Preproenkephalin DNA-binding proteins in the rat: 5' flanking region

Various extracellular signals (i.e. transmitters, hormones, growth factors, etc.), together with their respective second-messenger pathways, regulate transmitter biosynthesis and neuronal function by altering gene expression. In this study we validated a protocol for isolating rat striatum and adrenal medullary nuclei for the purpose of extracting, identifying, and characterizing, nuclear regulatory factors which may serve a functional role in signal-transduction processes. Through gel retardation studies using a 299 base pair (bp) XmnI-SacI 32P-labeled probe (derived from the 5' untranslated region of the rat preproenkephalin gene), we show that different patterns of retained bands result from nuclear extracts derived from rat adrenal medulla and striatum (as well as from other tissue). These tissue differences may have biological significance since rat adrenal medullae have low basal enkephalin levels while the striatum has high levels of this peptide and its respective mRNA. Additionally, certain retained bands were common to both cytosolic and nuclear compartments, suggesting binding factors may be located in either cell space. An initial biochemical characterization of these factors was also undertaken. Generally, salt levels of 100 mM or more reduced factor binding while 10-50 mM sodium ion levels showed preferentially enhanced bands. Binding activity appeared optimal at pH 6.8. As all retained bands were abrogated by proteinase K treatment, these factors appear to have a significant protein component. Finally, of particular interest is that this 299 bp region contains many sequences showing over 80% sequence identity with several previously characterized transcriptional control elements (i.e. cAMP and phorbol ester inducible enhancers, GCN4, AP1, Sp1, CCAAT binding factor, ATF, and AP2). If binding is confirmed (footprint analysis) and function validated (transfection studies), the evolutionary significance of the apparent presence of gene regulatory sequences and functional element divergence of the DNA region between different species can be evaluated.