Epigenetic alteration to activate Bmp2-Smad signaling in Raf-induced senescence

AIM: To investigate epigenomic and gene expression alterations during cellular senescence induced by oncogenic Raf. METHODS: Cellular senescence was induced into mouse embryonic fibroblasts(MEFs) by infecting retrovirus to express oncogenic Raf(RafV 600E). RNA was collected from RafV 600 E cells as well as MEFs without infection and MEFs with mock infection, and a genome-wide gene expression analysis was performed using microarray. The epigenomic status for active H3K4me3 and repressive H3K27me3 histone marks was analyzed by chromatin immunoprecipitation-sequencing for RafV 600 E cells on day 7 and for MEFs without infection. These data for Raf-induced senescence were compared with data for Ras-induced senescence that were obtained in our previous study. Gene knockdown and overexpression were done by retrovirus infection. RESULTS: Although the expression of some genes including secreted factors was specifically altered in either Ras- or Raf-induced senescence, many genes showed similar alteration pattern in Raf- and Ras-induced senescence. A total of 841 commonly upregulated 841 genes and 573 commonly downregulated genes showed a significant enrichment of genes related to signal and secreted proteins, suggesting the importance of alterations in secreted factors. Bmp2, a secreted protein to activate Bmp2-Smad signaling, was highly upregulated with gain of H3K4me3 and loss of H3K27me3 during Raf-induced senescence, as previously detected in Ras-induced senescence, and the knockdown of Bmp2 by sh RNA lead to escape from Raf-induced senescence. Bmp2-Smad inhibitor Smad6 was strongly repressed with H3K4me3 loss in Raf-induced senescence, as detected in Ras-induced senescence, and senescence was also bypassed by Smad6 induction in Raf-activated cells. Different from Ras-induced senescence, however, gain of H3K27me3 did not occur in the Smad6 promoter region during Raf-induced senescence. When comparing genome-wide alteration between Ras- and Raf-induced senescence, genes showing loss of H3K27me3 during senescence significantly overlapped; genes showing H3K4me3 gain, or those showing H3K4me3 loss, also well-overlapped between Ras- and Raf-induced senescence. However, genes with gain of H3K27me3 overlapped significantly rarely, compared with those with H3K27me3 loss, with H3K4me3 gain, or with H3K4me3 loss.CONCLUSION: Although epigenetic alterations are partly different, Bmp2 upregulation and Smad6 repression occur and contribute to Raf-induced senescence, as detected in Ras-induced senescence.     

Age-dependent resistance to lethal alphavirus encephalitis in mice: analysis of gene expression in the central nervous system and identification of a novel interferon-inducible protective gene,mouse ISG12

Several different mammalian neurotropic viruses produce an age-dependent encephalitis characterized by more severe disease in younger hosts. To elucidate potential factors that contribute to age-dependent resistance to lethal viral encephalitis, we compared central nervous system (CNS) gene expression in neonatal and weanling mice that were either mock infected or infected intracerebrally with a recombinant strain, dsTE12Q, of the prototype alphavirus Sindbis virus. In 1-day-old mice, infection with dsTE12Q resulted in rapidly fatal disease associated with high CNS viral titers and extensive CNS apoptosis, whereas in 4-week-old mice, dsTE12Q infection resulted in asymptomatic infection with lower CNS virus titers and undetectable CNS apoptosis. GeneChip expression comparisons of mock-infected neonatal and weanling mouse brains revealed developmental regulation of the mRNA expression of numerous genes, including some apoptosis regulatory genes, such as the proapoptotic molecules caspase-3 and TRAF4, which are downregulated during development, and the neuroprotective chemokine, fractalkine, which is upregulated during postnatal development. In parallel with increased neurovirulence and increased viral replication, Sindbis virus infection in 1-day-old mice resulted in both a greater number of host inflammatory genes with altered expression and greater changes in levels of host inflammatory gene expression than infection in 4-week-old mice. Only one inflammatory response gene, an expressed sequence tag similar to human ISG12, increased by a greater magnitude in infected 4-week-old mouse brains than in infected 1-day-old mouse brains. Furthermore, we found that enforced neuronal ISG12 expression results in a significant delay in Sindbis virus-induced death in neonatal mice. Together, our data identify genes that are developmentally regulated in the CNS and genes that are differentially regulated in the brains of different aged mice in response to Sindbis virus infection.     

