PLoS Genet

In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1(KO)Sox9(cKO) ()), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1(KO)Sox9(cKO) mice. Moreover, since testis development occurs in XY Rspo1(KO)Sox9(cKO) mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9(cKO) mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised

Global analysis reveals differential regulation of mRNA decay in human induced pluripotent stem cells

2013 Fall.Includes bibliographical references.Induced Pluripotent Stem (iPS) cells are able to proliferate indefinitely while maintaining the capacity for unlimited differentiation and these properties are reflected by global changes in gene expression required for reprogramming of differentiated cells. Although the rate of transcription is an important regulator of steady-state mRNA levels, mRNA decay also plays a significant role in modulating the expression of cell-specific genes. The contribution of regulated mRNA decay towards establishing and maintaining pluripotency is largely unknown. To address this, we sought to determine global mRNA decay rates in iPS cells and the genetically-matched fibroblasts (HFFs) they were derived from. Using a microarray based approach, we determined half-lives for 5,481 mRNAs in both cell lines and identified three classes of mRNAs whose decay is differentially regulated in iPS cells compared to HFFs. We found that replication-dependent histone mRNAs are more abundant and more stable in iPS cells, resulting in increased histone protein abundances. This up-regulation of histone expression may facilitate the unique chromatin dynamics of pluripotent cells. A large set of C2H2 ZNF mRNAs are also stabilized in iPS cells compared to HFFs, possibly through reduced expression of miRNAs that target their coding regions. As many of these mRNAs encode transcriptional repressors, stabilization of these transcripts may support the overall increased expression of C2H2 ZNF transcription factors in early embryogenesis. Finally, we found that mRNAs containing C-rich elements in their 3'UTR are destabilized in iPS cells compared to HFFs and many of these mRNAs encode factors important for development. Interestingly, we also identified the Poly(C)-Binding Protein (PCBP) family as differentially regulated in iPS cells and investigated their possible involvement in regulation of the mRNAs in our dataset identified as destabilized in iPS cells and having C-rich 3'UTR elements. Thus, we identified several interesting classes of mRNAs whose decay is differentially regulated in iPS cells compared to HFFs and our results highlight the importance of post-transcriptional control in stem cell gene expression. Coordinated control of mRNA decay is evident in pluripotency and characterization of the mechanisms involved would further contribute to our limited understanding of pluripotent gene expression and possibly identify additional targets for reprogramming

Determination of p53 role in cancer biology and therapy through interactome development and analysis

Cancer is a heterogeneous pathology of cell and tissue type, involving multi- dysregulation of pathways that govern fundamental cellular processes. Chemotherapy efficacy is highly affected by these molecular deviations and the majority of patients are non-responsive. The stress responsive transcription factor, p53 is a powerful tumour suppressor implicated in over 50 % of all human cancers, and the chemo- therapeutic applications of p53 have been well described. However, the vast literature base of p53 and complexity of its interactions makes it a challenging system for integration of this diverse information. Computational methodologies are novel tools for integration of diverse molecular information into a coherent framework. The in silico Boolean PKT206 p53–DNA damage model has previously demonstrated good predictive capability for p53 wildtype and null tumours. Here, we have expanded PKT206 to generate a more clinically robust representation of p53-cancer dynamics. The Boolean PMH260 and PMH302 p53 models were constructed to consider 260 nodes and 980 interactions (PMH260) and 302 nodes with 1398 interactions (PMH302). Processes of angiogenesis, DNA repair and cell cycle arrest were amalgamated into both models with an additional input of hypoxia included into PMH302. For greater representation we further constructed a logical model that was more relevant to a specific cancer and integrated 61 deregulated genes considered as important to mesothelioma (Meso-PMH61 model), and superimposed microarray data generated in our laboratory of mesothelioma cells treated with etoposide and 1 % O2 oxygen. We analysed all models, in silico, using CellNetAnalyzer. In silico knockout analysis of various nodes mimicking in vivo mutations revealed 98 and 514 potential novel predictions (PNPs) for PMH260 and PMH302 respectively, some were validated by laboratory and literature verification. Validation of 4 PNPs were investigated in our laboratory using transient gene and protein knockdown, q-PCR and western blot analysis. Of these, 2 PNPs were in agreement with the models prediction. For further validation we superimposed various human cancer transcriptome in vitro profiles and compared omics results to in silico LSSA data. Greater correct predictions were achieved than the earlier p53 logical models (PKT206 and PMH260) when using the expanded p53 interactomes (PMH302 and Meso-PMH61) resulting in 68.5 - 83 % dependant on model and simulation. We further tested the models capability to predict gene expression changes on a clinical and individual patient basis, and superimposed patient derived in vivo transcriptome tumour profiles with a p53 mutant and wild type status. Correct predictions were again in the majority ranging between 57 - 61.5 % for NSCLC and between 56-63 % for biphasic and epithelioid mesothelioma tumours. Gene expression analysis of individual tumour profiles identified several significantly over-represented pathways and deregulated genes common and unique contributing to these tumours, correctly predicted by the model. In particular, we highlight pathways, MAPK, Erbb and Ca+ as contributing to non-small cell lung cancer dependant on patient. For malignant pleural mesothelioma, we highlight cell cycle and MAPK pathways and the cell cycle genes; CCNB1, CCNB2 along with KIF14, PDGFRB and SULF1. These offer potential for further investigation that could be exploited for greater therapeutic efficacy in sarcomatoid, and CYP24A1, HIPK4 and PEG3 in biphasic p53 (+/+) malignant mesothelioma tumours. Drug profile analysis of deregulated genes identified by the model highlight the need for individualised therapeutic approaches, and we offer putative combined targeted therapeutic suggestions dependant on tumour profile. In summary, we have generated the largest p53 signalling model to date, and have successfully identified overall system attributes when compared to in vitro and in vivo patient derived data. We show the importance of individualised therapies and highlight the enlarged p53 interactome as a promising predictive tool for further investigation into personalised anti-cancer therapies with clinical relevance

