Functional Characterization of a Promoter Region in the Human MEN1 Tumor Suppressor Gene

Our previous studies on the human MEN1 (multiple endocrine neoplasia type 1) gene revealed heterogeneity of MEN1 2.8 kb transcripts related to variation in their 5′ UTR only. Six distinct exons 1 (e1A–e1F) were isolated that suggested the existence of multiple but not already identified transcriptional start sites (TSS) and of a complex transcriptional control. Identification of a minimal promoter region and its adjacent regulatory regions appears an inescapable step to the understanding of MEN1 gene transcriptional regulation in normal and pathological situations. For this purpose, we subcloned the ∼2000 bp region situated directly upstream of the exon 2 in front of a luciferase reporter gene, and we analyzed functional consequences of 5′ and 3′ serial deletions, comparatively in a series of endocrine versus non-endocrine cell lines. Primer extension and RPA experiments demonstrate that in HEK293 cells transcription initiated simultaneously at several points in endogenous MEN1 promoter as well as in transfected promoter fragments in reporter plasmids, mainly in Inr elements that are efficiently employed to synthetize previously described exons e1A–e1D. Functional consequences of TSS deletion are directly related to cellular context. The minimal promoter region is localized between −135 and −36. Five large adjacent cis-regulatory regions (UR1–UR5) exist upstream of this minimal promoter region, whose activity depend not only on the cellular context but also on the presence of a downstream sequence DR1. Five small cis-regulatory elements (C1–C5) are localized between −325 and −107. Overexpression of exogenous menin, the MEN1 geneʹs product, in mouse embryonic fibroblasts from Men1−/− knock-out mice dose-dependently decreases MEN1 promoter activity, through sequences surrounding the minimal promoter. Our data highlight the existence of a complex transcriptional regulation of the MEN1 gene, whose activity is clearly modulated depending not only on the cellular context but also on menin intracellular levels. They are the molecular bases required for a future understanding of a potential specific transcription control in endocrine cells.