Protein factors and 5' flanking sequences involved in the expression of the mouse myelin basic protein gene.

Myelin Basic Protein is a major structural protein of vertebrate myelin. The gene that codes for MBP is contained within the golli-MBP complex. This gene complex consists of two overlapping transcription units, golli and MBP, which are regulated by two distinct promoters. The golli unit is expressed in cells of the oligodendrocyte lineage (Central Nervous System), neurons, B and T lymphocytes, testis and thymus. However, the MBP unit is expressed exclusively in oligodendrocytes and Schwann cells (Peripheral Nervous System). The expression of the MBP unit is regulated mainly at the level of transcription by proteins that bind in a specific manner to DNA sequences located within its promoter region. The identification of these proteins and DNA sequences is essential to understanding the mechanisms that regulate the transcription of the MBP unit.This project was initiated by the isolation of the putative promoter region of the mouse myelin basic protein (MBP) gene. To achieve this the Hind III - Sac I fragment of pEX1 plasmid was subcloned in the vector pBluescript. The cloned insert, which corresponds to the region between nucleotides -1319 and +227 relative to the transcription start site of the mouse MBP gene, was subsequently sequenced manually using the chain termination method. Sequence analysis revealed a number of putative binding sites for transcription factors. The region -609 to -577 was selected for further studies because work published by other groups suggested that it contains a cell-type specific (for oligodendrocytes) transcription activator. The presence of protein factors specifically binding to the region -609 to -577 was demonstrated by electrophoretic mobility shift assay (EMSA).For this purpose, nuclear extracts were prepared from rodent brain or established glial cell lines e.g. C6 glioma cells. Extracts from tissues and cell lines, which do not express myelin basic protein e.g., HeLa cells served as a control. Nuclei were isolated by Dounce homogenisation of cultured cells or brain tissue. The proteins were then isolated by high salt extraction of the nuclei followed by ammonium sulphate fractionation.Putative protein(s) binding to the region located between nucleotides -609 to -577 of the myelin basic protein gene promoter were identified using the yeast one-hybrid system. This assay is based on the interaction between a specific protein DNA binding domain and the target DNA sequence. Proteins are expressed as fusions to the GAL4 activation domain (AD) in the yeast reporter strain in which the target sequence has been inserted upstream of the HIS3 gene minimal promoter. Binding of AD fusions to the target sequence increases activity of the HIS3 promoter enabling growth on medium lacking histidine. In this work a yeast reporter strain containing four copies of the -609 to -577 region tandemly repeated upstream of the HIS3 gene minimal promoter was constructed. A library containing rat brain cDNAs fused to the activation domain of GAL4 was screened using this strain as a host. Seven clones were obtained on medium lacking histidine in the presence of 30 mM 3-aminotriazole. DNA from these clones was automatically sequenced and analysed for sequence homology with known transcription factors by comparing the nucleotide and protein sequences to EMBL/Genbank and Swissprot/Swall databases using theFastA and Blast search tools. From the results of the homology searches the clones were identified as follows: the activating transcription factor 2 (ATF-2), the pituitary specific positive transcription factor 1 (Pit-1) or general transcription factor 2i, the E2F family transcription factor, the PASK protein and two of the clones were identified as c-jun. One clone, however, remains unidentified and this could be a novel transcription factor.