Abstract
The identification of transcriptional regulatory modules within mammalian
genomes is a prerequisite to understanding the mechanisms controlling
regulated gene expression. While high-throughput microarray- and
sequencing-based approaches have been used to map the genomic locations
of sites of nuclease hypersensitivity or target DNA sequences bound
by specific protein factors, the identification of regulatory elements
using functional assays, which would provide important complementary
data, has been relatively rare. Here we present a method that permits
the functional identification of active transcriptional regulatory
modules using a simple procedure for the isolation and analysis of
DNA derived from nucleosome-free regions (NFRs), the 2% of the cellular
genome that contains these elements. The more than 100 new active
regulatory DNAs identified in this manner from F9 cells correspond
to both promoter-proximal and distal elements, and display several
features predicted for endogenous transcriptional regulators, including
localization within DNase-accessible chromatin and CpG islands, and
proximity to expressed genes. Furthermore, comparison with published
ChIP-seq data of ES-cell chromatin shows that the functional elements
we identified correspond with genomic regions enriched for H3K4me3,
a histone modification associated with active transcriptional regulatory
elements, and that the correspondence of H3K4me3 with our promoter-distal
elements is largely ES-cell specific. The majority of the distal
elements exhibit enhancer activity. Importantly, these functional
DNA fragments are an average 149 bp in length, greatly facilitating
future applications to identify transcription factor binding sites
mediating their activity. Thus, this approach provides a tool for
the high-resolution identification of the functional components of
active promoters and enhancers.
Users
Please
log in to take part in the discussion (add own reviews or comments).