Characterization of the cystatin B gene promoter harboring the dodecamer repeat expanded in progressive myoclonus epilepsy, EPM1

Mutations in the gene encoding cystatin B (CSTB) are responsible for the pr imary defect in progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1). A novel and unique type of disease-causing mutation, an unstable do decamer repeat expansion, accounts for the majority of EPM1 patients world- wide. This minisatellite repeat expansion, located in the putative promoter of CSTB 175 bp upstream from the translation initiation codon, appears to downregulate CSTB gene expression in vivo. We report here the characterizat ion of the CSTB promoter using different promoter-luciferase gene construct s. Transient transfections of cultured mammalian cells suggest that the reg ion from -670 to -1 bp from the translation initiation codon functions as t he CSTB promoter. Active binding to five Sp1 and four AP1 sites as well as weak binding to an androgen response element (ARE) half site was demonstrat ed by electrophoretic mobility shift assays. The effect of the minisatellit e expansion on the promoter activity was evaluated by comparing the activit y of constructs containing wild-type and expanded alleles. An increase in t he number of dodecamer units from three to 19 repeats lowered transcription in vitro by 10-fold. Northern analysis of lymphoblastoid RNA from individu als with 'premutation' length dodecamer repeat (12-17 copies) expansions sh owed decreased levels of CSTB mRNA expression. These data indicate that exp ansion of the dodecamer repeat located in the proximal promoter of CSTB sev erely disrupts the function of the promoter and thereby reduces transcripti on of CSTB. (C) 2000 Published by Elsevier Science B.V. All rights reserved .