Gene expression in atherosclerotic lesion of ApoE deficient mice

BACKGROUND: Atherosclerosis, the major cause of mortality and invalidity in industrialized countries, is a multifactorial disease associated with high plasma cholesterol levels and inflammation in the vessel wall. Many different genes have previously been demonstrated in atherosclerosis, although limited numbers of genes are dealt with in each study. In general, data on dynamic gene expression during disease progress is limited and large-scale evaluation of gene expression patterns during atherogenesis could lead to a better understanding of the key events in the pathogenesis of atherosclerosis. We have therefore applied a mouse gene filter array to analyze gene expression in atherosclerotic ApoE-deficient mice. MATERIALS AND METHODS: ApoE-deficient mice were fed atherogenic western diet for 10 or 20 weeks and aortas isolated. C57BL/6 mice on normal chow were used as controls. The mRNAs of 15 animals were pooled and hybridized onto commercially available Clontech mouse gene array filters. RESULTS: The overall gene expression in the ApoE-deficient and control mice correlated well at both time points. Gene expression profiling showed varying patterns including genes up-regulated at 10 or 20 weeks only. At 20 weeks of diet, an increasing number of up-regulated genes were found in ApoE-deficient mice. CONCLUSIONS: The gene expression in atherogenesis is not a linear process with a maximal expression at advanced lesion stage. Instead, several genes demonstrate a dynamic expression pattern with peaks at the intermediate lesions stage. Thus, detailed evaluation of gene expression at several time points should help understanding the development of atherosclerosis and establishment of preventive intervention.     

Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection

Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit.     

Gene expression profile of mouse white adipose tissue during inflammatory stress: age‐dependent upregulation of major procoagulant factors

Tolerance to physiological stress resulting from inflammatory disease decreases significantly with age. High mortality rates, increased cytokine production, and pronounced thrombosis are characteristic complications of aged mice with acute systemic inflammation induced by injection with lipopolysaccharide (LPS). As adipose tissue is now recognized as an important source of cytokines, we determined the effects of aging on visceral white adipose tissue gene expression during LPS‐induced inflammation in male C57BL/6 mice. Microarray analysis revealed that the expression of 6025 genes was significantly changed by LPS; of those, the expression of 667 showed an age‐associated difference. Age‐associated differences were found in many genes belonging to the inflammatory response and blood clotting pathways. Genes for several procoagulant factors were upregulated by LPS; among these, tissue factor, thrombospondin‐1, and plasminogen activator inhibitors‐1 and ‐2, exhibited age‐associated increases in expression which could potentially contribute to augmented thrombosis. Further analysis by qRT–PCR, histological examination, and cell fraction separation revealed that most inflammatory and coagulant‐related gene expression changes occur in resident stromal cells rather than adipocytes or infiltrating cells. In addition, basal expression levels of 303 genes were altered by aging, including increased expression of component of Sp100‐rs (Csprs). This study indicates that adipose tissue is a major organ expressing genes for multiple inflammatory and coagulant factors and that the expression of many of these is significantly altered by aging during acute inflammation. Data presented here provide a framework for future studies aimed at elucidating the impact of adipose tissue on age‐associated complications during sepsis and systemic inflammation.     

Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo.

