Impact of Chromatin Structures on DNA Processing for Genomic Analyses

Leonid Teytelman,Michael B Eisen,Michael Snyder,Oliver Zill,Philippe Lefrançois,Bilge Özaydın,Jasper Rine,Rodolfo Aramayo

doi:10.1371/journal.pone.0006700

Abstract

Chromatin has an impact on recombination, repair, replication, and evolution of DNA. Here we report that chromatin structure also affects laboratory DNA manipulation in ways that distort the results of chromatin immunoprecipitation (ChIP) experiments. We initially discovered this effect at the Saccharomyces cerevisiae HMR locus, where we found that silenced chromatin was refractory to shearing, relative to euchromatin. Using input samples from ChIP-Seq studies, we detected a similar bias throughout the heterochromatic portions of the yeast genome. We also observed significant chromatin-related effects at telomeres, protein binding sites, and genes, reflected in the variation of input-Seq coverage. Experimental tests of candidate regions showed that chromatin influenced shearing at some loci, and that chromatin could also lead to enriched or depleted DNA levels in prepared samples, independently of shearing effects. Our results suggested that assays relying on immunoprecipitation of chromatin will be biased by intrinsic differences between regions packaged into different chromatin structures - biases which have been largely ignored to date. These results established the pervasiveness of this bias genome-wide, and suggested that this bias can be used to detect differences in chromatin structures across the genome.

Highlights

Chromatin packaging affects transcription, replication, and recombination in eukaryotic organisms [1,2,3,4]
If broad domains of shearing-resistant chromatin exist in the yeast genome, we expected that such regions would be under-represented among the sequence reads in the input controls of chromatin immunoprecipitation (ChIP)-Seq experiments in which the sheared formaldehyde-crosslinked chromatin has not yet been fractionated by an antibody against a protein or modification of interest
We mapped twelve million input-Seq reads to the S. cerevisiae genome

Summary

Introduction

Replication, and recombination in eukaryotic organisms [1,2,3,4]. Given the influence of chromatin on so many biochemical processes in vivo, we wondered how the chromatin state of a locus might affect its behavior in experimental procedures. The analysis was motivated by our prior results regarding DNA shearing at the silenced mating locus HMR of Saccharomyces cerevisiae. Silenced mating cassettes at the HML and HMR loci are the yeast version of heterochromatin. A complex biological state of chromatin in vivo exercised an impact on physical manipulations of chromatin in vitro. This result led us to ask whether chromatin structures influence experimental results only at silenced mating cassettes, or more broadly in other heterochromatic regions, or even in euchromatin across the genome

Methods

Results

Conclusion