In vitro hematopoietic differentiation of mouse embryonic stem cells requires the tumor suppressor menin and is mediated by Hoxa9

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 induces apoptosis in murine embryonic fibroblasts

Multiple endocrine neoplasia type I (MEN1) is a hereditary tumor syndrome characterized by multiple endocrine and occasionally non-endocrine tumors. The tumor suppressor gene Men1, which is frequently mutated in MEN1 patients, encodes the nuclear protein menin. Although many tumor suppressor genes are involved in the regulation of apoptosis, it is unclear whether menin facilitates apoptosis. Here we show that ectopic overexpression of menin via adenoviruses induces apoptosis in murine embryonic fibroblasts. The induction of apoptosis depends on Bax and Bak, two proapoptotic proteins. Moreover, loss of menin expression compromises apoptosis induced by UV irradiation and tumor necrosis factor-alpha (TNF-alpha), whereas complementation of menin-null cells with menin restores sensitivity to UV- and TNF-alpha-induced apoptosis. Interestingly, loss of menin reduces the expression of procaspase 8, a critical protease that is essential for apoptosis induced by death-related receptors, whereas complementation of the menin-null cells up-regulates the expression of procaspase 8. Furthermore, complementation of menin-null cells with menin increases the activation of caspase 8 in response to TNF-alpha treatment. These results suggest a proapoptotic function for menin that may be important in suppressing the development of MEN1.     

MEN1胰岛瘤中细胞周期蛋白cyclin B2高表达的作用和机制

多发性内分泌肿瘤1-(multiple endocrine neoplasia type 1,MEN1)是一种常染色体显性遗传的肿瘤综合征,患者常表现出多发性的内分泌器官肿瘤,包括垂体瘤、甲状旁腺瘤和胰岛瘤.抑癌基因Men1的突变导致MENl的发生,其编码的蛋白为核蛋白menin.Menin可以抑制包括胰岛β细胞在内的...     

Microarray analysis of gene expression in Men1 knockout embryoid body reveals genetic events involved in early mouse embryonic development

The Men1 gene has been identified as the gene responsible for MEN1, a hereditary syndrome transmitted with an autosomal dominant trait. Disruption of the Men1 gene results in defects of multiple organs development, including the nervous system, heart, liver, cranium, and face. In this study, we used embryoid bodies (EBs) formed from wild-type and Men1-/- ES cells as a model system to investigate effect of Men1 gene on the embryo development. We characterized in detail gene expression profile of these Men1-/- EBs by microarray techniques and identified a series of putative menin targeted genes, including genes involved in development of bone (e.g., Postn, Runx2, and Msx2), liver (e.g., KDR), blood (e.g., Hox9 and Kitl), and pancreatic islet (e.g., Sox4, Foxa1, Btc, Igf2, and Nfatc1). Further studies may shed light onto the underlying mechanisms of the interplay between menin and these genes.     

Menin, a gene product responsible for multiple endocrine neoplasia type 1, interacts with the putative tumor metastasis suppressor nm23

Although the gene responsible for multiple endocrine neoplasia type 1 (MEN1) has been identified, the function of its gene product, menin, is unknown. To examine the biological role of the MEN1 gene, we searched for associated proteins with a yeast two-hybrid system using the MEN1 cDNA fragment as bait. On screening a rat fetal brain embryonic day 17 library, in which a high level of MEN1 expression was detected, we identified a putative tumor metastasis suppressor nm23/nucleoside diphosphate (NDP) kinase as an associated protein. This finding was confirmed by in vitro interaction assays based on glutathione S-transferase pull down experiments. The association required almost the entire menin protein, and several missense MEN1 mutations reported in MEN1 patients caused a loss of the binding activity for nm23. This result suggests that this interaction may play important roles in the biological functions of the menin protein, including tumor suppressor activity.     

Genome-wide characterization of menin-dependent H3K4me3 reveals a specific role for menin in the regulation of genes implicated in MEN1-like tumors

