InteractiveFly: GeneBrief

Menin 1: Biological Overview | References

Menin is a tumor suppressor required to prevent multiple endocrine neoplasia in humans. Mammalian menin protein is associated with chromatin modifying complexes and has been shown to bind a number of nuclear proteins, including the transcription factor JunD. Menin shows bidirectional effects acting positively on c-Jun and negatively on JunD. Protein null alleles of Drosophila menin (mnn1) have been produced and the Mnn1 protein was overexpressed. Flies homozygous for protein-null mnn1 alleles are viable and fertile. Localized over-expression of Mnn1 causes defects in thoracic closure, a phenotype that sometimes results from insufficient Jun activity. Complex genetic interactions were observed between mnn1 and jun in different developmental settings. These data support the idea that one function of menin is to modulate Jun activity in a manner dependent on the cellular context (Cerrato, 2006).

Human multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome characterized by tumors occurring prevalently in endocrine tissues. Common features of most MEN1 tumors are low proliferation rates, well-differentiated morphology and excessive hormone secretion. Hereditary tumors arise in individuals heterozygous for a loss-of-function MEN1 allele followed by somatic loss of wild type alleles. Sporadic tumors also show bi-allelic loss of MEN1 (Agarwal, 2004). The MEN1 locus encodes menin, a nuclear protein with two nuclear-localization sites at the C-terminal quarter of the protein, but no other overt sequence motifs. Menin is ubiquitously expressed, but only shows a loss of heterozygosity phenotype in a highly restricted set of cells (Scacheri, 2004). This context dependency suggests that regulated co-factors or modifiers act in conjunction with menin for cell-type specific function. Menin has also been found in a SET1-like histone methylation complex (Hughes, 2004; Karnik, 2005; Milne, 2005; Yokoyama, 2004; Yokoyama, 2005). The mouse menin gene is required for embryonic viability and, like in humans, inactivation of both alleles results in endocrine tumors (Crabtree, 2001; Crabtree, 2003). Therefore, menin is a classic tumor suppressor in the endocrine system. Interestingly, there is also recent evidence that menin is an oncogenic co-factor in Mixed Lineage Leukemia (Yokoyama, 2005). The nature of this dual growth suppressing and enhancing role in the regulation of proper cell number and differentiation has not been clarified (Cerrato, 2006).

Multiple potential transcription factor partners for mammalian menin protein have been identified (Agarwal, 2004) including JunD, which has been shown to interact directly with menin (Agarwal, 1999). It is unclear how these protein-protein interactions relate to menin in the SET-1 like histone methylation complex, although it is possible that menin association with many different nuclear proteins helps target the complex to appropriate regions of chromatin. Experiments performed in immortalized mouse embryo fibroblasts have shown that menin binding to JunD is necessary for JunD to act as a growth suppressor (Agarwal, 2003). Menin functions to reduce JunD activity and has been shown to inhibit the accumulation of active phosphorylated JunD or c-Jun. Even though menin does not directly bind c-Jun, it augments the transcriptional activity of this transcriptional factor (Knapp, 2000). Thus, menin is strongly implicated in regulating Jun function. Interestingly, according to the potential roles of menin to promote or suppress tumorigenesis, menin can act in turn negatively on JunD or positively on c-Jun function (Cerrato, 2006).

Jun and Fos heterodimers are well-known regulators of tumorigenesis, differentiation, apoptosis, immune and stress responses in both vertebrates and Drosophila. There are a number of mammalian homodimers and heterodimers consisting in c-Jun, JunB or JunD and c-Fos, FosB, Fra1 or Fra2 combinations. Unlike mammals, Drosophila has a single Jun and a single Fos. Drosophila Jun has features of both JunD and c-Jun. This makes Drosophila a good reductionist model for learning more about Jun/menin interactions. While experiments to see if Drosophila menin binds Jun have been negative (Guru, 2001), genetic interactions have not been explored. This study specifically investigated the functional connection between Drosophila menin and Jun (Cerrato, 2006).

The Drosophila menin protein (Mnn1) is 47% identical to the human protein, including 69% of the amino acid residues that are required for tumor suppression in human endocrine tissues (Guru, 2001; Maruyama, 2000). The ongoing sequencing of multiple species in Drosophila reveals that menin is highly conserved. Despite this high degree of conservation, menin is not required for viability in Drosophila. Flies lacking mnn1 expression are viable and fertile (Busygina, 2004; Papaconstantinou, 2005). One report suggests that mnn1 is required for a wild type life span and some aspect of either chromosome stability or DNA repair (Busygina, 2004), while another report (Papaconstantinou, 2005) suggests that mnn1 is required for a robust response to various types of stress (Cerrato, 2006).

Two protein-null mnn1 alleles were isolated and transgenic flies were generated for the controlled over-expression of Drosophila Mnn1 protein. mnn1⁻ flies are viable and fertile. It has been reported that uniform over-expression of mnn1 has no effect on development or viability in flies (Papaconstantinou, 2005). This study found that over-expression results in pharate-adult phenotype, proboscis ablation and a cleft thorax. These over-expression phenotypes are modified by both gain-of-function and loss-of-function alleles of jun. Dominant-negative alleles of fos are enhanced by loss-of-function alleles of mnn1. The finding that both Drosophila and mammalian menin (Agarwal, 1999; Agarwal, 2003) are capable of interacting with Jun suggests that an evolutionarily conserved menin function in normal development and disease is linked to the Jun/Fos family of transcriptional regulators. Interestingly, as in mammals, Drosophila menin shows bidirectional modulation of Jun function (Cerrato, 2006).

The mnn1 locus is tightly flanked upstream by the milton gene (milt) and the CG31907 gene is nested in a mnn1 intron. Previously identified deletion alleles of mnn1 (Busygina, 2004; Papaconstantinou, 2005) are likely to disrupt the function of flanking genes in addition to mnn1. The mnn1^e200 allele is also mutant for milt (Busygina, 2004). The mnn1^e173 allele potentially disrupts milt. Both mnn1^e173 and mnn1^e30 delete sequences that approach the 3' end of CG31907 (Papaconstantinou, 2005). A P-element, P{wHy}^30G01, inserted in the 5'UTR of the mnn1 locus was mobilized, and small deletions were sought in order to identify new alleles of mnn1 that would not affect other genes (Cerrato, 2006).

FlyBase annotates two mnn1 transcripts, but neither of these transcripts have been isolated in previous molecular studies of the mnn1 locus (Guru, 2001; Maruyama, 2000). Maruyama and Guru, independently, describe mnn1 transcripts that differ from the two FlyBase annotations in the UTRs and in the terminal coding exon. A developmental profile of mnn1 expression has revealed two mnn1 transcripts (Guru, 2001) that are due to alternative poly-A sites. The shorter transcript annotated in FlyBase (mnn1-RB) may have been primed from an A-rich sequence in the intron of an unprocessed message rather than from the poly-A tail. Additionally, none of the 18 amino acids specific to Mnn1-PB are present in each of mnn1 genes of the Drosophila species that this study has reported. Thus, the Muruyama and Guru gene models are the reference for the work described in this study (Cerrato, 2006).

