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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 -
Cell Apr 2018Fetal hemoglobin (HbF, αγ) level is genetically controlled and modifies severity of adult hemoglobin (HbA, αβ) disorders, sickle cell disease, and β-thalassemia....
Fetal hemoglobin (HbF, αγ) level is genetically controlled and modifies severity of adult hemoglobin (HbA, αβ) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.
Topics: Base Sequence; Binding Sites; Carrier Proteins; Cell Line; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Erythroid Cells; Fetal Hemoglobin; Gene Editing; Humans; Nuclear Proteins; Promoter Regions, Genetic; Protein Isoforms; Repressor Proteins; Zinc Fingers; beta-Globins; beta-Thalassemia; gamma-Globins
PubMed: 29606353
DOI: 10.1016/j.cell.2018.03.016 -
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 -
Neuron Jun 2015Neuronal diversity is essential for mammalian brain function but poses a challenge to molecular profiling. To address the need for tools that facilitate...
Neuronal diversity is essential for mammalian brain function but poses a challenge to molecular profiling. To address the need for tools that facilitate cell-type-specific epigenomic studies, we developed the first affinity purification approach to isolate nuclei from genetically defined cell types in a mammal. We combine this technique with next-generation sequencing to show that three subtypes of neocortical neurons have highly distinctive epigenomic landscapes. Over 200,000 regions differ in chromatin accessibility and DNA methylation signatures characteristic of gene regulatory regions. By footprinting and motif analyses, these regions are predicted to bind distinct cohorts of neuron subtype-specific transcription factors. Neuronal epigenomes reflect both past and present gene expression, with DNA hyper-methylation at developmentally critical genes appearing as a novel epigenomic signature in mature neurons. Taken together, our findings link the functional and transcriptional complexity of neurons to their underlying epigenomic diversity.
Topics: Animals; Cell Nucleolus; Chromatin Immunoprecipitation; DNA Methylation; Epigenomics; Gene Expression Profiling; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Neocortex; Nerve Tissue Proteins; Neurons; Proto-Oncogene Proteins c-myc
PubMed: 26087164
DOI: 10.1016/j.neuron.2015.05.018 -
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 -
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 -
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 -
ELife Dec 2020Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved...
Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.
Topics: Acetylation; Binding Sites; Chromatin; DNA; Epigenesis, Genetic; High-Throughput Nucleotide Sequencing; Histones; Humans; K562 Cells; Nucleic Acid Conformation; Nucleosomes; Proof of Concept Study; Protein Conformation; Protein Processing, Post-Translational; Single Molecule Imaging; Site-Specific DNA-Methyltransferase (Adenine-Specific); Transcription Factors
PubMed: 33263279
DOI: 10.7554/eLife.59404