-
Nature Methods Mar 2016High-throughput sequencing technologies have allowed many gene locus-level molecular biology assays to become genome-wide profiling methods. DNA-cleaving enzymes such as...
High-throughput sequencing technologies have allowed many gene locus-level molecular biology assays to become genome-wide profiling methods. DNA-cleaving enzymes such as DNase I have been used to probe accessible chromatin. The accessible regions contain functional regulatory sites, including promoters, insulators and enhancers. Deep sequencing of DNase-seq libraries and computational analysis of the cut profiles have been used to infer protein occupancy in the genome at the nucleotide level, a method introduced as 'digital genomic footprinting'. The approach has been proposed as an attractive alternative to the analysis of transcription factors (TFs) by chromatin immunoprecipitation followed by sequencing (ChIP-seq), and in theory it should overcome antibody issues, poor resolution and batch effects. Recent reports point to limitations of the DNase-based genomic footprinting approach and call into question the scope of detectable protein occupancy, especially for TFs with short-lived chromatin binding. The genomics community is grappling with issues concerning the utility of genomic footprinting and is reassessing the proposed approaches in terms of robust deliverables. Here we summarize the consensus as well as different views emerging from recent reports, and we describe the remaining issues and hurdles for genomic footprinting.
Topics: Algorithms; Chromosome Mapping; DNA; DNA Footprinting; Genome, Human; High-Throughput Nucleotide Sequencing; Humans
PubMed: 26914206
DOI: 10.1038/nmeth.3766 -
Bioinformatics (Oxford, England) Jul 2021High-throughput chromatin immunoprecipitation (ChIP) sequencing-based assays capture genomic regions associated with the profiled transcription factor (TF). ChIP-exo is...
MOTIVATION
High-throughput chromatin immunoprecipitation (ChIP) sequencing-based assays capture genomic regions associated with the profiled transcription factor (TF). ChIP-exo is a modified protocol, which uses lambda exonuclease to digest DNA close to the TF-DNA complex, in order to improve on the positional resolution of the TF-DNA contact. Because the digestion occurs in the 5'-3' orientation, the protocol produces directional footprints close to the complex, on both sides of the double stranded DNA. Like all ChIP-based methods, ChIP-exo reports a mixture of different regions associated with the TF: those bound directly to the TF as well as via intermediaries. However, the distribution of footprints are likely to be indicative of the complex forming at the DNA.
RESULTS
We present ExoDiversity, which uses a model-based framework to learn a joint distribution over footprints and motifs, thus resolving the mixture of ChIP-exo footprints into diverse binding modes. It uses no prior motif or TF information and automatically learns the number of different modes from the data. We show its application on a wide range of TFs and organisms/cell-types. Because its goal is to explain the complete set of reported regions, it is able to identify co-factor TF motifs that appear in a small fraction of the dataset. Further, ExoDiversity discovers small nucleotide variations within and outside canonical motifs, which co-occur with variations in footprints, suggesting that the TF-DNA structural configuration at those regions is likely to be different. Finally, we show that detected modes have specific DNA shape features and conservation signals, giving insights into the structure and function of the putative TF-DNA complexes.
AVAILABILITY AND IMPLEMENTATION
The code for ExoDiversity is available on https://github.com/NarlikarLab/exoDIVERSITY.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Binding Sites; Chromatin Immunoprecipitation; DNA; DNA Footprinting; Exonucleases; Protein Binding; Sequence Analysis, DNA
PubMed: 34252930
DOI: 10.1093/bioinformatics/btab274 -
Journal of the American Chemical Society Jul 2004A novel DNA footprinting method employing strong semiquinone radical species generated from a dipeptide-hydroquinone conjugate is described.
A novel DNA footprinting method employing strong semiquinone radical species generated from a dipeptide-hydroquinone conjugate is described.
Topics: Benzoquinones; Binding Sites; DNA; DNA Footprinting; Deoxyribonuclease I; Dipeptides; Electrophoresis, Polyacrylamide Gel; Molecular Structure
PubMed: 15225037
DOI: 10.1021/ja040051m -
Methods (San Diego, Calif.) Feb 1997Analysis of the interaction of proteins with either DNA or RNA sequences by in vivo footprinting involves two steps: (i) the in situ modification of nucleic acids by the... (Comparative Study)
Comparative Study
Analysis of the interaction of proteins with either DNA or RNA sequences by in vivo footprinting involves two steps: (i) the in situ modification of nucleic acids by the footprinting reagent and (ii) the visualization of the footprints. Ligation-mediated PCR (LM-PCR) procedures provide a level of sensitivity and specificity that is suitable for visualization of footprints of single-copy genes or low-abundance mRNAs in higher eukaryotes. In this article, we discuss several of the technical aspects of these multistep procedures that contribute to the quality of the results, particularly the parameters that affect the specificity and fidelity of the reactions: (i) the design of the primers, which is important to achieve optimal specificity; (ii) the choice of polymerases so that the amplified material represents faithfully the initial nucleic acid population; and (iii) the impact of the plateau effect within the PCR on the interpretation of the data. We then discuss aspects of in vivo nucleic acid manipulation that may affect the quality of the footprinting image, in particular the choice of the footprinting reagent and its condition of use (e.g., on intact or permeabilized cells or prepared nuclei) and the extent of nucleic acid modification. Finally, we provide detailed experimental procedures corresponding to the techniques we have developed or modified: LM-PCR, reverse ligation-mediated PCR, and nuclease treatment of RNAs in vivo.
Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Line; DNA; DNA Footprinting; DNA Primers; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Deoxyribonuclease I; Humans; Liver Neoplasms; Molecular Sequence Data; Nucleic Acid Conformation; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA; RNA, Messenger; RNA-Binding Proteins; Receptors, Transferrin; Sensitivity and Specificity; Taq Polymerase
PubMed: 8993027
DOI: 10.1006/meth.1996.0401 -
Bioorganic & Medicinal Chemistry Apr 2010Allostery in the binding of peptides to DNA has been studied by quantitative DNase I footprinting using four newly designed peptides containing the XP(Hyp)RK motif and...
Allostery in the binding of peptides to DNA has been studied by quantitative DNase I footprinting using four newly designed peptides containing the XP(Hyp)RK motif and N-methylpyrrole (Py) moieties. Apparent binding constants in the micromolar range as well as Hill coefficients were determined for each peptide. The results, together with previous studies on five other peptides support the proposal that interaction network cooperativity is highly preferred in DNA-peptide interactions that involve multiple recognition sites. It is envisaged that interstrand bidentate interactions participate in the relay of conformational changes between recognition sites on the complementary strands. Models for interpreting DNA allostery based upon interaction networks are outlined. Circular dichroism experiments involving the titration of peptides against a short oligonucleotide duplex indicate that some of these peptides bind in a dimeric manner to DNA via the minor groove, inducing characteristic conformational changes. These insights should prompt the design of new DNA-binding peptides for investigating allosteric interactions between peptides and DNA, as well as novel interaction networks, and ultimately may shed light upon the fundamental chemical rules that govern allostery in more complex biological process such as DNA-protein interaction networks.
Topics: Autoradiography; Circular Dichroism; DNA; DNA Footprinting; Deoxyribonuclease I; Ligands; Oligonucleotides; Peptides; Protein Binding; Protein Conformation; Reverse Transcriptase Polymerase Chain Reaction; Structure-Activity Relationship
PubMed: 20338768
DOI: 10.1016/j.bmc.2010.02.047 -
Journal of Chemical Information and... 2005The sequence selectivity of small molecules binding to the minor groove of DNA can be predicted by "in silico footprinting". Any potential ligand can be docked in the...
The sequence selectivity of small molecules binding to the minor groove of DNA can be predicted by "in silico footprinting". Any potential ligand can be docked in the minor groove and then moved along it using simple simulation techniques. By applying a simple scoring function to the trajectory after energy minimization, the preferred binding site can be identified. We show application to all known noncovalent binding modes, namely 1:1 ligand:DNA binding (including hairpin ligands) and 2:1 side-by-side binding, with various DNA base pair sequences and show excellent agreement with experimental results from X-ray crystallography, NMR, and gel-based footprinting.
Topics: Computer Simulation; Crystallography, X-Ray; DNA; DNA Footprinting; Drug Evaluation, Preclinical; Ligands; Magnetic Resonance Spectroscopy; Nucleic Acid Conformation
PubMed: 16309297
DOI: 10.1021/ci050153b -
BioTechniques Nov 2000Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and...
Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving (i) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescein; (ii) brief exposure of the fragment to a DNA-binding protein and then DNase I; (iii) spin-column purification; and (iv) analysis of partial digestion products on the ABI Prism 310 capillary DNA sequencer/genetic analyzer. Very detailed and sensitive footprints of large (> 400 bp) DNA fragments can be easily obtained, as illustrated by our use of this method to characterize binding of PhcA, a LysR-type activator, to two sites greater than 100 bp apart in the 5' untranslated region of xpsR, one of its regulated target genes. The advantages of this new method are that it (i) uses long-lived, safe and easy-to-make fluorescently labeled target fragments; (ii) uses sensitive, robust and highly reproducible fragment analysis using an automated DNA sequencer, instead of gel electrophoresis and autoradiography; and (iii) is cost effective.
