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Briefings in Bioinformatics Sep 2019Deoxyribonuclease I (DNase I)-hypersensitive site sequencing (DNase-seq) has been widely used to determine chromatin accessibility and its underlying regulatory lexicon.... (Review)
Review
Deoxyribonuclease I (DNase I)-hypersensitive site sequencing (DNase-seq) has been widely used to determine chromatin accessibility and its underlying regulatory lexicon. However, exploring DNase-seq data requires sophisticated downstream bioinformatics analyses. In this study, we first review computational methods for all of the major steps in DNase-seq data analysis, including experimental design, quality control, read alignment, peak calling, annotation of cis-regulatory elements, genomic footprinting and visualization. The challenges associated with each step are highlighted. Next, we provide a practical guideline and a computational pipeline for DNase-seq data analysis by integrating some of these tools. We also discuss the competing techniques and the potential applications of this pipeline for the analysis of analogous experimental data. Finally, we discuss the integration of DNase-seq with other functional genomics techniques.
Topics: Computational Biology; DNA Footprinting; Data Management; Deoxyribonuclease I; Quality Control; Sequence Analysis, DNA
PubMed: 30010713
DOI: 10.1093/bib/bby057 -
Nucleic Acids Research Sep 2017Nucleosomes are the most abundant protein-DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA...
Nucleosomes are the most abundant protein-DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical footprinting (HRF) of protein-DNA complexes is a chemical technique that probes nucleosome organization in solution with a high precision unattainable by other methods. In this work we propose an integrative modeling method for constructing high-resolution atomistic models of nucleosomes based on HRF experiments. Our method precisely identifies DNA positioning on nucleosome by combining HRF data for both DNA strands with the pseudo-symmetry constraints. We performed high-resolution HRF for Saccharomyces cerevisiae centromeric nucleosome of unknown structure and characterized it using our integrative modeling approach. Our model provides the basis for further understanding the cooperative engagement and interplay between Cse4p protein and the A-tracts important for centromere function.
Topics: Algorithms; Centromere; Chromosomal Proteins, Non-Histone; DNA; DNA Cleavage; DNA Footprinting; DNA-Binding Proteins; Hydroxyl Radical; Models, Molecular; Nucleic Acid Conformation; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 28934480
DOI: 10.1093/nar/gkx616 -
Molecules (Basel, Switzerland) Aug 2020Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this...
Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.
Topics: Amino Acid Sequence; Basic-Leucine Zipper Transcription Factors; Binding Sites; Calorimetry, Differential Scanning; Catalysis; Chromatography, High Pressure Liquid; Click Chemistry; Copper; DNA; DNA Damage; DNA, Single-Stranded; Molecular Dynamics Simulation; Nucleic Acid Conformation; Peptides; Protein Domains; Saccharomyces cerevisiae Proteins; Spectrometry, Mass, Electrospray Ionization; Transition Temperature
PubMed: 32784992
DOI: 10.3390/molecules25163630 -
Bioinformatics (Oxford, England) Jun 2017The computational investigation of DNA binding motifs from binding sites is one of the classic tasks in bioinformatics and a prerequisite for understanding gene...
MOTIVATION
The computational investigation of DNA binding motifs from binding sites is one of the classic tasks in bioinformatics and a prerequisite for understanding gene regulation as a whole. Due to the development of sequencing technologies and the increasing number of available genomes, approaches based on phylogenetic footprinting become increasingly attractive. Phylogenetic footprinting requires phylogenetic trees with attached substitution probabilities for quantifying the evolution of binding sites, but these trees and substitution probabilities are typically not known and cannot be estimated easily.
RESULTS
Here, we investigate the influence of phylogenetic trees with different substitution probabilities on the classification performance of phylogenetic footprinting using synthetic and real data. For synthetic data we find that the classification performance is highest when the substitution probability used for phylogenetic footprinting is similar to that used for data generation. For real data, however, we typically find that the classification performance of phylogenetic footprinting surprisingly increases with increasing substitution probabilities and is often highest for unrealistically high substitution probabilities close to one. This finding suggests that choosing realistic model assumptions might not always yield optimal predictions in general and that choosing unrealistically high substitution probabilities close to one might actually improve the classification performance of phylogenetic footprinting.
AVAILABILITY AND IMPLEMENTATION
The proposed PF is implemented in JAVA and can be downloaded from https://github.com/mgledi/PhyFoo.
CONTACT
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Animals; Binding Sites; Computational Biology; DNA-Binding Proteins; Gene Regulatory Networks; Humans; Phylogeny; Sequence Analysis, DNA; Sequence Analysis, Protein; Software
PubMed: 28130227
DOI: 10.1093/bioinformatics/btx033 -
PNAS Nexus Sep 2022The tumor suppressor p53 functions as a pioneer transcription factor that binds a nucleosomal target DNA sequence. However, the mechanism by which p53 binds to its...
The tumor suppressor p53 functions as a pioneer transcription factor that binds a nucleosomal target DNA sequence. However, the mechanism by which p53 binds to its target DNA in the nucleosome remains elusive. Here we report the cryo-electron microscopy structures of the p53 DNA-binding domain and the full-length p53 protein complexed with a nucleosome containing the 20 base-pair target DNA sequence of p53 (p53BS). In the p53-nucleosome structures, the p53 DNA-binding domain forms a tetramer and specifically binds to the p53BS DNA, located near the entry/exit region of the nucleosome. The nucleosomal position of the p53BS DNA is within the genomic p21 promoter region. The p53 binding peels the DNA from the histone surface, and drastically changes the DNA path around the p53BS on the nucleosome. The C-terminal domain of p53 also binds to the DNA around the center and linker DNA regions of the nucleosome, as revealed by hydroxyl radical footprinting. These results provide important structural information for understanding the mechanism by which p53 binds the nucleosome and changes the chromatin structure for gene activation.
