Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing.
Microarray and next-generation sequencing techniques which allow whole genome analysis of chromatin structure and sequence-specific protein binding are revolutionizing our view of chromosome architecture and function. However, many current methods in this field rely on biochemical purification of highly specific fractions of DNA prepared from chromatin digested with either micrococcal nuclease or DNaseI and are restricted in the parameters they can measure. Here, we show that a broad size-range of genomic DNA species, produced by partial micrococcal nuclease digestion of chromatin, can be sequenced using paired-end mode next-generation technology. The paired sequence reads, rather than DNA molecules, can then be size-selected and mapped as particle classes to the target genome. Using budding yeast as a model, we show that this approach reveals position and structural information for a spectrum of nuclease resistant complexes ranging from transcription factor-bound DNA elements up to mono- and poly-nucleosomes. We illustrate the utility of this approach in visualizing the MNase digestion landscape of protein-coding gene transcriptional start sites, and demonstrate a comparative analysis which probes the function of the chromatin-remodelling transcription factor Cbf1p.
Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AX, UK. kentn@cardiff.ac.uk
Nicholas A
NA
Kent
Steffan
S
Adams
Alex
A
Moorhouse
Konrad
K
Paszkiewicz
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000032:analysis; D002843:Chromatin / Q000737:chemistry; D042002:Chromatin Assembly and Disassembly; D004268:DNA-Binding Proteins / Q000032:analysis; D059014:High-Throughput Nucleotide Sequencing; D008836:Micrococcal Nuclease; D009707:Nucleosomes / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis; D017422:Sequence Analysis, DNA / Q000379:methods*; D024363:Transcription Initiation Site
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D002843:Chromatin; D004268:DNA-Binding Proteins; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins; D008836:Micrococcal Nuclease