Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

Abstract

BackgroundDifferential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays.ResultsHere we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition.ConclusionsUsing an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1104) contains supplementary material, which is available to authorized users.

Biased visibility in Hi-C datasets marks dynamically regulated condensed and decondensed chromatin states genome-wide

“…To study the dynamics of gene regulatory activity in response to environmental and developmental clues, mapping chromatin accessibility remained an important task over decades. While DNase-I has been a preferred choice to digest the open chromatin due to relatively lesser sequence specificity of the enzyme, only few studies attempted restriction endonucleases to identify accessible regions in the chromatin [46,54,55]. On the contrary, restriction endonucleases have been extensively used to digest the chromatin, presumably in an unbiased manner, in proximity ligation based techniques like 3C, 4C, 5C and Hi-C [6][7][8][9].…”

“…Regional depletions in read counts obtained from digested chromatin when compared with the reads obtained from digested naked DNA would mark the biased restriction digestion of chromatin. Towards this, we obtained the 'Restriction Endonuclease Digestion coupled with sequencing' (REDseq) data of in-situ restriction digested chromatin and in-solution restriction digested naked DNA of mouse embryonic stem cells (mESC) from Chen et al [46]. We calculated the read counts for 10 kb bins of the mouse genome and normalized by the total reads.…”

Biased visibility in HiC datasets marks dynamically regulated condensed and decondensed chromatin states genome-wide

“…To study the dynamics of gene regulatory activity in response to environmental and developmental clues, mapping chromatin accessibility remained an important task over decades. While DNase-I had been a preferred choice to digest the chromatin due to relatively lesser sequence specificity of the enzyme, only few studies had attempted restriction endonucleases to identify accessible regions in the chromatin 41,47,48 . On the contrary, restriction endonucleases have been extensively used to digest the chromatin, presumably in an unbiased manner, in proximity ligation based techniques like 3C, 4C, 5C and HiC [6][7][8][9] .…”

“…We first showed that the in-situ restriction digestion of chromatin was not uniform in the genome. Towards this, we obtained the sequencing data for in-situ restriction digested chromatin and in-solution restriction digested naked DNA of mouse embryonic stem cells (mESC) 41 . We calculated the read counts for 10 kb bins of the mouse genome and normalized by the total reads.…”

Deconvolving nuclesome binding energy from experimental errors

“…Other experimental techniques measure nucleosome binding genome-wide, e.g. MPE-Seq (Ishii, Kadonaga, and Ren 2015), RED-Seq (Chen et al 2014), NOMe-Seq (Kelly et al 2012); nucleosome binding at individual loci, e.g. by electron microscopy (Brown et al 2013), by DNA methylation (Small et al 2014); or nucleosome binding to short DNA fragments in vitro, e.g.…”