Two deletion alleles, mnn1^Δ46 and mnn1^Δ79 were generated. RT-PCR and sequence analysis indicate that the mnn1^Δ46 allele has a deletion of 573 bp of the mnn1 locus missing 70 bp downstream of the first ATG of the mnn1 ORF (Agarwal, 2004), while the mnn1^Δ79 allele has a more extensive deletion of 186 bp downstream of the start codon. However, all distal genes appeared to be intact, including CG31907 which is nested in mnn1 intron 4. A third new allele, Df(2L)mnn1^Δ65 deletes at least 14 kb proximal to the 3' end of the Muruyama and Guru mnn1 gene model. This deletion, along with Df(2L)JH, removes several additional complementation groups required for viability (Cerrato, 2006).

Flies homozygous for either mnn1^Δ46 or mnn1^Δ79 are viable and fertile and can be readily maintained as homozygous stocks. Hemizygous mnn1^Δ46 or mnn1^Δ79 flies are also viable and fertile over Df(2L)mnn1 or Df(2L)JH. In addition to the mutant alleles, two precise excision lines, mnn1⁺⁸⁴ and mnn1⁺¹¹³, were selected as wild type isogenic controls for further experiments. The mnn1^Δ46 and mnn1^Δ79 chromosomes were extensively backcrossed to control y⁻ w⁻ flies to remove any undetected mutations associated with transposon mobilization (Cerrato, 2006).

Mnn1 mRNA isoforms are expressed in wild type early embryos and in adult females. The longer isoform is detected throughout development (Guru, 2001). To determine if the deletion alleles express mnn1 mRNA, as might be expected given the presence of residual promoter region and upstream sequences, RT-PCR reactions were performed on total RNA extracts from mnn1^Δ46 to mnn1^Δ79 homozygous adult females and on perfect excision lines using multiple primer pairs also overlapping the ATG sequence and spanning mnn1 intronic sequences. The use of intron-spanning primers in the absence of reverse transcriptase allowed distinguishing between transcripts and any contaminating genomic DNA. RT-PCR results obtained on wild type flies supported the first exon structure in the Muruyama and Guru gene model. While no transcripts were detected with primers directed against deleted sequence in homozygous mnn1^Δ46 and mnn1^Δ79, transcripts were detected using primers downstream from those deletions. These results indicate that mnn1 mRNAs are produced from the mutant alleles (Cerrato, 2006).

Both mnn1^Δ46 and mnn1^Δ79 alleles delete mnn1 sequence coding for residues homologous to those known to be required for menin function in humans (Agarwal, 2004). In both mnn1^Δ46 or mnn1^Δ79, the first two in-frame ATGs of mnn1 are deleted, such that homologs of at least five amino acids required to prevent disease in humans are deleted due to a downstream translational start site utilization. To determine if mnn1^Δ46 and mnn1^Δ79 alleles encode defective menin proteins initiated from downstream AUGs in the mutant mRNAs, immunoblots and cell staining experiments were performed. While these putative mutant polypeptides would be missing critical Mnn1 residues, they might retain some function (wild type or even dominant negative). Mnn1 proteins initiated by alternative downstream AUGs present in mnn1 mutant mRNAs should migrate faster on SDS-PAGE. Immunoblot analysis performed with an antibody produced against an epitope mapping in exon 4 showed a species migrating at ~ 95 kDa in extracts from wild type flies and extracts from bacteria expressing Drosophila Mnn1, but not from homozygous mnn1^Δ46 or mnn1^Δ79 flies. These results indicate that the antibody recognizes wild type Mnn1. Protein extracts were analyzed of whole adult females or males, third instar larvae and Central Nervous System (CNS) from third instar larvae, and in no case was a shorter isoform detected. Thus, in mnn1^Δ46 or mnn1^Δ79 larvae or adults, there is no evidence of N-terminally truncated Mnn1 proteins. Western blot results therefore simultaneously confirm that the bands in the wild type lanes correspond to endogenous Mnn1, not a cross-reacting species of similar mobility, and that the deletion alleles encode undetectable levels of N-terminally deleted Mnn1 protein. It is concluded that the mnn1^Δ46 and mnn1^Δ79 alleles are protein nulls. Previously reported mnn1 alleles are also likely to be protein nulls (Busygina,2004; Papaconstantinou, 2005). In no case has mnn1 been shown to be required for viability or fertility. All these data strongly suggest that mnn1 is not an essential gene in Drosophila (Cerrato, 2006).

Human menin is a nuclear protein (Guru, 1998). The Drosophila Mnn1 protein has a potential nuclear localization signal (KRTRR) in the region corresponding to the NLS-2 of human menin (Guru, 2001). The mnn1 gene is broadly expressed throughout development (Guru, 2001). As expected, Mnn1 immunoreactivity was detected in the nuclei of the central nervous system, and many other larval tissues of wild type flies. Such staining was absent in flies homozygous for mnn1^Δ46 or mnn1^Δ79. The weak anti-Mnn1 staining of endogenous protein was enriched in the nucleus and showed sub nuclear localization. To further evaluate the cellular localization of Drosophila Mnn1, over-expressed Mnn1 was detected following induction of UAS-mnn1 with any number of Gal4 drivers (e.g. AB1, 69B, How24, dilp2 and OK6). In all cases, over-expressed Mnn1 is nuclear. This staining is robust, again suggesting that endogenous Mnn1 is not abundant. Over-expressed Mnn1 from Drosophila extracts also co-migrates with bacterially expressed Mnn1 at ~ 95 kDa. These data suggest that, like mammalian menin, Drosophila menin is nuclear (Cerrato, 2006).

Flies lacking mnn1 are viable and fertile as also shown by others (Busygina, 2004; Papaconstantinou, 2005). The mutants used in this study show no overt and consistent phenotype as homozygote, trans-heterozygote or in trans to Df(2L)mnn1^Δ65 or Df(2L)J-H. As expected for a protein null allele, they behave as genetic amorphs, with the amorphic condition being viable, fertile, with no visible phenotype. Mnn1^e200 flies were stated to have a reduced life span (Busygina, 2004). The mnn1^e200 allele is deleted for both mnn1 and milt, and the milt locus is required for viability. Thus, incomplete rescue with a milt⁺ transgene could cause a reduction in life span (Busygina, 2004). However, current observations support the idea that mnn1 is required for a wild type life span. A slight, but highly significant reduction in viability was observed both in homozygous mnn1⁻ flies and in the trans-allelic mnn1⁻ flies. There were no significant differences between different genotypes of mnn1⁻. Interestingly, the reduction in viability was due to a constant rate of early mortality of mnn1⁻ males in days 1-30 (Cerrato, 2006).