Nuclear pore component Nup98 is a potential tumor suppressor and regulates posttranscriptional expression of select p53 target genes

The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3\u27UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3sigma) to be similarly regulated by Nup98. The expression of Nup98 is reduced in murine and human hepatocellular carcinomas (HCCs) and correlates with p21 expression in HCC patients. Our study elucidates a previously unrecognized function of wild-type Nup98 in regulating select p53 target genes that is distinct from the well-characterized oncogenic properties of Nup98 fusion proteins

Seneszenz-Signalweg im Prozess der Gefäßverkalkung sowohl in vitro als auch ex vivo

Vascular calcification (VC) is an independent predictor of cardiovascular events and is associated with significant morbidity and mortality. The increased risk of these associa-tions in chronic kidney disease (CKD) patients is more powerful. Current diagnostic and therapeutic approaches to VC are relatively limited and we need better insight into the underlying pathological mechanisms of VC as well as more effective intervention strate-gies. Previous studies have shown that cellular senescence is involved in VC and can promote the osteogenic transformation of vascular smooth muscle cells (VSMCs). Therefore, the first part of this study was to establish a proper multiplex Western blot protocol for subsequent analysis of the signaling pathway in later experiments. The second and third parts of this study were designed to investigate the effect of the cellu-lar senescence signaling pathway (p21) on vascular calcification in vitro and ex vivo. DOX induced senescence in VSMCs and activated the p21 senescence pathway while stimulating the expression of bone-associated molecules, with similar results obtained in ex vivo tissues stimulated by high phosphate. This study provides further hints for the p21-involved senescence pathway in vascular calcification in vitro and ex vivo and that it is accompanied by increased expression of multiple bone-associated molecules.Die Gefäßverkalkung (VC) ist ein unabhängiger Prädiktor für kardiovaskuläre Ereignisse und wird mit erheblicher Morbidität und Mortalität in Verbindung gebracht. Das erhöhte Risiko dieser Assoziationen bei Patienten mit chronischer Nierenerkrankung (CKD) ist noch stärker ausgeprägt. Die derzeitigen diagnostischen und therapeutischen Ansätze für VC sind relativ begrenzt. Deswegen brauchen wir einen besseren Einblick in die zugrunde liegenden pathologischen Mechanismen von VC und wirksamere Interven-tionsstrategien. Frühe Studien haben gezeigt, dass zelluläre Seneszenz an VC beteiligt ist und die osteogene Transformation von vaskulären glatten Muskelzellen (VSMCs) fördern kann. Daher bestand der erste Teil dieser Studie darin, ein geeignetes Multiplex-Westernblot-Protokoll zu erstellen, um im weiteren Verlauf die Signalkaskade und insbesondere die Proteinexpression weiter zu untersuchen. Im zweiten und dritten Teil dieser Studie sollte die Wirkung des zellulären Seneszenz-Signalwegs (p21) auf die Gefäßverkalkung in vitro und ex vivo untersucht werden. DOX induzierte Seneszenz in VSMCs und aktiv-ierte den p21-Seneszenz-Signalweg bei gleichzeitiger Stimulierung der Expression von knochenassoziierten Molekülen, wobei ähnliche Ergebnisse in ex vivo Geweben erzielt wurden, die durch hohen Phosphatgehalt stimuliert wurden. Diese Studie liefert weitere Hinweise dafür, dass der p21-Seneszenz-Stoffwechselweg in vivo und in vitro an der Gefäßverkalkung beteiligt ist und mit einer erhöhten Expression mehrerer knochenassoziierter Moleküle einhergeht

