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.