Topics: Automation; Bacterial Proteins; Binding Sites; DNA; DNA Footprinting; DNA-Binding Proteins; Deoxyribonuclease I; Electrophoresis, Capillary; Fluoresceins; Fluorescent Dyes; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Binding; Repressor Proteins; Response Elements; Sequence Analysis, DNA; Transcription Factors
PubMed: 11084866
DOI: 10.2144/00295st05 -
Methods in Molecular Biology (Clifton,... 2001
Topics: Animals; DNA; DNA Footprinting; DNA-Binding Proteins; Deoxyribonuclease I; Electrophoresis, Polyacrylamide Gel; Indicators and Reagents; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Xenopus laevis
PubMed: 11357594
DOI: 10.1385/1-59259-208-2:031 -
Journal of Molecular Biology Nov 1998Formation of many site-specific protein-nucleic acid complexes involves sequential conformational changes subsequent to initial binding which create functionally active...
Quantitative analysis of multiple-hit footprinting studies to characterize DNA conformational changes in protein-DNA complexes: application to DNA opening by Esigma70 RNA polymerase.
Formation of many site-specific protein-nucleic acid complexes involves sequential conformational changes subsequent to initial binding which create functionally active assemblies. Characterization of population distributions and structural characteristics of intermediate and product conformations is necessary to understand both the mechanisms and the thermodynamics of these processes. For these purposes, here we develop the quantitative method of multiple hit footprinting (MHF), where chemical or enzymatic probing is performed as a function of either concentrations of the footprinting agent and/or time of exposure to it, in the multiple hit regime where many of the population or subpopulation of reactive DNA molecules are modified at more than one site. Properly controlled MHF experiments yield both the population distribution of different conformers and reactivity rate constants of the footprinting agent at all reactive positions in each conformer, which may be interpreted in terms of the accessibility of the site or the local concentration of the reagent. MHF experiments are particularly well-suited for dissecting effects at sites where unbound DNA is non-reactive and bound DNA is reactive with base-specific probes (e.g. KMnO4, DMS). We suggest that this method will also be applicable to analysis of enhancements in reactivity of other footprinting agents (e.g. DNase I, HO.). To demonstrate the utility of the MHF analysis, we quantify fragment distributions and individual site reactivities from multiple-hit KMnO4 footprinting of the non-template strand of Esigma70 RNA polymerase-lambdaPR promoter DNA complexes populated at binding equilibrium at 37 degreesC and transiently populated at a fixed time after a temperature downshift from 37 degreesC to 0 degreesC. For this system, a MHF analysis directly addresses the following questions: (i) what fraction of the population of promoter DNA molecules is open in the vicinity of the transcription start site (RPo) both at 37 degreesC and (transiently) after a downshift to 0 degreesC; (ii) does opening of the start site region in RPo occur entirely in one mechanistic step at the lambdaPR promoter and (iii) does the structure of RPo vary with temperature? In addition, we use the MHF-determined population distribution of KMnO4-reactive (RPo) and non-reactive promoter DNA to normalize the biphasic kinetics of decay of RPo to free promoter DNA after a 37 degrees to 0 degreesC temperature downshift, and thereby characterize the kinetics of the conformational changes involved in forming RPo.
Topics: DNA; DNA Footprinting; DNA-Directed RNA Polymerases; Escherichia coli; Kinetics; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Potassium Permanganate; Temperature
PubMed: 9790838
DOI: 10.1006/jmbi.1998.2130 -
Nature Methods Apr 2009The orchestrated binding of transcriptional activators and repressors to specific DNA sequences in the context of chromatin defines the regulatory program of eukaryotic...
The orchestrated binding of transcriptional activators and repressors to specific DNA sequences in the context of chromatin defines the regulatory program of eukaryotic genomes. We developed a digital approach to assay regulatory protein occupancy on genomic DNA in vivo by dense mapping of individual DNase I cleavages from intact nuclei using massively parallel DNA sequencing. Analysis of >23 million cleavages across the Saccharomyces cerevisiae genome revealed thousands of protected regulatory protein footprints, enabling de novo derivation of factor binding motifs and the identification of hundreds of new binding sites for major regulators. We observed striking correspondence between single-nucleotide resolution DNase I cleavage patterns and protein-DNA interactions determined by crystallography. The data also yielded a detailed view of larger chromatin features including positioned nucleosomes flanking factor binding regions. Digital genomic footprinting should be a powerful approach to delineate the cis-regulatory framework of any organism with an available genome sequence.
Topics: Algorithms; Amino Acid Sequence; Base Sequence; Binding Sites; DNA; DNA Footprinting; Molecular Sequence Data; Protein Binding; Protein Interaction Mapping; Sequence Analysis, DNA; Transcription Factors
PubMed: 19305407
DOI: 10.1038/nmeth.1313