Naturally arising CD25+CD4+ regulatory T cells (T(R) cells) are engaged in the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses, such as autoimmune disease and allergy. T(R) cells specifically express the transcription factor Foxp3, a key regulator of T(R)-cell development and function. Ectopic expression of Foxp3 in conventional T cells is indeed sufficient to confer suppressive activity, repress the production of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and upregulate T(R)-cell-associated molecules such as CD25, cytotoxic T-lymphocyte-associated antigen-4, and glucocorticoid-induced TNF-receptor-family-related protein. However, the method by which Foxp3 controls these molecular events has yet to be explained. Here we show that the transcription factor AML1 (acute myeloid leukaemia 1)/Runx1 (Runt-related transcription factor 1), which is crucially required for normal haematopoiesis including thymic T-cell development, activates IL-2 and IFN-gamma gene expression in conventional CD4+ T cells through binding to their respective promoters. In natural T(R) cells, Foxp3 interacts physically with AML1. Several lines of evidence support a model in which the interaction suppresses IL-2 and IFN-gamma production, upregulates T(R)-cell-associated molecules, and exerts suppressive activity. This transcriptional control of T(R)-cell function by an interaction between Foxp3 and AML1 can be exploited to control physiological and pathological T-cell-mediated immune responses.

MEN1 is a tumor suppressor gene that is responsible for multiple endocrine neoplasia type 1 (MEN1) and that encodes a 610-amino-acid protein, called menin. While the majority of germ line mutations identified in MEN1 patients are frameshift and nonsense mutations resulting in truncation of the menin protein, various missense mutations have been identified whose effects on menin activity are unclear. For this study, we analyzed a series of menin proteins with single amino acid alterations and found that all of the MEN1-causing missense mutations tested led to greatly diminished levels of the affected proteins in comparison with wild-type and benign polymorphic menin protein levels. We demonstrate here that the reduced levels of the mutant proteins are due to rapid degradation via the ubiquitin-proteasome pathway. Furthermore, the mutants, but not wild-type menin, interact both with the molecular chaperone Hsp70 and with the Hsp70-associated ubiquitin ligase CHIP, and the overexpression of CHIP promotes the ubiquitination of the menin mutants in vivo. These findings reveal that MEN1-causing missense mutations lead to a loss of function of menin due to enhanced proteolytic degradation, which may be a common mechanism for inactivating tumor suppressor gene products in familial cancer.Multiple endocrine neoplasia type 1 (MEN1) is a dominantly inherited familial cancer syndrome characterized by the combined occurrence of tumors of the parathyroid gland, the pancreas, and the pituitary gland (2, 38). The responsible gene, MEN1, has been localized to chromosome 11q13 (21) and identified by positional cloning (7). The loss of heterozygosity of the MEN1 locus in tumors suggests that MEN1 is a tumor suppressor gene. MEN1 contains 10 exons and encodes a 610-amino-acid protein, called menin (7), which shows no similarity to any known protein. Previous studies have revealed that menin is found predominantly in the nucleus, contains two independent nuclear localization signals in the C terminus (amino acids 479 to 497 and 588 to 608) (14), and binds to transcription factors, including JunD (1), Smad3 (18) and NF-B (15), all of which suggest a role related to transcriptional regulation. Menin has also been shown to interact with the putative tumor metastasis suppressor/nucleoside diphosphate kinase nm23 (27), the homeobox protein Pem (23), GFAP and vimentin (24), and the 32-kDa subunit (RPA2) of replication protein A (33). However, the exact molecular mechanism for tumor suppression by menin is largely unknown.More than 400 independent germ line mutations distributed throughout the MEN1 coding region have been identified for familial and sporadic cases of MEN1 (13, 36). The majority of these are nonsense mutations or frame shifts, but missense mutations and short in-frame deletions-insertions have also been identified in about 30% of cases. Germ line MEN1 mutations are also found in a small subset of familial isolated hyperparathyroidism (FIHP), a syndrome consisting of genetically heterogeneous diseases that are characterized ...

