Identification of a positive regulatory element in the myelin-specific promoter of the PMP22 gene

Over- and under expression of the 22 kDa peripheral myelin protein (PMP22) results in dysmyelinating peripheral neuropathies, such as Charcot-Marie-To oth disease type 1A (CMT1A) and hereditary neuropathy, with the liability t o pressure palsies (HNPP). Expression of the PMP22 gene is driven by two al ternative promoters, P1 and P2, with transcripts originating from P1 associ ated with peripheral nerve myelination by Schwann cells. Transient transfec tions of constructs containing P1 (3.5 kb) or P2 (2.5 kb) resulted in high levels of reporter gene expression in the RT4-D6P2T schwannoma cell line. S erial deletions of P1 revealed that region Pl-A (-105 to -43), situated ups tream of the minimal promoter, contained a positive regulatory element. The 62 bp P1-A region conferred in cis a sevenfold increase in expression of l uciferase driven by a heterologous promoter in an orientation-dependent man ner. Interspecies comparison of the P1-A region revealed a 98% degree of id entity between the human, mouse, and rat sequences. A prominent sequence-de pendent DNA-protein complex (C-1) was detected in electrophoretic mobility shift assays with P1-A using RT4-D6P2T nuclear extract and was localized to a minimal 21 bp region within P1-A. Site-directed mutagenesis of this regi on revealed nucleotides at positions -46 to -43 as being necessary for form ation of C-1. Functional analysis of the mutated P1-A element indicated tha t positions -46 and -45 were essential for transactivation mediated by this element. Characterization of the transacting factor(s) interacting with th is key regulatory element will shed light on its role in regulating periphe ral nerve myelination. J. Neurosci. Res. 65: 508-519, 2001. (C) 2001 Wiley- Liss, Inc.