Genome-wide map of nucleosomes and sub-nucleosomes in human cells with a novel nuclease-based chromatin fragmentation strategy

In each cell, 2 meters of DNA is wrapped into about a 10 μM diameter space called the nucleus. DNA is coiled around histone octamers, collectively called a nucleosome. The position of nucleosomes determines what genes are accessible to nuclear proteins. We have devised a novel method in which a nuclease chews accessible DNA around these structures to determine their location in higher-resolution and to greater completion than previously published. A map of nucleosomes and sub-nucleosome particles genome-wide will provide a chromatin landscape for then surveying the location of transcription factors and otherchromatin complexes via nuclease-ChIP-exo (below)..

A streamlined ChIP-exo 6.1 assay measures same-molecule positional relationship between transcription factors and nucleosomes in vivo

Transcription factors (ssTFs) bind specific DNA sequences in promoter and enhancer regions in the context of an organized nucleosome architecture. There they recruit cofactors that regulate chromatin accessibility and transcription complex assembly. ChIP-exo is a high-resolution genome-wide assay that measures protein-DNA interactions with single-bp precision. Here we couple the precision of ChIP-exo with a nuclease chromatin fragmentation strategy that cleaves and marks rotationally-exposed DNA on nucleosomal surfaces. This maps the positional orientation of TFs with an adjacent nucleosome surface on the same DNA molecule in human cells. Additional modifications to the ChIP-exo assay present here (version 6.1) greatly simplify library construction over prior versions and provides a facile, high-precision means to map genomic TF binding sites..

Application of nuclease-ChIP-exo to mammalian tissues

Applying nuclease-ChIP-exo (6.1) to any clinical samples depends upon the ability to (1) grind different sized tissues, (2) remove embedding media if present, and (3) isolate nuclei. A method to grind <200 mg tissues while frozen was devised to minimize sample loss utilizing items available at any home improvement store. Removal of pathology-applied media is being studied as well as how to isolate nuclei from various organs. Quantifiable, purified nuclei will be an appropriate input for ChIP-exo to provide epigenomic data for clinical samples..