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Computational and Structural... 2022Cooperativity between transcription factors is important to regulate target gene expression. In particular, the binding grammar of TFs in relation to each other, as well...
Cooperativity between transcription factors is important to regulate target gene expression. In particular, the binding grammar of TFs in relation to each other, as well as in the context of other genomic elements, is crucial for TF functionality. However, tools to easily uncover co-occurrence between DNA-binding proteins, and investigate the regulatory modules of TFs, are limited. Here we present TF-COMB (Transcription Factor Co-Occurrence using Market Basket analysis) - a tool to investigate co-occurring TFs and binding grammar within regulatory regions. We found that TF-COMB can accurately identify known co-occurring TFs from ChIP-seq data, as well as uncover preferential localization to other genomic elements. With the use of ATAC-seq footprinting and TF motif locations, we found that TFs exhibit both preferred orientation and distance in relation to each other, and that these are biologically significant. Finally, we extended the analysis to not only investigate individual TF pairs, but also TF pairs in the context of networks, which enabled the investigation of TF complexes and TF hubs. In conclusion, TF-COMB is a flexible tool to investigate various aspects of TF binding grammar.
PubMed: 35983231
DOI: 10.1016/j.csbj.2022.07.025 -
Genome Biology Nov 2023Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing...
BACKGROUND
Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing their origin to evolutionary innovations first made in their archaeal ancestors. One of the most notable such features is nucleosomal chromatin, although archaeal histones and chromatin differ significantly from those of eukaryotes, not all archaea possess histones and it is not clear if histones are a main packaging component for all that do. Despite increased interest in archaeal chromatin in recent years, its properties have been little studied using genomic tools.
RESULTS
Here, we adapt the ATAC-seq assay to archaea and use it to map the accessible landscape of the genome of the euryarchaeote Haloferax volcanii. We integrate the resulting datasets with genome-wide maps of active transcription and single-stranded DNA (ssDNA) and find that while H. volcanii promoters exist in a preferentially accessible state, unlike most eukaryotes, modulation of transcriptional activity is not associated with changes in promoter accessibility. Applying orthogonal single-molecule footprinting methods, we quantify the absolute levels of physical protection of H. volcanii and find that Haloferax chromatin is similarly or only slightly more accessible, in aggregate, than that of eukaryotes. We also evaluate the degree of coordination of transcription within archaeal operons and make the unexpected observation that some CRISPR arrays are associated with highly prevalent ssDNA structures.
CONCLUSIONS
Our results provide the first comprehensive maps of chromatin accessibility and active transcription in Haloferax across conditions and thus a foundation for future functional studies of archaeal chromatin.
Topics: Chromatin; Histones; Haloferax volcanii; Nucleosomes; Biological Evolution; Eukaryota; Archaeal Proteins
PubMed: 37932847
DOI: 10.1186/s13059-023-03095-5 -
Scientific Reports Oct 2019Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of...
Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.
Topics: Adenosine Triphosphate; Animals; Cell-Free System; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; DNA Footprinting; DNA, Bacterial; Deoxyribonucleases, Type II Site-Specific; Fungal Proteins; Histones; Microscopy, Atomic Force; Multiprotein Complexes; Nucleosomes; Plasmids; RNA-Binding Proteins; Recombinant Proteins; Saccharomyces cerevisiae; Xenopus laevis
PubMed: 31578361
DOI: 10.1038/s41598-019-50572-8 -
Nature Communications May 2023Decades ago, it was shown that proteins binding to DNA can quantitatively alter the formation of DNA damage by UV light. This established the principle of UV...
Decades ago, it was shown that proteins binding to DNA can quantitatively alter the formation of DNA damage by UV light. This established the principle of UV footprinting for non-intrusive study of protein-DNA contacts in living cells, albeit at limited scale and precision. Here, we perform deep base-resolution quantification of the principal UV damage lesion, the cyclobutane pyrimidine dimer (CPD), at select human promoter regions using targeted CPD sequencing. Several transcription factors exhibited distinctive and repeatable damage signatures indicative of site occupancy, involving strong (up to 17-fold) position-specific elevations and reductions in CPD formation frequency relative to naked DNA. Positive damage modulation at some ETS transcription factor binding sites coincided at base level with melanoma somatic mutation hotspots. Our work provides proof of concept for the study of protein-DNA interactions at individual loci using light and sequencing, and reveals widespread and potent modulation of UV damage in regulatory regions.
Topics: Humans; DNA-Binding Proteins; Ultraviolet Rays; DNA Damage; Pyrimidine Dimers; DNA
PubMed: 37169761
DOI: 10.1038/s41467-023-38266-2 -
Molecules (Basel, Switzerland) Sep 2021Modulating the expression or function of the enigmatic MYC protein has demonstrated efficacy in an array of cancer types and a marked potential therapeutic index and...
