4221

MEN1

MEAI|SCG2;multiple endocrine neoplasia I;MEN1;multiple endocrine neoplasia I

menin

11q13

protein-coding

mutations of the MEN1 tumor suppressor gene cause the multiple endocrine neoplasia type 1 (MEN1) syndrome in humans, and they are involved in a variety of sporadic human endocrine tumors. We here characterize the MEN1 gene homologs of the mouse and rat. cDNA was isolated from a mouse phage library, and two alternative MEN1 mRNA transcripts containing variant 5 untranslated regions were identified by RT-PCR in several mouse and rat tissues. When compared to the human molecule, mouse and rat MEN1 (611 and 610 amino acids, respectively) show an overall identity of 96.5% and 97.0% at the protein level, delimiting four conservational domains (A-D). Mouse and rat MEN1 mRNA, as studied by template-calibrated quantitative RT-PCR, is non-exclusively expressed in hematopoietic and endocrine cells, with similar expression patterns found in fetal and adult tissues. Fluorescent in situ hybridization maps the single murine MEN1 locus to chromosome 19, region B. No MEN1 gene mutations were identified in endocrine islet tumor cell lines RIN 5AH (rat) and NIT-1 (mouse) as compared to wild type cDNA. Our data define mouse and rat MEN1 as widely expressed and highly conserved homologs of the human MEN1 tumor suppressor gene whose role in biology and endocrine tumorigenesis is due for experimental study.

Multiple endocrine neoplasia type 1 is an autosomal dominant tumor syndrome. Manifestations include neoplasms of the parathyroid glands, enteropancreatic neuroendocrine cells, and the anterior pituitary gland. The MEN1 tumor suppressor gene encodes menin, a 610 amino acid nuclear protein without sequence homology to other proteins. To elucidate menin function, we used immunoprecipitation to identify interacting proteins. The NF-kappaB proteins p50, p52 and p65 were found to interact specifically and directly with menin in vitro and in vivo. The region of NF-kappaB proteins sufficient for binding to menin is the N-terminus. Furthermore, amino acids 305-381 of menin are essential for this binding. Menin represses p65-mediated transcriptional activation on NF-kappaB sites in a dose-dependent and specific manner. Also, PMA (phorbol 12-myristate 13-acetate)-stimulated NF-kappaB activation is suppressed by menin. These observations suggest that menins ability to interact with NF-kappaB proteins and its modulation of NF-kappaB transactivation contribute to menins tumor suppressor function.

The multiple endocrine neoplasia type 1 gene (MEN1) is a tumor suppressor gene associated with the development of tumors in the parathyroids, the pituitary, and the pancreas and has also been linked to impaired germ cell production. The murine ortholog, Men1, is highly homologous to the human counterpart both at DNA and protein levels. The present study was undertaken to further approach the function of Men1 and its encoded protein menin. By 5 RACE and RT-PCR four alternative splice variants were identified, indicating a 5 heterogeneity of Men1 similar to the human counterpart. By mRNA in situ hybridization of embryonal and adult mouse tissues, all four splice variants were shown to be expressed, albeit at varying timepoints and levels in the different tissues. However, a putative isoform postulated from the DNA sequence, which would elongate the reading frame by 15 bases at the exon 2/intron 2 junction, was not found to occur in mouse. The strongest expression was detected in testis, both at the mRNA and protein level and was therefore further characterized by protein analysis of cells isolated from different stages of the spermatogenesis. Western blotting revealed a single protein of approximately 70 kDa detected in total testis, isolated pachytene spermatocytes and in haploid spermatids. Notably, no menin expression was detectable in the extracts from epididymis where the maturation of sperms is almost completed, suggesting that menin plays a crucial role during spermatogenesis.

Multiple endocrine neoplasia type 1 is an autosomal dominant cancer predisposition syndrome caused by mutations in the tumor-suppressor gene MEN1. The gene encodes a nuclear protein, menin, with no recognized functional motifs. Menin has been shown negatively to regulate transcriptional activation mediated by JunD, although the significance of this interaction in normal cell physiology and how the absence of menin leads to tumorigenesis are unknown. Menin is highly expressed in testes. We used immunocytochemistry to explore its role in meiosis and found that it localizes exclusively at telomeres. JunD was not found at telomeres in meiotic cells. In view of elevated telomerase activity or abnormal telomere structure in virtually all malignancies, regulation of telomere function would be an appealing role for a tumor suppressor. However, menin does not specifically associate with telomeres in somatic cells, as indicated by lack of co-localization with the known telomeric protein TRF2. Cells overexpressing menin had normal telomerase activity, and tumors with homozygous MEN1 mutations showed no aberrations in telomere length, indicating that menin does not directly regulate telomerase activity. The role of menin at meiotic telomeres appears to be independent of JunD and may not have a counterpart in somatic cells. These results suggest that menin may play different roles in different tissues through interactions with different proteins.