Molecular diversity through alternative splicing is important for cellular function and development. However, little is known about the factors that regulate alternative splicing. Here we demonstrate that one isoform of coactivator-associated arginine methyltransferase 1 (named CARM1-v3) associates with the U1 small nuclear RNP-specific protein U1C and affects 5 splice site selection of the pre-mRNA splicing. CARM1-v3 was generated by the retention of introns 15 and 16 of the primary transcript of CARM1. Its deduced protein lacks the C-terminal domain of the major isoform of CARM1 and instead has v3-specific sequences at the C terminus. CARM1-v3, but not the other isoforms, strongly stimulates a shift to the distal 5 splice site of the pre-mRNA when the adenoviral E1A minigene is used as a reporter and enhances the exon skips in the CD44 reporter. A CARM1-v3 mutant lacking the v3-specific sequences completely lost the ability to regulate the alternative splicing patterns. In addition, CARM1-v3 shows tissuespecific expression patterns distinct from those of the other isoforms. These results suggest that the transcriptional coactivator can affect the splice site decision in an isoform-specific manner.It has been estimated that about 60% of human genes undergo alternative splicing (1). Commonly, alternative splicing determines the inclusion of a portion of coding sequence in the mRNA, giving rise to protein isoforms that differ in their peptide sequence and hence chemical and biological activity. The mechanism of alternative splicing permits diversity of translatable mRNAs, thereby increasing the proteome diversity encoded by a limited number of genes. Genetic switches based on alternative splicing are known to be important in many cellular and developmental processes, including sex determination, apoptosis, axon guidance, and tissue-specific differentiation (2-4).Although a large variety of splicing decisions can be explained by the antagonistic effects of general splicing factors, such as serine/arginine-rich proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs), 1 it is most likely that tissue-specific or developmentally regulated splicing factors have an important role in the regulation of alternative splicing. In Drosophila melanogaster, sex-lethal functions as a regulator of alternative splicing in sex determination (2), and the embryonic lethal abnormal visual system is a gene-specific regulator of alternative pre-mRNA processing in neurons (3). However, in mammals, only a limited number of splicing regulators have been identified to date, despite the large diversity of the mammalian gene transcripts, and little is known about the mechanisms by which alternative splicing is regulated.Pre-mRNA splicing occurs in a large multicomponent ribonucleoprotein complex called the spliceosome, which is composed of U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein (snRNP) particles and many non-snRNP protein splicing factors (5). U1 snRNP recognizes the 5Ј splice site and is among the first factors to inter...

Heterozygous germline mutations of the tumor-suppressor gene MEN1 are responsible for multiple endocrine neoplasia type 1 (MEN1), a dominantly inherited familial cancer syndrome characterized by pituitary, parathyroid, and enteropancreatic tumors. Various mutations have been identified throughout the entire gene region in patients with MEN1 and related disorders. Neither mutation hot spot nor phenotype-genotype correlation has been established in MEN1 although some missense mutations may be specifically associated with a phenotype of familial isolated hyperparathyroidism. The gene product menin has been implicated in multiple roles, including gene transcription, maintenance of genomic integrity, and control of cell division and differentiation. These multiple functions are likely to be conferred by association with multiple protein factors. Occurrence of MEN1-causing missense mutations throughout menin also suggests the requirement of multiple binding factors for its full tumor-suppressive activity. The effect of menin depletion is highly tissue specific, but its underlying mechanism remains to be elucidated. A DNA test for MEN1 germline mutations is a useful tool for diagnosis of MEN1 although it needs further improvements. The germline mutations are heterozygous, and somatic loss of the normal MEN1 allele has been observed in the tumors arising in MEN1, in agreement with the Knudson's two-hit model. (11) Somatic inactivation of both MEN1 alleles has also been detected in some sporadic endocrine tumors, indicating involvement of this gene in the development of sporadic tumors. So far, MEN1 gene mutations have not been implicated in any other human diseases.The product of the MEN1 gene, menin, is a 610-amino acid protein that exhibits no apparent sequence similarity to any other known protein.(1) Thus, its biochemical function can not be deduced from its structure. In the last decade, a number of studies have demonstrated physical and functional associations between menin and diverse proteins of known function, shedding light on its molecular function. These menin-interacting proteins include nuclear proteins such as transcription factors, histone deacetylases, and histone methyltransferases, suggesting that menin is involved in gene transcription. Several lines of evidence also suggest that menin is involved in the maintenance of genomic integrity. Although menin is localized mainly to cell nuclei, possible extranuclear functions have not been excluded because various cytoplasmic proteins bind to menin.Although the molecular function of menin is still poorly understood, identification of the MEN1 gene has enabled a direct DNA test for predisposition to MEN1. In the present review, recent findings on the MEN1 gene are summarized and its mutations are discussed from basic and clinical points of view. Structure and expression of the MEN1 geneThe human MEN1 gene comprises 10 exons distributed over 7 kb in the chromosome region 11q13 and encodes mRNA of approximately 2.8 kb (Fig. 2).(1) Exons 2 through 10 encode th...

