Abstract. The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS). This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J. Gordon, and K. Khalili. 1993. Mol. Cell. Biol. 13:3103-3112). Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif. Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur a. Expression ofpur a cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brainderived 39-kD protein in band shift competition assays. Cotransfection of the recombinant pur ~ expressor plasmid with MBP promoter construct indicated that Pur t~ stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur c~ to DNA within the MB1 region is crucial for this activity. Moreover, transient expression of Pur a caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells. Thus, Pur or, a sequence-specific DNAbinding protein upon binding to MB 1 regulatory region may play a significant role in determining the cell typespecific expression of MBP in brain.M YELIN basic protein (MBP) 1 is a major component of the myelin sheath composing greater than 30% of the total myelin protein in the central nervous system (CNS) (6, for review see 5). This protein has several isoforms, all of which are encoded by alternative splicing of a single major transcript from a single gene on mouse chromosome 18 (7,23,33,35,36,43). MBP is expressed in a cell type-specific manner. In the CNS, myelin formation and MBP gene expression occur in oligodendrocytes, whereas in the peripheral nervous system This work represents equal contributions of the first two authors.

The myelin basic protein (MBP) gene is expressed only in oligodendrocytes and Schwann cells, and expression follows a tightly regulated developmental time course. Cell type- and developmental stage-specific expression of the MBP gene appears to be regulated by a series of cis-acting elements located upstream of the transcription start site. The proximal element of the MBP regulatory region (MB1), located between nucleotides -14 and -50, is one of several elements participating in the programmed expression of MBP. In this report, we describe the molecular cloning and characterization of myelin gene expression factor-2 (Myef-2), a protein isolated from mouse brain that binds specifically to single-stranded DNA derived from the MB1 element and represses transcription of the MBP gene in transient transfection assay. Myef-2 mRNA is developmentally regulated in mouse brain; its peak expression occurs at postnatal day 7, prior to the onset of MBP expression. The developmental pattern of Myef-2 mRNA expression coincides with that previously described for SCIP, a POU domain transcription factor that also represses myelin basic protein expression. The myef-2 gene maps to mouse chromosome 2. The relevance of these findings for regulation of MBP gene expression and oligodendrocyte differentiation is discussed.

Transcription of the myelin basic protein (MBP) gene is regulated in a cell-type-specific and developmental stage-specific manner during myelin formation in the murine central nervous system. The 5'-flanking region of the MBP gene contains several regulatory elements that differentially contribute to the cell-type-specific transcription of MBP in cells derived from the central nervous system. The proximal element, termed MB1, which is located between nucleotides -14 and -50 with respect to the RNA start site, has previously been shown to have characteristics of a cell-type-specific enhancer element. In this study, we used band shift and UV cross-linking assays to identify DNA-binding proteins in mouse brain nuclear extract which interact with the MB1 element. Fractionation of these extracts has allowed the identification of a 38-to 41-kDa nuclear protein, derived from mouse brain tissue at the peak of myelination, which specifically binds the MB1 DNA sequence. (7,12,35,45