Rapid In Vivo Mapping of Hematopoietic Cis-Regulatory Elements.

Cis-acting sequences play defining roles in the control of genes involved in hematopoiesis. However, for the vast majority of such genes, regulatory sequences remain to be defined. We describe a powerful, generic approach to identification of cis-regulatory sequences that may be applied to any gene locus in the context of any cell type. We used densely tiled primers and a novel real-time PCR-based assay to create continuous, high-resolution quantitative profiles of in vivo chromatin structure across entire gene domains. Such profiles can be analyzed using robust statistical algorithms to pinpoint disruptions in chromatin structure that are characteristic of cis-regulatory elements. We analyzed >1Mb of human genomic terrain from diverse gene loci in the context of several hematopoietic cell lines and cleanly delineated a spectrum of classical cis-regulatory activities including enhancers, promoters, insulators, and locus control regions. The approach displayed outstanding sensitivity (100%) and specificity (>99.6%) for known elements and was successful in defining novel elements even in heavily-explored terrain such as the alpha- and beta-globin loci. Since only small quantities of cells are required, the approach can be used readily in the context of hematopoietic progenitors. Systematic application of quantitative chromatin profiling to relevant genes promises to expand dramatically our understanding of the regulation of hematopoiesis.