It has also been reported that mnn1^e173 and mnn1^e30 mutants are sensitive to a range of stressors (Papaconstantinou, 2005), but again the results obtained might have been confounded by the more extensive deletions. mnn1 mutant alleles were tested for oxidative stress sensitivity using the herbicide paraquat, a powerful generator of reactive oxygen species. Flies either homozygous for the mnn1⁻ alleles or trans-heterozygous for those alleles appear to be slightly more resistant to paraquat than mnn1⁺ or mnn1⁺/mnn1⁻ flies. This observation is more striking given the increased mortality of young mnn1⁻ flies. The results appear to be in contrast to what was reported previously by Papaconstantinou indicating that mnn1^e178 or mnn1^e30 flies are more sensitive to paraquat, not resistant. Determining if this inconsistency is due to the different nature of the generated mutants will require further investigation. The salient agreement among all the mnn1 functional studies is that mnn1 is a non-essential gene in Drosophila. The lack of a developmental defect in the more streamlined Drosophila genome is surprising as mice homozygous for menin null alleles die as embryos (Crabtree, 2001). There are no obvious additional mnn1-like genes in Drosophila suggesting that the absence of a developmental defect is not due to the function of a second mnn1 gene. Perhaps menin has acquired non-conditional function only in the vertebrate lineage (Cerrato, 2006).

The consequences of excess mnn1 expression were explored by generating transgenic lines bearing the full-length mnn1⁺ cDNA under the control of the yeast Gal4 inducible UAS promoter and a wide range of Gal4 drivers. It has been reported that uniform over-expression of Mnn1 does not alter development or viability (Papaconstantinou, 2005). This study saw distinct and dramatic effects of Mnn1 over-expression in a subset of tissues (Cerrato, 2006).

To begin systematically exploring the effect of Mnn1 over-expression on Drosophila development, mnn1 expression was driven with Gal4 in a series of distinct spatiotemporal patterns. Because the distribution of endogenous Mnn1 is quite broad, this is likely to increase the levels of Mnn1 in cells, rather than altering the spatial distribution of Mnn1. The over-expression of Mnn1 protein (as determined by cell staining and/or immunoblotting) with any of five different Gal4 drivers resulted in a adult-pharate lethal phenotype. Interestingly, it was found that all of these drivers are expressed in subsets of neurons in addition to the reported expression patterns. Development was arrested during late pupal morphogenesis at stage P14. Dissection of dead pupae shows deletion of distal elements of the proboscis and a melanotic mass at that location. The melanotic mass is evident prior to lethality (stage P7). Exceptional flies that escape adult-pharate lethality when UAS-mnn1 is driven by How24-GAL4 (~ 5%) or 69B-GAL4 (~ 25%) show a melanotic mass at the anterior proboscis following eclosion. In the rare eclosing flies, the presence of a proboscis defect is not compatible with adult life, flies die 2-3 days later probably because of hindered intake of food and water. Wing inflation also failed in these escaping flies. Experiments performed at a lower temperature (22°) show an increased percentage of escaped flies and a reduced severity in the proboscis and wing defects. Gal4 is known to be less active at lower temperatures, suggesting that the level of Mnn1 induction correlates with the intensity of the phenotype observed (Cerrato, 2006).

Occasionally a cleft thorax phenotype was observed in flies when UAS-mnn1 is driven with 69B-GAL4. To further investigate the role of mnn1 in the developing Drosophila thorax, UAS-mnn1 was expressed using the pnr-GAL4 driver, which is expressed specifically in the leading edge cells of the wing disc and the medial region of the thorax in adults. These cells participate in thorax closure during metamorphosis. Mnn1 protein is expressed throughout the wing disc in wild type flies and is clearly over-expressed in the leading edge cells in pnr > mnn1. The thorax of adult flies carrying one copy each of both pnr-GAL4 and the UAS-mnn1 transgene always showed a dorsal cleft along the entire thorax with disrupted chaetae orientation whereas the thorax of flies carrying either one copy of pnr-GAL4 or one copy of UAS-mnn1 alone was wild type. This thoracic defect was 100% penetrant. Furthermore, the severity of the phenotype was modulated by the number of copies of UAS-mnn1 expressed in the thorax (more copies result in a more extreme phenotype) and by the growth temperature, indicating that the phenotype is proportional to the degree of Mnn1 over expression (Cerrato, 2006).

The cleft thorax phenotype raises the possibility that Drosophila menin can act in the jun/fos pathway. Thorax formation occurs by fusion of hemithoraces during pupal development as the result of spreading and fusion of two lateral groups of cells in the midline. This event requires the coordinated action of the Jun/Fos signaling pathway. Too little or too much Jun/Fos activity results in failure to properly suture imaginal discs during metamorphosis. Jun/Fos activity is also required for embryonic dorsal closure, but an overt dorsal closure phenotype associated with loss-of-function or over expression of mnn1 was never observed. The latter may be due in part to the abundant maternally deposited mnn1 transcript in embryos (Cerrato, 2006).

The cleft thorax phenotype is consistent with the idea that Mnn1 interacts with Jun/Fos, although this does not imply that the only function of mnn1 is in the jun/fos pathway. The idea of Mnn1 interacting with Jun/Fos was tested by using an extensive set of crosses designed to explore genetic interactions between mnn1 over-expressed with pnr-GAL4 and jun/fos alleles. Mnn1 was overexpressed in a heterozygous jun background (jra¹/+) and a more severe thoracic cleft phenotype was observed. Similarly, the contextual over-expression of a dominant-negative jun (UAS-jun^bZIP) and mnn1 enhanced the thorax defect. Also an effect of the induction of UAS-jun^bZIP was tested on the adult-pharate lethal phenotype observed in How24 > mnn1 flies. While flies over-expressing UAS-jun^bZIP were wild type, the flies expressing both mnn1 and jun^bZIP showed a much earlier arrest of the pupal development, rather than the adult-pharate phenotype seen when only mnn1 was over-expressed, again indicating that mnn1 over-expression is exacerbated by dominant negative jun activity (Cerrato, 2006).

The synergistic effect of mnn1 transgene and dominant negative alleles of jun along with the effect of altered jun dose suggests that menin acts to antagonize Jun protein function. If this is the case, then menin might suppress the effect of excess Jun activity. Expression of constitutively active, phosphomimetic jun (UAS-jun^asp) using pnr-GAL4 results in fully penetrant embryonic lethality. In contrast, the simultaneous expression of UAS-jun^asp and UAS-mnn1 driven from pnr-GAL4 results in 1%-5% of flies escaping to eclosion. Thus, expression of Mnn1 suppresses the lethality associated with constitutive expression of active Jun. These data are consistent with mnn1 acting as a negative regulator of jun function (Cerrato, 2006).