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.

Patients suffering from multiple endocrine neoplasia type 1 (MEN1) are predisposed to multiple endocrine tumors. The MEN1 gene product, menin, is expressed in many embryonic, as well as adult tissues, and interacts with several proteins in vitro and in vivo. However, the biological function of menin remains largely unknown. Here we show that disruption of the Men1 gene in mice causes embryonic lethality at E11.5-E13.5. The Men1 null mutant embryos appeared smaller in size, frequently with body haemorrhages and oedemas, and a substantial proportion of them showed disclosure of the neural tube. Histological analysis revealed an abnormal development of the nervous system and heart hypotrophy in some Men1 null embryos. Furthermore, Men1 null livers generally displayed an altered organization of the epithelial and hematopoietic compartments associated with enhanced apoptosis. Chimerism analysis of embryos generated by injection of Men1 null ES cells, showed that cells lacking menin do not seem to have a general cell-autonomous defect. However, primary Men1 null embryonic fibroblasts entered senescence earlier than their wild-type counterparts. Despite normal proliferation ability, Men1 null ES cells exhibited a deficiency to form embryoid bodies, suggesting an impaired differentiation capacity in these cells. The present study demonstrates that menin plays an important role in the embryonic development of multiple organs in addition to its proposed role in tumor suppression.

Telomerase expression is repressed in most somatic cells but is observed in stem cells and a high percentage of human cancers and has been hypothesized to contribute to tumorigenesis and maintenance of stem cell states. To explore telomerase regulation, we employed a general genetic screen to identify negative regulators of hTERT. We discovered three tumor suppressor/oncogene pathways involved in hTERT repression. One, the Mad1/c-Myc pathway, had been previously implicated in hTERT regulation. The second, SIP1, a transcriptional target of the TGF-beta pathway, mediates the TGF-beta regulated repression of hTERT. The third, the tumor suppressor Menin, is a direct repressor of hTERT. Depleting Menin immortalizes primary human fibroblasts and causes a transformation phenotype when coupled with expression of SV40 Large and Small T antigen and oncogenic ras. These studies suggest that multiple tumor suppressor/oncogene pathways coordinately repress hTERT expression and imply that telomerase is reactivated in human tumors through oncogenic mutations.

MEN1 is a likely tumor suppressor gene that encodes a novel protein, menin. Menin is a 610 amino-acid residue protein with as yet unknown function(s). We have used tandem affinity purification and mass spectroscopy to isolate and identify proteins associating with menin from cultured HeLa cell extracts. This strategy has resulted in the isolation and identification of nonmuscle myosin type II-A heavy chain (NMHC II-A) as a menin interacting protein. This interaction was confirmed by glutathione-S-transferase pulldown assays, by coimmunoprecipitation, and by actin selection of myosin. We have further identified the amino-terminal region of menin and the head domain of NMHC II-A to be regions required for this interaction. Moreover menin was seen to colocalize with this myosin isoform in the cleavage furrow of dividing cells by indirect immunofluoresence. These data indicate that menin through binding to NMHC II-A could participate in cell division and in other processes that involve NMHC II-A.

