Regulation of myelin basic protein gene transcription: identification of a distal cis-acting regulatory element.

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 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 MB3, 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 MB3 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 MB3 sequence interacts with nuclear proteins from mouse brain and forms specific major C1 and a minor C2 complex. Methylation interference experiments have allowed the identification of the G-residues within nucleotides -100 to -108, named MB3a, which are distinct from the NF1/CTF of MB3 that contact with nuclear proteins to form the major C1 complex. Results from band shift studies revealed assembly of the C1 complex upon incubation of MB3 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.