As mnn1 shows a genetic interaction with jun, tests were performed for interaction with fos (kay), the other component of the heterodimer. Heterozygosity for kay¹ had no effect on the mnn1 over-expression phenotype. However, expression of UAS-fos from pnr-GAL4 results in a weak cleft thorax phenotype and this phenotype is greatly enhanced by simultaneous expression from UAS-mnn1. Thus, excessive Fos is deleterious to Jun/Fos function, perhaps by affecting the homodimer/heterodimer ratio. These data suggest that menin and Fos have a negative synergistic effect on Jun/Fos function. However, genetic interactions between loss-of-function mnn1 alleles and dominant-negative alleles of fos suggest that the interaction is complex. In mnn1⁻/mnn1⁺ flies, over-expression of fos^bZIP with the How24 driver has a more dramatic effect on thorax closure, and in the complete absence of mnn1, How24-GAL4 induction of fos^bZIP results in pupal lethality. Thus, in some experiments, Mnn1 is behaving as a positive regulator of Jun/Fos and in other experiments Mnn1 is acting as a negative regulator of Jun/Fos (Cerrato, 2006).

Dominant interactions were tested between mnn1 over-expression and loss-of-function alleles of other members of the Jun kinase (JNK) cascade, hemipterous (hep) or basket (bsk), that lead to activated Jun and the negative regulator puckered (puc), but no interaction was seen. The phenotypic effects were tested of simultaneous over-expression of mnn1 with hep, bsk or puc, and no interaction was seen. Thus, there is no evidence that the interaction between mnn1 and jun/fos is mediated by these components of the JNK signaling pathway. However, this does not imply that there is direct contact between Mnn1 and Jun/Fos proteins, and indeed, direct testing for physical interaction between Mnn1 and Drosophila Jun or Fos has revealed no interaction (Guru, 2001; Cerrato, 2006).

Jun/Fos is also required for Drosophila eye morphogenesis. Analysis of interactions between Mnn1 and Fos in eye development also suggests that Mnn1 can negatively or positively modulate Jun/Fos. While no effect was observed of mnn1 over-expression in otherwise wild type eyes, an interaction was seen with Fos. Over-expression of a fos dominant negative, UAS-fos^bZIP, in the eye using ey-GAL4 results in a small rough eye phenotype. The simultaneous induction of mnn1 expression dramatically suppresses this severe small eye phenotype. Thus, even though loss-of-function and gain-of function of mnn1 are not overtly deleterious to eye development, interaction with dominant negative fos reveals a genetic interaction of mnn1 with Jun/Fos in the eye. Thus, wild type Mnn1 appears to augment Jun/Fos activity in the eye, or to negatively regulate the dominant negative activity of Fos^bZIP (Cerrato, 2006).

If Mnn1 is a positive regulator of Jun/Fos in the eye, then it might also enhance the effect of constitutive active jun in that tissue. The opposite was found. Induction of UAS-jun^asp by ey-GAL4 resulted in pre-pupae lethality, but this was partially rescued by contextual over-expression of Mnn1. Flies expressing both UAS-jun^asp and UAS-mnn1 driven by ey-GAL4 eclose (1%), suggesting that Mnn1 can inhibit active Jun/Fos (Cerrato, 2006).

It is concluded that Mnn1 is highly conserved in Drosophila. The experiments show that over-expressed Mnn1 can functionally interact with either wild type or over-expressed Jun/Fos. Furthermore, the absence of Mnn1 also modulates the activity of over-expressed Fos. Interestingly, these interactions result in defects consistent with both positive and negative influences of Mnn1 on Jun/Fos. While these results are unsatisfying for placing Mnn1 squarely in a particular and invariant position in the Jun/Fos pathway, it is clear that Jun/Fos is differently regulated in the eye and thorax of Drosophila. It is also clear that mammalian Menin can function as either a positive or negative regulator of Jun family members (Agarwal, 2003). It is suggested that there are contextual influences that allow Mnn1 to be both an activator or suppressor of Jun/Fos in Drosophila. This context-dependent effect might also underlie the opposing tumor suppressing (Chandrasekharappa, 1997; Crabtree, 2001) and tumor promoting (Yokoyama, 2005) effects of menin in mammals. Finally, while strong interactions were seen between mnn1 and jun/fos, this does not rule out a role for mnn1 in other nuclear events. Indeed, mammalian menin is in a complex which is involved in modifying the histones at a large number of genes (Hughes, 2004; Yokoyama, 2004) and a large number of transcription factors have been reported to physically contact menin (Agarwal, 2004). Why such a broad biochemical activity is associated with such modest phenotypic effects is not well understood in mammals or in Drosophila. Perhaps Mnn1 has a more subtle role in fine tuning gene expression (Cerrato, 2006).

Menin is a regulator of the stress response in Drosophila melanogaster

Menin, the product of the multiple endocrine neoplasia type I gene, has been implicated in several biological processes, including the control of gene expression and apoptosis, the modulation of mitogen-activated protein kinase pathways, and DNA damage sensing or repair. This study investigated the function of menin in Drosophila. Drosophila lines overexpressing menin or an RNA interference for this gene develop normally but are impaired in their response to several stresses, including heat shock, hypoxia, hyperosmolarity and oxidative stress. In the embryo subjected to heat shock, this impairment is characterized by a high degree of developmental arrest and lethality. The overexpression of menin enhances the expression of HSP70 in embryos and interfers with its down-regulation during recovery at the normal temperature. In contrast, the inhibition of menin with RNA interference reduced the induction of HSP70 and blocked the activation of HSP23 upon heat shock, Menin was recruited to the Hsp70 promoter upon heat shock and menin overexpression stimulated the activity of this promoter in embryos. A 70-kDa inducible form of menin was expressed in response to heat shock, indicating that menin is also regulated in conditions of stress. The induction of HSP70 and HSP23 was markedly reduced or absent in mutant embryos harboring a deletion of the menin gene. These embryos, which did not express the heat shock-inducible form of menin, were also hypersensitive to various conditions of stress. These results suggest a novel role for menin in the control of the stress response and in processes associated with the maintenance of protein integrity (Papaconstantinou, 2005).

The misexpression of menin impaired the response of Drosophila embryos, larvae, and flies to heat shock, hypoxia, hyperosmolarity, or oxidative stress. In embryos subjected to heat shock, this impairment resulted in a high degree of developmental arrest and lethality. The overexpression of menin enhanced the expression of HSP70 in response to heat shock and interfered with its repression during recovery at the normal temperature. In contrast, the induction of HSP70 and HSP23 was markedly reduced or absent when menin was down-regulated by RNAi or when embryos of two Mnn1 mutant lines were subjected to heat shock. These results indicate that menin is a positive regulator of heat shock protein expression in Drosophila melanogaster (Papaconstantinou, 2005).

