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Cell Jan 2016Nucleosome positioning varies between cell types. By deep sequencing cell-free DNA (cfDNA), isolated from circulating blood plasma, we generated maps of genome-wide in...
Nucleosome positioning varies between cell types. By deep sequencing cell-free DNA (cfDNA), isolated from circulating blood plasma, we generated maps of genome-wide in vivo nucleosome occupancy and found that short cfDNA fragments harbor footprints of transcription factors. The cfDNA nucleosome occupancies correlate well with the nuclear architecture, gene structure, and expression observed in cells, suggesting that they could inform the cell type of origin. Nucleosome spacing inferred from cfDNA in healthy individuals correlates most strongly with epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as the normal source of cfDNA. We build on this observation to show how nucleosome footprints can be used to infer cell types contributing to cfDNA in pathological states such as cancer. Since this strategy does not rely on genetic differences to distinguish between contributing tissues, it may enable the noninvasive monitoring of a much broader set of clinical conditions than currently possible.
Topics: CCCTC-Binding Factor; Cell Line; Chromatin Assembly and Disassembly; DNA; DNA Footprinting; Genome, Human; Genome-Wide Association Study; Humans; Neoplasms; Nucleosomes; Organ Specificity; Repressor Proteins; Sequence Analysis, DNA
PubMed: 26771485
DOI: 10.1016/j.cell.2015.11.050 -
Molekuliarnaia Biologiia 2018Ligand binding influences the dynamics of the DNA helix in both the binding site and adjacent regions. This, in particular, is reflected in the changing pattern of...
Ligand binding influences the dynamics of the DNA helix in both the binding site and adjacent regions. This, in particular, is reflected in the changing pattern of cleavage of complexes under the action of ultrasound. The specificity of ultrasound-induced cleavage of the DNA sugar-phosphate backbone was studied in actinomycin D (AMD) complexes with double-stranded DNA restriction fragments. After antibiotic binding, the cleavage intensity of phosphodiester bonds between bases was shown to decrease at the chromophore intercalation site and to increase in adjacent positions. The character of cleavage depended on the sequences flanking the binding site and the presence of other AMD molecules bound in the close vicinity. A comparison of ultrasonic and DNase I cleavage patterns of AMD-DNA complexes provided more detail on the local conformation and dynamics of the DNA double helix in both binding site and adjacent regions. The results pave the way for developing a novel approach to studies of the nucleotide sequence dependence of DNA conformational dynamics and new techniques to identify functional genome regions.
Topics: Base Sequence; Binding Sites; DNA; DNA Footprinting; DNA-Binding Proteins; Dactinomycin; Deoxyribonuclease I; Gene Expression; Intercalating Agents; Ligands; Nucleic Acid Conformation; Ultrasonic Waves
PubMed: 30113037
DOI: 10.1134/S0026898418040067 -
Nature Protocols Dec 2021Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to... (Review)
Review
Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to simultaneously resolve the occupancy of multiple proteins including transcription factors, RNA polymerase II and nucleosomes on single DNA molecules genome-wide. The technique combines the use of cytosine methyltransferases to footprint the genome with bisulfite sequencing to resolve transcription factor binding patterns at cis-regulatory elements. DNA footprinting is performed by incubating permeabilized nuclei with recombinant methyltransferases. Upon DNA extraction, whole-genome or targeted bisulfite libraries are prepared and loaded on Illumina sequencers. The protocol can be completed in 4-5 d in any laboratory with access to high-throughput sequencing. Analysis can be performed in 2 d using a dedicated R package and requires access to a high-performance computing system. Our method can be used to analyze how transcription factors cooperate and antagonize to regulate transcription.
Topics: Animals; Cell Nucleus; DNA; DNA Footprinting; DNA Modification Methylases; Gene Expression Regulation; Gene Library; Genome; High-Throughput Nucleotide Sequencing; Humans; Mice; Mouse Embryonic Stem Cells; Nucleosomes; RNA Polymerase II; Sequence Analysis, DNA; Single Molecule Imaging; Software; Transcription Factors
PubMed: 34773120
DOI: 10.1038/s41596-021-00630-1 -
Critical Reviews in Biochemistry and... 2015Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the... (Review)
Review
Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA cross-linking patterns by combining chromatin immunoprecipitation (ChIP) with 5' → 3' exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATAC-seq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches.
Topics: Animals; Chromatin; Chromatin Immunoprecipitation; Computational Biology; Computer Simulation; DNA; DNA Footprinting; DNA-Binding Proteins; Datasets as Topic; Exodeoxyribonucleases; Expert Systems; Genomics; Humans; Hydrolysis; Models, Molecular; Nucleic Acid Conformation; Nucleosomes; Protein Conformation; Protein Footprinting
PubMed: 26038153
DOI: 10.3109/10409238.2015.1051505 -
Nature Communications Aug 2017Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a...
Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present a fast and accurate method based on parallelized single molecule magnetic tweezers to detect the sequence selectivity and characterize the thermodynamics and kinetics of binding in a single assay. Mechanical manipulation of DNA hairpins with an engineered sequence is used to detect ligand binding as blocking events during DNA unzipping, allowing determination of ligand selectivity both for small drugs and large proteins with nearly base-pair resolution in an unbiased fashion. The assay allows investigation of subtle details such as the effect of flanking sequences or binding cooperativity. Unzipping assays on hairpin substrates with an optimized flat free energy landscape containing all binding motifs allows determination of the ligand mechanical footprint, recognition site, and binding orientation.Mapping the sequence specificity of DNA ligands remains a challenge, particularly for small drugs. Here the authors develop a parallelized single molecule magnetic tweezers approach using engineered DNA hairpins that can detect sequence selectivity, thermodynamics and kinetics of binding for small drugs and large proteins.
Topics: Base Sequence; Binding Sites; DNA; DNA Footprinting; Kinetics; Ligands; Magnetics; Models, Genetic; Nucleic Acid Conformation; Optical Tweezers; Thermodynamics
PubMed: 28824174
DOI: 10.1038/s41467-017-00379-w -
Acta Chimica Slovenica Dec 2016The synthesis and biological activity of a variety of analogues to the naturally occurring antibacterial and antifungal Distamycin A were explored by a number of... (Review)
Review
The synthesis and biological activity of a variety of analogues to the naturally occurring antibacterial and antifungal Distamycin A were explored by a number of authors. These compounds were subject to a large array of assays. Some of these compounds showed high activity against a range of Gram-positive, Gram-negative bacteria as well as fungi. To explore the anti-parasitic activity of this class of compounds, specific modifications had to be made. A number of these compounds proved to be active against Trypanosoma brucei. The binding of a number of these compounds to short sequences of DNA were also examined using footprinting assays as well as NMR spectroscopy. Computer modelling was employed on selected compounds to understand the way these compounds bind to specific DNA sequences. A large number of variations were made to the standard structure of Distamycin. These changes involved the replacement of the pyrrole moieties as well as the head and tail groups with a number of heterocyclic compounds. Some of these minor groove binders (MGBs) were also investigated for their capability for the treatment of cancer and in particular lung cancer.
Topics: Animals; Anti-Bacterial Agents; Computer Simulation; DNA; DNA Footprinting; Distamycins; Humans; Magnetic Resonance Spectroscopy; Trypanocidal Agents
PubMed: 28004090
DOI: 10.17344/acsi.2016.2775 -
Analytical Biochemistry Aug 2013The mapping of DNA footprints and affinity cleavage sites for small DNA ligands is affected by the choice of sequencing chemistry and end label, and the potential for...
The mapping of DNA footprints and affinity cleavage sites for small DNA ligands is affected by the choice of sequencing chemistry and end label, and the potential for indexing errors can be significant when mapping small ligand-DNA interactions. Described here is a mechanism for avoiding such errors based on a summary of standard labeling, cleavage, and indexing chemistries and a comparison among them for analysis of these interactions by capillary electrophoresis. The length dependence of the difference between Sanger and Maxam-Gilbert indexing is examined for a number of duplexes of mixed sequence.
Topics: Chromatography, Affinity; DNA Footprinting; Electrophoresis, Capillary; Hydroxides; Ligands
PubMed: 23608054
DOI: 10.1016/j.ab.2013.04.011 -
Methods (San Diego, Calif.) Jan 2009Bacterial promoter identification and characterization is not as straightforward as one might presume. Promoters vary widely in their similarity to the consensus... (Review)
Review
Bacterial promoter identification and characterization is not as straightforward as one might presume. Promoters vary widely in their similarity to the consensus recognition element sequences, in their activities, and in their utilization of transcription factors, and multiple approaches often must be used to provide a framework for understanding promoter regulation. Characterization of RNA polymerase-promoter complex formation in the absence of additional regulatory factors (basal promoter function) can provide a basis for understanding the steps in transcription initiation that are ultimately targeted by nutritional or environmental factors. Promoters can be localized using genetic approaches in vivo, but the detailed properties of the RNAP-promoter complex are studied most productively in vitro. We first describe approaches for identification of bacterial promoters and transcription start sites in vivo, including promoter-reporter fusions and primer-extension. We then describe a number of methods for characterization of RNAP-promoter complexes in vitro, including in vitro transcription, gel mobility shift assays, footprinting, and filter binding. Utilization of these methods can result in determination of not only basal promoter strength but also the rates of transcription initiation complex formation and decay.
Topics: Bacteria; Binding Sites; DNA Footprinting; DNA, Bacterial; DNA-Directed RNA Polymerases; Promoter Regions, Genetic; Protein Binding; Transcription Initiation Site; Transcriptional Activation
PubMed: 18952176
DOI: 10.1016/j.ymeth.2008.10.018 -
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 -
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