Modulating the expression or function of the enigmatic MYC protein has demonstrated efficacy in an array of cancer types and a marked potential therapeutic index and safety profile. Despite its high therapeutic value, specific and selective inhibitors or downregulating therapeutics have proven difficult to develop. In the current study, we expanded our work on a MYC promoter G-quadruplex (G4) stabilizing DNA clamp to develop an oligonucleotide interfering DNA (DNAi) therapeutic. We explored six DNAi for G4-stabilization through EMSA, DMS footprinting, and thermal stability studies, focusing on the DNAi 5T as the lead therapeutic. 5T, but not its scramble control 5Tscr, was then shown to enter the nucleus, modulate cell viability, and decrease MYC expression through G4-stabilization. DNAi 5T is thus described to be our lead DNAi, targeting MYC regulation through stabilization of the higher-order DNA G4 structure in the proximal promoter, and it is poised for further preclinical development as an anticancer therapeutic.
Topics: Down-Regulation; G-Quadruplexes; Humans; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc
PubMed: 34577013
DOI: 10.3390/molecules26185542 -
Scientific Reports Sep 2020We scrutinize the length dependency of the binding affinity of bacterial repressor TrpR protein to trpO (specific site) on DNA. A footprinting experiment shows that the...
We scrutinize the length dependency of the binding affinity of bacterial repressor TrpR protein to trpO (specific site) on DNA. A footprinting experiment shows that the longer the DNA length, the larger the affinity of TrpR to the specific site on DNA. This effect termed "antenna effect" might be interpreted as follows: longer DNA provides higher probability for TrpR to access to the specific site aided by one-dimensional diffusion along the nonspecific sites of DNA. We show that, however, the antenna effect cannot be explained while detailed balance holds among three kinetic states, that is, free protein/DNA, nonspecific complexes, and specific complex. We propose a working hypothesis that slow degree(s) of freedom in the system switch(es) different potentials of mean force causing transitions among the three states. This results in a deviation from detailed balance on the switching timescale. We then derive a simple reaction diffusion/binding model that describes the antenna effect on TrpR binding to its target operator. Possible scenarios for such slow degree(s) of freedom in TrpR-DNA complex are addressed.
Topics: Bacterial Proteins; Binding Sites; DNA, Bacterial; Escherichia coli; Models, Theoretical; Operator Regions, Genetic; Protein Binding; Repressor Proteins
PubMed: 32973254
DOI: 10.1038/s41598-020-71598-3 -
Genome Research Jul 2020Transcription is tightly regulated by -regulatory DNA elements where transcription factors (TFs) can bind. Thus, identification of TF binding sites (TFBSs) is key to...
Transcription is tightly regulated by -regulatory DNA elements where transcription factors (TFs) can bind. Thus, identification of TF binding sites (TFBSs) is key to understanding gene expression and whole regulatory networks within a cell. The standard approaches used for TFBS prediction, such as position weight matrices (PWMs) and chromatin immunoprecipitation followed by sequencing (ChIP-seq), are widely used but have their drawbacks, including high false-positive rates and limited antibody availability, respectively. Several computational footprinting algorithms have been developed to detect TFBSs by investigating chromatin accessibility patterns; however, these also have limitations. We have developed a footprinting method to predict TF footprints in active chromatin elements (TRACE) to improve the prediction of TFBS footprints. TRACE incorporates DNase-seq data and PWMs within a multivariate hidden Markov model (HMM) to detect footprint-like regions with matching motifs. TRACE is an unsupervised method that accurately annotates binding sites for specific TFs automatically with no requirement for pregenerated candidate binding sites or ChIP-seq training data. Compared with published footprinting algorithms, TRACE has the best overall performance with the distinct advantage of targeting multiple motifs in a single model.
Topics: Binding Sites; Cell Line; Chromatin; DNA Footprinting; Deoxyribonucleases; Humans; K562 Cells; Markov Chains; Nucleotide Motifs; Sequence Analysis, DNA; Transcription Factors
PubMed: 32660981
DOI: 10.1101/gr.258228.119 -
Journal of Chemical Information and... Jun 2023Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin to stimulate vital cellular processes. In this work, we dissect the...
Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin to stimulate vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and physiochemistry approaches, along with DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the base-specific Sox:DNA interactions (base reading) and Sox-induced DNA changes (shape reading) are concurrently required for sequence-specific nucleosomal DNA recognition. Among three different nucleosome positions located on the positive DNA arm, a sequence-specific reading mechanism is solely satisfied at the superhelical location 2 (SHL2). While SHL2 acts transparently for solvent-facing Sox binding, among the other two positions, SHL4 permits only shape reading. The final position, SHL0 (dyad), on the other hand, allows no reading mechanism. These findings demonstrate that Sox-based nucleosome recognition is essentially guided by intrinsic nucleosome properties, permitting varying degrees of DNA recognition.
