REGULATION OF MYELIN BASIC-PROTEIN GENE-TRANSCRIPTION - IDENTIFICATION OF A DISTAL CIS-ACTING REGULATORY ELEMENT

The myelin basic protein (MBP) gene contains sequences located upstrea m of its transcription start site which play a key role in glial-speci fic transcription of the MBP promoter. Earlier analysis of the 320 bp upstream regulatory sequence of MBP has revealed multiple cis-acting r egulatory motifs which differentially regulate transcription of a hete rologous promoter fused to a reporter gene in glial and nonglial cells . In the present study, we have focused on a region designated MB(3), which is located between -93 to -130 nucleotides with respect to the R NA start site, and contains a binding site for the NF1/CTF family of t ranscription activators. Results from DNase I footprint protection ana lysis of nuclear proteins prepared from mouse brain revealed a major r egion within the 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 r egion, we show that the double-stranded MB(3) sequence interacts with nuclear proteins from mouse brain and forms specific major C-1 and a m inor C-2 complex. Methylation interference experiments have allowed th e identification of the C-residues within nucleotides -100 to -108, na med MB(3a), which are distinct from the NF1/CTF of MB(3) that contact with nuclear proteins to form the major C-1 complex. Results from band shift studies revealed assembly of the C-1 complex upon incubation of MB(3) DNA with the nuclear proteins from various cells of glial origi n. Site-directed mutagenesis experiments revealed that the identified G-residues for DNA-protein interaction are important to confer transcr iptional activity to this domain in transiently transfected glial cell s. (C) 1995 Wiley-Liss, Inc.