Our previous studies on the human MEN1 (multiple endocrine neoplasia type 1) gene revealed heterogeneity of MEN1 2.8 kb transcripts related to variation in their 5 UTR only. Six distinct exons 1 (e1A-e1F) were isolated that suggested the existence of multiple but not already identified transcriptional start sites (TSS) and of a complex transcriptional control. Identification of a minimal promoter region and its adjacent regulatory regions appears an inescapable step to the understanding of MEN1 gene transcriptional regulation in normal and pathological situations. For this purpose, we subcloned the approximately 2000 bp region situated directly upstream of the exon 2 in front of a luciferase reporter gene, and we analyzed functional consequences of 5 and 3 serial deletions, comparatively in a series of endocrine versus non-endocrine cell lines. Primer extension and RPA experiments demonstrate that in HEK293 cells transcription initiated simultaneously at several points in endogenous MEN1 promoter as well as in transfected promoter fragments in reporter plasmids, mainly in Inr elements that are efficiently employed to synthetize previously described exons e1A-e1D. Functional consequences of TSS deletion are directly related to cellular context. The minimal promoter region is localized between -135 and -36. Five large adjacent cis-regulatory regions (UR1-UR5) exist upstream of this minimal promoter region, whose activity depend not only on the cellular context but also on the presence of a downstream sequence DR1. Five small cis-regulatory elements (C1-C5) are localized between -325 and -107. Overexpression of exogenous menin, the MEN1 genes product, in mouse embryonic fibroblasts from Men1(-/-) knock-out mice dose-dependently decreases MEN1 promoter activity, through sequences surrounding the minimal promoter. Our data highlight the existence of a complex transcriptional regulation of the MEN1 gene, whose activity is clearly modulated depending not only on the cellular context but also on menin intracellular levels. They are the molecular bases required for a future understanding of a potential specific transcription control in endocrine cells.

Menin, a gene product of multiple endocrine neoplasia type I (MEN1), is known to act as a tumor suppressor to repress JunD transcription factor. However, the mechanism by which Menin represses JunD transcriptional activity was still unclear. In this study, we found that Menin is a corepressor against JunD transcriptional activity via recruitment of histone deacetylases in an mSin3A-dependent manner. The amino acid search revealed that central domain of Menin includes a alpha-helical mSin3-interacting domain [SID (371-387)]. The SID mutation of Menin (L381P/A385P) abolished the interaction between mSin3A and paired amphipathic helix 2 domain of Menin and reduced its ability to repress JunD transcriptional activity, implicating that SID of Menin is important for recruiting an mSin3A-histone deacetylase complex to repress JunD transcriptional activity.

Activin, a member of the TGFbeta superfamily, is a negative regulator of cell growth and prolactin (PRL) production in pituitary lactotrope cells. However, the mechanisms by which this growth factor exerts its growth-inhibitory and -repressive effect on PRL remain unclear. In this study, we show that activin negatively regulates PRL expression at the transcriptional level through the Smad pathway and the multiple endocrine neoplasia type 1 gene product, menin. Our results also demonstrate that the tumor suppressor menin is required for activin-induced growth arrest of somatolactotrope cells. Moreover, we show that activin represses transcription and expression of Pit-1, a pituitary transcription factor that is essential for maintenance and development of lactotrope cells. We defined two Pit-1 DNA-binding sites in the proximal region of the PRL promoter as critical for the activin-mediated inhibition. Together, our results highlight the Smad pathway and the tumor suppressor menin as key regulators of activin effects on PRL and Pit-1 expression, as well as on cell growth inhibition, and emphasize the critical role of activin in the regulation of pituitary function.

Multiple endocrine neoplasia type I (MEN1) is an inherited tumor syndrome characterized by development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a nuclear protein, menin. Menin interacts with several transcription factors and inhibits their activities. However, it is unclear whether menin is essential for the repression of the expression of endogenous genes. Here, using menin-null cells, we show that menin is essential for repression of the endogenous IGFBP-2, a gene that can regulate cell proliferation. Additionally, complementation of menin-null cells with wild-type menin, but not with a MEN1 disease-related point mutant, restores the function of menin in repressing IGFBP-2. Consistent with this, the promoter of IGFBP-2 is repressed by wild-type menin, but not by a MEN1-related point mutant. Menin also alters the structure of the chromatin surrounding the promoter of the IGFBP-2 gene, as demonstrated by the deoxyribonuclease I hypersensitivity assay. Furthermore, nuclear localization signals in menin are crucial for repressing the expression of IGFBP-2. Together, these results suggest that menin regulates the expression of the endogenous IGFBP-2 gene at least in part through the promoter of IGFBP-2.

Menin is a tumor suppressor that is mutated in patients with multiple endocrine neoplasia type I (MEN1), an inherited tumor-prone syndrome. Because there is no obvious conserved structural domain in menin that suggests a biochemical function, little is known as to how menin suppresses tumorigenesis. Although menin interacts with a variety of nuclear proteins including transcription factors, it is unknown whether menin itself can directly bind DNA. Here we show that menin directly binds to double-stranded DNA. It also binds a variety of DNA structures, including Y-structures, branched structures, and 4-way junction structures. The COOH terminus of menin mediates binding to DNA, but MEN1 disease-derived mutations in the COOH terminus abolish the ability of menin to bind DNA. Importantly, these MEN1 disease-related menin mutants also fail to repress cell proliferation as well as cell cycle progression at the G2/M phase. Furthermore, detailed mutagenesis studies indicate that positively charged residues in two nuclear localization signals mediate direct DNA binding as well as repression of cell proliferation. Collectively, these results demonstrate, for the first time, a novel biochemical activity of menin, binding to DNA, and link its DNA binding to the regulation of cell proliferation.

