Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease

Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour

The miR-223 host non-coding transcript linc-223 induces IRF4 expression in acute myeloid leukemia by acting as a competing endogenous RNA

Alterations in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. Here, we identify the host transcript of miR-223, linc-223, as a novel functional long non-coding RNA (lncRNA) in AML. We show that from the primary nuclear transcript, the alternative production of miR-223 and linc-223 is finely regulated during monocytic differentiation. Moreover, linc-223 expression inhibits cell cycle progression and promotes monocytic differentiation of AML cells. We also demonstrate that endogenous linc-223 localizes in the cytoplasm and acts as a competing endogenous RNA for miR-125-5p, an oncogenic microRNA in leukemia. In particular, we show that linc-223 directly binds to miR-125-5p and that its knockdown increases the repressing activity of miR-125-5p resulting in the downregulation of its target interferon regulatory factor 4 (IRF4), which it was previously shown to inhibit the oncogenic activity of miR-125-5p in vivo. Furthermore, data from primary AML samples show significant downregulation of linc-223 in different AML subtypes. Therein, these findings indicate that the newly identified lncRNA linc-223 may have an important role in myeloid differentiation and leukemogenesis, at least in part, by cross-talking with IRF4 mRNA

Down-regulation of serotonergic genes expression in the raphe nuclei of midbrain under chronic social defeat stress in male mice

Background: 
There is ample experimental evidence supporting the hypothesis that the brain serotonergic system is involved in the control of chronic social defeat stress (CSDS), depression and anxiety. The study aimed to analyze mRNA levels of the serotonergic genes in the raphe nuclei of the midbrain that may be associated with chronic social defeats consistently shown by male mice in special experimental settings. 

Methodology/Principal Findings: 
The serotonergic genes were the Tph2, Sert, Maoa and Htr1a. The Bdnf, Creb, Cphn, Gapdh, Hprt, B2M, 18S and Actb genes were also studied. The experimental groups were composed of male mice with experience of defeats in 21 daily encounters and male mice with the same track record of defeats followed by a no-defeat period without agonistic interactions (relative rest for 14 days). It has been shown that mRNA levels of the Tph2, Maoa, Sert, Htr1a, Bdnf and Creb genes in the raphe nuclei of defeated mice are decreased as compared with the controls. Under CSDS the Cphn, Gapdh, Hprt, B2M, 18S, Actb genes are also down-regulated. The expression of the serotonergic genes as well as the Cphn and Creb genes is not restored to the control level after the 2 weeks of relative rest. mRNA levels of other genes are not recovered to the control levels, although some up-regulation was observed in rested losers. Significant positive correlations were found between the total time of avoidance behavior demonstrated by the 21-day defeaters in agonistic interactions and Sert, Maoa, Bdnf, Gapdh and 18S mRNA levels. 

Conclusions: 
CSDS experience inducing the development of mixed anxious/depression-like state in male mice down-regulates the serotonergic genes expression associated with the synthesis, inactivation and reception of serotonin. The Bdnf and Creb genes as well as the cell and metabolic Cphn, Gapdh, Hprt, B2M, Actb and 18S genes in the midbrain raphe nuclei are also down-regulated under CSDS. Period of relative rest is not enough for most genes to recover expression to the control levels

Immune-mediated loss of transgene expression from virally transduced brain cells is irreversible, mediated by IFNγ, perforin, and TNFα, and due to the elimination of transduced cells

The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4 and CD8 T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cellsFil: Zirger, Jeffrey M.. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Puntel, Mariana. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bergeron, Josee. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Wibowo, Mia. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Moridzadeh, Rameen. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Bondale, Niyati. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Barcia, Carlos. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Kroeger, Kurt M.. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Liu, Chunyan. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Castro, Maria Graciela. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos. University of Michigan; Estados UnidosFil: Lowenstein, Pedro R.. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos. University of Michigan; Estados Unido

Alternative polyadenylation of ZEB1 promotes its translation during genotoxic stress in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extremely poor prognosis. The standard chemotherapeutic drug, gemcitabine, does not offer significant improvements for PDAC management due to the rapid acquisition of drug resistance by patients. Recent evidence indicates that epithelial-to-mesenchymal transition (EMT) of PDAC cells is strictly associated to early metastasization and resistance to chemotherapy. However, it is not exactly clear how EMT is related to drug resistance or how chemotherapy influences EMT. Herein, we found that ZEB1 is the only EMT-related transcription factor that clearly segregates mesenchymal and epithelial PDAC cell lines. Gemcitabine treatment caused upregulation of ZEB1 protein through post-transcriptional mechanisms in mesenchymal PDAC cells within a context of global inhibition of protein synthesis. The increase in ZEB1 protein correlates with alternative polyadenylation of the transcript, leading to shortening of the 3' untranslated region (UTR) and deletion of binding sites for repressive microRNAs. Polysome profiling indicated that shorter ZEB1 transcripts are specifically retained on the polysomes of PDAC cells during genotoxic stress, while most mRNAs, including longer ZEB1 transcripts, are depleted. Thus, our findings uncover a novel layer of ZEB1 regulation through 3'-end shortening of its transcript and selective association with polysomes under genotoxic stress, strongly suggesting that PDAC cells rely on upregulation of ZEB1 protein expression to withstand hostile environments

