Different consequences of EGR2 mutants on the transactivation of human Cx32 promoter Marco Musso, b, * Piercesare Balestra, b Franco Taroni, a Emilia Bellone, b and Paola Mandich b a IRCCS National Neurological Institute C. Besta, Milan, Italy b Department of Neurosciences, Ophthalmology, and Genetics, Section of Medical Genetics, University of Genoa, c/o DIMI Viale Benedetto XV, 6-16132 Genova, Italy Received 22 July 2002; revised 7 November 2002; accepted 8 November 2002 Abstract The early growth response 2 (EGR2) transcription factor plays a crucial role in peripheral nerve myelination. Mutations of this gene are associated with a wide variety of demyelinating neuropathies differing from each other in the severity of nerve injury. Although the expression of EGR2 mutants inhibits the transactivation of myelin gene promoters, the exact molecular mechanism by which these mutations cause the alteration of the myelination process is still unknown. Recently, it was reported that EGR2 is directly involved in the transcriptional regulation of Connexin 32, a myelin gene frequently mutated in peripheral neuropathies. Here we describe the differential effect of two EGR2 mutants; while mutant D355V partially induces Cx32 promoter, mutant R381H does not. Furthermore, we show that a sequence located at -216, recognized by the wild-type and the mutant D355V recombinant proteins, is relevant for promoter transactivation. © 2003 Elsevier Science (USA). All rights reserved. Introduction Inherited peripheral neuropathies are characterized by a wide spectrum of phenotypic variations and age at onset. Several gene mutations were found in myelin protein zero (MPZ), connexin 32 (Cx32), peripheral myelin protein 22 (PMP22), and periaxin (Prx) (reviewed in Nelis et al., 1999; Guilbot et al., 2001). Lately, related clinical disorders rang- ing from CMT1 to congenital hypomyelination have been associated with mutations in the early growth factor 2 gene (EGR2). EGR2 (also known as Krox20) is a zinc-finger DNA- binding protein controlling Schwann cell myelination by modulating the expression of genes encoding myelin pro- teins and enzymes required for the synthesis of myelin lipids (Nagarajan et al., 2001). Although mice heterozygous for an EGR2 null allele do not exhibit any defect in axonal myelination, heterozygous mutations were found in patients affected by Charcot-Marie-Tooth (CMT) and Dejerine-Sot- tas (DSS) diseases. This suggests that human zinc-finger mutations such as D355V, R359W, R381H, R381C, S382R- D383Y, and R409W (Bellone et al., 1999; Timmerman et al., 1999; Pareyson et al., 2000; Yoshihara et al., 2001; Warner et al., 1998), rather than acting as loss-of-function alleles, might instead behave as gain-of-function alleles by binding to inappropriate targets or as dominant negative by sequestering cofactors. We previously reported that the mutation D355V, asso- ciated with the CMT1 classical phenotype, slightly affects the binding affinity for the EGR2 consensus sequence, while it drastically reduces the binding to a DNA sequence localized at nucleotide -93 in the Cx32 nerve-specific pro- moter (Musso et al., 2001). Despite this low binding affin- ity, the EGR2 mutant D355V still retains some capability to transactivate the Cx32 promoter, suggesting the presence of another cis-acting element. Recently, Bondurand et al. (2001) have shown that EGR2 transactivation of the Cx32 promoter was abolished when three putative EGR2-binding sites named E1 (from -224 to -216), E2 (from -187 to -179), and E3 (from -101 to -93) were mutated. Using constructs in which each element was singularly mutated, * Corresponding author. Fax: +39-10-353-8978. E-mail address: genetica.medica@unige.it (M. Musso). R Available online at www.sciencedirect.com Neurobiology of Disease 12 (2003) 89 –95 www.elsevier.com/locate/ynbdi 0969-9961/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0969-9961(02)00018-9