Multiple endocrine neoplasia type I (MEN1), a hereditary tumor syndrome, is characterized by the development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a tumor suppressor, menin. Overexpression of menin leads to inhibition of Ras-transformed cells. However, it is unclear whether menin is essential for repression of cell proliferation, and if it is, how it inhibits cell proliferation. Here, we show that targeted disruption of the Men1 gene leads to enhanced cell proliferation, whereas complementation of menin-null cells with menin reduces cell proliferation. Moreover, menin interacts with activator of S-phase kinase (ASK), a component of the Cdc7/ASK kinase complex that is crucial for cell proliferation, but does not appear to alter Cdc7 kinase activity in in vitro kinase assays. We identify the COOH terminus of menin as the domain that mediates the specific interaction with ASK. Notably, wild-type menin completely represses ASK-induced cell proliferation, although it does not obviously affect the steady-state cell cycle profile of ASK-infected cells. Interestingly, disease-related COOH-terminal menin mutants that do not interact with ASK completely fail to repress ASK-induced cell proliferation. Together, these findings demonstrate a functional link between menin and ASK in the regulation of cell proliferation.

mutations in the MEN1 gene are associated with the multiple endocrine neoplasia syndrome type 1 (MEN1), which is characterized by parathyroid hyperplasia and tumors of the pituitary and pancreatic islets. The mechanism by which MEN1 acts as a tumor suppressor is unclear. We have recently shown that menin, the MEN1 protein product, interacts with mixed lineage leukemia (MLL) family proteins in a histone methyltransferase complex including Ash2, Rbbp5, and WDR5. Here, we show that menin directly regulates expression of the cyclin-dependent kinase inhibitors p27Kip1 and p18Ink4c. Menin activates transcription by means of a mechanism involving recruitment of MLL to the p27Kip1 and p18Ink4c promoters and coding regions. Loss of function of either MLL or menin results in down-regulation of p27Kip1 and p18Ink4c expression and deregulated cell growth. These findings suggest that regulation of cyclin-dependent kinase inhibitor transcription by cooperative interaction between menin and MLL plays a central role in menins activity as a tumor suppressor.

The Mixed-Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.

Menin is the product of the tumor suppressor gene Men1 that is mutated in the inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1). Menin has been shown to interact with SET-1 domain-containing histone 3 lysine 4 (H3K4) methyltransferases including mixed lineage leukemia proteins to regulate homeobox (Hox) gene expression in vitro. Using conditional Men1 knockout mice, we have investigated the requirement for menin in hematopoiesis and myeloid transformation. Men1 excision causes reduction of Hoxa9 expression, colony formation by hematopoietic progenitors, and the peripheral white blood cell count. Menin directly activates Hoxa9 expression, at least in part, by binding to the Hoxa9 locus, facilitating methylation of H3K4, and recruiting the methylated H3K4 binding protein chd1 to the locus. Consistent with signaling downstream of menin, ectopic expression of both Hoxa9 and Meis1 rescues colony formation defects in Men1-excised bone marrow. Moreover, Men1 excision also suppresses proliferation of leukemogenic mixed lineage leukemia-AF9 fusion-protein-transformed myeloid cells and Hoxa9 expression. These studies uncover an important role for menin in both normal hematopoiesis and myeloid transformation and provide a mechanistic understanding of menins function in these processes that may be used for therapy.

