The developmental regulation of phenotypic diversity in the mammalian central nervous system (CNS) was studied using the enkephalin phenotype as a model at the molecular and cellular level with special emphasis: a) on the identification and function of cis- and trans-acting elements of the rat enkephalin (ENK) gene; b) on understanding the involvement of DNA- and nuclear structure in this regulation; and c) on identification of cellular interactions and extracellular signals that induce phenotypic differentiation. The three components of the putative early (E12-E16) phenotypic specifier complex of the ENK gene (septamer-binding complex) were identified and characterized; the cDNA of the 43 kD member (ABP-43) of the late repressor complex (E21-P2) of the ENK gene that binds to the motif (ATT)19 encodes a novel nuclear protein; the whole ATT binding protein complex was translocated from the soluble nuclear protein fraction to the nuclear matrix fraction in a development-specific manner; the predicted development- and cell-specific repressor function of this ATT-protein complex was verified in vivo using various DNA molecular decoys in primary differentiating neurons; a caudate nucleus specific protein (CSP) that binds to a novel activator motif of the ENK gene was identified; this protein specifically binds to intranuclear actin that can act as molecular chaperone for CSP. Genomic DNA was found to bind calcium with high affinity (DNA>calmodulin or BAPTA) and induce specific structural changes depending on the DNA's superhelical density; differentially phosphorylated intranuclear proteins were competing with the DNA for calcium binding. The enkephalin phenotype was selectively regulated by specific cell-cell interactions that mimicked major basal ganglia connectivities. A new method (in situ DNA binding assay-ISDBA) was developed that enables spatial analysis of specific protein-DNA interactions. Intranuclear calcium was quantified in differentiating neurons using X-ray-based element microanalysis at the EM level that revealed """"""""calcium hot spots"""""""" exclusive to heterochromatin. Myelin gene expression was found to be induced and regulated by electrical / neuronal activity in a frequency dependent manner in the differentiating CNS. Astroglia derived from the Down syndrome/Alzheimer model TS16 was found to depress cholinergic functions. ENK gene regulation in neonatal brain injury model was identified as age-dependent derepression through the (ATT) repeat. The HIV repressor protein (TDP-43) of unknown normal cellular function interacts with the (ATT) repeat of the ENK gene in a development-specific manner.