Using Drosophila lines harboring genomic deletions of Hsp70 genes, it was asked whether a 50% reduction in Hsp70 gene copy number reduced the lethality of heat-shocked flies overexpressing menin, but no changes were observed in the rate of lethality. These results suggest that Hsp70 genes are not the main target of menin or that menin is a more global regulator of the stress response, controlling the expression of several heat shock response genes, including Hsp70. The observation that HSP23 expression is also altered in conditions of menin misexpression supports the latter possibility. It is also possible that the remaining copies of the Hsp70 genes were sufficient to provide levels of HSP70 above the threshold level for lethality caused by menin overexpression in conditions of stress. Therefore, whether or not the lethality observed in conditions of menin misexpression reflects predominantly the aberrant induction of HSP70 remains to be determined (Papaconstantinou, 2005).

Menin was recruited to the Hsp70 promoter upon heat shock. Since antibodies recognized both the 83- and 70-kDa forms of menin, the identity of the menin species recruited on the Hsp70 promoter is presently unknown. The results of immunoprecipitation assays indicated that menin does not interact directly with the heat shock factor. Therefore, menin may be part of a multiprotein complex recruited to the Hsp70 promoter in response to stress. Other proteins, including DAXX, Spt5, Spt6, and elongin A, cooperate with heat shock factor in the regulation of heat shock proteins and, like menin, may play an important role in the survival of the organism facing adverse conditions. Since mammalian HSP70 is also part of a negative feedback loop controlling its own expression, it was asked whether menin interacts with HSP70 in Drosophila embryos, but no interactions were observed using immunoprecipitation and Western blotting analyses (Papaconstantinou, 2005).

A 70-kDa form of menin accumulated in response to heat shock, indicating that menin expression is itself regulated in conditions of stress. In contrast, the 83-kDa form of menin did not accumulate in control embryos subjected to heat shock (parental strains nos-GAL4 and Hsp70-GAL4) or in UAS-Mnn1 strains harboring the nos-GAL4 driver. Since the Mnn1 RNAi blocked the expression of the 70-kDa menin species and Mnn1 mutant embryos did not express this protein, full induction of HSP70, HSP23, and possibly other heat shock proteins may depend on this heat shock-inducible form of menin. Testing of this hypothesis will depend on the molecular characterization of the 70-kDa menin and awaits the development of reagents and Drosophila strains specific for single menin protein species (Papaconstantinou, 2005).

Two alternative forms of menin, differing at the C terminus and encoding proteins 763 and 530 amino acids in length, are predicted by the sequence of existing cDNA clones for Mnn1. Since the 70-kDa menin is recognized by antibodies generated against the central domain of the protein or the predicted C terminus of the 83-kDa form of menin (763 amino acids in length), it is not the 530-amino-acid menin protein encoded by the Mnn1-RB transcript. The expression of the 70-kDa menin is presently under investigation (Papaconstantinou, 2005).

This study did not investigate the expression or mechanism of action of menin in the response to hypoxia, hyperosmolarity, or oxidative stress. Since the expression of heat shock proteins is induced in response to several conditions of stress, it is probable that menin exerts a similar effect on the expression of HSP70 and HSP23 in response to these stresses. A recent study concluded that Drosophila larvae and embryos with a mutation of the menin gene are characterized by genome instability and are more susceptible to a variety of chemical mutagens (Busygina, 2004). Interestingly, the effect of several of these mutagens was only observed at 29°C. Whether or not these observations reflect the impairment of the stress response caused by the absence of menin remains to be investigated (Papaconstantinou, 2005).

The survival of organisms subjected to heat shock depends on the induction of stress-responsive genes. Interfering with the expression or activity of heat shock proteins results in increased lethality in organisms subjected to high temperatures. However, precise regulation of these genes is also required for survival since forced expression of heat shock proteins, such as HSP70, is toxic. This demonstrates that, in Drosophila, proper expression of heat shock proteins depends on menin gene function and that menin is itself regulated by heat shock. Consistent with these findings are recent reports describing a role for the BRCA1 tumor suppressor in the expression of HSP27 in mammals. In addition, BRCA1 was processed and inactivated at high temperatures, indicating that it is also regulated in response to heat stress. Similarly, Tid1, the mammalian homolog of the Drosophila tumor suppressor lethal (2) tumorous imaginal disks, is a cochaperone of HSP70 and therefore a candidate regulator of the stress response (Papaconstantinou, 2005).

The current experiments did not address whether this novel function of menin is relevant for its role as a tumor suppressor in humans. However, they are consistent with the idea that processes involved in the maintenance of protein integrity may also be important for ensuring the integrity of the genome. These processes may depend on a number of common factors, such as menin, BRCA1, and possibly Tid1 (Papaconstantinou, 2005).

Hypermutability in a Drosophila model for multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type I (MEN1) is an autosomal dominant cancer predisposition syndrome, the gene for which encodes a nuclear protein, menin. The biochemical function of this protein has not been completely elucidated, but several studies have shown a role in transcriptional modulation through recruitment of histone deacetylase. The mechanism by which MEN1 mutations cause tumorigenesis is unknown. The Drosophila homolog of MEN1, Mnn1, encodes a protein 50% identical to human menin. In order to further elucidate the function of MEN1, a null allele of this gene was generated in Drosophila; homozygous inactivation results in morphologically normal flies that are hypersensitive to ionizing radiation and two DNA cross-linking agents, nitrogen mustard and cisplatinum. The spectrum of agents to which mutant flies are sensitive and analysis of the molecular mechanisms of this sensitivity suggest a defect in nucleotide excision repair. Drosophila Mnn1 mutants have an elevated rate of both sporadic and DNA damage-induced mutations. In a genetic background heterozygous for lats, a Drosophila and vertebrate tumor suppressor gene, homozygous inactivation of Mnn1 enhances somatic mutation of the second allele of lats and formation of multiple primary tumors. These data indicate that Mnn1 is a novel member of the class of autosomal dominant cancer genes that function in maintenance of genomic integrity, similar to the BRCA and HNPCC genes (Busygina, 2004).

Kinzler and Vogelstein introduced the term 'gatekeeper' to describe a class of genes that directly control cellular proliferation and whose dysregulation is necessary to cause neoplastic growth in specific tissues (Kinzler, 1997). Gatekeepers include many classical tumor suppressors, such as RB1 and APC. Inactivation of both homologs of these genes, often through mutation of one copy and deletion of the second copy, is found both in hereditary forms of cancer and in the great majority of sporadic tumors of the types seen in the hereditary disease (Busygina, 2004).