PubMed: 36714865
DOI: 10.1093/pnasnexus/pgac177 -
Molecular Cell Mar 2023Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA...
Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA demethylation. However, whether this epigenetic remodeling is necessary for enhancer activation is unknown. Here, we adapted single-molecule footprinting to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their chromatin accessibility in multiple cell lineages. Although reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where DNA methylation antagonizes the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding require active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at active enhancers.
Topics: Animals; DNA Methylation; Enhancer Elements, Genetic; Chromatin; Transcription Factors; Gene Expression Regulation; Mammals
PubMed: 36758546
DOI: 10.1016/j.molcel.2023.01.017 -
Methods in Molecular Biology (Clifton,... 2019DNA G-quadruplexes are globular nucleic acid secondary structures which occur throughout the human genome under physiological conditions. There is accumulating evidence...
DNA G-quadruplexes are globular nucleic acid secondary structures which occur throughout the human genome under physiological conditions. There is accumulating evidence supporting G-quadruplex involvement in a number of important aspects of genome functions, including transcription, replication, and genomic stability, and that protein and enzyme recognition of G-quadruplexes may represent a key event to regulate physiological or pathological pathways. Two important techniques to study G-quadruplexes and their protein interactions are the electrophoretic mobility shift assay (EMSA) and dimethyl sulfate (DMS) footprinting assay. EMSA, one of the most sensitive and robust methods for studying the DNA-protein interactions, can be used to determine the binding parameters and relative affinities of a protein for the G-quadruplex. DMS footprinting is a powerful assay for the initial characterization of G-quadruplexes, which can be used to deduce the guanine bases involved in the formation of G-tetrads under physiological salt conditions. DMS footprinting can also reveal important information in G-quadruplex-protein complexes on protein contacts and regional changes in DNA G-quadruplex upon protein binding. In this paper, we will provide a detailed protocol for the EMSA and DMS footprinting assays for characterization of G-quadruplexes and G-quadruplex-protein complexes. Expected outcomes and references to extensions of the method will be further discussed.
Topics: Electrophoretic Mobility Shift Assay; G-Quadruplexes; Nucleic Acid Conformation; Sulfuric Acid Esters
PubMed: 31444751
DOI: 10.1007/978-1-4939-9666-7_11 -
Journal of Inorganic Biochemistry Jan 2022The investigation of compounds capable of strongly and selectively interacting with DNA comprises a field of research in constant development. In this work, we...
The investigation of compounds capable of strongly and selectively interacting with DNA comprises a field of research in constant development. In this work, we demonstrate that a trinuclear coordination complex based on a dinuclear Fe(III)Zn(II) core designed for biomimicry of the hydrolytic enzyme kidney bean purple acid phosphatase, containing an additional pendant arm coordinating a Pd(II) ion, has the ability to interact with DNA and to promote its hydrolytic cleavage. These results were found through analysis of plasmid DNA interaction and cleavage by the trinuclear complex 1 and its derivatives 2 and 3, in addition to the analysis of alteration in the DNA structure in the presence of the complexes through circular dichroism and DNA footprinting techniques. The suggested covalent interaction of the palladium-containing complex with DNA was analysed using an electrophoretic mobility assay, circular dichroism, high resolution gel separation techniques and kinetic analysis. This is a new and promising metal complex targeted to nucleic acids and acting in two separate ways: strong DNA interaction and hydrolytic cleavage.
Topics: Coordination Complexes; DNA Cleavage; Deoxyribonucleases; Metals; Plasmids
PubMed: 34717251
DOI: 10.1016/j.jinorgbio.2021.111631 -
Nucleic Acids Research Apr 2017Transcription initiation by bacterial RNA polymerase (RNAP) requires a variable σ subunit that directs it to promoters for site-specific priming of RNA synthesis. The...
Transcription initiation by bacterial RNA polymerase (RNAP) requires a variable σ subunit that directs it to promoters for site-specific priming of RNA synthesis. The principal σ subunit responsible for expression of house-keeping genes can bind the transcription elongation complex after initiation and induce RNAP pausing through specific interactions with promoter-like motifs in transcribed DNA. We show that the stationary phase and stress response σ38 subunit can also induce pausing by Escherichia coli RNAP on DNA templates containing promoter-like motifs in the transcribed regions. The pausing depends on σ38 contacts with the DNA template and RNAP core enzyme and results in formation of backtracked transcription elongation complexes, which can be reactivated by Gre factors that induce RNA cleavage by RNAP. Our data suggest that σ38 can bind the transcription elongation complex in trans but likely acts in cis during transcription initiation, by staying bound to RNAP and recognizing promoter-proximal pause signals. Analysis of σ38-dependent promoters reveals that a substantial fraction of them contain potential pause-inducing motifs, suggesting that σ38-depended pausing may be a common phenomenon in bacterial transcription.
Topics: Bacterial Proteins; DNA Footprinting; DNA-Directed RNA Polymerases; Escherichia coli; Promoter Regions, Genetic; Sigma Factor; Templates, Genetic; Transcription Elongation, Genetic; Transcription Initiation, Genetic
PubMed: 27928053
DOI: 10.1093/nar/gkw1213 -
Nature Methods Oct 2015Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging...
Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.
Topics: Base Sequence; Binding Sites; Carrier Proteins; DNA Breaks, Double-Stranded; DNA Footprinting; DNA Repair; Enhancer Elements, Genetic; Erythrocytes; Erythropoiesis; Genome, Human; Genomics; Humans; Mutation; Nuclear Proteins; Regulatory Sequences, Nucleic Acid; Repressor Proteins; Transcription Factors
PubMed: 26322838
DOI: 10.1038/nmeth.3554