POSITIONAL CLONING OF THE MEN1 GENE

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We have shown earlier that mutations in the MEN1 gene are responsible for the Multiple endocrine neoplasia type 1 (MEN1) syndrome, characterized by multiple tumors of the parathyroid, anterior pituitary and GI endocrine tissues. The MEN1 encoded nuclear protein, Menin, binds the transcription factors JunD and NFkB, and can repress JunD and NFkB-induced transcription. By expressing WT or mutant JunD in mouse fibroblast cell lines that are null for menin and JunD, we find that interaction with menin is required for the growth suppressor function(s) of JunD. We have developed both conventional and conditional mouse knockout models, which yield phenotypes that are remarkably similar to the human MEN1 disease, and have allowed us to delineate the stages in tumor development. Conditional knockout of menin in liver was well tolerated, a tissue not affected in MEN1 syndrome whereas similar loss in parathyroid or pancreatic islets resulted in tumors of the respective tissues. In addition, we have developed tissue specific menin-inducible transgenic mouse models. Expression changes associated with menin in cell lines and during tumorigenesis, and specifically identification of the promoters of the genes with which menin is associated are being studied, using ChIP on chip approach, to understand the biology of menin. This is particularly relevant in light of the recent demonstration that menin is a critical component of a huge protein complex that includes MLL (mixed lineage leukemia), which plays key role in transcriptional regulation by methylation of Histone H3. MLL plays a critical role in hematopoiesis, and therefore we tested hematopoietic differentiation of mouse ES (embryonic stem) cells lacking menin and found that this process was severely affected. We are exploring the role of menin in hematopoiesis and other early developmental processes in zebrafish and Xenopus laevis, which are excellent models for these studies. Injection of antisense oligonucleotides (morpholino derivatives) designed to complement splice junctions of Men1 to 1-2 cell stage embryos resulted in altered splicing of the Men1 mRNA and reduction in the levels of menin protein. The effect of such a reduction in menin levels in early development of zebrafish and Xenopus will be followed. In addition, tissue specific transgenic expression and knockout models for MEN1 are developed in Drosophila. These models should help to understand the functional role(s) of menin.