Exogenous retinoic acid (RA) has teratogenic effects on vertebrate embryos and alters Hox-C gene expression in vivo and in vitro. We wish to examine whether RA has a role in the normal regulation of Hox-C genes, and whether altered Hox-C gene expression in response to RA leads to abnormal morphology. The expression of 3' Hox-2 genes (Hox-2.9, Hox-2.8, Hox-2.6 and Hox-2.1) and a 5' gene (Hox-2.5) were examined by whole-mount in situ hybridization on embryos 4 hours after maternal administration of teratogenic doses of RA on embryonic day 7 to 9. The expression of the 3' Hox-2 genes was found to be ectopically induced in anterior regions in a stage-specific manner. The Hox-2.9 and Hox-2.8 genes were induced anteriorly in the neurectoderm in response to RA on day 7 but not at later stages. Expression of Hox-2.6 and Hox-2.1 was ectopically induced anteriorly in neurectoderm in response to RA on day 8. Hox-2.1 remained responsive on day 9, whereas Hox-2.6 was no longer responsive at this stage. The expression of the 5' gene Hox-2.5 was not detectably altered at any of these stages by RA treatments. We also examined the response of other genes whose expression is spatially regulated in early embryos. The expression of En-2 and Wnt-7b was not detectably altered by RA, whereas RAR beta expression was induced anteriorly by RA on day 7 and 8. Krox-20 expression was reduced in a stage- and region-specific manner by RA. The ectopic anterior expression of Hox-2.8 and Hox-2.9 induced by RA on day 7 was persistent to day 8, as was the altered expression of Krox-20. The altered pattern of expression of these genes in response to RA treatment on day 7 may be indicative of a transformation of anterior hindbrain to posterior hindbrain, specifically, a transformation of rhombomeres 1 to 3 towards rhombomere 4 identity with an anterior expansion of rhombomere 5. The ectopic expression of the 3' Hox-2 genes in response to RA is consistent with a role for these genes in mediating the teratogenic effects of RA; the rapid response of the Hox-C genes to RA is consistent with a role for endogenous RA in refining 3' Hox-C gene expression boundaries early in development.     

Gene expression change in the Müllerian duct of the mouse fetus exposed to diethylstilbestrol in utero

In utero exposure to diethylstilbestrol (DES) induces various abnormalities in the Müllerian duct of the mouse. In order to understand the underlying molecular mechanisms associated with DES-induced abnormalities of the Müllerian duct, gene expression was examined on Gestation Day (GD) 19 in mouse fetuses exposed to DES (67 microg/kg body weight) from GDs 10 to 18. Microarray analysis revealed that 387, 387, and 225 genes were upregulated and 177, 172, and 75 genes were downregulated by DES in the oviduct, uterus, and vagina, respectively. DES exposure in utero commonly upregulated 72 genes and downregulated 15 genes in these three organs. The present study demonstrated that organ-specific gene expression patterns in the mouse Müllerian duct were altered by in utero DES exposure. DES-induced changes in expression of genes such as Dkk2, Nkd2, and sFRP1 as well as changes in genes of the Hox, Wnt, and Eph families in the female mouse fetal reproductive tract could be the basis for various abnormalities in reproductive tracts following exposure to this estrogenic drug.     

Gene Expression Profile Analysis of Type 2 Diabetic Mouse Liver

Liver plays a key role in glucose metabolism and homeostasis, and impaired hepatic glucose metabolism contributes to the development of type 2 diabetes. However, the precise gene expression profile of diabetic liver and its association with diabetes and related diseases are yet to be further elucidated. In this study, we detected the gene expression profile by high-throughput sequencing in 9-week-old normal and type 2 diabetic db/db mouse liver. Totally 12132 genes were detected, and 2627 genes were significantly changed in diabetic mouse liver. Biological process analysis showed that the upregulated genes in diabetic mouse liver were mainly enriched in metabolic processes. Surprisingly, the downregulated genes in diabetic mouse liver were mainly enriched in immune-related processes, although all the altered genes were still mainly enriched in metabolic processes. Similarly, KEGG pathway analysis showed that metabolic pathways were the major pathways altered in diabetic mouse liver, and downregulated genes were enriched in immune and cancer pathways. Analysis of the key enzyme genes in fatty acid and glucose metabolism showed that some key enzyme genes were significantly increased and none of the detected key enzyme genes were decreased. In addition, FunDo analysis showed that liver cancer and hepatitis were most likely to be associated with diabetes. Taken together, this study provides the digital gene expression profile of diabetic mouse liver, and demonstrates the main diabetes-associated hepatic biological processes, pathways, key enzyme genes in fatty acid and glucose metabolism and potential hepatic diseases.     

