Luciferase Assay System TECHNICAL NOTE : High-throughput functional analysis of 528 STAT binding sites in the human genome

The ENCODE consortium has generated binding maps for the STAT1 and STAT2 transcription factors during interferon stimulation using the ChIP-seq method. In this study we wished to functionally characterize and map the functional motifs of all of the STAT1 and STAT2 binding sites in the human genome identified by ChIP-seq. To accomplish this we used 528 reporter constructs representing all of the STAT binding sites. We identified nearly 200 DNA elements that were inducible by either interferon alpha or gamma. Furthermore we mutagenized nearly 300 5-10 bp motifs to map at high resolution the functional motifs involved in STAT signaling. The results show that there are STAT-bound elements that respond preferentially to either IFNa or IFNg. The results also show that the strongest interferon responses come from proximal promoter regions that are closest to transcription start sites of genes. Lastly, we used high-throughput site-directed mutagenesis to map the functional motifs with base pair resolution.