Mapping accessible chromatin regions using Sono-Seq

Abstract

Disruptions in local chromatin structure often indicate features of biological interest such as regulatory regions. We find that sonication of cross-linked chromatin, when combined with a sizeselection step and massively parallel short-read sequencing, can be used as a method (Sono-Seq) to map locations of high chromatin accessibility in promoter regions. Sono-Seq sites frequently correspond to actively transcribed promoter regions, as evidenced by their co-association with RNA Polymerase II ChIP regions, transcription start sites, histone H3 lysine 4 trimethylation (H3K4me3) marks, and CpG islands; signals over other sites, such as those bound by the CTCF insulator, are also observed. The pattern of breakage by Sono-Seq overlaps with, but is distinct from, that observed for FAIRE and DNase I hypersensitive sites. Our results demonstrate that Sono-Seq can be a useful and simple method by which to map many local alterations in chromatin structure. Furthermore, our results provide insights into the mapping of binding sites by using ChIP-Seq experiments and the value of reference samples that should be used in such experiments.ChIP-Seq ͉ ENCODE ͉ formaldehyde cross-linking ͉ sonication ͉ DNA sequencing T he accessibility of regulatory elements in chromatin is critical for many aspects of gene regulation. Nucleosomes positioned over regulatory elements inhibit access of transcription factors to DNA; deprotection of the DNA arises from local changes in chromatin conformation. Previous methods for mapping chromatin accessibility include mapping DNase I hypersensitivity sites or formaldehyde-assisted isolation of regulatory elements (FAIRE) regions and analyzing the DNA using microarrays or DNA sequencing (1-3). These methods have mapped many open chromatin sites to promoters of actively transcribed genes as well as to enhancers.The in vivo mapping of regulatory elements is often performed by chromatin immunoprecipitating of a factor of interest followed by analyzing the associated DNA (4-6). Chromatin complexes are preserved through cell fixation with formaldehyde, the chromatin is fragmented, and protein-bound DNA regions are isolated by using antibodies to a specific DNA-associated protein. DNA fragments are purified and used to probe DNA microarrays (ChIPchip) or, more recently, identified by high-throughput DNA sequencing (ChIP-Seq), thereby locating transcription factor binding sites (TFBSs) on a genome-wide scale (4-7). In ChIP experiments, significant targets representing binding regions are found by analyzing signal levels produced by an experimental sample relative to a reference sample. Although several automated scoring algorithms exist for ChIP-Seq data (6,(8)(9)(10)(11), an appreciation of the characteristics and biases inherent to different reference DNA samples and preparation methods is important for understanding the significance of the results obtained.In the work presented here, we examine the signal distributions of commonly used reference samples including sonicated chromatin and investigate the a...

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