Gene expression profiling of mouse embryonic stem cell subpopulations

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.     

Early Maternal Alcohol Consumption Alters Hippocampal DNA Methylation,Gene Expression and Volume in a Mouse Model

The adverse effects of alcohol consumption during pregnancy are known, but the molecular events that lead to the phenotypic characteristics are unclear. To unravel the molecular mechanisms, we have used a mouse model of gestational ethanol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first 8 days of gestation (GD 0.5-8.5). Early neurulation takes place by the end of this period, which is equivalent to the developmental stage early in the fourth week post-fertilization in human. During this exposure period, dynamic epigenetic reprogramming takes place and the embryo is vulnerable to the effects of environmental factors. Thus, we hypothesize that early ethanol exposure disrupts the epigenetic reprogramming of the embryo, which leads to alterations in gene regulation and life-long changes in brain structure and function. Genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and three miRNAs in ethanol-exposed, adolescent offspring at postnatal day (P) 28. We confirmed this result by using two other tissues, where three candidate genes are known to express actively. Interestingly, we found a similar trend of upregulated gene expression in bone marrow and main olfactory epithelium. In addition, we observed altered DNA methylation in the CpG islands upstream of the candidate genes in the hippocampus. Our MRI study revealed asymmetry of brain structures in ethanol-exposed adult offspring (P60): we detected ethanol-induced enlargement of the left hippocampus and decreased volume of the left olfactory bulb. Our study indicates that ethanol exposure in early gestation can cause changes in DNA methylation, gene expression, and brain structure of offspring. Furthermore, the results support our hypothesis of early epigenetic origin of alcohol-induced disorders: changes in gene regulation may have already taken place in embryonic stem cells and therefore can be seen in different tissue types later in life.     

Cholesterol diet enhances daily rhythm of Pai-1 mRNA in the mouse liver

Myocardial infarction frequently occurs in the morning, a phenomenon in part resulting from the downregulation of fibrinolytic activity. Plasminogen activator inhibitor-1 (PAI-1) is a key factor behind fibrinolytic activity, and its gene expression is controlled under the circadian clock gene in the mouse heart and liver. Hypercholesterolemia has been associated with impaired fibrinolysis due to enhanced PAI-1 activity, which has also been implicated in atherosclerosis. The aim of this study was to decipher whether the Pai-1 gene is still expressed daily with hypercholesterolemia. Hypercholesterolemia (1% cholesterol diet) did not significantly affect the daily expression of clock genes (Per2 and Bmal1) and clock-controlled genes (Dbp and E4bp4) in the liver (P > 0.05); however, daily expression of the Pai-1 gene and Pai-1 promoter regulating factor genes such as Nr4a1 was significantly upregulated (P < 0.01). Daily restricted feeding for 4 h during the day reset the gene expression of Per2, Pai-1, Nr4a1, and Tnf-alpha. Lesion of the suprachiasmatic nucleus, the location of the main clock system, led to loss of Per2 and Pai-1 daily expression profiles. In the present experiments, we demonstrated that hypercholesterolemia enhanced daily expression of the Pai-1, Tnf-alpha, and Nr4a1 genes in the mouse liver without affecting clock and clock-controlled genes. Therefore, the risk or high frequency of acute atherothrombotic events in the morning still seems to be a factor that may be augmented under conditions of hypercholesterolemia.     

