Functional studies of the multiple endocrine neoplasia type 1 gene

Abstract

The multiple endocrine neoplasia type 1 (MEN1, OMIM 131 100) syndrome is an autosomal dominant trait with a near 100% penetrance and with an estimated prevalence of 2-20/100 000. The syndrome is characterized by neoplasia affecting the parathyroid glands (90-97%), enteropancreatic endocrine tissues (30-80%) and the anterior pituitary gland (15-50%) but tumors arc also found in other, both endocrine and non endocrine tissues. The gene MEN1 was identified in 1997, and its protein menin was found highly conserved during evolution but with no similarity to any other known protein. Although a variety of possible functions has been assigned to menin, it is yet unclear what links the functional loss of menin to the development of endocrine tumors, and what controls the expression of the gene. To identify the transcription start site and to search for alternative transcripts we performed RACE analysis on RNA from human tissues and cell lines (paper I). The 3´end was homogenous while we detected six alternative first, untranslated exons. In a nonendocrine cell line the most abundantly expressed transcript was the splice form exon 1b, identical with the most common mouse transcript. In thymus and a thyroid cell line a second splice form, exon 1d, was equally highly expressed. In the region closely upstream of exon lb we found core promoter activity in human and mouse. Despite lack of sequence similarity between human and mouse, the promoter in both was preceded by a silencer region, and further upstream by an enhancer region that completely cancelled the effect of the silencer. The MEN1 promoter showed self-regulating capacity since lower expression of menin activated the promoter. The promoter region contains no classical transcription factor sites that may indicate how the promoter is regulated (paper II). In order to investigate the effect of altered menin expression, we developed two systems, one for inducible menin overexpression from a transgene, and one for menin downregulation using RNA interference. The overexpressing cell line, a derivative of HEK293, showed no changes in proliferation, cell cycle distribution or chromosomal stability. Substantial changes in the chromosomal makeup of 293-sublines were demonstrated as the effect of clonal expansion (paper III). We have downregulated menin in the cell lines HeLa and BON-1 by RNA interference and developed a strategy to distinguish the effects of menin downregulation from unspecific effects of RNAi. Hela cells were reduced in the S and/or G2/M (i.e. the proliferating) phase of the cell cycle whereas BON-1 cells were less affected. The altered expression of a number of genes was revealed by a microarray study and confirmed by Real-Time PCR (paper IV).