Genome-wide mapping of direct protein-DNA interactions in vivo by UV-laser ChIP-seq
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PROVIDER: PRJNA144871 | ENA |
REPOSITORIES: ENA
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Project description:Genome-wide mapping of transcription factor binding is generally performed by chemical protein-DNA crosslinking, followed by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Here we present the ChIP-seq technique based on photochemical crosslinking of protein-DNA interactions by high-intensity ultraviolet (UV) laser irradiation in living mammalian cells (UV-ChIP-seq). UV laser irradiation induces efficient and instant formation of covalent “zero-length” crosslinks exclusively between nucleic acids and proteins that are in immediate contact, thus resulting in a “snapshot” of direct protein-DNA interactions in their natural environment. We applied UV-ChIP-seq for genome-wide profiling of the sequence-specific transcriptional repressor B-cell lymphoma 6 (BCL6) in human diffuse large B-cell lymphoma (DLBCL) cells. Our approach resulted in sensitive and precise protein-DNA binding profiles, highly enriched in canonical BCL6 DNA sequence motifs. UV-ChIP-seq also revealed numerous previously undetectable BCL6 binding sites, particularly in more condensed, inaccessible areas of chromatin.
2017-11-06 | GSE103125 | GEO
Project description:Transcription factors (TFs) regulate target genes by specific interaction with DNA sequences. Detecting and understanding these interactions on molecular level is of fundamental interest in both biological and clinical context. Crosslinking mass spectrometry is a powerful tool to assist structure prediction of protein complexes by providing direct evidence for physical contacts on sub-molecular level. Yet, until now it has been limited to the study of protein-protein and protein-RNA interactions. Here, we developed a femtosecond laser induced crosslinking mass spectrometry (fLiX-MS) workflow, which allows mapping of protein-DNA contacts at single nucleotide and up to single amino acid resolution. Applying the method to in-vitro assembled nucleosomes and recombinant TF-DNA complexes, our method proved both versatile and highly specific for mapping DNA binding domains. While for Nuclear factor 1 (NF1) the detected crosslinks were in perfect agreement with previous biochemical data on DNA binding, crosslinks for the TATA box binding protein (TBP) fitted to the known crystal structure. We further identified several protein-DNA interactions for the nucleosome and TBP, which were rather distant in the crystal-structure, indicating, that our method is capable to detect structural flexibility reflected in distinct conformational states. Moreover, applying the fLiX-MS workflow to cells, we detected several bona-fide crosslinks on DNA-binding domains, proving the applicability of the method for future large scale in-vivo experiments.
2020-06-22 | PXD014898 | Pride
Project description:We report changes in the S. cerevisiae transcriptome in response to 30s irradiation with a UV laser
2019-06-18 | GSE129319 | GEO
Project description:UV laser-induced, time-resolved transcriptome resonses of Saccharomyces cerevisiae
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Project description:In vivo protein-DNA interactions connect each transcription factor with its direct targets to form a gene network scaffold. To map these protein-DNA interactions comprehensively across entire mammalian genomes, we developed a large-scale chromatin immunoprecipitation assay (ChIPSeq) based on direct ultrahigh-throughput DNA sequencing. This sequence census method was then used to map in vivo binding of the neuron-restrictive silencer factor (NRSF; also known as REST, for repressor element–1 silencing transcription factor) to 1946 locations in the human genome. The data display sharp resolution of binding position [±50 base pairs (bp)], which facilitated our finding motifs and allowed us to identify noncanonical NRSF-binding motifs. These ChIPSeq data also have high sensitivity and specificity [ROC (receiver operator characteristic) area ≥ 0.96] and statistical confidence (P < 10−4), properties that were important for inferring new candidate interactions. These include key transcription factors in the gene network that regulates pancreatic islet cell development.
2008-10-24 | GSE13047 | GEO
Project description:Identification of RNA-RNA interactions in Bacillus Subtilis by in vivo UV crosslinking
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Project description:Protein–DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein–DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by UV light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein–RNA and DNA interactions in one single experiment, we project that it will be possible to obtain completely new insights into chromatin and its regulation in the future.
