High-resolution mapping of transcription factor binding sites on native chromatin
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ABSTRACT:
PROVIDER: PRJNA195609 | ENA |
REPOSITORIES: ENA
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Project description:Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites. Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.
2013-12-14 | E-GEOD-45672 | biostudies-arrayexpress
Project description:Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites.
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Project description:Mapping ultra high resolution of Brachyury:DNA interaction would provide us with valuable new mechanistic insights into complex molecular transactions at Brachyury-bound enhancers. Embryonic stem cells were differentiated into Brachyury-positive mesoendoderm cells. And, ChIP-exo experiment was then performed to identify detailed Brachyury-DNA binding profiles.
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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).
2014-02-06 | GSE40664 | GEO
Project description:Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, Cut-and-Run is performed in situ, allowing for both high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, Cut-and-Run yielded precise transcription factor profiles while avoiding cross-linking and solubilization issues. Cut-and-Run is simple to perform and at low temperatures is inherently robust, with extremely low backgrounds that make it especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, Cut-and-Run mapped high-resolution 3D directional interactions. We conclude that Cut- and-Run is a suitable complement or replacement for ChIP-seq that can also provide 3D mapping information.
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Project description:Polycomb-mediated chromatin repression modulates gene expression during development in metazoans. Binding of multiple sequence-specific factors at discrete Polycomb Response Elements (PREs) is thought to recruit repressive complexes that spread across an extended chromatin domain. To dissect the structure of PREs, we applied high-resolution mapping of non-histone chromatin proteins in native chromatin of Drosophila cells. Analysis of occupied sites reveal cooperative interactions between transcription factors that stabilize Polycomb anchoring to DNA, and implicate the general transcription factor Adf1 as a novel PRE component. By comparing two Drosophila cell lines with differential chromatin states, we provide evidence that repression is accomplished at multiple steps in transcription, including inactivation of distant enhancers, enhanced Polycomb recruitment to PREs and target promoters, and elevated stalling of RNAPII in repressed genes. These results suggest that the stability of complexes bridging promoters, enhancers, and PREs is a crucial aspect of developmentally regulated gene expression. Native chromatin immunoprecipitation of histones, transcription factors and Polycomb protein in Drosophila cell lines.
2014-02-08 | E-GEOD-47829 | biostudies-arrayexpress