensus CCW6GG), and how different MADS-domain proteins achieve DNA-binding specificity, are still largely unknown. We used information from in vivo ChIP-seq experiments, in vitro DNA-binding data and evolutionary conservation to address these important questions. We found that structural characteristics of the DNA play an important role in the DNA binding of plant MADS-domain proteins. The central region of the CArG-box largely resembles a structural motif called ‘A-tract’, which is characterized by a narrow minor groove and may assist bending of the DNA by MADS-domain proteins. Periodically spaced A-tracts outside the CArG-box suggest additional roles for this structure in the process of DNA binding of these transcription factors. Structural characteristics of the CArG-box not only play an important role in DNA-binding site recognition of MADS-domain proteins, but also partly explain differences in DNA-binding specificity of different members of this transcription factor family and their heteromeric complexes.

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