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 approximately 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 gene's 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.

Transcription regulation of the multiple endocrine neoplasia type 1 gene in human and mouse.

Multiple endocrine neoplasia type I (MEN1) is an autosomal dominant tumor syndrome, with the presence of tumors in parathyroid, pancreatic, and anterior pituitary. The tumor suppressor gene MEN1, located on chromosome 11q13, encodes a 610 amino acid, 68-kDa protein, menin. Menin is conserved among species but has no similarity with any known protein. To investigate how the expression is regulated in both man and mouse, we assayed a greater than 1-kb region upstream of the second exon for promoter activity in luciferase reporter vectors. The basic promoter was located closely upstream the most commonly expressed first exon. The region further upstream modified the activity. Repetitive elements of the short interspersed/Alu type covered the entire human upstream regulatory region and were the only apparent motif in common with its murine ortholog. Previous studies have indicated a compensatory induction of the second allele because of inactivation of the first allele. We found that overexpression of menin in an inducible cell culture system down-regulated the proximal promoter. In response to down-regulation of MEN1 expression by RNA interference, the regulatory region activated the promoter in a compensatory manner. Our data confirm that the expression of the MEN1 gene is regulated by a feedback from its product menin.

Menin interacting proteins as clues toward the understanding of multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized mostly by tumors of the parathyroids, pancreas and anterior pituitary. The gene responsible, MEN1, encodes Menin, a 610 aminoacid nuclear protein with no sequence homology to other proteins. Although a mouse knock-out model is available, the function of Menin is still elusive. Proteins of known function are shown to interact with Menin: JunD, nuclear factor-KappaB, Smad3, Pem, Nm23H1, glial fibrillary acidic protein, Vimentin, and probably P53. Their partnership with Menin may correspond to a regulation of their activity, but their relevance to the various traits of MEN1 pathogenicity is not established. This raises fundamental issues on the regulation pathways implicated in this complex endocrine disease.