Target Genes and DNA-Binding Sites of the Response Regulator PhoR from Corynebacterium glutamicum

ABSTRACT The two-component signal transduction system PhoRS of Corynebacterium glutamicum is involved in the phosphate (Pi) starvation response. To analyze the binding of unphosphorylated and phosphorylated PhoR to the promoters of phosphate starvation-inducible (psi) genes, this response regulator and the kinase domain of its cognate sensor, PhoS (MBP-PhoSΔ1-246), were overproduced and purified. MBP-PhoSΔ1-246 showed constitutive autophosphorylation activity, and a rapid phosphoryl group transfer from phosphorylated MBP-PhoSΔ1-246 to PhoR was observed. Gel mobility shift assays revealed that phosphorylation increases the DNA-binding affinity of PhoR. The affinity of PhoR∼P to different promoters varied and decreased in the order pstSCAB > phoRS > phoC > ushA > porB > ugpA > pitA > nucH and phoH1 > glpQ1. The binding sites in front of pstSCAB and phoRS were localized at positions −194 to −176 and −61 to −43 upstream of the transcriptional start sites, respectively. Alignment of these two 19-bp binding sites revealed a high identity in the 5′-terminal part, but not in the 3′-terminal part. As many OmpR-type response regulators bind to direct repeats, the 19-bp sequence might be interpreted as a loosely conserved 8-bp direct repeat separated by 3 bp. This idea was supported by the fact that the highest binding affinity was observed with a perfect 8-bp direct repeat of the sequence CCTGTGAAaatCCTGTGAA. Inspection of the other target promoters revealed sequences with some similarity to this binding motif, which might represent PhoR binding sites. The in vivo relevance of the PhoR-binding site within the phoRS promoter was supported by reporter gene studies.

Download Full-text

EPEN-41. C11orf95-RELA FUSION REGULATES ABERRANT GENE EXPRESSION THROUGH THE UNIQUE GENOMIC BINDING SITES FOR EPENDYMOMA FORMATION

Neuro-Oncology ◽

10.1093/neuonc/noaa222.175 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii316-iii316

Author(s):

Tatsuya Ozawa ◽

Syuzo Kaneko ◽

Mutsumi Takadera ◽

Eric Holland ◽

Ryuji Hamamoto ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Sites ◽

Motif Discovery ◽

Target Genes ◽

Cultured Cells ◽

De Novo ◽

Binding Motif ◽

Aberrant Gene Expression ◽

Aberrant Gene

Abstract A majority of supratentorial ependymoma is associated with recurrent C11orf95-RELA fusion (RELAFUS). The presence of RELA as one component of the RELAFUS leads to the suggestion that NF-kB activity is involved in the ependymoma formation, thus being a viable therapeutic target in these tumors. However, the oncogenic role of another C11orf95 component in the tumorigenesis is not still determined. In this study, to clarify the molecular mechanism underlying tumorigenesis of RELAFUS, we performed RELAFUS-ChIP-Seq analysis in cultured cells expressing the RELAFUS protein. Genomic profiling of RELAFUS binding sites pinpointed the transcriptional target genes directly regulated by RELAFUS. We then identified a unique DNA binding motif of the RELAFUS different from the canonical NF-kB motif in de novo motif discovery analysis. Significant responsiveness of RELAFUS but not RELA to the motif was confirmed in the reporter assay. An N-terminal portion of C11orf95 was sufficient to localize in the nucleus and recognizes the unique motif. Interestingly, the RELAFUS peaks concomitant with the unique motif were identified around the transcription start site in the RELAFUS target genes as previously reported. These observations suggested that C11orf95 might have served as a key determinant for the DNA binding sites of RELAFUS, thereby induced aberrant gene expression necessary for ependymoma formation. Our results will give insights into the development of new ependymoma therapy.

Download Full-text

A Unique DNA Binding Domain Converts T-Cell Factors into Strong Wnt Effectors

Molecular and Cellular Biology ◽

10.1128/mcb.02132-06 ◽

2007 ◽

Vol 27 (23) ◽

pp. 8352-8363 ◽

Cited By ~ 82

Author(s):

Fawzia A. Atcha ◽

Adeela Syed ◽

Beibei Wu ◽

Nate P. Hoverter ◽

Noriko N. Yokoyama ◽

...