Topics: Nucleosomes; Transcription Factors; DNA; Gene Expression Regulation
PubMed: 37307148
DOI: 10.1021/acs.jcim.2c01520 -
Journal of Bacteriology Jun 2020HutC is known as a transcriptional repressor specific for histidine utilization () genes in Gram-negative bacteria, including SBW25. However, its precise mode of...
HutC is known as a transcriptional repressor specific for histidine utilization () genes in Gram-negative bacteria, including SBW25. However, its precise mode of protein-DNA interactions hasn't been examined with purified HutC proteins. Here, we performed electrophoretic mobility shift assay (EMSA) and DNase I footprinting using His-tagged HutC and biotin-labeled probe of the promoter (P). Results revealed a complex pattern of HutC oligomerization, and the specific protein-DNA interaction is disrupted by urocanate, a histidine derivative, in a concentration-dependent manner. Next, we searched for putative HutC-binding sites in the SBW25 genome. This led to the identification of 143 candidate targets with a value less than 10 HutC interaction with eight selected candidate sites was subsequently confirmed by EMSA analysis, including the type IV pilus assembly protein PilZ, phospholipase C (PlcC) for phosphatidylcholine hydrolyzation, and key regulators of cellular nitrogen metabolism (NtrBC and GlnE). Finally, an isogenic deletion mutant was subjected to transcriptome sequencing (RNA-seq) analysis and phenotypic characterization. When bacteria were grown on succinate and histidine, deletion caused upregulation of 794 genes and downregulation of 525 genes at a value of <0.05 with a fold change cutoff of 2.0. The mutant displayed an enhanced spreading motility and pyoverdine production in laboratory media, in addition to the previously reported growth defect on the surfaces of plants. Together, our data indicate that HutC plays global regulatory roles beyond histidine catabolism through low-affinity binding with operator sites located outside the locus. HutC in is a representative member of the GntR/HutC family of transcriptional regulators, which possess a N-terminal winged helix-turn-helix (wHTH) DNA-binding domain and a C-terminal substrate-binding domain. HutC is generally known to repress expression of histidine utilization () genes through binding to the P promoter with urocanate (the first intermediate of the histidine degradation pathway) as the direct inducer. Here, we first describe the detailed molecular interactions between HutC and its P target site in a plant growth-promoting bacterium, SBW25, and further show that HutC possesses specific DNA-binding activities with many targets in the SBW25 genome. Subsequent RNA-seq analysis and phenotypic assays revealed an unexpected global regulatory role of HutC for successful bacterial colonization .
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Histidine; Promoter Regions, Genetic; Pseudomonas fluorescens; Repressor Proteins; Transcription, Genetic
PubMed: 32291279
DOI: 10.1128/JB.00792-19 -
Frontiers in Molecular Biosciences 2020Site-specific recombination is a DNA breaking and reconstructing process that plays important roles in various cellular pathways for both prokaryotes and eukaryotes....
Site-specific recombination is a DNA breaking and reconstructing process that plays important roles in various cellular pathways for both prokaryotes and eukaryotes. This process requires a site-specific recombinase and direct or inverted repeats. Some tyrosine site-specific recombinases catalyze DNA inversions and regulate subpopulation diversity and phase variation in many bacterial species. In , the PsrA tyrosine recombinase was shown to control DNA inversions in the three DNA methyltransferase genes of the type I restriction-modification locus. Such DNA inversions are mediated by three inverted repeats (IR1, IR2, and IR3). In this work, we purified an untagged form of the PsrA protein and studied its DNA-binding and catalytic features. Gel retardation assays showed that PsrA binds to linear and supercoiled DNAs, containing or not inverted repeats. Nevertheless, DNase I footprinting assays showed that, on linear DNAs, PsrA has a preference for sites that include an IR1 sequence (IR1.1 or IR1.2) and its boundary sequences. Furthermore, on supercoiled DNAs, PsrA was able to generate DNA inversions between specific inverted repeats (IR1, IR2, and IR3), which supports its ability to locate specific target sites. Unlike other site-specific recombinases, PsrA showed reliance on magnesium ions for efficient catalysis of IR1-mediated DNA inversions. We discuss that PsrA might find its specific binding sites on the bacterial genome by a mechanism that involves transitory non-specific interactions between protein and DNA.
PubMed: 32266289
DOI: 10.3389/fmolb.2020.00043