A viable mouse model of factor X deficiency provides evidence for maternal transfer of factor X.

BackgroundActivated factor X (FXa) is a vitamin K-dependent serine protease that plays a pivotal role in blood coagulation by converting prothrombin to thrombin. There are no reports of humans with complete deficiency of FX, and knockout of murine F10 is embryonic or perinatal lethal.ObjectiveWe sought to generate a viable mouse model of FX deficiency.MethodsWe used a socket-targeting construct to generate F10-knockout mice by eliminating F10 exon 8 (knockout allele termed F10(tm1Ccmt), abbreviated as '-'; wild-type '+'), and a plug-targeting construct to generate mice expressing a FX variant with normal antigen levels but low levels of FX activity [4-9% normal in humans carrying the defect, Pro343-->Ser, termed FX Friuli (mutant allele termed F10(tm2Ccmt), abbreviated as F)].ResultsF10 knockout mice exhibited embryonic or perinatal lethality. In contrast, homozygous Friuli mice [F10 (F/F)] had FX activity levels of approximately 5.5% (sufficient to rescue both embryonic and perinatal lethality), but developed age-dependent iron deposition and cardiac fibrosis. Interestingly, F10 (-/F) mice with FX activity levels of 1-3% also showed complete rescue of lethality. Further study of this model provides evidence supporting a role of maternal FX transfer in the embryonic survival.ConclusionsWe demonstrate that, while complete absence of FX is incompatible with murine survival, minimal FX activity as low as 1-3% is sufficient to rescue the lethal phenotype. This viable low-FX mouse model will facilitate the development of FX-directed therapies as well as investigation of the FX role in embryonic development

DNMTs are required for delayed genome instability caused by radiation

This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited - Copyright @ 2012 Landes Bioscience.The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.This study is funded by NOTE, BBSRC and the Royal Society Dorothy Hodgkin Research Fellowship

The Mbd1-Atf7ip-Setdb1 pathway contributes to the maintenance of X chromosome inactivation.

BackgroundX chromosome inactivation (XCI) is a developmental program of heterochromatin formation that initiates during early female mammalian embryonic development and is maintained through a lifetime of cell divisions in somatic cells. Despite identification of the crucial long non-coding RNA Xist and involvement of specific chromatin modifiers in the establishment and maintenance of the heterochromatin of the inactive X chromosome (Xi), interference with known pathways only partially reactivates the Xi once silencing has been established. Here, we studied ATF7IP (MCAF1), a protein previously characterized to coordinate DNA methylation and histone H3K9 methylation through interactions with the methyl-DNA binding protein MBD1 and the histone H3K9 methyltransferase SETDB1, as a candidate maintenance factor of the Xi.ResultsWe found that siRNA-mediated knockdown of Atf7ip in mouse embryonic fibroblasts (MEFs) induces the activation of silenced reporter genes on the Xi in a low number of cells. Additional inhibition of two pathways known to contribute to Xi maintenance, DNA methylation and Xist RNA coating of the X chromosome, strongly increased the number of cells expressing Xi-linked genes upon Atf7ip knockdown. Despite its functional importance in Xi maintenance, ATF7IP does not accumulate on the Xi in MEFs or differentiating mouse embryonic stem cells. However, we found that depletion of two known repressive biochemical interactors of ATF7IP, MBD1 and SETDB1, but not of other unrelated H3K9 methyltransferases, also induces the activation of an Xi-linked reporter in MEFs.ConclusionsTogether, these data indicate that Atf7ip acts in a synergistic fashion with DNA methylation and Xist RNA to maintain the silent state of the Xi in somatic cells, and that Mbd1 and Setdb1, similar to Atf7ip, play a functional role in Xi silencing. We therefore propose that ATF7IP links DNA methylation on the Xi to SETDB1-mediated H3K9 trimethylation via its interaction with MBD1, and that this function is a crucial feature of the stable silencing of the Xi in female mammalian cells

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway.

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application