A micro-evolutionary change in target binding sites as key determinant of Ultrabithorax function in Drosophila.

All Hox proteins are known to recognize, in vitro, similar DNA-binding sites containing a TAAT core sequence. This poor DNA-binding specificity is in sharp contrast with their specific functions in vivo. Here we report a new binding motif with TAAAT core sequence to which the Hox protein Ultrabithorax (Ubx) binds with higher affinity and specificity. Using transgenic and luciferase assays, we show that this new motif is critical for Ubx-mediated regulation of a target gene in Drosophila melanogaster. Interestingly, this new motif with TAAAT core sequences is not associated with the targets of Ubx in the honeybee, Apis mellifera, wherein hindwings are nearly identical to the forewings. We show that introduction of TAAAT motif in the place of TAAT motif is sufficient to bring an enhancer of a wing-promoting gene of A. mellifera under the regulation of Ubx. Our results, thus, suggest that binding motifs with a TAAAT core sequence may help identify functionally relevant direct targets of Ubx in D. melanogaster and the emergence of these binding sites may be crucial for Hox-mediated morphological changes during insect evolution.

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Accurate and sensitive quantification of protein-DNA binding affinity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714376115 ◽

2018 ◽

Vol 115 (16) ◽

pp. E3692-E3701 ◽

Cited By ~ 32

Author(s):

Chaitanya Rastogi ◽

H. Tomas Rube ◽

Judith F. Kribelbauer ◽

Justin Crocker ◽

Ryan E. Loker ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Sites ◽

Biophysical Model ◽

Regulatory Sequences ◽

Binding Modes ◽

Perfect Agreement ◽

Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

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An A257V Mutation in the Bacillus subtilis Response Regulator Spo0A Prevents Regulated Expression of Promoters with Low-Consensus Binding Sites

Journal of Bacteriology ◽

10.1128/jb.00590-09 ◽

2009 ◽

Vol 191 (17) ◽

pp. 5489-5498 ◽

Cited By ~ 3

Author(s):

Steve D. Seredick ◽

Barbara M. Seredick ◽

David Baker ◽

George B. Spiegelman

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Transcription Initiation ◽

Molecular Basis ◽

Response Regulator ◽

Mutant Protein ◽

Wild Type ◽

Dna Binding Sites

ABSTRACT In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

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Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.861 ◽

1995 ◽

Vol 15 (2) ◽

pp. 861-871 ◽

Cited By ~ 110

Author(s):

K M Catron ◽

H Zhang ◽

S C Marshall ◽

J A Inostroza ◽

J M Wilson ◽

...

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Transcriptional Repression ◽

Binding Domain ◽

Specific Cell ◽

Homeodomain Protein ◽

Dna Binding Domain ◽

Dna Binding Sites

This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.

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Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

Journal of Developmental Biology ◽

10.3390/jdb9010006 ◽

2021 ◽

Vol 9 (1) ◽

pp. 6

Author(s):

Narendra Pratap Singh ◽

Bony De Kumar ◽

Ariel Paulson ◽

Mark E. Parrish ◽

Carrie Scott ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Embryonic Stem ◽

Binding Motif ◽

Binding Assays ◽

Histone Marks ◽

Genome Wide ◽

Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

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The HOX Homeodomain Proteins Block CBP Histone Acetyltransferase Activity

Molecular and Cellular Biology ◽

10.1128/mcb.21.21.7509-7522.2001 ◽

2001 ◽

Vol 21 (21) ◽

pp. 7509-7522 ◽

Cited By ~ 73

Author(s):

Wei-fang Shen ◽

Keerthi Krishnan ◽

H. J. Lawrence ◽

Corey Largman

Keyword(s):

Dna Binding ◽

Protein Function ◽

Histone Acetyltransferase ◽

Gene Activation ◽

Homeodomain Proteins ◽

Hox Proteins ◽

Reporter Assays ◽

Hox Protein

ABSTRACT Despite the identification of PBC proteins as cofactors that provide DNA affinity and binding specificity for the HOX homeodomain proteins, HOX proteins do not demonstrate robust activity in transient-transcription assays and few authentic downstream targets have been identified for these putative transcription factors. During a search for additional cofactors, we established that each of the 14 HOX proteins tested, from 11 separate paralog groups, binds to CBP or p300. All six isolated homeodomain fragments tested bind to CBP, suggesting that the homeodomain is a common site of interaction. Surprisingly, CBP-p300 does not form DNA binding complexes with the HOX proteins but instead prevents their binding to DNA. The HOX proteins are not substrates for CBP histone acetyltransferase (HAT) but instead inhibit the activity of CBP in both in vitro and in vivo systems. These mutually inhibitory interactions are reflected by the inability of CBP to potentiate the low levels of gene activation induced by HOX proteins in a range of reporter assays. We propose two models for HOX protein function: (i) HOX proteins may function without CBP HAT to regulate transcription as cooperative DNA binding molecules with PBX, MEIS, or other cofactors, and (ii) the HOX proteins may inhibit CBP HAT activity and thus function as repressors of gene transcription.

