Odd-skipped related 2 regulates genes related to proliferation and development

Cell proliferation is a biological process in which chromosomes replicate in one cell and equally divide into two daughter cells. Our previous findings suggested that Odd-skipped related 2 (Osr2) plays an important role in cellular quiescence and proliferation under epigenetic regulation. However, the mechanism used by Osr2 to establish and maintain proliferation is unknown. To examine the functional role of Osr2 in cell proliferation, we analyzed its downstream target genes using microarray analysis following adenovirus-induced overexpression of Osr2 as well as knockdown with Osr2 siRNA, which showed that Osr2 regulates a multitude of genes involved in proliferation and the cell cycle, as well as development. Additional proliferation assays also indicated that Osr2 likely functions to elicit cell proliferation. Together, these results suggest that Osr2 plays important roles in proliferation and development.     

Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney

The mammalian metanephric kidney is derived from the intermediate mesoderm. In this report, we use molecular fate mapping to demonstrate that the majority of cell types within the metanephric kidney arise from an Osr1⁺ population of metanephric progenitor cells. These include the ureteric epithelium of the collecting duct network, the cap mesenchyme and its nephron epithelia derivatives, the interstitial mesenchyme, vasculature and smooth muscle. Temporal fate mapping shows a progressive restriction of Osr1⁺ cell fates such that at the onset of active nephrogenesis, Osr1 activity is restricted to the Six2⁺ cap mesenchyme nephron progenitors. However, low-level labeling of Osr1⁺ cells suggests that the specification of interstitial mesenchyme and cap mesenchyme progenitors occurs within the Osr1⁺ population prior to the onset of metanephric development. Furthermore, although Osr1⁺ progenitors give rise to much of the kidney, Osr1 function is only essential for the development of the nephron progenitor compartment. These studies provide new insights into the cellular origins of metanephric kidney structures and lend support to a model where Osr1 function is limited to establishing the nephron progenitor pool.     

Characterisation of CpG methylation in the upstream control region of mouse Nat2: Evidence for a gene–environment interaction in a polymorphic gene implicated in folate metabolism

Human arylamine N-acetyltransferase 1 (NAT1), a polymorphic xenobiotic metabolising enzyme, has been investigated in relation to susceptibility and prognosis in certain types of cancer. Both human NAT1 and its murine equivalent NAT2 have previously been shown to play roles in the catabolism of folate, which is required for the synthesis of S-adenosylmethionine, the methyl donor for cellular methylation reactions. We have tested whether the expression of mouse Nat2 is subject to epigenetic regulation, specifically CpG methylation in the promoter region, by determining levels of 5-methylcytosine by bisulphite sequencing and methylation-specific PCR. Under normal conditions, methylation levels of the Nat2 promoter were low, and varied in different tissues. However, CpG methylation was significantly increased by dietary folate supplementation, and increased methylation corresponded to decreased use of the core promoter. Functional deletion of the Nat2 gene gave rise to a significant increase in Nat2 methylation, extending our previous observations that folate catabolism is decreased in Nat2 null mice. Mouse NAT2 is likely to influence epigenetic gene control, particularly of its own locus, and this is consistent with recent evidence associating aberrant mouse Nat2/human NAT1 gene expression with certain developmental malformations and cancers.     

Demethylation of CpG islands in the 5' upstream regions mediates the expression of the human testis-specific gene MAGEB16 and its mouse homolog Mageb16

Tissue-specific gene expression is regulated by epigenetic modification involving trans-acting factors. Here, we identified that the human MAGEB16 gene and its mouse homolog, Mageb16, are only expressed in the testis. To investigate the mechanism governing their expression, the promoter methylation status of these genes was examined in different samples. Two CpG islands (CGIs) in the 5'' upstream region of MAGEB16 were highly demethylated in human testes, whereas they were methylated in cells without MAGEB16 expression. Similarly, the CGI in Mageb16 was hypomethylated in mouse testes but hypermethylated in other tissues and cells without Mageb16 expression. Additionally, the expression of these genes could be activated by treatment with the demethylation agent 5''-aza-2''-deoxycytidine (5''-aza-CdR). Luciferase assays revealed that both gene promoter activities were inhibited by methylation of the CGI regions. Therefore, we propose that the testis-specific expression of MAGEB16 and Mageb16 is regulated by the methylation status of their promoter regions. [BMB Reports 2014; 47(2): 86-91]     

The C. elegans M3 neuron guides the growth cone of its sister cell M2 via the Krüppel-like zinc finger protein MNM-2

