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.

Direct interaction between transcription factors may provide a mechanism for the regulatory function of these proteins on transcription of the responsive genes. These interactions may be facilitated if the target DNA sequences for the participant regulatory proteins are overlapped or positioned in close proximity to each other within the promoter of the responsive genes. In earlier studies, we identified a cellular protein, named Puralpha, which upon binding to the MB1 regulatory DNA sequence of the myelin basic protein (MBP) gene, stimulates its transcription in central nervous system (CNS) cells. Here, we provide evidence for binding of the ubiquitous DNA binding transcription factor, Sp1, to the MB1 DNA motif at the region that partially overlaps with the Puralpha binding site. We demonstrate that binding of Puralpha to its target sequence is enhanced by inclusion of Sp1 in the binding reaction. Under this condition, binding of Sp1 to the MB1 regulatory sequence remained fairly unchanged, and no evidence for the formation of Puralpha:MB1:Sp1 was observed. This observation suggests that transient interaction of Puralpha and Sp1 may result in stable association of Puralpha and the MB1 element. In support of this notion, results from immunoprecipitation/Western blot studies have established association of Puralpha and Sp1 in nuclear extracts from mouse brain. Of interest, Puralpha appears to bind to the phosphorylated form of Sp1 which is developmentally regulated and that coincides with the periods when MBP gene expression is at its maximum level. Results from cotransfection studies revealed that ectopic expression of Puralpha and Sp1 synergistically stimulates MBP promoter activity in CNS cells. The importance of these findings in stage-specific expression of MBP during brain development is discussed.

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Myelin basic protein (MBP) is a major component of the myelin sheath whose production is developmentally controlled during myelinogenesis. Earlier studies have indicated that programmed expression of the MBP gene is regulated at the level of transcription. Evidently, the MB1 regulatory motif located between nucleotides -14 to -50 plays an important role in transcription of the MBP promoter in both in vivo systems. The MB1 element contains binding sites for the activator protein MEF-1/Pur alpha and the repressor protein MyEF-2. In this study we use bandshift assays with purified MEF-1/Pur alpha and MyEF-2 and demonstrate that binding of MyEF-2 to its target sequence is inhibited by MEF-1/Pur alpha. Under similar conditions, MyEF-2 enhances the association of MEF-1/Pur alpha with MB1 DNA. MEF-1/Pur alpha binds to MB1 in mono- and dimeric forms. Inclusion of MyEF-2 in the binding reaction increases the dimeric association of MEF-1/Pur alpha with the MB1 sequence. The use of MEF-1/Pur alpha variants in the bandshift assay suggests that two distinct regions of this protein may be involved in its binding to the MB1 sequences, and its ability to block MyEF-2 interaction with the MB1 sequence. Based on previous studies on the programmed expression of MEF-1/Pur alpha and MyEF-2 during myelination and the current findings on their interplay for binding to the MB1 motif, a model is proposed for their involvement in transcriptional regulation of the MBP gene during the course of brain development.