The Rbm38-p63 feedback loop is critical for tumor suppression and longevity.

The RNA-binding protein Rbm38 is a target of p63 tumor suppressor and can in-turn repress p63 expression via mRNA stability. Thus, Rbm38 and p63 form a negative feedback loop. To investigate the biological significance of the Rbm38-p63 loop in vivo, a cohort of WT, Rbm38-/-, TAp63+/-, and Rbm38-/-;TAp63+/- mice were generated and monitored throughout their lifespan. While mice deficient in Rbm38 or TAp63 alone died mostly from spontaneous tumors, compound Rbm38-/-;TAp63+/- mice had an extended lifespan along with reduced tumor incidence. We also found that loss-of-Rbm38 markedly decreased the percentage of liver steatosis in TAp63+/- mice. Moreover, we found that Rbm38 deficiency extends the lifespan of tumor-free TAp63+/- mice along with reduced expression of senescence-associated biomarkers. Consistent with this, Rbm38-/-;TAp63+/- MEFs were resistant, whereas Rbm38-/- or TAp63+/- MEFs were prone, to cellular senescence. Importantly, we showed that the levels of inflammatory cytokines (IL17D and Tnfsf15) were significantly reduced by Rbm38 deficiency in senescence-resistant Rbm38-/-;TAp63+/- mouse livers and MEFs. Together, our data suggest that Rbm38 and p63 function as intergenic suppressors in aging and tumorigenesis and that the Rbm38-p63 loop may be explored for enhancing longevity and cancer management

Dietary Restriction Ameliorates Age-Related Increase in DNA Damage, Senescence and Inflammation in Mouse Adipose Tissuey.

Ageing is associated with redistribution of fat around the body and saturation of visceral adipose depots. Likewise, the presence of excess fat in obesity or during ageing places extra stress on visceral depots, resulting in chronic inflammation and increased senescence. This process can contribute to the establishment of the metabolic syndrome and accelerated ageing. Dietary restriction (DR) is known to alleviate physiological signs of inflammation, ageing and senescence in various tissues including adipose tissue. OBJECTIVES:Our pilot study aimed to analyse senescence and inflammation parameters in mouse visceral fat tissue during ageing and by short term, late-onset dietary restriction as a nutritional intervention. Design, measurements: In this study we used visceral adipose tissue from mice between 5 and 30 months of age and analysed markers of senescence (adipocyte size, γH2A.X, p16, p21) and inflammation (e.g. IL-6, TNFα, IL-1β, macrophage infiltration) using immuno-staining, as well as qPCR for gene expression analysis. Fat tissues from 3 mice per group were analysed. RESULTS:We found that the amount of γH2A.X foci as well as the expression of senescence and inflammation markers increased during ageing but decreased with short term DR. In contrast, the increase in amounts of single or aggregated macrophages in fat depots occurred only at higher ages. Surprisingly, we also found that adipocyte size as well as some senescence parameters decreased at very high age (30 months). CONCLUSIONS:Our results demonstrate increased senescence and inflammation during ageing in mouse visceral fat while DR was able to ameliorate several of these parameters as well as increased adipocyte size at 17.5 months of age. This highlights the health benefits of a decreased nutritional intake over a relatively short period of time at middle age