Another class of tumor predisposition genes, the 'caretakers' does not directly affect growth control. The loss of caretakers causes genomic instability and promotes mutation of gatekeepers and other genes directly involved in growth control. Mutations in caretakers are not often found in sporadic cancers. For example, patients with hereditary breast cancer due to BRCA1 or BRCA2 have germline mutations in one copy of the gene and frequent allelic loss of the other copy in tumor tissue, but mutations in the BRCA genes are almost never seen in sporadic breast cancer. Likewise, a high proportion of colorectal tumors arising in hereditary non-polyposis colorectal cancer (HNPCC) patients have loss of both copies of HNPCC genes, but only a minority of sporadic colorectal cancers arise through this mechanism. These genes have often been referred to as tumor suppressors but a more appropriate term for them might be 'mutation suppressors' (Busygina, 2004).

Tumors in MEN1 patients arise through a two-hit mechanism, but the majority of sporadic tumors of the types seen in this disease arise without MEN1 mutations. This discrepancy in the rate of mutations in hereditary versus sporadic tumors could indicate that there are alternative pathways of pathogenesis that lead to the tumors seen in MEN1. Parathyroid adenomas, for example, can arise with loss of MEN1 or with translocations between the Parathormone gene promoter region and Cyclin D. Alternatively, the low frequency of MEN1 mutations in sporadic tumors may indicate that the gene functions as a caretaker and leads to cancer through a role in the maintenance of genomic integrity (Busygina, 2004).

The present work investigated the function of MEN1 in vivo by examining the effects of the loss of its Drosophila homolog, Mnn1. Mnn1 is not an essential gene in Drosophila, and its inactivation does not lead to lethality, sterility or morphologic abnormalities. These findings provide some evidence against a direct role for Mnn1 in regulation of cell growth and differentiation in Drosophila (Busygina, 2004).

Mnn1 mutant flies showed sensitivity to certain types of DNA damage, and the data are consistent with an elevated mutation rate. Flies without menin function and heterozygous for the lats tumor suppressor are more likely than wild-type to develop tumors both spontaneously and in response to ionizing radiation and chemical mutagens. This effect is likely attributable to a role of Mnn1 in tumor initiation, loss of the gene increasing the somatic mutation rate of the wild-type copy of lats. Alternatively, Mnn1 loss could have a tumor promoter effect, i.e. enhancement of tumor formation by increasing the rate of tumor growth after initiation. However, somatic loss of both copies of lats in Drosophila is sufficient to initiate overgrowth, and virtually all Drosophila cells that lose both copies of lats develop into tumors, arguing against a role for Mnn1 in tumor promotion. Furthermore, if loss of menin had a tumor promoter effect, larger tumors might be expected in mutants compared to wild-type flies, while in fact the size range of tumors was indistinguishable between these two groups. Therefore a role for Mnn1 as a caretaker gene is favored (Busygina, 2004).

Mnn1 mutants were moderately sensitive to ionizing radiation, which causes double strand breaks and a variety of other types of DNA damage. The sharp rise in the mutation frequency after exposure to nitrogen mustard suggests that menin participates in repair of the type of damage caused by this agent more specifically. Nitrogen mustard leads to the formation of interstrand cross-links through the alkylation of the N7 position in guanine residues. Interestingly, cisplatinum has a mechanism of action similar to that of nitrogen mustard, and Mnn1 mutants show considerable sensitivity to cisplatinum as well. In contrast, both DEB and Mitomycin C, to which Mnn1 mutants are only marginally sensitive, more often affect the N2 position in guanine residues as the site of nucleophilic attack. It is therefore possible that only certain types of DNA interstrand cross-links require menin for successful resolution. The repair of interstrand cross-links is not fully understood, but it is thought to involve a combination of nucleotide excision repair, homologous recombination, and non-homologous end joining. This analysis showed that Mnn1 mutants were not more sensitive than wild type flies to double strand breaks, suggesting that the mechanisms involved in repair of this type of damage are intact. Furthermore, at least at the level of meiosis, homologous recombination, an important component of double strand break repair, was normal in Mnn1 mutants. Taken together, these data suggest that menin might be involved in the pathways that lead to nucleotide excision repair (Busygina, 2004).

Interestingly, menin has been shown to interact with FANCD2, a protein that plays a central role in repair of DNA cross-links (Jin, 2003). Sensitivity to DNA cross-linking agents is characteristic of cells from Fanconi anemia patients. Recent studies showed that monoubiquitination of FANCD2 in response to DNA damage promotes the interaction of FANCD2 and BRCA2 and loading of BRCA2 on chromatin in sites of damage. This interaction, in turn, promotes the assembly of damage-inducible Rad51 foci possibly through conformational changes in BRCA2 that allow it to release Rad51 onto DNA. The monoubiquitination of FANCD2 protein in response to damage caused by DNA cross-linking agents is controlled by ATR. It is noteworthy that another menin interactor, RPA, is required for association of ATR and its binding partner, ATRIP, with damaged DNA. It is possible that menin assists in bringing together different components of cross-link repair machinery. Alternatively, menin's association with histone deacetylases (Gobl, 1999) such as mSin3A (see Drosophila Sin3A), a subunit of Sin3/Rpd3 HDAC complex, suggests that menin may have a more general effect on chromatin remodeling, allowing the repair machinery to access the damaged DNA (Busygina, 2004).

Although Drosophila studies favor a role for Mnn1 as a caretaker gene, data from mouse models of Men1 have been interpreted as indicating a more direct role in growth control. For example, homozygous Men1 knockout mice die during embryogenesis (Crabtree, 2001; Bertolino, 2003), suggesting that menin has a role in control of growth or differentiation in the murine embryo. Likewise, tissue-specific gene inactivation in pancreatic islet cells of the mouse leads to widespread hyperplasia and cellular atypia in adults, consistent with a similar role for the gene postnatally. It is possible that menin functions differently in Drosophila and mammals, but several studies have provided evidence that menin plays a role in genomic integrity in humans and mice as well as flies. Targeted disruption of Men1 in mouse embryonic fibroblasts caused moderate sensitivity to DEB and an increase in chromosomal aberrations in response to MMC treatment (Jin, 2003). Similarly, the peripheral blood lymphocytes from MEN1 patients show increased chromosomal abnormalities after DEB treatment. Numerous chromosomal abnormalities are also found in MEN1-related pancreatic islet tumors (Busygina, 2004 and references therein).