Keyword(s):

T Cell ◽

Dna Binding ◽

Binding Sites ◽

Target Genes ◽

Regulation Of Gene Expression ◽

Dna Interaction ◽

Binding Motif ◽

Double Stranded Dna ◽

Alternatively Spliced ◽

Equilibrium Dissociation

ABSTRACT Wnt regulation of gene expression requires binding of LEF/T-cell factor (LEF/TCF) transcription factors to Wnt response elements (WREs) and recruitment of the activator β-catenin. There are significant differences in the abilities of LEF/TCF family members to regulate Wnt target genes. For example, alternatively spliced isoforms of TCF-1 and TCF-4 with a C-terminal “E” tail are uniquely potent in their activation of LEF1 and CDX1. Here we report that the mechanism responsible for this unique activity is an auxiliary 30-amino-acid DNA interaction motif referred to here as the “cysteine clamp” (or C-clamp). The C-clamp contains invariant cysteine, aromatic, and basic residues, and surface plasmon resonance (SPR) studies with recombinant C-clamp protein showed that it binds double-stranded DNA but not single-stranded DNA or RNA (equilibrium dissociation constant = 16 nM). CASTing (Cyclic Amplification and Selection of Targets) experiments were used to test whether this motif influences WRE recognition. Full-length LEF-1, TCF-1E, and TCF-1E with a mutated C-clamp all bind nearly identical WREs (TYYCTTTGATSTT), showing that the C-clamp does not alter WRE specificity. However, a GC element downstream of the WRE (RCCG) is enriched in wild-type TCF-1E binding sites but not in mutant TCF-1E binding sites. We conclude that the C-clamp is a sequence-specific DNA binding motif. C-clamp mutations destroy the ability of β-catenin to regulate the LEF1 promoter, and they severely impair the ability of TCF-1 to regulate growth in colon cancer cells. Thus, E-tail isoforms of TCFs utilize two DNA binding activities to access a subset of Wnt targets important for cell growth.

Download Full-text

Regulatory Targets of the Response Regulator RR_1586 from Clostridioides difficile Identified Using a Bacterial One-Hybrid Screen

Journal of Bacteriology ◽

10.1128/jb.00351-18 ◽

2018 ◽

Vol 200 (23) ◽

Cited By ~ 1

Author(s):

Skyler D. Hebdon ◽

Smita K. Menon ◽

George B. Richter-Addo ◽

Elizabeth A. Karr ◽

Ann H. West

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Regulatory Networks ◽

Target Genes ◽

Response Regulator ◽

Self Regulation ◽

Transport Systems ◽

Content Type ◽

Clostridioides Difficile ◽

Two Component

ABSTRACT The Clostridioides difficile R20291 genome encodes 57 response regulator proteins that, as part of two-component signaling pathways, regulate adaptation to environmental conditions. Genomic and transcriptomic studies in C. difficile have been limited, due to technical challenges, to the analysis of either high-throughput screens or high-priority targets, such as primary regulators of toxins or spore biology. We present the use of several technically accessible and generally applicable techniques to elucidate the putative regulatory targets of a response regulator, RR_1586, involved in sporulation of the hypervirulent C. difficile strain R20291. A DNA-binding specificity motif for RR_1586 was determined using a bacterial one-hybrid assay originally developed for Drosophila transcription factors. Comparative bioinformatics approaches identified and in vitro experiments confirmed RR_1586 binding sites upstream of putative target genes, including those that encode phosphate ion transporters, spermidine/putrescine biosynthesis and transport pathways, ABC type transport systems, known regulators of sporulation, and genes encoding spore structural proteins. Representative examples of these regulatory interactions have been tested and confirmed in Escherichia coli-based reporter assays. Finally, evidence of possible regulatory mechanisms is also presented. A working model includes self-regulation by RR_1586 and phosphorylation-dependent and -independent DNA binding at low- and high-fidelity binding sites, respectively. Broad application of this and similar approaches is anticipated to be an important catalyst for the study of gene regulation by two-component systems from pathogenic or technically challenging bacteria. IMPORTANCE Clostridioides difficile spores survive under harsh conditions and can germinate into actively dividing cells capable of causing disease. An understanding of the regulatory networks controlling sporulation and germination in C. difficile could be exploited for therapeutic advantage. However, such studies are hindered by the challenges of working with an anaerobic pathogen recalcitrant to genetic manipulation. Although two-component response regulators can be identified from genetic sequences, identification of their downstream regulatory networks requires further development. This work integrates experimental and bioinformatic approaches, which provide practical advantages over traditional transcriptomic analyses, to identify the putative regulon of the C. difficile response regulator RR_1586 by first screening for protein-DNA interactions in E. coli and then predicting regulatory outputs in C. difficile.

