Project description:Genome-wide mapping of proteinM-bM-^@M-^SDNA interactions is essential for a full understanding of transcriptional regulation. A precise map of binding sites for transcription factors, core transcriptional machinery is vital for deciphering the gene regulatory networks that underlie various biological processes. Chromatin immunoprecipitation followed by sequencing (ChIPM-bM-^@M-^Sseq) is a technique for genome-wide profiling of DNA-binding proteins. However, our conventional ChIPM-bM-^@M-^Sseq occasionally gives wider peaks which might be due to overlapping binding sites of two or more transcription factors. Therefore, to improve the resolution of our conventional ChIPM-bM-^@M-^Sseq which have DNA-protein footprint of ~100 bp, we decreased the size of DNA-protein footprint to ~ 50 bp by DNaseI digestion of whole cell extract (WCE). ChIP-seq for Twist transcription factor in Drosophila embryos
Project description:Base-pair level resolution of DNase susceptibility of the native chromatin state. These maps represent and are dependent upon the nature and the specificity of interaction of the DNA with the regulatory/modulatory proteins binding at specific loci in the genome; thus they represent the native chromatin state of the genome under investigation. The deep sequencing approach has been used to define the footprint landscape of the genome by identifying DNA motifs that interact with known or novel DNA-binding proteins 2 replicates of the erythroid cells were generated and profiled by Dnase I.
Project description:Genome-wide mapping of protein–DNA interactions is essential for a full understanding of transcriptional regulation. A precise map of binding sites for transcription factors, core transcriptional machinery is vital for deciphering the gene regulatory networks that underlie various biological processes. Chromatin immunoprecipitation followed by sequencing (ChIP–seq) is a technique for genome-wide profiling of DNA-binding proteins. However, our conventional ChIP–seq occasionally gives wider peaks which might be due to overlapping binding sites of two or more transcription factors. Therefore, to improve the resolution of our conventional ChIP–seq which have DNA-protein footprint of ~100 bp, we decreased the size of DNA-protein footprint to ~ 50 bp by DNaseI digestion of whole cell extract (WCE).
Project description:Base-pair level resolution of DNase susceptibility of the native chromatin state. These maps represent and are dependent upon the nature and the specificity of interaction of the DNA with the regulatory/modulatory proteins binding at specific loci in the genome; thus they represent the native chromatin state of the genome under investigation. The deep sequencing approach has been used to define the footprint landscape of the genome by identifying DNA motifs that interact with known or novel DNA-binding proteins
Project description:Nucleosomes are the basic unit of packaging of eukaryotic chromatin, and nucleosome positioning can differ substantially between cell types. Here, we sequence 14.5 billion plasma-borne cell-free DNA (cfDNA) fragments (700-fold coverage) to generate genome-wide maps of in vivo nucleosome occupancy. We identify 13 million local maxima of nucleosome protection, spanning 2.53 gigabases (Gb) of the human genome, whose positions and spacings correlate with nuclear architecture, gene structure and gene expression. We further show that short cfDNA fragments - poorly recovered by standard protocols - directly footprint the in vivo occupancy of DNA-bound transcription factors such as CTCF. The sequence composition of cfDNA has previously been used to noninvasively monitor cancer, pregnancy and organ transplantation, but a key limitation of this paradigm is its dependence on genotypic differences to distinguish between contributing tissues. We show that nucleosome spacing in gene bodies and cis-regulatory elements, inferred from cfDNA in healthy individuals, correlates most strongly with transcriptional and 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 in vivo nucleosome footprints can be used to infer the cell types that contribute to circulating cfDNA in pathological states such as cancer. Because it does not rely on genotypic differences, this strategy may enable the noninvasive cfDNA-based monitoring of a much broader set of clinical conditions than is currently possible. Sequencing of cfDNA libraries from healthy individuals, pooled healthy individuals and individuals with disease for the identification of nucleosomes and protection from other DNA binding proteins.
Project description:DNA are packaged into nucleosomes and chromatin. We performed incomplete MNase digestion of chromatin to identify nucleosome-free regions that may indicate active promoters and regulatory regions. Additionally, HpaII digestion was performed which cleaves CCGG sites when the internal C remains unmodified. The hypomethylation state and nuclease sensitivity of the chromatin are indicators of transcription regulatory regions.
Project description:The Pseudomonas aeruginosa quorum-sensing (QS) systems contribute to bacterial homeostasis and pathogenicity. Although many regulators have been characterized to control the production of virulence factors and QS signaling molecules, its detailed regulatory mechanisms still remain elusive. Here, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) on 10 key QS regulators. The direct regulation of these genes by corresponding regulator has been confirmed by Electrophoretic mobility shift assays (EMSAs) and quantitative real-time polymerase chain reactions (qRT-PCR). Binding motifs are found by using MEME suite and verified by footprint assays in vitro. Collectively, this work provides new cues to better understand the detailed regulatory networks of QS systems. ChIP-seq of 10 QS regulators in Pseudomonas aeruginosa
Project description:B-cell lineage acute lymphoblastic leukemia (B-ALL) is comprised of diverse molecular subtypes and while transcriptional and DNA methylation profiling of B-ALL subtypes has been extensively examined, the accompanying chromatin landscape is not well characterized for many subtypes. We therefore mapped chromatin accessibility using ATAC-seq for 10 B-ALL molecular subtypes in primary ALL cells from 154 patients. Comparisons with B-cell progenitors identified candidate B-ALL cell-of-origin and AP-1-associated cis-regulatory rewiring in B-ALL. Cis-regulatory rewiring promoted B-ALL-specific gene regulatory networks impacting oncogenic signaling pathways that perturb normal B-cell development. We also identified that over 20% of B-ALL accessible chromatin sites exhibit strong subtype enrichment, with transcription factor (TF) footprint profiling identifying candidate TFs that maintain subtype-specific chromatin architectures. Over 9000 inherited genetic variants were further uncovered that contribute to variability in chromatin accessibility among individual patient samples. Overall, our data suggest that distinct chromatin architectures are driven by diverse TFs and inherited genetic variants which promote unique gene regulatory networks that contribute to transcriptional differences among B-ALL subtypes.