The APC tumor suppressor controls the stability and nuclear export of beta-catenin (beta-cat), a transcriptional coactivator of LEF-1/TCF HMG proteins in the Wnt/Wg signaling pathway. We show here that beta-cat and APC have opposing actions at Wnt target genes in vivo. The beta-cat C-terminal activation domain associates with TRRAP/TIP60 and mixed-lineage-leukemia (MLL1/MLL2) SET1-type chromatin-modifying complexes in vitro, and we show that beta-cat promotes H3K4 trimethylation at the c-Myc gene in vivo. H3K4 trimethylation in vivo requires prior ubiquitination of H2B, and we find that ubiquitin is necessary for transcription initiation on chromatin but not nonchromatin templates in vitro. Chromatin immunoprecipitation experiments reveal that beta-cat recruits Pygopus, Bcl-9/Legless, and MLL/SET1-type complexes to the c-Myc enhancer together with the negative Wnt regulators, APC, and betaTrCP. Interestingly, APC-mediated repression of c-Myc transcription in HT29-APC colorectal cancer cells is initiated by the transient binding of APC, betaTrCP, and the CtBP corepressor to the c-Myc enhancer, followed by stable binding of the TLE-1 and HDAC1 corepressors. Moreover, nuclear CtBP physically associates with full-length APC, but not with mutant SW480 or HT29 APC proteins. We conclude that, in addition to regulating the stability of beta-cat, APC facilitates CtBP-mediated repression of Wnt target genes in normal, but not in colorectal cancer cells.

Multiple endocrine neoplasia type I (MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menins role as a tumor suppressor and gain insights into the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to approximately 20,000 promoters in HeLa S3, HepG2, and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis. We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes but do not always bind together. These data suggest that menin functions as a general regulator of transcription. We also found that factor occupancy generally correlates with high gene expression but that the loss of menin does not result in significant changes in most transcript levels. One exception is the developmentally programmed transcription factor, HLXB9, which is overexpressed in islets in the absence of menin. Our findings expand the realm of menin-targeted genes several hundred-fold beyond that previously described and provide potential insights to the endocrine tumor bias observed in MEN1 patients.

Multiple endocrine neoplasia type 1 (MEN1), an inherited tumor syndrome affecting endocrine organs including pancreatic islets, results from mutation of the tumor suppressor gene Men1 that encodes protein menin. Although menin is known to be involved in regulating cell proliferation in vitro, it is not clear how menin regulates cell cycle and whether mutation of Men1 acutely promotes pancreatic islet cell proliferation in vivo. Here we show that excision of the floxed Men1 in mouse embryonic fibroblasts (MEF) accelerates G(0)/G(1) to S phase entry. This accelerated S-phase entry is accompanied by increased cyclin-dependent kinase 2 (CDK2) activity as well as decreased expression of CDK inhibitors p18(Ink4c) and p27(Kip1). Moreover, Men1 excision results in decreased expression of p18(Ink4c) and p27(Kip1) in the pancreas. Furthermore, complementation of menin-null cells with wild-type menin represses S-phase entry. To extend the role of menin in repressing cell cycle in cultured cells to in vivo pancreatic islets, we generated a system in which floxed Men1 alleles can be excised in a temporally controllable manner. As early as 7 days following Men1 excision, pancreatic islet cells display increased proliferation, leading to detectable enlargement of pancreatic islets 14 days after Men1 excision. These observations are consistent with the notion that an acute effect of Men1 mutation is accelerated S-phase entry and enhanced cell proliferation in pancreatic islets. Together, these results suggest a molecular mechanism whereby menin suppresses MEN1 tumorigenesis at least partly through repression of G(0)/G(1) to S transition.

Multiple endocrine neoplasia type 1 (MEN-1) is a heritable syndrome typified by tumors in multiple endocrine organs, including the pituitary, parathyroids, and pancreatic islets. MEN-1 is attributable to mutations in the MEN1 tumor-suppressor gene that encodes the menin protein. Recent studies have implicated menin in transcriptional regulation and in covalent histone modification; however, little is known about modifications of the menin protein. Here, we report that menin is subject to phosphorylation on serine residues, including Ser543 and Ser583. Phosphorylation-defective mutants of either or both of these residues retain the associated histone methyltransferase activity of menin, as well as binding to the trithorax complex members Ash2L, Rbbp5, and MLL2 and to RNA polymerase II. Chromatin immunoprecipitation experiments reveal that binding of menin to the Hoxc8 locus is not affected by phosphorylation on Ser543 or Ser583.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized primarily by endocrine tumors of the parathyroids, anterior pituitary, and enteropancreatic endocrine tissues. Affected individuals carry a germ-line loss-of-function mutation of the MEN1 gene, and tumors arise after loss of the second allele. Homozygous loss of Men1 in the germ line of mice results in early embryonic lethality, with defective development of neural tube, heart, liver, and craniofacial structures. We generated immortalized wild-type (WT) and menin-null mouse embryo fibroblast (MEF) cell lines and evaluated their characteristics, including global expression patterns. The WT and menin-null cell lines were aneuploid, and the nulls did not display tumorigenic characteristics in soft agar assay. expression arrays in menin-null MEFs revealed altered expression of several extracellular matrix proteins that are critical in organogenesis. Specifically, transcripts for fibulin 2 (Fbln2), periostin (Postn), and versican [chondroitin sulfate proteoglycan (Cspg2)], genes critical for the developing heart and known to be induced by transforming growth factor-beta (TGF-beta), were decreased in their expression in menin-null MEFs. Fbln2 expression was the most affected, and the reduction in menin-null MEFs for Fbln2, Postn, and Cspg2 was 16.18-, 5.37-, and 2.15-fold, respectively. Menin-null MEFs also showed poor response to TGF-beta-induced Smad3-mediated transcription in a reporter assay, supporting a role for menin in this pathway. Postn and Cspg2 expression in WT, unlike in null MEFs, increased on TGF-beta treatment. The expression changes associated with the loss of the tumor suppressor menin provide insights into the defective organogenesis observed during early embryonic development in Men1-null mouse embryos.

