Quantitative ChIP-Seq Normalization Reveals Global Modulation of the Epigenome

Abstract

Epigenomic profiling by chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is a prevailing methodology used to investigate chromatin-based regulation in biological systems such as human disease, but the lack of an empirical methodology to enable normalization among experiments has limited the precision and usefulness of this technique. Here, we describe a method called ChIP with reference exogenous genome (ChIP-Rx) that allows one to perform genome-wide quantitative comparisons of histone modification status across cell populations using defined quantities of a reference epigenome. ChIP-Rx enables the discovery and quantification of dynamic epigenomic profiles across mammalian cells that would otherwise remain hidden using traditional normalization methods. We demonstrate the utility of this method for measuring epigenomic changes following chemical perturbations and show how reference normalization of ChIP-seq experiments enables the discovery of disease-relevant changes in histone modification occupancy.

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

“…Recently, Pol III ChIP-seq from mouse cells under different experimental conditions was normalized by adding internal standard chromatin isolated from human cells (Bonhoure et al, 2014). Similarly, H3K79me2 ChIP from human cell lines treated with a specific Dot1L inhibitor was recently calibrated with Drosophila chromatin (Orlando et al, 2014). Although these approaches have value in making comparative measurements, the internal controls are not well defined.…”

“…Herein we demonstrate that ICeChIP measurements of histone modification density are precise, accurate, and reproducible, rendering ChIP robust to handling differences and preserving quantitative relationships between samples. Unlike other attempts to calibrate ChIP with chromatin from other organisms (Bonhoure et al, 2014; Orlando et al, 2014), our method measures actual amounts of marks using defined standards and can provide in situ evaluation of the specificity of the immunoprecipitation and a means of data correction. ICeChIP also enables direct and unbiased comparison amongst experiments: we present quantitative analyses of patterns of histone marks that reinterprets the nature of bivalent domains (Bernstein et al, 2006; Mikkelsen et al, 2007)), and suggest a correlation between the mark symmetry of promoter nucleosomes and transcriptional output.…”

Direct interrogation of the role of H3K9 in metazoan heterochromatin function

“…2E). Consequently, normalizing to sequencing depth in this case misrepresents the relative signal between the two genotypes (Orlando et al 2014). We therefore developed an alternative procedure that uses Drosophila virilis chromatin as an internal normalization control (see the Supplemental Material).…”

The Overlooked Fact: Fundamental Need for Spike-In Control for Virtually All Genome-Wide Analyses

“…As described above, a recent study successfully used the Drosophila epigenome as a normalization control for ChIP-seq analysis of H3K79me2 in human cells (7).…”