The myelin basic protein (MBP) gene contains sequences located upstream of its transcription start site which play a key role in glial-specific transcription of the MBP promoter. Earlier analysis of the (320 \mathrm{bp}) upstream regulatory sequence of MBP has revealed multiple cis-acting regulatory motifs which differentially regulate transcription of a heterologous promoter fused to a reporter gene in glial and nonglial cells. In the present study, we have focused on a region designated (M B_{3}), which is located between -93 to -130 nucleotides with respect to the RNA start site, and contains a binding site for the NF1/CTF family of transcription activators. Results from DNase I footprint protection analysis of nuclear proteins prepared from mouse brain revealed a major region within the (\mathrm{MB}{3}) regulatory element that specifically interacts with the proteins derived from mouse brain at various stages of brain development. Using synthetic oligonucleotides spanning the protected region, we show that the double-stranded (M B{3}) sequence interacts with nuclear proteins from mouse brain and forms specific major (\mathrm{C}{1}) and a minor (\mathrm{C}{2}) complex. Methylation interference experiments have allowed the identification of the G-residues with in nucleotides -100 to -108 , named (\mathrm{MB}{3 a}), which are distinct from the (\mathrm{NF} 1 / \mathrm{CTF}) of (\mathrm{MB}{3}) that contact with nuclear proteins to form the major (\mathrm{C}{1}) complex. Results from band shift studies revealed assembly of the (C{1}) complex upon incubation of (\mathrm{MB}_{3} \mathrm{DNA}) with the nuclear proteins from various cells of glial origin. Site-directed mutagenesis experiments revealed that the identified G-residues for DNA-protein interaction are important to confer transcriptional activity to this domain in transiently transfected glial cells. (C) 1995 Wiley-Liss, Inc.