Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide

Abstract

SUMMARY Chromatin immunoprecipitation (ChIP) serves as a central experimental technique in epigenetics research, yet there are serious drawbacks: it is a relative measurement, which untethered to any external scale obscures fair comparison amongst experiments; it employs antibody reagents that have differing affinities and specificities for target epitopes that vary in abundance; and it is frequently not reproducible. To address these problems, we developed Internal Standard Calibrated ChIP (ICeChIP), wherein a native chromatin sample is spiked 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 reveals unique insight into the nature of bivalent domains. This technology provides in situ assessment of the immunoprecipitation step, accommodating for many experimental pitfalls, as well as providing a critical examination of untested assumptions inherent to conventional ChIP.

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

“…IP-MS, ICeChIP, and ChIP-seq were performed following published methods (11,28,29). Further details on the materials and methods used in this study are described in SI Appendix.…”

ChIP-seq in Studying Epigenetic Mechanisms of Disease and Promoting Precision Medicine: Progresses and Future Directions

“…Therefore, it is often difficult to directly compare different datasets due to the variability in antibody sensitivity, specificity and reproducibility, epitope 10.2217/epi-2016-0053 www.futuremedicine.com future science group ChIP-seq in studying epigenetic mechanisms of disease & promoting precision medicine: progresses & future directions Perspective abundance, as well as experimental inconsistency [64,71]. To allow comparison across different samples, mapped reads are typically normalized to the same library size, like reads or fragments per million.…”

“…A more complicated spike-in method, called ICeChIP-seq for Internal Standard Calibrated ChIPseq, adds reconstituted nucleosomes bearing a given histone modification in a concentration series (barcoded by different DNA sequences) before immunoprecipitation [71]. ICeChIP-seq estimates the spike-in enrichment based on the calibration curve of IP versus input counts from the concentration series, and uses this spike-in enrichment to calculate per-base histone modification density.…”

“…MOWChIP-seq also uses microfluidic device in ChIP [79]. Chromatin fragments are flowed [71] Add a pool of semisynthetic nucleosomes that bear a given mark in a DNA-barcoded concentration series…”

Spurlose Synthese von asymmetrisch modifizierten, bivalenten Nukleosomen

“…[17] In dieser Studie haben wir eine Reihe von asymmetrischen Nukleosomen hergestellt und dazu verwendet, den Regulationsmechanismus von PRC2 durch die Kombination von H3K4me3 und H3K27me3 zu untersuchen. Die Ergebnisse zeigen, dass H3K4me3, wenn es bereits vorher auf einem Histon vorhanden ist, PRC2 lokal inhibiert, aber dass dieser inhibitorische Effekt teilweise durch benachbartes H3K27me3 aufgehoben werden kann.…”