The molecular mechanism of phenotypic establishment was investigated by studying embryonic specific protein-DNA interactions and structural changes of the DNA during neuronal differentiation using the enkephalin (ENK) gene as model gene. DNA methylation interference footprinting was used to identify a novel (TTTGCAT) octamer-like motif (sept) and its binding protein which is dissimilar to oct-1 or oct-2, phosphorylated, brain and neuron-specific and showed strong correlation with the ontogeny of brain regions. The effect of Ca2+ on the DNA structure containing the di-nucleotide repeat (TG/AC)28 was further investigated by osmium tetroxide (OsO4) footprinting and supported earlier findings that Ca2+ could induce a concentration-dependent change in DNA structure mediated by (TG/AC)n motif which are abundant in the genome. Using rapid freeze fixation of embryonic neurons followed by electron probe X-ray microanalysis the intranuclear physiological Ca2+ concentration was determined at around 1 mM. Two brain- and development-specific (TG/AC)n repeat binding proteins were identified, one present only in E14 striatal primordium, the other is present in all regions of the embryonic brain between embryonic ages E14 and E18. A novel magnetic bead based assay system was developed for studying protein based far-distant DNA-DNA interactions. A far-upstream region (-2000 to -2800 bp relative to the start site of the rat ENK gene) showed development- and brain region-- specific, protein based interaction with the core promoter/TATA region. Within this region a novel trinucleotide repeat (ATT)19 located around - 2450 bp of rat ENK gene was identified by DNAse I footprinting interacting with the proximal promoter/TATA region. Mobility shift assay demonstrated that the (ATT)19 repeat binding protein is brain-, neuron-, development- and brain region specific and furthermore negatively correlates with ENK expression. An RT PCR based assay was established to follow the developmental changes in the expression pattern of various markers in different regions of the rat brain. A primary developing neuronal culture system in conjunction with lipofectin-based DNA transfer was established to study cis-requirements of the ENK gene and to load neurons with double stranded decoy DNA to investigate the role of identified motifs in ENK expression. Developmental changes in the methylation of the rat ENK gene were studied by PCR and a development- and tissue-specific change in DNA methylation was established. Genomic PCR analysis of various mouse and human phenotypes was performed and showed unusually low extent of polymorphism of the (TG/AC)n repeat of both mouse and human ENK gene.
