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Calibrating ChIP-seq with nucleosomal internal standards to measure histone modification density genome-wide

ABSTRACT: Despite serving as a central experimental technique in epigenetics research, chromatin immunoprecipitation (ChIP) suffers from several serious drawbacks: it is a relative measurement untethered to any external scale that obviates fair comparison amongst experiments; it employs antibody reagents that have differing affinity and specificity for target epitopes, which are in turn variable in abundance; and it is frequently not reproducible. To address these problems, we developed internal standard calibrated ChIP (ICeChIP), a method of spiking a native chromatin sample with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons and revealing a correlation between the apparent symmetry of H3K4me3 in promoter nucleosomes and gene expression. Direct in situ assessment of immunoprecipitation accommodates for a number of experimental pitfalls, and provides a critical examination of untested assumptions inherent in conventional ChIP.

ORGANISM(S): Mus musculus synthetic construct Homo sapiens Drosophila melanogaster

PROVIDER: GSE60378 | GEO | 2015/05/07

SECONDARY ACCESSION(S): PRJNA258581

REPOSITORIES: GEO

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Calibrating ChIP-seq with nucleosomal internal standards to measure histone modification density genome-wide

Project description:Despite serving as a central experimental technique in epigenetics research, chromatin immunoprecipitation (ChIP) suffers from several serious drawbacks: it is a relative measurement untethered to any external scale that obviates fair comparison amongst experiments; it employs antibody reagents that have differing affinity and specificity for target epitopes, which are in turn variable in abundance; and it is frequently not reproducible. To address these problems, we developed internal standard calibrated ChIP (ICeChIP), a method of spiking a native chromatin sample with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons and revealing a correlation between the apparent symmetry of H3K4me3 in promoter nucleosomes and gene expression. Direct in situ assessment of immunoprecipitation accommodates for a number of experimental pitfalls, and provides a critical examination of untested assumptions inherent in conventional ChIP. Examination of spiked-in semi-synthetic nucleosomes in ICeChIP-seq experiments performed for HEK293, mESC E14 and DM S2 cell line

2015-05-07 | E-GEOD-60378 | biostudies-arrayexpress

ChIP-Seq analysis of estrogen deprived MCF7 cells treated with H3B-5942 and standards of care compounds

Project description:The goal of this experiment was to interrogate the potential transcriptional impact of H3B-5942 by investigating its influence on genome-wide DNA-binding modes of ERa in MCF7 cells using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-Seq). Affects of H3B-5942 on chromatin recruitment were compared to results from treatment with E2, 4-OHT, and fulvestrant.

2018-06-12 | GSE115607 | GEO

Quantitative ChIP-MS analysis of proteins associated with H3K4me1- and H3K4me3-enriched mono-nucleosomes purified from crosslinked IMR-90 cells

Project description:Here we used ChIP-MS to quantitatively profile chromatin-associated proteins that are specifically associated with H3K4me1- and H3K4me3-modified nucleosomes in IMR-90 chromatin.

2023-12-10 | PXD042826 | Pride

Genome-wide mapping of histone marks at single-nucleosome resolution

Project description:Histone post-translational modifications (PTM) encode much of genome's regulatory information. However, regular chromatin immunoprecipitation followed by sequencing (ChIP-seq) protocols trace histone PTM to genomic regions spanning several nucleosomes, making it difficult to study how different histone PTM combine within individual nucleosomes to regulate the genome. Here, we devised computational and statistical methods to map H3K4me3, H3K27Ac, H3K9me3, and H3K27me3 to individual nucleosomes using MNase digestion of chromatin coupled with ChIP-seq. A significant number of nucleosomes were marked by two or more of histone marks. Nucleosomes marked simultaneously by H3K4me3 and H3K27me3 were prevalent among bivalent domains compared to the genomic background whereas nucleosomes having the repressive marks H3K27me3 and H3K9me3 were enriched at the transcription staring site of highly active genes only if they were also co-localized with the activating mark H3K27Ac. Inclusion of alternatively spliced exons on the final mRNA is correlated with nucleosomes marked by H3K4me3, H3K27Ac, and H3K27me3, but is largely unaffected by nucleosomes marked by H3K9me3. Together, these findings reveal that combinatorial patterns of histone PTM within individual nucleosomes are fundamental to encode regulatory information.

2018-09-14 | GSE73004 | GEO

ChIP-Seq of SA1, SA2, and Rad21 in RPE-1 and CHM13 cells

Project description:The biorientation of sister chromatids on the mitotic spindle, essential for accurate sister chromatid segregation, relies on critical centromere components including cohesin, the centromere-specific H3 variant CENP-A, and centromeric DNA. Centromeric DNA is highly variable between chromosomes yet must accomplish a similar function. Moreover, how the 50 nm cohesin ring, proposed to encircle sister chromatids, accommodates inter-sister centromeric distances of hundreds of nanometers on the metaphase spindle is a conundrum. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We used ChIP-seq and super-resolution microscopy to examine the geometry of essential centromeric components on human chromosomes. ChIP-seq of SA1, SA2, and Rad21 in human cells demonstrates that cohesin subunits are depleted in -satellite arrays where CENP-A nucleosomes and kinetochores assemble. Cohesin is instead enriched at pericentromeric DNA. Structured illumination microscopy of sister centromeres is consistent, revealing a non-overlapping pattern of CENP-A and cohesin.

2023-10-11 | GSE240957 | GEO

Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq

Project description:Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein’s distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C.glabrata cells (the calibration genome) with S.cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this “internal standard” calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using PCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics. Develop a method for quantitative ChIP-seq

2015-07-10 | E-GEOD-69907 | biostudies-arrayexpress

Asymmetric nucleosomes flank promoters in the budding yeast genome

Project description:Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that nucleosomes at 5% of budding yeast nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling. We have analyzed the chromatin landscape of the yeast genome using paired-end MNase-seq and the chromatin binding of yeast remodelers Swr1, Ino80 and RSC at base-pair resolution using native chromatin immunoprecipitation followed by sequencing (N-ChIP-seq).

2014-12-04 | E-GEOD-59523 | biostudies-arrayexpress

Substrate selectivity for semisynthetic CK2 proteins

Project description:This SuperSeries is composed of the following subset Series: GSE33149: Substrate selectivity for semisynthetic CK2 proteins with various posttranslational modifications GSE33150: Substrate selectivity for semisynthetic CK2 proteins with Pin1 Refer to individual Series

2011-10-24 | E-GEOD-33151 | biostudies-arrayexpress

Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq

2015-07-10 | GSE69907 | GEO

Mus musculus strain:ICR | breed:Japan_SLC

Project description:ChIP-seq of moue sperm nucleosomes

| PRJNA348162 | ENA