2020-10-20 | PXD020290 | Pride
Project description:In vivo protein-DNA interactions connect each transcription factor with its direct targets to form a gene network scaffold. To map these protein-DNA interactions comprehensively across entire mammalian genomes, we developed a large-scale chromatin immunoprecipitation assay (ChIPSeq) based on direct ultrahigh-throughput DNA sequencing. This sequence census method was then used to map in vivo binding of the neuron-restrictive silencer factor (NRSF; also known as REST, for repressor element–1 silencing transcription factor) to 1946 locations in the human genome. The data display sharp resolution of binding position [±50 base pairs (bp)], which facilitated our finding motifs and allowed us to identify noncanonical NRSF-binding motifs. These ChIPSeq data also have high sensitivity and specificity [ROC (receiver operator characteristic) area ≥ 0.96] and statistical confidence (P < 10−4), properties that were important for inferring new candidate interactions. These include key transcription factors in the gene network that regulates pancreatic islet cell development. Experiment Overall Design: Exam NRSF binding in Jurkat T cell line
2009-08-29 | E-GEOD-13047 | biostudies-arrayexpress
Project description:SARS-CoV-2 coronavirus is one of the largest RNA viruses (26-32kb) and has emerged as a major threat to global public health. The resulting pandemic has caused global societal and economic disruptions. Here, we investigate the intramolecular and intermolecular RNA interactions of wildtype and a 382 deletion SARS-CoV-2 virus inside cells. We identified twelve potentially functional structural elements along the SARS-CoV-2 genome and observed that subgenomic viral RNA (sgRNAs) contains different structures that could be important for their functions using direct RNA sequencing. Proximity ligation sequencing experiments identified hundreds of intramolecular and intermolecular pair-wise interactions within the virus genome and between virus and host cellular RNAs. SARS-CoV-2 binds strongly to mitochondrial and nucleolar RNAs including COX1 and SNORD27, and contains 130 2’O-methylation sites along its genome. 2’O-methylation sites along the virus RNA are enriched in the UTRs and associated with increased pair-wise interactions. SARS-CoV-2 infection also results in a global decrease of 2’O-methylation sites on host mRNAs, suggesting that binding of SARS-CoV-2 to snoRNAs could be a pro-viral mechanism to sequester methylation machinery away from host RNAs to methylate the virus genome. Collectively, these studies deepen our understanding of the molecular basis of SARS-CoV-2 pathogenicity, its cellular host factors and provides a platform for targeting the virus.
2021-07-05 | GSE165005 | GEO
Project description:UV-induced DNA lesions are an important contributor to mutagenesis and cancer, but it is not fully understood how the chromosomal landscape influences UV lesion formation and repair. We have used a novel high-throughput sequencing method to precisely map UV-induced cyclobutane pyrimidine dimers (CPDs) at nucleotide resolution throughout the yeast genome. Analysis of CPD formation reveals that nucleosomal DNA having an inward rotational setting is protected from CPD lesions. In strongly positioned nucleosomes, this nucleosome 'photofootprint' overrides intrinsic dipyrimidine sequence preferences for CPD formation. CPD formation is also inhibited by DNA-bound transcription factors, in effect protecting important DNA elements from UV damage. Analysis of CPD repair revealed a clear signature of efficient transcription-coupled nucleotide excision repair. Repair was less efficient at translational positions near a nucleosome dyad and at heterochromatic regions in the yeast genome. These findings define the roles of nucleosomes and transcription factors in UV damage formation and repair. UV mapping data was analyzed for yeast cells irradiated with 125J/m2 and allowed to repair for 0hr (2 samples), 20 minutes, 1 hour, or 2 hours. Data is also included for naked DNA irradiated with UV 60 or 90 J/m2
2016-08-19 | E-GEOD-79977 | biostudies-arrayexpress