Isolation and analysis of DNA derived from nucleosome-free regions.

Precise regulation of the levels and timing of gene expression is fundamental to all biological processes and is largely determined by the activity of cis-regulatory modules, containing the binding sites for transcription factors, within promoters and enhancers. The global identification of these transcriptional regulatory elements within mammalian genomes, and understanding when and where they are active, is an important effort that will require the development and implementation of several different technologies. Here we detail a means for the identification of transcriptional regulatory elements using functional assays. The success of this approach relies on focusing the functional assay on DNA derived from nucleosome-free regions (NFRs), i.e., the 2% of the genome within a given cell in which regulatory elements reside. Accordingly, we present a simple method for isolating NFR DNA, and a functional assay that can be used for the identification of promoter and enhancers components within this population.