Sample and probe: a novel approach for identifying development-specific cis-elements of the enkephalin gene.

We have developed a novel `sample and probe' approach as a means to identifying specific DNA elements of the enkephalin gene that control differentiation of the enkephalinergic phenotype during neurodevelopment. The approach is a systematic spatiotemporal analysis of protein–DNA interactions; soluble nuclear proteins (`samples') prepared from microdissected regions of the developing brain are `probed' with radiolabeled DNA fragments representing various regulatory regions of the enkephalin gene. The resulting spatiotemporal `molecular maps', i.e. characteristic patterns of protein–DNA complexes showed DNA regions that harbor potential cis-elements regulating differentiation of the enkephalin phenotype at various stages of neurodevelopment. DNase I footprint analysis of such a DNA region identified a binding site (GACGGGAGATCGCTCGT) which is similar to the motif for a lymphoid-specific, developmentally regulated transcription factor, Ikaros, suggesting that the developing brain expresses Ikaros-like transcription factor(s) in a spatiotemporally defined manner. In summary, our approach offers a unique view into the chronology of coordinated protein–DNA interactions and will greatly facilitate identifying DNA elements and isolating development-specific transcription factors.