The invariant cell-cell interactions occurring during C. elegans development offer unique opportunities to discover how growing axons may receive guidance cues from neighboring cells. The mnm-2 mutant was isolated because of its defects in the axon trajectory of the bilateral M2 pharyngeal neurons in C. elegans. We found that mnm-2 enhances the effects of many growth cone guidance mutations on these axons, suggesting that it performs a novel function during axon guidance. We cloned mnm-2 and found that it encodes a protein with three C2H2 zinc finger domains related to the Krüppel-like Factor protein family. mnm-2 is expressed only transiently in the M2 neuron, but exhibits a sustained expression in its sister cell, the M3 neuron. Strikingly, the expression of mnm-2 is not sustained in the M3 cell of the mnm-2 mutant, indicating that this gene positively regulates itself in that cell. Electropharyngeograms also indicate that the M3 cell is functionally impaired in the mnm-2 mutant. We used an M3-specific promoter to show that the M2 axon defect can be rescued by expression of mnm-2 in its sister cell M3. The same promoter was used to express the pro-apoptotic gene egl-1 to kill the M3 cell, which resulted in an M2 axon guidance defect similar to that found in the mnm-2 mutant. Our results suggest an M2 axon guidance model in which the M3 cell provides an important signal to the growth cone of its sister M2 and that this signal and the proper differentiation of M3 both depend on mnm-2 expression. This mechanism of axon guidance regulation allows fine-tuning of trajectories between sister cells.     

CEP2 Attenuates Myoblast Differentiation But Does Not Affect Proliferation

CEP2 (CDC42EP2) is a member of the CDC42 subfamily that belongs to the Rho family. The Rho family plays an important role in a variety of cellular processes including skeletal myogenesis. Here, we find the expression of CEP2 increased significantly during C2C12 myogenesis. Overexpression of CEP2 could attenuate myoblast differentiation, while knockdown of CEP2 by siRNA results in enhancing myogenesis. Furthermore, we demonstrate for the first time that CEP2 attenuates myoblast differentiation via suppression of muscle regulatory factors (MRFs) rather than influencing myoblast proliferation. These results indicate that CEP2 acts as a repressor during myogenesis, which provides new insights into the role of CEP2 in muscle development.     

Epigenetic inactivation of SLIT2 in human hepatocellular carcinomas

Recent findings have shown that SLIT2 appears to function as a novel tumor suppressor gene. In addition, hypermethylation of its promoter region has been detected in various cancers, including breast and lung cancer, colorectal carcinoma, and gliomas. Here, we report for the first time that there is epigenetic silencing of SLIT2 in human hepatocellular carcinoma (HCC). Downregulation of SLIT2 was detected in 6 of 8 (75%) HCC cell lines by quantitative real-time RT-PCR (qRT-PCR), and the downregulation of SLIT2 was generally dependent on the degree of methylation at the promoter region. Furthermore, expression of SLIT2 was restored in relatively low-expressing cell lines after treatment with 5-aza-2-deoxycytidine (5-Aza-dC). Downregulation of SLIT2 expression was also detected in 45 of 54 primary HCC samples (83.3%), and the decrease in expression was significantly correlated with CpG island hypermethylation. This decrease of SLIT2 expression was also associated with lymph node metastasis in HCC. Moreover, overexpression of SLIT2 in SMMC-7721 cells induced by recombinant adenovirus suppressed cell growth, migration, and invasion, These results suggest that epigenetic inactivation of SLIT2 in HCC may be important in the development and progression of HCC. Thus, SLIT2 may be useful as a therapeutic target in the treatment of HCC.     

Conditional Deletion of the Pten Gene in the Mouse Prostate Induces Prostatic Intraepithelial Neoplasms at Early Ages but a Slow Progression to Prostate Tumors

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, Pten^LoxP:Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of Pten^LoxP/LoxP:Osr1-Cre mice as early as 2 weeks of age. Intriguingly, Pten^LoxP/LoxP:Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. Pten^LoxP/+:Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of Pten^LoxP/LoxP:Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated Pten^LoxP/LoxP:Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.     

A Conserved GEF for Rho-Family GTPases Acts in an EGF Signaling Pathway to Promote Sleep-like Quiescence in Caenorhabditis elegans

Sleep is evolutionarily conserved and required for organism homeostasis and survival. Despite this importance, the molecular and cellular mechanisms underlying sleep are not well understood. Caenorhabditis elegans exhibits sleep-like behavioral quiescence and thus provides a valuable, simple model system for the study of cellular and molecular regulators of this process. In C. elegans, epidermal growth factor receptor (EGFR) signaling is required in the neurosecretory neuron ALA to promote sleep-like behavioral quiescence after cellular stress. We describe a novel role for VAV-1, a conserved guanine nucleotide exchange factor (GEF) for Rho-family GTPases, in regulation of sleep-like behavioral quiescence. VAV-1, in a GEF-dependent manner, acts in ALA to suppress locomotion and feeding during sleep-like behavioral quiescence in response to cellular stress. Additionally, VAV-1 activity is required for EGF-induced sleep-like quiescence and normal levels of EGFR and secretory dense core vesicles in ALA. Importantly, the role of VAV-1 in promoting cellular stress–induced behavioral quiescence is vital for organism health because VAV-1 is required for normal survival after cellular stress.