REGULATION OF MYELIN BASIC PROTEIN-ENCODING GENE-TRANSCRIPTION IN RAT OLIGODENDROCYTES

The myelin (My) basic protein-encoding gene (MBP) is specifically expressed by oligodendrocytes (OL) in the central nervous system (CNS) and by Schwann cells in the peripheral nervous system (PNS). To define cell-type-specific regulatory elements, a series of chimaeric constructs containing varying lengths of the 5'-flanking region and exon 1 of MBP linked to the eat reporter gene was transfected into several cell types, including primary cultures of differentiated rat OL, Schwann cells and kidney cells, as well as neuronal and non-neuronal cell lines. All the constructs generated variable levels of chloramphenicol acetyltransferase (CAT) activity in all cell types, except in primary OL and Schwann cells, where distinct positive (PRE) and negative regulatory elements (NRE) were found to be involved in regulating cat expression. The nucleotide (nt) -53/+70 construct gave maximal activity in all cell types, except OL and Schwann cells, where sequences upstream from nt -53 were necessary for maximal promoter activity. The sequences located at nt -655 to -397 and nt -394 to -54 showed enhancer and repressor effects, respectively, in OL. In Schwann cells, sequences from -394 to -253 showed positive regulatory effects, while those between -655 to -397 and -253 to -54 showed negative regulatory effects. In the downstream region of the promoter, sequences from +20 to +70 and +70 to +200 showed strong silencer and enhancer activities specifically in OL. In gel-retardation assays using nuclear extracts prepared from several cell types and the -253 to -53 repressor sequences, specific DNA-protein complexes unique to OL were identified. Similarly, DNase I footprinting analysis of the -253 to -53 region revealed seven protected sites (I-VII) using OL nuclear extracts, but none were detected using primary baby rat kidney cell extracts. Protected sites VII (-220/-183) and V (-135/-120) showed 90% sequence homology to elements in the 5'-flanking regions of other My-encoding genes and the NRE of the mouse renin-encoding gene, respectively. The expression of MBP is therefore controlled by basal PRE which function in different cell types, but PRE and NRE located on either side of the basal promoter may play an essential role in regulating MBP transcription in My-forming OL.