Download Full-text

Conserved DNA-binding motif loci reflect functional transcription factor binding sites

10.21203/rs.2.15866/v1 ◽

2019 ◽

Author(s):

Akihiro Kuno ◽

Satoru Takahashi

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Target Genes ◽

Regulatory Elements ◽

Binding Motif ◽

Genomic Locus ◽

Critical Pathway ◽

Conserved Motif ◽

Dna Elements ◽

Functional Dna

Abstract Background Transcription factors (TF) regulate gene expression by binding to specific DNA elements called DNA-binding motifs, or motifs. The motifs at a certain genomic locus can offer clues for predicting TFs that control target genes. However, since motifs are short nucleotide sequences and many TFs share similar motifs, they are distributed across the whole genome, making it difficult to investigate candidate functional DNA elements. Because previous studies have reported that TF binding sites are highly conserved among mammals, we focused on the conserved motif loci between humans and mice to identify functional DNA elements. Results Our results showed that conserved motif loci overlapping considerably with TF binding sites at both promoter and intergenic regions compared to nonconserved motif loci. In addition, conserved motif loci were significantly enriched in enhancer regions and enabled us to predict GATA4 binding to the heart-specific enhancer. Moreover, the integration of ChIP-seq and RNA-seq glioblastoma data revealed that genes with ASCL1 binding at the conserved ASCL1-related motifs were associated with NOTCH signaling, which is a critical pathway in glioblastoma. Conclusions These results suggest that conserved motif loci can reflect gene regulatory elements and can be utilized to predict candidate TFs that regulate genes of interest. The conserved motif data are publicly available at https://osf.io/jhfnb/?view_only=1c82bc9963ee45b799be42512abb08d1.

Download Full-text

Analysis of the Campylobacter jejuni FlgR Response Regulator Suggests Integration of Diverse Mechanisms To Activate an NtrC-Like Protein

Journal of Bacteriology ◽

10.1128/jb.01827-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2422-2433 ◽

Cited By ~ 26

Author(s):

Stephanie N. Joslin ◽

David R. Hendrixson

Keyword(s):

Dna Binding ◽

Campylobacter Jejuni ◽

Target Genes ◽

Response Regulator ◽

Binding Motif ◽

Flagellar Motility ◽

Receiver Domain ◽

Terminal Domain ◽

Biochemical Analyses ◽

Flagellar Genes

ABSTRACT Flagellar motility in Campylobacter jejuni mediates optimal interactions with human or animal hosts. σ54 and the FlgSR two-component system are necessary for the expression of many C. jejuni flagellar genes. The FlgR response regulator is homologous to the NtrC family of transcriptional activators. These regulators usually contain an N-terminal receiver domain, a central domain that interacts with σ54 and hydrolyzes ATP, and a DNA-binding C-terminal domain. Most often, phosphorylation of the receiver domain influences its inherent ability to either positively or negatively control the activity of the regulator. In this study, we performed genetic and biochemical analyses to understand how FlgR activity is controlled to culminate in the expression of σ54-dependent flagellar genes. Our data suggest that the FlgR receiver domain has the capacity for both positive and negative regulation in controlling the activation of the protein. Analysis of the C-terminal domain of FlgR revealed that it lacks a DNA-binding motif and is not required for σ54-dependent flagellar gene expression. Further analysis of FlgR lacking the C-terminal domain indicates that this protein is partially functional in the absence of the cognate sensor kinase, FlgS, but its activity is still dependent on the phosphorylated residue in the receiver domain, D51. We hypothesize that the C-terminal domain may not function to bind DNA but may ensure the specificity of the phosphorylation of FlgR by FlgS. Our results demonstrate that FlgR activation mechanisms are unusual among characterized NtrC-like proteins and emphasize that various means are utilized by the NtrC family of proteins to control the transcription of target genes.

Download Full-text

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.

Download Full-text

Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0018 ◽

2013 ◽

Vol 368 (1632) ◽

pp. 20130018 ◽

Cited By ~ 67

Author(s):

Andrea I. Ramos ◽

Scott Barolo

Keyword(s):

Transcription Factor ◽

Binding Affinity ◽

Binding Sites ◽

Target Genes ◽

Gene Activation ◽

Transcriptional Response ◽

Morphogen Gradients ◽

Weak Binding ◽

Initial Hypothesis

In the era of functional genomics, the role of transcription factor (TF)–DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. Here, we investigate the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients . We noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to our initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp , wingless and stripe , by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, we found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, we observed patterning defects in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. We propose that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes.

Download Full-text