During pregnancy, maternal pancreatic islets grow to match dynamic physiological demands, but the mechanisms regulating adaptive islet growth in this setting are poorly understood. Here we show that menin, a protein previously characterized as an endocrine tumor suppressor and transcriptional regulator, controls islet growth in pregnant mice. Pregnancy stimulated proliferation of maternal pancreatic islet beta-cells that was accompanied by reduced islet levels of menin and its targets. Transgenic expression of menin in maternal beta-cells prevented islet expansion and led to hyperglycemia and impaired glucose tolerance, hallmark features of gestational diabetes. Prolactin, a hormonal regulator of pregnancy, repressed islet menin levels and stimulated beta-cell proliferation. These results expand our understanding of mechanisms underlying diabetes pathogenesis and reveal potential targets for therapy in diabetes.

Loss of menin, a tumor suppressor coded by the MEN1 gene, is a key factor in the pathogenesis of multiple endocrine neoplasia type I and in a percentage of sporadic endocrine tumors of the pancreas and parathyroid glands. This study investigated expression of the menin protein in the normal exocrine pancreas and in pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic tumor. Immunofluorescence (IF) analyses showed that menin is expressed at high levels in normal acinar and duct cells. Examination of 24 clinical samples of PDAC revealed a pronounced decrease in menin expression in all tumors examined. To identify alterations underlying this defect, we searched for disruption and epigenetic silencing of the MEN1 gene. Analysis of nine laser-microdissected tumors revealed loss of heterozygosity of intragenic (one tumor) or adjacent (three tumors) MEN1 microsatellite markers. Methylation of CpG sites in the MEN1 promoter was documented in five of 24 tumors. IF analyses also revealed low to undetectable menin expression in the PDAC cell lines MiaPaCa-2 and Panc-1. Ectopic expression of menin in these cells resulted in a marked alteration of the cell cycle, with an increase in the G1/S+G2 ratio. These findings represent the first evidence that the MEN1 gene is a target of mutation and methylation in PDAC and that menin influences the cell cycle profile of duct cells.

Inactivating mutations in the tumor suppressor gene MEN1 cause the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1). The ubiquitously expressed MEN1 encoded protein, menin, interacts with MLL (mixed-lineage leukemia protein), and together they are essential components of a multiprotein complex with histone methyl transferase activity. MLL is also essential for hematopoiesis, and plays a critical role in leukemogenesis via epigenetic regulation of Hoxa9 expression that also requires menin. Therefore we chose to explore the role of menin in hematopoiesis. We generated Men1(-/-) embryonic stem (ES) cell lines, and induced them to differentiate in vitro. While these cells were able to form embryoid bodies (EBs) expressing the early markers Flk-1 and c-Kit, their ability to further differentiate into hematopoietic colonies was compromised. The Men1(-/-) ES cells show reduced expression of Hoxa9 that can be recovered by reexpression of Menin. We demonstrate that the block in differentiation of Men1(-/-) ES cell lines can be rescued not only by the expression of menin but also that of Hoxa9. These results suggest that, similar to MLL, menin is required for hematopoiesis, and this requirement may be mediated through regulation of Hoxa9 expression.

