POSITIONAL CLONING OF THE MEN1 GENE

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Our laboratory employs genomic technologies for use in identifying disease-causing genes. We identified the gene (MEN1) responsible for multiple endocrine neoplasia type 1 (MEN1) syndrome in 1997. MEN1 is a familial cancer syndrome characterized by tumors in multiple endocrine tissues, primarily in the parathyroid, enteropancreas, and anterior pituitary. The amino acid sequence of the MEN1-encoded protein (menin) does not contain any identifiable structural or functional motifs. Our studies using mouse MEN1 models have revealed that the heterozygous loss of Men1 results in a tumor spectrum similar to that in humans, thus explaining the role of menin in tumorigenesis. Interestingly, homozygous loss results in early embryonic lethality (E12.5) indicating the importance of menin in early development. We have found that menin resides primarily in the nucleus, and is involved in transcriptional regulation. We are trying to elucidate the functional roles of menin in tissue differentiation, organogenesis, and development by identifying the overall changes in expression associated with the loss or overexpression of menin itself. To this end, we have generated cell lines that are null for menin in two cell types: mouse embryo fibroblasts (MEFs) and mouse embryonic stem cells (ES). Menin-null MEFs showed a significant decrease in the expression of several extracellular matrix/cell adhesion protein genes including fibulin-2 (Fbln2), periostin (Postn), and versican (Cspg2, chondroitin sulfate proteoglycan). All three of these proteins, which showed reduced expression in the absence of menin, are critical for the developing heart, and thus explain the defective heart development observed in the homozygous knockout mouse embryos. In addition, these three genes are also known targets of TGF-&#946;. Consistently, we find that TGF-&#946; responsive PAI-1 promoter is less responsive to TGF-&#946; in menin-null MEFs. Expression arrays were used to catalog the genes responding to TGF-&#946; in the presence or absence of menin. Menin interacts with MLL, the mixed lineage leukemia protein, and both are components of a COMPASS-like protein complex, regulating expression of Hox genes among others. MLL-null mouse embryonic stem (ES) cells are defective hematopoietic differentiation via dysregulation of Hox genes. Thus, it appears that menin may play a role in hematopoiesis. We generated three menin-null ES clones, and found that they were deficient in their ability to generate mature hematopoietic colonies. Formation of embryoid bodies (EB), an initial step in ES cell differentiation, was normal in menin-null cells. However, their subsequent differentiation into mature colonies was affected by the absence of menin. Re-expression of either Men1 or Hoxa9 could rescue the defect. Global expression changes in menin-null ES cells during different stages of hematopoietic differentiation are being studied to evaluate the molecular changes mediated by menin in this differentiation process leading to mesodermal lineages. We also evaluated whether loss of menin affects differentiation into endodermal lineages, using the pluripotent mouse P19 embryonic stem cells, in which differentiation processes are very well studied. We find that menin expression is induced by a natural morphogen, the retinoic acid (RA), which, at 10 nM concentration, induces endodermal differentiation in P19 cells. Interestingly, cells with shRNA-mediated reduced menin expression were resistant to endodermal differentiation when stimulated with RA. These cells proliferated at a higher rate and retained the stem cell-specific antigen SSEA-1 as in the untreated cells. On the other hand, menin overexpressing cells displayed the characteristic of an endodermal phenotype by the acquisition of cytokeratin Endo A expression, a marker for the primitive endoderm, with a concomitant loss of SSEA-1. Menins ability to induce endodermal differentiation in the absence of RA implied that menin could substitute RA by inducing a set of target genes that are RA-responsive. We have used high-density SNP arrays to molecularly define the precise deletion boundaries in patients with 2q37 deletion syndrome, characterized by several distinct physical features, including short stature, round faces, subcutaneous ossifications, brachydactyly, and other skeletal anomalies. Developmental delay, obesity, hypotonia, and autism have been reported in some cases. We found the smallest deletion (<2 Mb) in a patient with minimal clinical characteristics. We have observed three cases (out of ten) where the 2q terminal deletion is also associated with duplication of the adjacent region. A detailed evaluation of the deletion intervals for distinct clinical phenotype is being pursued. Using high-resolution BAC arrays for Array CGH, we had identified a novel gene, TRMT12, amplified and overexpressed in breast cancer cell lines and tumors. TRMT12 is a human homolog of a yeast gene encoding an enzyme that catalyzes a step in the posttranscriptional modification of a G to a highly modified base, yW (wybutosine), present in tRNAPhe. We find that human TRMT12 can substitute for the homologous yeast gene in the yW biosynthetic pathway in yeast. However, yW modification was unaffected in tRNAPhe from mouse mammary tumor model overexpressing TRMT12. Our efforts to identify the consequence of TRMT12 over expression lead to the discovery that TRMT12 regulates the expression of SMG-1, a new member of the well-known PIKK kinase family. We have demonstrated that siRNA-mediated reduced expression of TRMT12 results in increased SMG-1 expression. SMG-1 regulates non-sense mediated mRNA decay (NMD) as well as a key player in p53 mediated genotoxic stress. The functional role(s) of TRMT12 in NMD and cellular response to DNA damage is being pursued.