The multiple endocrine neoplasia type 1 (MEN1) gene is a tumor suppressor gene identified by positional cloning by an NIH collaborative group including members of MDB. Germline mutations in the gene are found in affected subjects of MEN1 kindreds, and somatic mutations in the gene have been identified in sporadic endocrine and other tumors. The gene encodes a 610 residue protein termed menin without homology to other known proteins. We have initiated a series of studies aimed at defining the structure, function, subcellular localization, and range of expression of menin. We have generated a series of polyclonal peptide antisera that have proved useful in immunoblot and immunoprecipitation studies. Furthermore,these antibodies detect the expression of recombinant forms of menin to be used for structural and biochemical analyses. Subcellular fractionation and immunoblotting of 293 cells transfected with the cDNA encoding menin, in conjunction with studies of GFP-tagged menin conducted by our collaborators in NHGRI have shown that menin is primarily localized to the nucleus. A yeast-two-hybrid screen identified junD as a menin interacting partner. Mutagenesis of junD was done to identify specific residues involved in menin interaction. The biologic properties of junD mutants indicate that they function as oncogenes rather than as a tumor suppressor. Purified recombinant menin expressed in E coli is being used for biochemical and structural (X-ray crystallography) studies. Determination of 3-D structure should offer insights into functional domains for protein interaction and the mechanism whereby many naturally occurring missense mutations cause loss of function. The drosophila ortholog of menin has been identified from genomic sequence submitted to Genebank. This has allowed us to employ drosophila genetics (collaboration with Brian Oliver/NIDDK) to study the function of menin and its interacting proteins. Results to date support menin's functional interaction with drosophila jun/fos. Transgenic mice expressing cre recombinase in target tissues relevant to MEN1 such as parathyroid and pancreatic islets have been generated for crosses with mice (generated by NHGRI collaborators)with a conditional knockout of the MEN1 gene. Mice expressing cre in parathyroid and homozygous for conditional KO of MEN1 gene develop hypercalcemia and parathyroid neoplasia by 8 months of age. These mice provide a model of hyperparathyroidism in which treatments can be tested, and should offer insights into the role of menin in tumor formation. The phenotype of mice with tissue-specific KO of menin that are crossed with mice with other defined genetic lesions will be studied to help define interactions between menin and other growth regulating genes. Work from two other laboratories has identified menin as a member of the MLL (histone methyltransferase) nuclear complex, and shown that menin is necessary for expression of HoxC8 and HoxA9 genes. Given the key role of MLL and HoxA9 genes in hematopoietic stem cell differentiation, we are testing the ability of mouse embryonic stem cells null for MEN1 to differentiate along the hematopoietic lineage (E. Novotny/NHGRI). We are using two approaches to probe menin's role in gene regulation: CHIP on chip (P. Scacheri/NHGRI) and SACO (serial analysis of chromatin occupancy) (S. Agarwal/NIDDK in collaboration with R. Goodman/Vollum Institute). Results to date show widespread occupancy of hox genes by menin. Further studies are underway to elucidate critical genes regulated by menin in endocrine cells where menin loss leads to hyperplasia. Finally, we are testing possible biochemical functions of menin related to histone modifications. Our overall goals are to understand the biochemical function of menin, how this function relates to gene regulation, and how changes in specific gene expression lead to the endocrine neoplasia phenotype in mouse models and humans.
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