Menin, the product of the MEN1 (multiple endocrine neoplasia type 1) tumor suppressor gene, is involved in activation of gene transcription as part of an MLL1 (mixed-lineage leukemia 1)/MLL2 (KMT2A/B)-containing protein complex which harbors methyltransferase activity for lysine 4 of histone H3 (H3K4). As MEN1 patients frequently develop lipomas and peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in several MEN1-related tumor types, we investigated regulation of PPARgamma activity by menin. We found that menin is required for adipocyte differentiation of murine 3T3-L1 cells and PPARgamma-expressing mouse embryonic fibroblasts. Menin augments PPARgamma target gene expression through recruitment of H3K4 methyltransferase activity. Menin interacts directly with the activation function 2 transcription activation domain of PPARgamma in a ligand-independent fashion. Ligand-dependent coactivation, however, is dependent on the LXXLL motif of menin and the intact helix 12 of PPARgamma. We propose that menin is an important factor in PPARgamma-mediated adipogenesis and that loss of PPARgamma function may contribute to lipoma development in MEN1 patients.

Hibernomas are benign tumors with morphological features resembling brown fat. They consistently display cytogenetic rearrangements, typically translocations, involving chromosome band 11q13. Here we demonstrate that these aberrations are associated with concomitant deletions of AIP and MEN1, tumor suppressor genes that are located 3 Mb apart and that underlie the hereditary syndromes pituitary adenoma predisposition and multiple endocrine neoplasia type I. MEN1 and AIP displayed a low expression in hibernomas whereas the expression of genes up-regulated in brown fat--PPARA, PPARG, PPARGC1A, and UCP1--was high. Thus, loss of MEN1 and AIP is likely to be pathogenetically essential for hibernoma development. Simultaneous loss of two tumor suppressor genes has not previously been shown to result from a neoplasia-associated translocation. Furthermore, in contrast to the prevailing assumption that benign tumors harbor relatively few genetic aberrations, the present analyses demonstrate that a considerable number of chromosome breaks are involved in the pathogenesis of hibernoma.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder associated mainly with tumors of multiple endocrine organs. mutations in the MEN1 gene that encodes for the menin protein are the predominant cause for hereditary MEN1 syndrome. Though menin is a tumor suppressor, its molecular mechanism of action has not been defined. Here, we report that menin interacts with AKT1 in vitro and in vivo. Menin downregulates the level of active AKT and its kinase activity. Through interaction with AKT1, menin suppresses both AKT1-induced proliferation and antiapoptosis in nonendocrine and endocrine cells. Confocal microscopy analysis revealed that menin regulates AKT1 in part by reducing the translocation of AKT1 from the cytoplasm to the plasma membrane during growth factor stimulation. Our findings may be generalizable to other cancers, insofar as we found that loss of menin expression was also associated with AKT activation in a mouse model of pancreatic islet adenoma. Together, our results suggest menin as an important novel negative regulator of AKT kinase activity.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) is a heritable cancer syndrome characterized by tumors of the pituitary, pancreas and parathyroid. Menin, the product of the MEN1 gene, is a tumor suppressor protein that functions in part through the regulation of transcription mediated by interactions with chromatin modifying enzymes. PRINCIPAL FINDINGS: Here we show menin association with the 5 regions of DNA damage response genes increases after DNA damage and is correlated with RNA polymerase II association but not with changes in histone methylation. Furthermore, we were able to detect significant levels of menin at the 3 regions of CDKN1A and GADD45A under conditions of enhanced transcription following DNA damage. We also demonstrate that menin is specifically phosphorylated at Ser394 in response to several forms of DNA damage, Ser487 is dynamically phosphorylated and Ser543 is constitutively phosphorylated. Phosphorylation at these sites however does not influence the ability to interact with histone methyltransferase activity. In contrast, the interaction between menin and RNA polymerase II is influenced by phosphorylation, whereby a phospho-deficient mutant had a higher affinity for the elongating form of RNA polymerase compared to wild type. Additionally, a subset of MEN1-associated missense point mutants, fail to undergo DNA damage dependent phosphorylation. CONCLUSION: Together, our findings suggest that the menin tumor suppressor protein undergoes DNA damage induced phosphorylation and participates in the DNA damage transcriptional response.