Gene expression profiles in genetically different mice infected with Toxoplasma gondii: ALDH1A2, BEX2, EGR2, CCL3 and PLAU

Toxoplasma gondii can modulate host cell gene expression; however, determining gene expression levels in intermediate hosts after T. gondii infection is not known much. We selected 5 genes (ALDH1A2, BEX2, CCL3, EGR2 and PLAU) and compared the mRNA expression levels in the spleen, liver, lung and small intestine of genetically different mice infected with T. gondii. ALDH1A2 mRNA expressions of both mouse strains were markedly increased at day 1-4 postinfection (PI) and then decreased, and its expressions in the spleen and lung were significantly higher in C57BL/6 mice than those of BALB/c mice. BEX2 and CCR3 mRNA expressions of both mouse strains were significantly increased from day 7 PI and peaked at day 15-30 PI (P<0.05), especially high in the spleen liver or small intestine of C57BL/6 mice. EGR2 and PLAU mRNA expressions of both mouse strains were significantly increased after infection, especially high in the spleen and liver. However, their expression patterns were varied depending on the tissue and mouse strain. Taken together, T. gondii-susceptible C57BL/6 mice expressed higher levels of these 5 genes than did T. gondii-resistant BALB/c mice, particularly in the spleen and liver. And ALDH1A2 and PLAU expressions were increased acutely, whereas BEX2, CCL3 and EGR2 expressions were increased lately. Thus, these demonstrate that host genetic factors exert a strong impact on the expression of these 5 genes and their expression patterns were varied depending on the gene or tissue.     

Differential expression of orthologous Dlx genes in zebrafish and mice: implications for the evolution of the Dlx homeobox gene family

Dlx homeobox genes of vertebrates are often organised as physically linked pairs in which the two genes are transcribed convergently (tail-to-tail arrangement). Three such Dlx pairs have been found in mouse, human, and zebrafish and are thought to have originated from the duplication of an ancestral gene pair. These pairs include Dlx1/Dlx2, Dlx7/Dlx3, and Dlx6/Dlx5 (the zebrafish orthologue of Dlx5 is named dlx4). Expression patterns of physically linked Dlx genes overlap extensively. Furthermore, orthologous Dlx genes often show highly similar expression patterns. We analysed Dlx expression during the gastrula and early somitogenesis of the mouse and zebrafish. It was found that expression of the mouse Dlx6 gene takes place in the rostral ectoderm and presumptive olfactory and otic placodes with patterns similar to the previously reported expression of the physically linked Dlx5 gene. However, we observed only very weak expression of the mouse Dlx3 gene at the same stage. This contrasts with the expression of dlx genes in zebrafish where dlx3 and dlx7, but not dlx4 and dlx6 are expressed during gastrulation in the rostral ectoderm and presumptive placodes. Thus, Dlx expression patterns at early stages are better conserved between paralogous pairs of physically linked genes than between orthologous pairs. This suggests that early expression of Dlx genes existed prior to the duplications that led to the multiple pairs of physically linked genes but was differentially conserved in different paralogs in zebrafish and mice.     

Global gene expression changes including drug metabolism and disposition induced by three-dimensional culture of HepG2 cells-Involvement of microtubules

Constitutive upregulation and a higher degree of induction of drug metabolism and disposition-related genes were found in a three-dimensional HepG2 culture. The upregulated genes are believed to be regulated by different regulatory factors. Global gene expression analysis using the Affymetrix GeneChip indicated that altered expression of microtubule-related genes may change the expressed levels of drug metabolizing and disposition genes. Stabilization of microtubule molecules with docetaxel, a tubulin-stabilizing agent, in the two-dimensional culture showed gene expression patterns similar to those found in the three-dimensional culture, indicating that the culture environment affects drug metabolism functions in HepG2 cells.