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Science (New York, N.Y.) Oct 2018We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709...
We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709 transposase-accessible DNA elements that substantially extend the compendium of known cis-regulatory elements. Integration of ATAC-seq (the assay for transposase-accessible chromatin using sequencing) with TCGA multi-omic data identifies a large number of putative distal enhancers that distinguish molecular subtypes of cancers, uncovers specific driving transcription factors via protein-DNA footprints, and nominates long-range gene-regulatory interactions in cancer. These data reveal genetic risk loci of cancer predisposition as active DNA regulatory elements in cancer, identify gene-regulatory interactions underlying cancer immune evasion, and pinpoint noncoding mutations that drive enhancer activation and may affect patient survival. These results suggest a systematic approach to understanding the noncoding genome in cancer to advance diagnosis and therapy.
Topics: Chromatin; DNA Footprinting; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Genetic Loci; Genetic Predisposition to Disease; Humans; Immunity; Neoplasms; Regulatory Sequences, Nucleic Acid; Transcription Factors; Transposases
PubMed: 30361341
DOI: 10.1126/science.aav1898 -
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 -
Nature Jul 2020The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better...
The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE and Roadmap Epigenomics data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.
Topics: Animals; Chromatin; DNA; DNA Footprinting; DNA Methylation; DNA Replication Timing; Databases, Genetic; Deoxyribonuclease I; Genome; Genome, Human; Genomics; Histones; Humans; Mice; Mice, Transgenic; Molecular Sequence Annotation; RNA-Binding Proteins; Registries; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transposases
PubMed: 32728249
DOI: 10.1038/s41586-020-2493-4 -
Nature Methods Mar 2016The advent of DNA footprinting with DNase I more than 35 years ago enabled the systematic analysis of protein-DNA interactions, and the technique has been instrumental... (Review)
Review
The advent of DNA footprinting with DNase I more than 35 years ago enabled the systematic analysis of protein-DNA interactions, and the technique has been instrumental in the decoding of cis-regulatory elements and the identification and characterization of transcription factors and other DNA-binding proteins. The ability to analyze millions of individual genomic cleavage events via massively parallel sequencing has enabled in vivo DNase I footprinting on a genomic scale, offering the potential for global analysis of transcription factor occupancy in a single experiment. Genomic footprinting has opened unique vistas on the organization, function and evolution of regulatory DNA; however, the technology is still nascent. Here we discuss both prospects and challenges of genomic footprinting, as well as considerations for its application to complex genomes.
Topics: Algorithms; Chromosome Mapping; DNA; DNA Footprinting; Genome, Human; High-Throughput Nucleotide Sequencing; Humans
PubMed: 26914205
DOI: 10.1038/nmeth.3768 -
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 Aug 2021The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters, yet our understanding of their regulation...
The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.
Topics: Animals; Base Sequence; Cell Cycle Proteins; Cell Line, Tumor; Chromatin; Chromatin Assembly and Disassembly; CpG Islands; DNA Methylation; DNA-Binding Proteins; Gene Expression Regulation; Genes, Essential; Humans; Mice; Nuclear Proteins; Single Molecule Imaging
PubMed: 34234345
DOI: 10.1038/s41586-021-03689-8 -
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 -
Methods in Molecular Biology (Clifton,... 2022In-gel footprinting enables the precise identification of protein binding sites on the DNA after separation of free and protein-bound DNA molecules by gel...
In-gel footprinting enables the precise identification of protein binding sites on the DNA after separation of free and protein-bound DNA molecules by gel electrophoresis in native conditions and subsequent digestion by the nuclease activity of the 1,10-phenanthroline-copper ion [(OP)-Cu] within the gel matrix. Hence, the technique combines the resolving power of protein-DNA complexes in the electrophoretic mobility shift assay (EMSA) with the precision of target site identification by chemical footprinting. This approach is particularly well suited to characterize distinct molecular assemblies in a mixture of protein-DNA complexes and to identify individual binding sites within composite operators, when the concentration-dependent occupation of binding sites, with a different affinity, results in the generation of complexes with a distinct stoichiometry and migration velocity in gel electrophoresis.
Topics: Binding Sites; DNA; Electrophoretic Mobility Shift Assay; Protein Binding; Proteins
PubMed: 35922628
DOI: 10.1007/978-1-0716-2413-5_11 -
Plant Methods Jul 2021DNA-protein interactions are essential for several molecular and cellular mechanisms, such as transcription, transcriptional regulation, DNA modifications, among others.... (Review)
Review
DNA-protein interactions are essential for several molecular and cellular mechanisms, such as transcription, transcriptional regulation, DNA modifications, among others. For many decades scientists tried to unravel how DNA links to proteins, forming complex and vital interactions. However, the high number of techniques developed for the study of these interactions made the choice of the appropriate technique a difficult task. This review intends to provide a historical context and compile the methods that describe DNA-protein interactions according to the purpose of each approach, summarise the respective advantages and disadvantages and give some examples of recent uses for each technique. The final aim of this work is to help in deciding which technique to perform according to the objectives and capacities of each research team. Considering the DNA-binding proteins characterisation, filter binding assay and EMSA are easy in vitro methods that rapidly identify nucleic acid-protein binding interactions. To find DNA-binding sites, DNA-footprinting is indeed an easier, faster and reliable approach, however, techniques involving base analogues and base-site selection are more precise. Concerning binding kinetics and affinities, filter binding assay and EMSA are useful and easy methods, although SPR and spectroscopy techniques are more sensitive. Finally, relatively to genome-wide studies, ChIP-seq is the desired method, given the coverage and resolution of the technique. In conclusion, although some experiments are easier and faster than others, when designing a DNA-protein interaction study several concerns should be taken and different techniques may need to be considered, since different methods confer different precisions and accuracies.
PubMed: 34301293
DOI: 10.1186/s13007-021-00780-z -
Nucleic Acids Research Oct 2018Escherichia coli McrA (EcoKMcrA) acts as a methylcytosine and hydroxymethylcytosine dependent restriction endonuclease. We present a biochemical characterization of...
Escherichia coli McrA (EcoKMcrA) acts as a methylcytosine and hydroxymethylcytosine dependent restriction endonuclease. We present a biochemical characterization of EcoKMcrA that includes the first demonstration of its endonuclease activity, small angle X-ray scattering (SAXS) data, and a crystal structure of the enzyme in the absence of DNA. Our data indicate that EcoKMcrA dimerizes via the anticipated C-terminal HNH domains, which together form a single DNA binding site. The N-terminal domains are not homologous to SRA domains, do not interact with each other, and have separate DNA binding sites. Electrophoretic mobility shift assay (EMSA) and footprinting experiments suggest that the N-terminal domains can sense the presence and sequence context of modified cytosines. Pyrrolocytosine fluorescence data indicate no base flipping. In vitro, EcoKMcrA DNA endonuclease activity requires Mn2+ ions, is not strictly methyl dependent, and is not observed when active site variants of the enzyme are used. In cells, EcoKMcrA specifically restricts DNA that is modified in the correct sequence context. This activity is impaired by mutations of the nuclease active site, unless the enzyme is highly overexpressed.
Topics: Amino Acid Sequence; Binding Sites; Catalytic Domain; Cytosine; DNA Restriction Enzymes; DNA-Binding Proteins; Escherichia coli; Gene Expression Regulation, Enzymologic; Protein Binding; Protein Structure, Tertiary; Scattering, Small Angle
PubMed: 30107581
DOI: 10.1093/nar/gky731