Inactivating mutations in the MEN1 gene predisposing to the multiple endocrine neoplasia type 1 (MEN1) syndrome can also cause sporadic pancreatic endocrine tumors. MEN1 encodes menin, a subunit of MLL1/MLL2-containing histone methyltransferase complexes that trimethylate histone H3 at lysine 4 (H3K4me3). The importance of menin-dependent H3K4me3 in normal and transformed pancreatic endocrine cells is unclear. To study the role of menin-dependent H3K4me3, we performed in vitro differentiation of wild-type as well as menin-null mouse embryonic stem cells (mESCs) into pancreatic islet-like endocrine cells (PILECs). Gene expression analysis and genome-wide H3K4me3 ChIP-Seq profiling in wild-type and menin-null mESCs and PILECs revealed menin-dependent H3K4me3 at the imprinted Dlk1-Meg3 locus in mESCs, and all four Hox loci in differentiated PILECs. Specific and significant loss of H3K4me3 and gene expression was observed for genes within the imprinted Dlk1-Meg3 locus in menin-null mESCs and the Hox loci in menin-null PILECs. Given that the reduced expression of genes within the DLK1-MEG3 locus and the HOX loci is associated with MEN1-like sporadic tumors, our data suggests a possible role for menin-dependent H3K4me3 at these genes in the initiation and progression of sporadic pancreatic endocrine tumors. Furthermore, our investigation also demonstrates that menin-null mESCs can be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null mESC-derived specialized cell types for genome-wide high-throughput studies.     

Heterozygous Men1 mutant mice develop a range of endocrine tumors mimicking multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary syndrome characterized by the occurrence of multiple endocrine tumors of the parathyroid, pancreas, and anterior pituitary in patients. To study tumorigenesis related to the MEN1 syndrome, we have generated Men1 knockout mice using the gene targeting approach. Heterozygous Men1 mutant mice developed the same range of major endocrine tumors as is seen in MEN1 patients, affecting the parathyroid, pancreatic islets, pituitary and adrenal glands, as well as the thyroid, and exhibiting multistage tumor progression with metastatic potential. In particular, extrapancreatic gastrinoma, pancreatic glucagonoma, and mixed hormone-producing tumors in islets were observed. In addition, there was a high incidence of gonadal tumors of endocrine origin, i.e. Leydig cell tumors, and ovary sex-cord stromal cell tumors in heterozygous Men1 mutant mice. Hormonal disturbance, such as abnormal PTH and insulin levels, was also observed in these mice. These tumors were associated with loss of heterozygosity of the wild-type Men1 allele, suggesting that menin is involved in suppressing the development of these endocrine tumors. All of these features are reminiscent of MEN1 symptoms in humans and establish heterozygous Men1 mutant mice as a suitable model for this disease.     

Menin represses malignant phenotypes of melanoma through regulating multiple pathways

Substantial genetic evidence suggests that chromosome 11q is involved in regulating initiation and progression of malignant melanomas. Mutations of the MEN1 gene, located in chromosome 11q13, predispose individuals to the multiple endocrine neoplasia type 1 (MEN1) familial syndrome. MEN1 patients develop primary malignant melanoma, suggesting a potential link between MEN1 syndrome and development of melanomas, but the precise molecular mechanism is poorly understood. Here we show that the MEN1 gene suppresses malignant phenotypes of melanoma cells through multiple signalling pathways. Ectopic expression of menin, the product of MEN1 gene, significantly inhibited melanoma cell proliferation and migration in vitro and in vivo. The inhibition was partly achieved through suppressing expression of growth factor pleiotrophin (PTN) and receptor protein tyrosine phosphatase (RPTP) β/ζ, accompanied with the reduced expression of phosphatidylinositol 3-kinase (pI3K) and decreased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2). Interestingly, reduced expression of menin was associated with hypermethylation of the CpG islands of the MEN1 promoter in melanoma cells. Taken together, these findings suggest a previously unappreciated function for menin in suppressing malignant phenotypes of melanomas and unravel a novel mechanism involving in regulating PTN signalling by menin in development and progression of melanomas.     

Menin and JunD regulate gastrin gene expression through proximal DNA elements

Mutations in the MEN1 gene correlate with multiple endocrine neoplasia I (MEN1). Gastrinomas are the most malignant of the neuroendocrine tumors associated with MEN1. Because menin and JunD proteins interact, we examined whether JunD binds to and regulates the gastrin gene promoter. Both menin and JunD are ubiquitous nuclear proteins that we showed colocalize in the gastrin-expressing G cells of the mouse antrum. Transfection with a JunD expression vector alone induced endogenous gastrin mRNA in AGS human gastric cells, and the induction was blocked by menin overexpression. We mapped repression by menin to both a nonconsensus AP-1 site and proximal GC-rich elements within the human gastrin promoter. Chromatin immunoprecipitation assays, EMSAs, and DNA affinity precipitation assays documented that JunD and Sp1 proteins bind these two elements and are both targets for menin regulation. Consistent with menin forming a complex with histone deacetylases, we found that repression of gastrin gene expression by menin was reversed by trichostatin A. In conclusion, proximal DNA elements within the human gastrin gene promoter mediate interactions between JunD, which induces gastrin gene expression and menin, which suppresses JunD-mediated activation.