tumor suppressor menin, the product of the MEN1 gene, plays a key role in controlling histone 3 lysine 4 trimethylation (H3K4me3) and gene transcription, which can regulate proliferation, apoptosis, and differentiation. However, little is known as to whether menin controls gene expression and cell proliferation and survival via regulating Polycomb group (PcG) protein complex/H3K27me3. Here we show that menin specifically represses transcription factor Paired box gene 2 (Pax2) through PcG-mediated H3K27me3 and Wilms tumor suppressor protein (WT1), a zinc finger domain-containing DNA-binding protein. Menin does not directly bind to the Pax2 locus, instead, it up-regulates WT1 expression. WT1 recruits PcG complex to the Pax2 promoter and represses expression of Pax2 through PcG-dependent H3K27me3. Moreover, WT1 also interacts with DNA methyltransferase 1 (DNMT1), and recruits DNMT1 to the Pax2 promoter, resulting in hypermethylation of CpG in the Pax2 promoter. Together, these studies have uncovered a novel epigenetic mechanism whereby menin regulates H3K27me3 and promoter DNA methylation via WT1 and suggest that WT1 protein plays an important, yet previously unappreciated role in regulating the function of the menin/PcG axis, H3K27 methylation, and DNA methylation, resulting in repression of gene transcription.

Menin is a tumor suppressor protein that is encoded by the MEN1 (multiple endocrine neoplasia 1) gene and controls cell growth in endocrine tissues. Importantly, menin also serves as a critical oncogenic cofactor of MLL (mixed lineage leukemia) fusion proteins in acute leukemias. Direct association of menin with MLL fusion proteins is required for MLL fusion protein-mediated leukemogenesis in vivo, and this interaction has been validated as a new potential therapeutic target for development of novel anti-leukemia agents. Here, we report the first crystal structure of menin homolog from Nematostella vectensis. Due to a very high sequence similarity, the Nematostella menin is a close homolog of human menin, and these two proteins likely have very similar structures. Menin is predominantly an alpha-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two alpha-helical bundles and covered by a beta-sheet motif. A very interesting feature of menin structure is the presence of a large central cavity that is highly conserved between Nematostella and human menin. By employing site-directed mutagenesis, we have demonstrated that this cavity constitutes the binding site for MLL. Our data provide a structural basis for understanding the role of menin as a tumor suppressor protein and as an oncogenic co-factor of MLL fusion proteins. It also provides essential structural information for development of inhibitors targeting the menin-MLL interaction as a novel therapeutic strategy in MLL-related leukemias.

Oncogenic activation of the NF-kappaB signaling pathway is common in hepatocellular carcinoma (HCC). However, the molecular mechanisms remain largely unexplored. Previous studies have demonstrated that menin, a tumor suppressor protein, could interact with NF-kappaB protein and repress p65-mediated transcriptional activation. In the present study, we found that expression of menin was frequently down-regulated in HCC tissues and cells. Furthermore, menin could repress p65 acetylation through recruitment of Sirt1, an enzyme that deacetylases p65 in lysine 310 (K310). Indeed, Sirt1 inhibitor or its specific small interfering RNA abolished the inhibitory roles of menin. Together, these observations suggest that the interaction between menin and Sirt1 is required for the tumor suppressor function of menin in HCC.

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic beta cells for people with diabetes and for targeting menin-sensitive endocrine tumors.

Classical familial adenomatous polyposis (FAP) is a high-penetrance autosomal dominant disease that predisposes to hundreds or thousands of colorectal adenomas and carcinoma and that results from truncating mutations in the APC gene. A variant of FAP is attenuated adenomatous polyposis coli, which results from germ-line mutations in the 5 and 3 regions of the APC gene. Attenuated adenomatous polyposis coli patients have "multiple" colorectal adenomas (typically fewer than 100) without the florid phenotype of classical FAP. Another group of patients with multiple adenomas has no mutations in the APC gene, and their phenotype probably results from variation at a locus, or loci, elsewhere in the genome. Recently, however, a missense variant of APC (I1307K) was described that confers an increased risk of colorectal tumors, including multiple adenomas, in Ashkenazim. We have studied a set of 164 patients with multiple colorectal adenomas and/or carcinoma and analyzed codons 1263-1377 (exon 15G) of the APC gene for germ-line variants. Three patients with the I1307K allele were detected, each of Ashkenazi descent. Four patients had a germ-line E1317Q missense variant of APC that was not present in controls; one of these individuals had an unusually large number of metaplastic polyps of the colorectum. There is increasing evidence that there exist germ-line variants of the APC gene that predispose to the development of multiple colorectal adenomas and carcinoma, but without the florid phenotype of classical FAP, and possibly with importance for colorectal cancer risk in the general population.