Albert Silva

Mutation of Tumor Suppressor Gene Men1 Acutely Enhances Proliferation of Pancreatic Islet Cells

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.

Tumor suppressor menin: the essential role of nuclear localization signal domains in coordinating gene expression

Menin is encoded by the tumor suppressor gene MEN1 that is mutated in patients with an inherited tumor syndrome, multiple endocrine neoplasia type 1 (MEN1). Although menin is a nuclear protein and directly binds to DNA through its nuclear localization signals (NLSs), the precise role for each of the NLSs in nuclear translocation and gene expression remains to be elucidated. Here, we show that point mutations in three individual NLSs, NLS1, NLS2, and a novel accessory NLS, NLSa, do not block nuclear translocation, but compromise the ability of menin to repress expression of the endogenous insulin-like growth factor binding protein-2 (IGFBP-2) gene. This repression is not released by an inhibitor of histone deacetylases. Although subtle mutations in menin NLSs do not affect menin association with chromatin, they abolish menin binding to the IGFBP-2 promoter in vivo. Furthermore, each of the NLSs is also crucial for menin-mediated induction of caspase 8 expression. Together, these results suggest that menin may act as a scaffold protein in coordinating activation and repression of gene transcription and that its NLSs play a more important role in controlling gene transcription than merely targeting menin into the nucleus.

Functional Interaction between Tumor Suppressor Menin and Activator of S-Phase Kinase

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.

Menin-mediated caspase 8 expression in suppressing multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is a familial tumor syndrome linked to mutation of the MEN1 gene, which encodes a tumor suppressor, menin. We previously reported that menin up-regulates the caspase 8 expression and promotes TNF-α-induced apoptosis. However, it remains unclear how menin up-regulates caspase 8 expression and whether menin-mediated caspase 8 expression plays a role in repressing MEN1 development. Here we show that menin binds the 5′-untranslated region (5′-UTR) of the Caspase 8 locus in vivo and activates transcription of a reporter gene through the 5′-UTR. Menin directly binds the 5′-UTR in a sequence-independent manner in vitro. Moreover, Men1 ablation in cells reduces acetylation of histones H3 and H4 at the 5′-UTR of the caspase 8 locus bound by menin in vivo. Notably, the MEN1-derived menin point mutants lose their ability to bind the caspase 8 locus and fail to induce caspase 8 expression and TNF-α-mediated apoptosis. Consistent with these observations, the expression level of caspase 8 is markedly reduced in insulinomas from Men1+/– mice. Together, our results indicate that menin enhances the caspase 8 expression by binding the caspase 8 locus, and suggest that menin suppresses MEN1 tumorigenesis, at least in part, by up-regulating caspase 8 expression.

Cdx4 and Menin Co-Regulate Hoxa9 Expression in Hematopoietic Cells

Background Transcription factor Cdx4 and transcriptional coregulator menin are essential for Hoxa9 expression and normal hematopoiesis. However, the precise mechanism underlying Hoxa9 regulation is not clear. Methods and Findings Here, we show that the expression level of Hoxa9 is correlated with the location of increased trimethylated histone 3 lysine 4 (H3K4M3). The active and repressive histone modifications co-exist along the Hoxa9 regulatory region. We further demonstrate that both Cdx4 and menin bind to the same regulatory region at the Hoxa9 locus in vivo, and co-activate the reporter gene driven by the Hoxa9 cis-elements that contain Cdx4 binding sites. Ablation of menin abrogates Cdx4 access to the chromatin target and significantly reduces both active and repressive histone H3 modifications in the Hoxa9 locus. Conclusion These results suggest a functional link among Cdx4, menin and histone modifications in Hoxa9 regulation in hematopoietic cells.

Energetics of Lizard Embryos Are Not Canalized by Thermal Acclimation

In some species of ectotherms, temperature has little or no effect on the amount of energy expended during embryonic development. This phenomenon can result from either of two mechanisms: (1) a shorter incubation period at higher temperatures, which offsets the expected increase in metabolic rate, or (2) a compensatory decrease in the rate at which embryos expend energy for maintenance. To distinguish the relative importance of these two mechanisms, we quantified the acute and chronic effects of temperature on embryonic metabolism in the eastern fence lizard (Sceloporus undulatus). First, we measured metabolic rates of individual embryos at 27°, 31°, and 34°C. Second, we examined the capacity for thermal acclimation by measuring the metabolic rates of embryos at 30°C, after a period of incubation at either 28° or 32°C. As with adult reptiles, the metabolic rates of embryos increased with an acute increase in temperature; the Q10 of metabolic rate from 27° to 34°C was 2.1 (±0.2). No evidence of thermal acclimation was observed either early or late in development. In S. undulatus, a shorter incubation period at higher temperatures appears to play the primary role in canalizing the energy budget of an embryo, but a reduction in the cost of growth could play a secondary role.