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The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes

Development ◽

10.1242/dev.110.1.141 ◽

1990 ◽

Vol 110 (1) ◽

pp. 141-149 ◽

Cited By ~ 1

Author(s):

F. Payre ◽

S. Noselli ◽

V. Lefrere ◽

A. Vincent

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Polytene Chromosomes ◽

Duplication Event ◽

Amino Acid Sequences ◽

Evolutionary Divergence ◽

Coding Region ◽

Protein Coding

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12–13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

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Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1405 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1405-1421 ◽

Cited By ~ 197

Author(s):

C C Adams ◽

J L Workman

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Sites ◽

Specific Binding ◽

General Mechanism ◽

Nf Kappa B ◽

Nucleosomal Dna ◽

Nucleosome Binding

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

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An effective approach for identification of in vivo protein-DNA binding sites from paired-end ChIP-Seq data

BMC Bioinformatics ◽

10.1186/1471-2105-11-81 ◽

2010 ◽

Vol 11 (1) ◽

pp. 81 ◽

Cited By ~ 16

Author(s):

Congmao Wang ◽

Jie Xu ◽

Dasheng Zhang ◽

Zoe A Wilson ◽

Dabing Zhang

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Dna Binding Sites

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In Vitro Analysis of Predicted DNA-Binding Sites for the Stl Repressor of the Staphylococcus aureus SaPIBov1 Pathogenicity Island

PLoS ONE ◽

10.1371/journal.pone.0158793 ◽

2016 ◽

Vol 11 (7) ◽

pp. e0158793 ◽

Cited By ~ 7

Author(s):

Veronika Papp-Kádár ◽

Judit Eszter Szabó ◽

Kinga Nyíri ◽

Beata G. Vertessy

Keyword(s):

Staphylococcus Aureus ◽

Dna Binding ◽

Binding Sites ◽

Pathogenicity Island ◽

Dna Binding Sites ◽

Vitro Analysis ◽

In Vitro Analysis

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The homeodomain region controls the phenotype of HOX-induced murine leukemia

Blood ◽

10.1182/blood-2011-10-384685 ◽

2012 ◽

Vol 120 (19) ◽

pp. 4018-4027 ◽

Cited By ~ 13

Author(s):

Constanze Breitinger ◽

Emanuel Maethner ◽

Maria-Paz Garcia-Cuellar ◽

Robert K. Slany

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Expression Patterns ◽

Hox Proteins ◽

Hematopoietic Development ◽

N Terminus ◽

Fast Acting ◽

Murine Leukemia

Abstract HOX proteins are widely involved in hematopoietic development. These transcription factors combine a conserved DNA-binding homeobox with a divergent N-terminus that mediates interaction with variable cofactors. The resulting combinatorial diversity is thought to be responsible for mammalian HOX specificity. Contrasting this proposed mechanism for normal HOX function, here we demonstrate that, in the context of hematopoietic immortalization and leukemogenesis, individual HOX properties are governed almost exclusively by the homeodomain. Swap experiments between HOXA1 and HOXA9, 2 members of nonrelated paralog groups, revealed that gene expression patterns of HOX transformed cells in vitro are determined by the nature of the homeodomain. Similar results were seen in vivo during HOX-mediated leukemogenesis. An exchange of the homeodomains was sufficient to convert the slow, low-penetrance phenotype of HOXA1-induced leukemia to the aggressive fast-acting disease elicited by HOXA9 and vice versa. Mutation and deletion studies identified several subregions within the DNA binding domain responsible for paralog specificity. Previously defined binding sites for PBX cofactors within the exchangeable, nonhomeobox segment were dispensable for in vitro oncogenic HOX activity but affected in vivo disease development. The transcriptional activator domain shared by HOXA1 and HOXA9 at the very N-terminus proved essential for all transformation.

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