scholarly journals DNA-binding properties of the MADS-domain transcription factor SEPALLATA3 and mutant variants characterized by SELEX-seq

2021 ◽  
Vol 105 (4-5) ◽  
pp. 543-557
Author(s):  
Sandra Käppel ◽  
Ralf Eggeling ◽  
Florian Rümpler ◽  
Marco Groth ◽  
Rainer Melzer ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
High Throughput Sequencing ◽  
Binding Specificity ◽  
Binding Mode ◽  
Arginine Residue ◽  
Single Amino Acid ◽  
Binding Profile ◽  
Carg Box

Abstract Key message We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. Abstract MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved arginine residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved arginine residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

scholarly journals The floral homeotic protein SEPALLATA3 recognizes target DNA sequences by shape readout involving a conserved arginine residue in the MADS-domain

2017 ◽  
Author(s):  
Sandra Gusewski ◽  
Rainer Melzer ◽  
Florian Rüempler ◽  
Christian Gafert ◽  
Güenter Theiβen
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Specificity ◽  
Regulatory Elements ◽  
Arginine Residue ◽  
Single Amino Acid ◽  
Dna Minor Groove ◽  
Carg Box ◽  
Dna Shape

ABSTRACTSEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor and a central player in flower development. MADS-domain proteins bind as dimers to AT-rich sequences termed ‘CArG-boxes’ which share the consensus 5’-CC(A/T)6GG-3’. Since only a fraction of the abundant CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-box sequences into genuine recognition sites. Here, we investigated to which extent the shape of the DNA contributes to the DNA-binding specificity of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to a variety of DNA probes which all contain the CArG-box motif, but differ in their DNA shape characteristics. We found that binding affinity correlates well with certain DNA shape features associated with ‘A-tracts’. Analysis of SEPALLATA3 proteins with single amino acid substitutions in the DNA-binding MADS-domain further revealed that a highly conserved arginine residue, which is expected to contact the DNA minor groove, contributes significantly to the shape readout. Our studies show that the specific recognition of cis-regulatory elements by plant MADS-domain transcription factors heavily depend on shape readout mechanisms and that the absence of a critical arginine residue in the MADS-domain impairs binding specificity.

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

1997 ◽  
Vol 17 (5) ◽  
pp. 2876-2887 ◽  
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.

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

2013 ◽  
Vol 42 (4) ◽  
pp. 2138-2146 ◽  
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.

scholarly journals In vitro DNA-binding profile of transcription factors: methods and new insights

2011 ◽  
Vol 210 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Jinke Wang ◽  
Jie Lu ◽  
Guangming Gu ◽  
Yingxun Liu
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Target Genes ◽  
Binding Specificity ◽  
Cell Physiology ◽  
Binding Profile ◽  
Dna Binding Specificity

The DNA-binding specificity of transcription factors (TFs) has broad impacts on cell physiology, cell development and in evolution. However, the DNA-binding specificity of most known TFs still remains unknown. The specificity of a TF protein is determined by its relative affinity to all possible binding sites. In recent years, the development of several in vitro techniques permits high-throughput determination of relative binding affinity of a TF to all possible k bp-long DNA sequences, thus greatly promoting the characterization of DNA-binding specificity of many known TFs. All DNA sequences that can be bound by a TF with various binding affinities form their DNA-binding profile (DBP). The DBP is important to generate an accurate DNA-binding model, identify all DNA-binding sites and target genes of TFs in the whole genome, and build transcription regulatory network. This study reviewed these techniques, especially two master techniques: double-stranded DNA microarray and systematic evolution of ligands by exponential enrichment in combination with parallel DNA sequencing techniques (SELEX-seq).

scholarly journals An interpretable bimodal neural network characterizes the sequence and preexisting chromatin predictors of induced transcription factor binding

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Divyanshi Srivastava ◽  
Begüm Aydin ◽  
Esteban O. Mazzoni ◽  
Shaun Mahony
Keyword(s):  
Neural Network ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Specificity ◽  
Transcription Factor Binding ◽  
Cell Type ◽  
Factor Binding ◽  
Genome Wide

Abstract Background Transcription factor (TF) binding specificity is determined via a complex interplay between the transcription factor’s DNA binding preference and cell type-specific chromatin environments. The chromatin features that correlate with transcription factor binding in a given cell type have been well characterized. For instance, the binding sites for a majority of transcription factors display concurrent chromatin accessibility. However, concurrent chromatin features reflect the binding activities of the transcription factor itself and thus provide limited insight into how genome-wide TF-DNA binding patterns became established in the first place. To understand the determinants of transcription factor binding specificity, we therefore need to examine how newly activated transcription factors interact with sequence and preexisting chromatin landscapes. Results Here, we investigate the sequence and preexisting chromatin predictors of TF-DNA binding by examining the genome-wide occupancy of transcription factors that have been induced in well-characterized chromatin environments. We develop Bichrom, a bimodal neural network that jointly models sequence and preexisting chromatin data to interpret the genome-wide binding patterns of induced transcription factors. We find that the preexisting chromatin landscape is a differential global predictor of TF-DNA binding; incorporating preexisting chromatin features improves our ability to explain the binding specificity of some transcription factors substantially, but not others. Furthermore, by analyzing site-level predictors, we show that transcription factor binding in previously inaccessible chromatin tends to correspond to the presence of more favorable cognate DNA sequences. Conclusions Bichrom thus provides a framework for modeling, interpreting, and visualizing the joint sequence and chromatin landscapes that determine TF-DNA binding dynamics.

scholarly journals Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure.

1994 ◽  
Vol 14 (11) ◽  
pp. 7557-7568 ◽  
Author(s):  
J Zuo ◽  
R Baler ◽  
G Dahl ◽  
R Voellmy
Keyword(s):  
Transcription Factor ◽  
Heat Stress ◽  
Heat Shock ◽  
Dna Binding ◽  
Hydrophobic Interactions ◽  
Coiled Coil ◽  
Heat Shock Transcription Factor ◽  
Single Amino Acid ◽  
Binding Ability ◽  
Heat Shock Element

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.

scholarly journals DNA-binding specificity of GATA family transcription factors.

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

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.

scholarly journals Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

2015 ◽  
Vol 113 (2) ◽  
pp. 326-331 ◽  
Author(s):  
William H. Hudson ◽  
Bradley R. Kossmann ◽  
Ian Mitchelle S. de Vera ◽  
Shih-Wei Chuo ◽  
Emily R. Weikum ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Amino Acid ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Binding Specificity ◽  
Conformational Space ◽  
Amino Acid Substitutions ◽  
Ancestral Gene ◽  
Response Elements ◽  
Binding Interface

Many genomes contain families of paralogs—proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD’s ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors’ response element-binding specificity were far from the proteins’ DNA-binding interface and interacted epistatically to change the DBD’s function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

1993 ◽  
Vol 13 (7) ◽  
pp. 3850-3859
Author(s):  
T A Coleman ◽  
C Kunsch ◽  
M Maher ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Fusion Protein ◽  
Binding Specificity ◽  
Single Amino Acid ◽  
Nf Kappa B ◽  
Dna Binding Specificity ◽  
Dna Motif ◽  
Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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