Ž . Molecular Brain Research 52 1997 98–111 Research report Sample and probe: a novel approach for identifying development-specific cis-elements of the enkephalin gene Albert Dobi a , Miklos Palkovits b, ) , Mary A. Ring a , Alex Eitel a , Christina G. Palkovits a , Francis Lim a , Denes v. Agoston a a Unit on Molecular Control of Neurodifferentiation, LDN, NICHD, NIMH, NIH, Building 49, Room 5A38, Bethesda, MD 20892, USA b Section of Genetics, NIMH, NIH, Bethesda, MD 20892, USA Accepted 1 July 1997 Abstract 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 Ž . 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 Ž . region identified a binding site GACGGGAGATCGCTCGT which is similar to the motif for a lymphoid-specific, developmentally Ž. regulated transcription factor, Ikaros, suggesting that the developing brain expresses Ikaros-like transcription factor s in a spatiotempo- rally 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. q 1997 Elsevier Science B.V. Keywords: Enkephalin gene; Regulation; Development; Microdissection; Protein–DNA interaction 1. Introduction A fundamental question in developmental neurobiology is how seemingly identical multipotent neuronal precursors differentiate into the large number of diverse phenotypes Ž w x. of the adult brain e.g. 19,36,37 . More specifically, what are the genetic and epigenetic clues that initiate the differ- entiation program? What are the signaling pathways and molecules that guide? And finally, at the level of gene transcription, which DNA-binding proteins and their cog- nate DNA elements are involved in executing phenotypic differentiation? One of the major obstacles in elucidating the mecha- nism of transcriptional regulation in the developing mam- malian brain is its extraordinary complexity. In order to identify cis- and trans-acting elements that regulate the development of this complexity, we have developed a Ž . novel approach using the enkephalin ENK gene as a ) Ž . Corresponding author. Fax: q1 301 402-3149; E-mail: vagoston@helix.nih.gov model. Our working hypothesis is that successive protein– DNA interactions are necessary to restrict ENK gene expression to specific subsets of developing neurons, and Ž some of these interactions correlate negatively or posi- . tively to ENK expression. Consequently, our method is based on a systematic spatiotemporal analysis of develop- Ž mentally-specific protein–DNA interactions ‘sample and . Ž . probe’ as detected by mobility shift assays MSA and DNase I footprinting. The various ENK peptides are neurotransmitters mediat- Ž ing diverse physiological functions i.e. reward, motiva- w x. tion, neuroimmune interactions, etc.; for review, see 41 and pathological conditions such as Huntington’s and w x Parkinson’s diseases 5,29,40,47 , schizophrenia w x w x w x 5,29,40,47 , autism 24 , alcoholism 17,22,23,39,45,54 w x and drug addiction 27,32 . Enkephalinergic neurons populate the brain in spatially well defined manner, i.e. the caudate nucleus, hypothala- mus contain large population of enkephalinergic neurons medulla oblongata, spinal cord, hippocampus and the cere- bral cortex contain medium to low numbers and globus 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.