Many of the apparent discrepancies between mouse and Drosophila may reflect phenotypic differences at the level of the organism that nevertheless have the same biological basis in both organisms at the level of the cell. For example, the embryonic lethal phenotype in mice is due to defects in several organs, but there is little evidence for a cell-autonomous defect in growth control or differentiation. The most important abnormality is in liver organogenesis, and the main cellular phenotype observed in vivo is increased apoptosis in hepatocyte precursors. By analogy to BRCA1 and BRCA2, which are known to function in maintenance of genomic integrity, cell death resulting in embryonic lethality can be a manifestation of genomic instability. Drosophila embryonic cells may be less sensitive to the effects of menin loss on genomic integrity and less likely to undergo apoptosis (Busygina, 2004 and references therein).

Tissue-specific gene inactivation indicates that, at the level of individual cells, loss of menin may have little effect on growth or differentiation in juvenile and adult mice. In particular, mice with RIP-driven loss of Men1 had normal pituitary glands and, for the most part, normal pancreatic islet cells at four month of age. Frank tumorigenesis was not seen until 6-12 months of age despite early loss of menin, strongly suggesting that additional somatic events were necessary for tumorigenesis. This model is consistent with a function for menin in maintenance of genomic integrity. In contrast, widespread pancreatic islet hyperplasia was seen in both heterozygotes and tissue specific knockout mice in some studies, suggesting a direct role in growth control (Crabtree, 2003). There may be overlap between the role of the gene in maintenance of genomic integrity and its role in control of cell growth. BRCA1 was initially thought to function by directly inhibiting growth of breast and ovarian epithelial cells, a finding that reflects one of the roles of the gene in the broader context of its role in maintenance of genomic integrity (Busygina, 2004).

In conclusion, this work shows a role for the Drosophila homolog of MEN1 in maintenance of genomic integrity and suggests that tumor pathogenesis in the human disease may relate to genomic instability, comparable to that seen in BRCA-related breast cancer and HNPCC. Generation of a MEN1 mutant in Drosophila creates an important resource for the in vivo study of menin's role in development and carcinogenesis and sets the stage for developing and testing potential cancer therapies for MEN1-related tumors (Busygina, 2004).

Characterization of a MEN1 ortholog from Drosophila melanogaster

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized by tumors of the parathyroid, entero-pancreatic neuroendocrine and pituitary tissues and caused by inactivating mutations in the MEN1 gene. Menin, the 610-amino acid nuclear protein encoded by MEN1, binds to the transcription factor JunD and can repress JunD-induced transcription. This study reports the identification of a MEN1 ortholog in Drosophila, Menin1, that encodes a 763 amino acid protein sharing 46% identity with human menin. Additionally, 69% of the missense mutations and in-frame deletions reported in MEN1 patients appear in amino acid residues that are identical in the Drosophila and human protein, suggesting the importance of the conserved regions. Drosophila Menin1 gene transcripts use alternative polyadenylation sites resulting in 4.3 and 5-kb messages. The 4.3-kb transcript appears to be largely maternal, while the 5-kb transcript appears mainly zygotic. The binding of Drosophila menin to human JunD or Drosophila Jun could not be demonstrated by the yeast two-hybrid analysis. The identification of the MEN1 ortholog from Drosophila will provide an opportunity to utilize Drosophila genetics to enhance understanding of the function of human menin (Guru, 2001).

Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression

MLL (for mixed-lineage leukemia) is a proto-oncogene that is mutated in a variety of human leukemias. Its product, a homolog of Drosophila melanogaster trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression. This study reports the biochemical purification of MLL and demonstrates that it associates with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including a homolog of Ash2, another Trx-G group protein. Two other members of the novel MLL complex identified in this study are host cell factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties. Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1-MDa MLL complex. Abrogation of menin expression phenocopies loss of MLL and reveals a critical role for menin in the maintenance of Hox gene expression. Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components. These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis (Yokoyama, 2007).

The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis

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. 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 (Yokoyama, 2005).

Menin is a product of the MEN1 tumor suppressor gene, whose inactivation is associated with a variety of heritable and sporadic endocrine tumors. It functions as a classic tumor suppressor protein for the endocrine lineage as confirmed by genetic studies in humans and mice. Menin is a component of a macromolecular complex containing MLL, which is a leukemic protooncogenic protein, and that this complex participates in maintenance of Hox gene expression. The unexpected biochemical association of a tumor suppressor protein with a protooncogenic protein raised important questions regarding their respective roles in endocrine and hematologic cancers. The issue specifically addressed in the current study was whether MLL oncoproteins require, or alternatively bypass, menin molecular function in leukemia pathogenesis. The results demonstrate that MLL oncoproteins are critically dependent on menin, indicating that in addition to its tumor suppressor role, menin also provides an essential function on an oncogenic pathway. Loss of the ability to associate with menin abrogates the oncogenic properties of MLL in vitro and in vivo, and MLL-transformed cells require the continued presence of menin, indicating that it is necessary for both the initiation and maintenance of MLL-mediated oncogenic transformation. This does not reflect a generalized requirement for menin in myeloid progenitor transformation since the unrelated E2A-HLF oncoprotein is not dependent on menin for its oncogenic effects under identical experimental conditions. Therefore, menin is a specific and essential oncogenic cofactor for MLL-mediated leukemogenesis (Yokoyama, 2005).

The demonstration that menin associates with MLL oncoproteins on the HOXA9 promoter in leukemia cells suggested that they work together for transcriptional regulation of biologically relevant target genes, thereby providing a molecular basis for a menin requirement in MLL-mediated oncogenesis. Their colocalization on Hox promoters suggests that the menin requirement for transformation is unlikely to simply reflect its dominant-negative sequestration by MLL oncoproteins, although more complex scenarios that may in part involve menin sequestration cannot be unequivocally rule out . In support of a cofactor role for menin, expression of several Hoxa cluster genes is dramatically reduced in cells transduced with MLL oncoproteins lacking a menin binding motif and in MLL-transformed cells acutely depleted of menin. Hoxa9 in particular influences the incidence and/or phenotype of MLL leukemias, demonstrating that constitutive expression of Hox genes by menin/MLL fusion protein complexes is an important aspect of MLL leukemogenesis. Furthermore, a simple dominant-negative mechanism should not be affected by menin deletion, which would otherwise be redundant with MLL-mediated sequestration. Thus, menin appears to be a transcriptional and oncogenic cofactor that is required for transformation of myeloid progenitors as well as transcriptional misregulation of Hox genes by MLL oncoproteins (Yokoyama, 2005).

Although there are clear examples of oncoproteins (e.g., Mdm2) whose transforming activities are mediated by functional inactivation of subordinate tumor suppressor proteins, the dependence of an oncoprotein on direct physical interaction with a tumor suppressor protein as a necessary cofactor for its oncogenic activity has no precedent. Current data support a provisional model for menin as a component of the MLL macromolecular HMT complex, which localizes to Hox and other critical target genes to participate in their normal dynamic regulation in response to upstream growth and differentiation signals. MLL oncoproteins harboring gain-of-function mutations retain a critical dependence on menin but are devoid of other known MLL-associated factors and may no longer respond to upstream signals, thus maintaining constitutively high levels of subordinate gene expression likely through transcriptional effector functions of their fusion partners. Misregulation of genes such as HoxA9, which has oncogenic properties when hyperexpressed in hematopoietic progenitors, would complete an oncogenic circuit (Yokoyama, 2005).

