scholarly journals An improved bind-n-seq strategy to determine protein-DNA interactions validated using the bacterial transcriptional regulator YipR

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Shi-qi An ◽  
Miguel A. Valvano ◽  
Yan-hua Yu ◽  
Jeremy S. Webb ◽  
Guillermo Lopez Campos
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Computational Algorithm ◽  
Dna Recombination ◽  
Transcriptional Regulator ◽  
Sequence Motif ◽  
Dependent Manner ◽  
Dna Interactions ◽  
Promoter Regions ◽  
Protein Dna Interactions

Abstract Background Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein–DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc). Results The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC_2633 in a CCCTCTC motif-dependent manner. Conclusion We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.

scholarly journals HcaR Ligand and DNA Interactions in the Regulation of Catabolic Gene Expression

2014 ◽  
Vol 70 (a1) ◽  
pp. C203-C203
Author(s):  
Andrzej Joachimiak ◽  
Grazyna Joachimiak ◽  
Lance Bigelow ◽  
Garrett Cobb ◽  
Youngchang Kim
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Aromatic Compounds ◽  
Environmental Research ◽  
Structural Basis ◽  
Dna Interactions ◽  
Catabolic Genes ◽  
Protein Dna Interactions ◽  
Acinetobacter Sp

Precise tuning of gene expression by transcriptional regulators determines the response to internal and external chemical signals and adjusts the metabolic machinery for many cellular processes. As a part of ongoing efforts by the Midwest Center for Structural Genomics, a number of transcription factors were selected to study protein-ligand and protein-DNA interactions. HcaR, a new member of the MarR/SlyA family of transcription regulators from soil bacteria Acinetobacter sp. ADP1, is an evolutionarily atypical regulator and represses hydroxycinnamate (hca) catabolic genes. Hydroxycinnamates containing an aromatic ring play diverse, critical roles in plant architecture and defense. HcaR regulates the expression of the hca catabolic operon, allowing this and related bacterial strains to utilize hydroxycinnamates: ferulate, p-coumarate, and caffeate as sole sources of carbon and energy. HcaR appears to be capable of responding to multiple aromatic ligands. These aromatic compounds bind to HcaR and reduce its affinity to the specific DNA sites. As a result, the transcription of genes encoding several catabolic enzymes is up-regulated. The HcaR structures of the apo-form and in a complex with several ligands: ferulic acid, 3,4 dihydroxybenzoic acid, vanillin and p-coumaric acid have been determined to understand how HcaR accommodates various aromatic compounds using the same binding pocket. We also have identified a potential DNA site for HcaR in the regulatory region upstream of the genes of the hca catabolic operon in Acinetobacter sp. ADP1 and have confirmed DNA binding by EMSA. The co-crystal structure of HcaR and palindromic 24-mer DNA has been determined for this DNA site. The crystal structures of HcaR, the apo-form, ligand-bound forms, and the specific DNA-bound form provide critical structural basis of protein-ligand (substrates or product) and protein-DNA interactions to understand the regulation of the expression of hydroxycinnamate (hca) catabolic genes. Our studies allow for better understanding of DNA-binding and regulation by this important group of transcription factors belonging to the MarR/SlyA families. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

scholarly journals Multicomponent differentiation-regulated transcription factors in F9 embryonal carcinoma stem cells.

1991 ◽  
Vol 11 (3) ◽  
pp. 1686-1695 ◽  
Author(s):  
M K Shivji ◽  
N B La Thangue
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Dna Binding ◽  
Embryonal Carcinoma ◽  
Regulatory Sequence ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Dna Sequence Specificity ◽  
Cell Extracts

Murine F9 embryonal carcinoma (F9 EC) stem cells have an E1a-like transcription activity that is down-regulated as these cells differentiate to parietal endoderm. For the adenovirus E2A promoter, this activity requires at least two sequence-specific transcription factors, one that binds the cyclic AMP-responsive element (CRE) and the other, DRTF1, the DNA-binding activity of which is down-regulated as F9 EC cells differentiate. Here we report the characterization of several binding activities in F9 EC cell extracts, referred to as DRTF 1a, 1b and 1c, that recognize the DRTF1 cis-regulatory sequence (-70 to -50 region). These activities can be chromatographically separated but are not distinguishable by DNA sequence specificity. Activity 1a is a detergent-sensitive complex in which DNA binding is regulated by phosphorylation. In contrast, activities 1b and 1c are unaffected by these treatments but exist as multicomponent protein complexes even before DNA binding. Two sets of DNA-binding polypeptides, p50DR and p30DR, affinity purified from F9 EC cell extracts produce complexes 1b and 1c. Both polypeptides appear to be present in the same DNA-bound protein complex and both directly contact DNA. These affinity-purified polypeptides activate transcription in vitro in a binding-site-dependent manner. These data indicate the in F9 EC stem cells, multicomponent differentiation-regulated transcription factors contribute to the cellular E1a-like activity.

scholarly journals High similarity among ChEC-seq datasets

2021 ◽  
Author(s):  
Chitvan Mittal ◽  
Matthew J. Rossi ◽  
B. Franklin Pugh
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Regulators ◽  
Micrococcal Nuclease ◽  
High Similarity ◽  
Dna Interactions ◽  
Promoter Regions ◽  
Genome Wide ◽  
A Genome ◽  
Protein Dna Interactions ◽  
Negative Controls

AbstractChEC-seq is a method used to identify protein-DNA interactions across a genome. It involves fusing micrococcal nuclease (MNase) to a protein of interest. In principle, specific genome-wide interactions of the fusion protein with chromatin result in local DNA cleavages that can be mapped by DNA sequencing. ChEC-seq has been used to draw conclusions about broad gene-specificities of certain protein-DNA interactions. In particular, the transcriptional regulators SAGA, TFIID, and Mediator are reported to generally occupy the promoter/UAS of genes transcribed by RNA polymerase II in yeast. Here we compare published yeast ChEC-seq data performed with a variety of protein fusions across essentially all genes, and find high similarities with negative controls. We conclude that ChEC-seq patterning for SAGA, TFIID, and Mediator differ little from background at most promoter regions, and thus cannot be used to draw conclusions about broad gene specificity of these factors.

scholarly journals Protein–DNA Interactions: The Story so Far and a New Method for Prediction

2003 ◽  
Vol 4 (4) ◽  
pp. 428-431 ◽  
Author(s):  
Susan Jones ◽  
Janet M. Thornton
Keyword(s):  
Dna Binding ◽  
Protein Structures ◽  
Accessible Surface Area ◽  
New Method ◽  
Dna Interactions ◽  
Binding Function ◽  
Protein Dna Interactions ◽  
Template Library ◽  
Accessible Surface ◽  
Complete Protein

This review describes methods for the prediction of DNA binding function, and specifically summarizes a new method using 3D structural templates. The new method features the HTH motif that is found in approximately one-third of DNAbinding protein families. A library of 3D structural templates of HTH motifs was derived from proteins in the PDB. Templates were scanned against complete protein structures and the optimal superposition of a template on a structure calculated. Significance thresholds in terms of a minimum root mean squared deviation (rmsd) of an optimal superposition, and a minimum motif accessible surface area (ASA), have been calculated. In this way, it is possible to scan the template library against proteins of unknown function to make predictions about DNA-binding functionality.

scholarly journals Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters.

1992 ◽  
Vol 12 (7) ◽  
pp. 3273-3287 ◽  
Author(s):  
A J van Wijnen ◽  
F M van den Ent ◽  
J B Lian ◽  
J L Stein ◽  
G S Stein
Keyword(s):  
Cell Cycle ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Consensus Sequence ◽  
Proximal Promoter ◽  
Gene Promoters ◽  
Histone H4 ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Diploid Cells

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.

scholarly journals High Fractional Occupancy of a Tandem MARE and its Role in Long-Range β-globin Gene Regulation

2015 ◽  
pp. MCB.00723-15 ◽  
Author(s):  
Jared R. Stees ◽  
Mir A. Hossain ◽  
Tomoki Sunose ◽  
Yasushi Kudo ◽  
Carolina E. Pardo ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Globin Gene ◽  
Gene Promoter ◽  
Erythroid Cell ◽  
Dna Interactions ◽  
Promoter Regions ◽  
Recognition Element ◽  
Protein Dna Interactions ◽  
Erythroid Cell Differentiation ◽  
Transcription Complexes

Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using MAPit (methyltransferase accessibility protocol for individual templates). The data show that a tandem MARE (Maf recognition element) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused reduction in the association of MARE binding proteins and transcription complexes at LCR HS2 and the adult βmaj-globin gene promoter but did not affect expression of the βmin-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmaj-globin gene promoter during erythroid cell differentiation.

scholarly journals Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters

1992 ◽  
Vol 12 (7) ◽  
pp. 3273-3287
Author(s):  
A J van Wijnen ◽  
F M van den Ent ◽  
J B Lian ◽  
J L Stein ◽  
G S Stein
Keyword(s):  
Cell Cycle ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Consensus Sequence ◽  
Proximal Promoter ◽  
Gene Promoters ◽  
Histone H4 ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Diploid Cells

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.

scholarly journals The geometric influence on the Cys2His2 zinc finger domain and functional plasticity

2020 ◽  
Vol 48 (11) ◽  
pp. 6382-6402
Author(s):  
April L Mueller ◽  
Carles Corbi-Verge ◽  
David O Giganti ◽  
David M Ichikawa ◽  
Jeffrey M Spencer ◽  
...  
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Zinc Fingers ◽  
Binding Specificity ◽  
Synthetic Approach ◽  
Dna Interactions ◽  
Dna Binding Specificity ◽  
Mechanistic Insight ◽  
Natural Geometry ◽  
Protein Dna Interactions

Abstract The Cys2His2 zinc finger is the most common DNA-binding domain expanding in metazoans since the fungi human split. A proposed catalyst for this expansion is an arms race to silence transposable elements yet it remains poorly understood how this domain is able to evolve the required specificities. Likewise, models of its DNA binding specificity remain error prone due to a lack of understanding of how adjacent fingers influence each other's binding specificity. Here, we use a synthetic approach to exhaustively investigate binding geometry, one of the dominant influences on adjacent finger function. By screening over 28 billion protein–DNA interactions in various geometric contexts we find the plasticity of the most common natural geometry enables more functional amino acid combinations across all targets. Further, residues that define this geometry are enriched in genomes where zinc fingers are prevalent and specificity transitions would be limited in alternative geometries. Finally, these results demonstrate an exhaustive synthetic screen can produce an accurate model of domain function while providing mechanistic insight that may have assisted in the domains expansion.

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