POSITIONAL CLONING OF THE MEN1 GENE

Human Genome Research

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Abstract

We have shown earlier that mutations in the MEN1 gene are responsible for the familial cancer syndrome, Multiple endocrine neoplasia type 1 (MEN1). MEN1 is characterized by multiple tumors of the parathyroid, anterior pituitary and GI endocrine tissues. We found that the MEN1 encoded protein, menin, resides primarily in the nucleus. We discovered that menin binds the transcription factor JunD, and this interaction is required for the growth suppressor function(s) of JunD. Menin interacts with another transcription factor NFkb, and in both instances, the interaction results in the repression of transcription driven by JunD or NFkB. Menin has been found to be a component of a huge protein complex that includes MLL (mixed lineage leukemia), which plays key role in transcriptional regulation by methylation of Histone H3. Menin is a critical component of this complex as it is required for the optimal methylase activity of MLL. Prompted by the interaction of menin with transcriptional regulators, using ChIP on chip approach, we have identified thousands of promoters of the genes with which menin is associated. This study has allowed us to identify the promoters that are also associated with MLL alone, and with both MLL and menin. MLL plays a critical role in hematopoiesis, and therefore we tested whether menin is required for this differentiation process. We generated mouse ES (embryonic stem) cells lacking menin, and induced them to undergo hematopoietic differentiation. We found that the ability to generate hematopoietic colonies was severely affected in cells lacking menin. MLL mediates its role in hematopoiesis by regulating the expression of Hox family members such as Hoxa9 and Hoxc6, and we find decreased expression of these genes in menin-null ES cells. Reduction of menin by injections of antisense oligonucleotides (morpholino derivatives) in embryos allowed us to evaluate the role of menin in zebrafish hematopoiesis; the reduction did not appear to adversely affect blood development in embryos or alter the expression of the critical genes in this process such as Hoxa9 and Gata1. Now that we have isolated a zebrafish menin missense mutant, induced by chemical mutagenesis, efforts are underway to evaluate the role of menin, if any, in zebrafish growth and development. We continue to explore the biological function(s) of menin using other model systems such as Xenopus and Drosophila: transgenic expression and knockout models for MEN1 in Drosophila helped us identify the genetic interactions between menin and Jun/Fos. 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. We have developed high-density BAC arrays and demonstrated their usefulness in evaluating the genomic changes in tumors by the identification of several novel regions amplification and deletion in breast cancer: We intend to use these arrays to study the genomic alterations associated with MEN1 tumors. In addition to the role in tumorigeneis, menin appears to play a critical role in early development as homozygous loss of Men1 alleles in mice results in embryonic lethality (E11.5-E13.5) with defects in the development of several organs including heart. We have established mouse embryo fibroblast (MEF) cell lines lacking menin, and evaluated their characteristic including their expression patterns. Menin-null MEFs display alteration in expression of several genes encoding extracellular matrix proteins. These genes, regulated by TGF-b, are known to be required for early development of heart. We also found that MEF cell lines lacking menin respond poorly to TGF-b. Reexpression of menin in menin-null MEFs, either transiently or stably, resulted in the identification genes regulated by menin. Evaluation of the expression changes associated with menin, and the usefulness of the variety of animal models are likely to reveal the basic biological function(s) of menin.