In endocrine cancers, on the other hand, menin loss of function results from truncating or missense mutations, several of which have been shown to abrogate the ability of menin to coprecipitate with HMT activity (Hughes, 2004). Loss of menin function, as opposed to MLL gain of function in leukemias, has been shown to compromise subordinate Hox gene expression, such as Hoxc8 and HOXA9 in menin-deficient MEFs and HeLa cells, respectively (Hughes, 2004; Yokoyama, 2004). Although HOX gene misregulation may contribute to endocrine neoplasia, menin-MLL HMT complexes also positively regulate genes encoding anti-proliferation factors, such as p27Kip1 and p18Ink4c. Their expression is compromised in the absence of menin in immortalized fibroblasts (Milne, 2005), although not in myeloid progenitors transformed by MLL oncoproteins. Menin is also implicated in negative gene regulation through association with the Sin3/HDAC corepressor complex. Menin-mediated tethering of this chromatin-modifying machinery to JunD alters its transcriptional properties, converting JunD from a growth promoter to a growth suppressor. Thus, the consequences of menin loss are complex and apparently cell context, target gene, and coregulator dependent. Taken together, these observations suggest the broader hypothesis that contrasting functions of menin as a tumor suppressor versus oncogenic cofactor may be different manifestations of its critical roles in tethering or targeting a subset of chromatin modifying complexes to specific promoters (Yokoyama, 2005).

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, the role of menin in hematopoiesis was investigated. Men1^-/- embryonic stem (ES) cell lines were induced 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. 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 (Novotny, 2009).

Search PubMed for articles about Drosophila Menin

Agarwal, S. K., et al. (1999). Menin interacts with the AP1 transcription factor JunD and represses JunD-activated transcription. Cell 96: 143-152. PubMed ID: 9989505

Agarwal, S. K., et al. (2003) Transcription factor JunD, deprived of menin, switches from growth suppressor to growth promoter. Proc. Natl. Acad. Sci. 100: 10770-10775. PubMed ID: 12960363

Agarwal, S. K., et al. (2004). Molecular pathology of the MEN1 gene. Ann. N. Y. Acad. Sci. 1014: 189-198. PubMed ID: 15153434

Bertolino, P., Radovanovic, I., Casse, H., Aguzzi, A., Wang, Z. Q. and Zhang, C. X. (2003). Genetic ablation of the tumor suppressor menin causes lethality at mid-gestation with defects in multiple organs. Mech. Dev. 120: 549-560. PubMed ID: 12782272

Busygina, V., et al. (2004). Hypermutability in a Drosophila model for multiple endocrine neoplasia type 1. Hum. Mol. Genet. 13: 2399-2408. PubMed ID: 15333582

Cerrato, A., Parisi, M., Santa Anna, S., Missirlis, F., Guru, S., Agarwal, S., Sturgill, D., Talbot, T., Spiegel, A., Collins, F., Chandrasekharappa, S., Marx, S. and Oliver, B. (2006). Genetic interactions between Drosophila melanogaster menin and Jun/Fos. Dev. Biol. 298(1): 59-70. PubMed ID: 16930585

Chandrasekharappa, S. C., et al. (1997). Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 276: 404-407. PubMed ID: 9103196

Crabtree, J. S., et al. (2001). A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors. Proc. Natl. Acad. Sci. 98: 1118-1123. PubMed ID: 9103196

Crabtree, J. S., et al. (2003). Of mice and MEN1: insulinomas in a conditional mouse knockout. Mol. Cell Biol. 23: 6075-6085. PubMed ID: 12917331

Gobl, A. E., Berg, M., Lopez-Egido, J. R., Oberg, K., Skogseid, B. and Westin, G. (1999). Menin represses JunD-activated transcription by a histone deacetylase-dependent mechanism. Biochim. Biophys. Acta 1447: 51-56. PubMed ID: 10500243

Guru, S. C., et al. (1998). Menin, the product of the MEN1 gene, is a nuclear protein. Proc. Natl. Acad. Sci. 95: 1630-1634. PubMed ID: 9465067

Guru, S. C., et al. (2001). Characterization of a MEN1 ortholog from Drosophila melanogaster. Gene 263: 31-38. PubMed ID: 11223240

Hughes, C. M. et al. (2004). Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus. Mol. Cell 13: 587-597. PubMed ID: 14992727

Jin, S., Mao, H., Schnepp, R. W., Sykes, S. M., Silva, A. C., D'Andrea, A. D. and Hua, X. (2003). Menin associates with FANCD2, a protein involved in repair of DNA damage. Cancer Res. 63: 4204-4210. PubMed ID: 12874027

Karnik, S. K., et al. (2005). Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27Kip1 and p18INK4c. Proc. Natl. Acad. Sci. 102: 14659-14664. PubMed ID: 16195383

Kinzler, K. W. and Vogelstein, B. (1997) Cancer-susceptibility genes. Gatekeepers and caretakers. Nature 386: 761-763. PubMed ID: 9126728

Maruyama, K., et al. (2000). Complementary DNA structure and genomic organization of Drosophila menin. Mol. Cell Endocrinol. 168: 135-140. PubMed ID: 11064160

Milne, T. A., et al. 2005 Menin and MLL cooperatively regulate expression of cyclin-dependent kinase inhibitors. Proc. Natl. Acad. Sci. 102: 749-754. PubMed ID: 15640349

Novotny, E., et al. (2009). In vitro hematopoietic differentiation of mouse embryonic stem cells requires the tumor suppressor menin and is mediated by Hoxa9. Mech. Dev. 126(7): 517-22. PubMed ID: 19393316

Papaconstantinou, M., et al. (2005). Menin is a regulator of the stress response in Drosophila melanogaster. Mol. Cell Biol. 25(22): 9960-72. PubMed ID: 16260610

Scacheri, P. C., et al. (2004). Homozygous loss of menin is well tolerated in liver, a tissue not affected in MEN1. Mamm. Genome. 15: 872-877. PubMed ID: 15672591

Yokoyama, A., et al. (2004). Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol. Cell Biol. 24: 5639-5649. PubMed ID: 15199122

Yokoyama, A., et al. (2005). The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis. Cell 123: 207-218. PubMed ID: 16239140

Yokoyama, A., et al. (2007). Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol. Cell. Biol. 24(13): 5639-49. PubMed ID: 15199122

date revised: 10 September 2009

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