Germline mutations of the tumor suppressor gene MEN1 are found not only in typical multiple endocrine neoplasia type 1 (MEN1) but also in its incomplete forms such as familial isolated hyperparathyroidism (FIHP) and apparently sporadic parathyroid tumor (ASPT). No definitive genotype-phenotype correlation has been established between these clinical forms and MEN1 gene mutations. We previously demonstrated that mutant menin proteins associated with MEN1 are rapidly degraded by the ubiquitin-proteasome pathway. To examine whether the intracellular stability of mutant menin is correlated with clinical phenotypes, we developed a method of evaluating menin stability and examined 20 mutants associated with typical MEN1 (17 missense, two in-frame deletion, one nonsense) and 21 mutants associated with FIHP or ASPT (19 missense, two in-frame deletion). All tested mutants associated with typical MEN1 showed reduced stability. Some missense and in-frame deletion mutants (G28A, R171W, T197I, E255K, E274A, Y353del and E366D) associated with FIHP or ASPT were almost as stable as or only slightly less stable than wild-type menin, while others were as unstable as those associated with typical MEN1. Some stable mutants exhibited substantial biological activities when tested by JunD-dependent transactivation assay. These findings suggest that certain missense and in-frame mutations are fairly stable and retain intrinsic biological activity, and might be specifically associated with incomplete clinical phenotypes. The menin stability test will provide useful information for the management of patients carrying germline MEN1 mutations especially when they have missense or in-frame variants of ambiguous clinical significance. (Cancer Sci 2011; 102: 2097-2102 M enin is a tumor suppressor protein encoded by MEN1, a gene responsible for multiple endocrine neoplasia type 1 (MEN1), a familial cancer syndrome typically characterized by the development of multiple tumors in the pituitary, parathyroid and enteropancreatic endocrine tissues.(1,2) Menin is a nuclear protein having C-terminal nuclear localizing signal sequences, (3) and exhibits modulatory activity on gene expression such as repression of JunD-dependent transcription. (4) Diverse roles have been implicated for menin, including cell cycle control, cell differentiation and DNA repair, which are likely to be conferred by interaction with various menin-binding proteins.(2) Menin is considered to be a scaffold protein tethering such proteins to specific gene loci.(5) Although the mechanism of how menin is involved in gene regulation has been elucidated to some extent, the basis for tissue-specific tumorigenesis in MEN1 remains unknown.Heterozygous germline mutations of the MEN1 gene are found in 70-90% of patients clinically diagnosed as MEN1. (6) More than 500 different loss-of-function mutations have been identified, which are distributed throughout the gene with no apparent hot spot.(2,6,7) Germline MEN1 mutations have also been found in a subset of familial isolated hyperparathy...

Objective: Dopamine agonists normalize prolactin (PRL) levels and reduce tumour size in responsive prolactinoma. However, several cases have shown resistance to dopamine agonists upon initial treatment. Infrequently, prolactinoma initially responds, but then becomes refractory to prolonged treatment (secondary resistance). We investigated the possible mechanisms of resistance to dopamine agonists. Subjects and methods: Twelve cases of prolactinoma were surgically resected and classified according to the responsiveness of PRL levels and tumour size to dopamine agonists: good responders (nZ5), poor responders (nZ5), or secondary resistance (nZ2). We examined the expression of dopamine D 2 receptor (D 2 R) isoform (short: D 2 S and long: D 2 L) mRNA and protein. We investigated DNA methylation patterns in the promoter region of the DRD2 gene. Results: The predominant D 2 R isoform expressed in prolactinoma was D 2 L. Levels of D 2 L mRNA were significantly lower in secondary resistance and poor responders than in good responders. Expression of D 2 R protein was variable among cases. Almost no CpG sites of the DRD2 gene promoter region were methylated. Conclusion: Resistance of prolactinoma to dopamine agonists is correlated with a reduction in D 2 L isoform mRNA levels. Silencing of the DRD2 gene by methylation in the promoter region is unlikely to play a role in dopamine agonist resistance in prolactinoma.

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In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins.