Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals.

The increasing worldwide prevalence of Hepatocellular carcinoma (HCC), characterized by resistance to conventional chemotherapy, poor prognosis and eventually mortality, place it as a prime target for new modes of prevention and treatment. Hepatitis C Virus (HCV) is the predominant risk factor for HCC in the US and Europe. Multiple epidemiological studies showed that sustained virological responses (SVR) following treatment with the powerful direct acting antivirals (DAAs), which have replaced interferon-based regimes, do not eliminate tumor development. We aimed to identify an HCV-specific pathogenic mechanism that persists post SVR following DAAs treatment. We demonstrate that HCV infection induces genome-wide epigenetic changes by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) for histone post-translational modifications that are epigenetic markers for active and repressed chromatin. The changes in histone modifications correlate with reprogramed host gene expression and alter signaling pathways known to be associated with HCV life cycle and HCC. These epigenetic alterations require the presence of HCV RNA or/and expression of the viral proteins in the cells. Importantly, the epigenetic changes induced following infection persist as an "epigenetic signature" after virus eradication by DAAs treatment, as detected using in vitro HCV infection models. These observations led to the identification of an 8 gene signature that is associated with HCC development and demonstrate persistent epigenetic alterations in HCV infected and post SVR liver biopsy samples. The epigenetic signature was reverted in vitro by drugs that inhibit epigenetic modifying enzyme and by the EGFR inhibitor, Erlotinib. This epigenetic "scarring" of the genome, persisting following HCV eradication, suggest a novel mechanism for the persistent pathogenesis of HCV after its eradication by DAAs. Our study offers new avenues for prevention of the persistent oncogenic effects of chronic hepatitis infections using specific drugs to revert the epigenetic changes to the genome.

Dysregulation of the cohesin subunit RAD21 by Hepatitis C virus mediates host-virus interactions.

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV possesses an RNA genome and its replication is confined to the cytoplasm. Yet, infection with HCV leads to global changes in gene expression, and chromosomal instability (CIN) in the host cell. The mechanisms by which the cytoplasmic virus affects these nuclear processes are elusive. Here, we show that HCV modulates the function of the Structural Maintenance of Chromosome (SMC) protein complex, cohesin, which tethers remote regions of chromatin. We demonstrate that infection of hepatoma cells with HCV leads to up regulation of the expression of the RAD21 cohesin subunit and changes cohesin residency on the chromatin. These changes regulate the expression of genes associated with virus-induced pathways. Furthermore, siRNA downregulation of viral-induced RAD21 reduces HCV infection. During mitosis, HCV infection induces hypercondensation of chromosomes and the appearance of multi-centrosomes. We provide evidence that the underlying mechanism involves the viral NS3/4 protease and the cohesin regulator, WAPL. Altogether, our results provide the first evidence that HCV induces changes in gene expression and chromosome structure of infected cells by modulating cohesin.

Assessing the involvement of the placental microbiome and virome in preeclampsia using non coding RNA sequencing.

OBJECTIVES: Preeclampsia is a dangerous pregnancy complication. The source of preeclampsia is unknown, though the placenta is believed to have a central role in its pathogenesis. An association between maternal infection and preeclampsia has been demonstrated, yet the involvement of the placental microbiome in the etiology of preeclampsia has not been determined. In this study, we examined whether preeclampsia is associated with an imbalanced microorganism composition in the placenta. METHODS: To this end, we developed a novel method for the identification of bacteria/viruses based on sequencing of small non-coding RNA, which increases the microorganism-to-host ratio, this being a major challenge in microbiome methods. We validated the method on various infected tissues and demonstrated its efficiency in detecting microorganisms in samples with extremely low bacterial/viral biomass. We then applied the method to placenta specimens from preeclamptic and healthy pregnancies. Since the placenta is a remarkably large and heterogeneous organ, we explored the bacterial and viral RNA at each of 15 distinct locations. RESULTS: Bacterial RNA was detected at all locations and was consistent with previous studies of the placental microbiome, though without significant differences between the preeclampsia and control groups. Nevertheless, the bacterial RNA composition differed significantly between various areas of the placenta. Viral RNA was detected in extremely low quantities, below the threshold of significance, thus viral abundance could not be determined. CONCLUSIONS: Our results suggest that the bacterial and viral abundance in the placenta may have only limited involvement in the pathogenesis of preeclampsia. The evidence of a heterogenic bacterial RNA composition in the various placental locations warrants further investigation to capture the true nature of the placental microbiome.

Differential microRNAs expression in calcified versus rheumatic aortic valve disease.

BACKGROUND: The aortic valve (AV) is the most commonly affected valve in valvular heart diseases (VHDs). The objective of the study is to identify microRNA (miRNA) molecules expressed in VHDs and the differential expression patterns of miRNA in AVs with either calcification or rheumatism etiologies. METHODS: Human AVs were collected during valve replacement surgery. RNA was extracted and miRNA containing libraries were prepared and sequenced using the next generation sequencing (NGS) approach. miRNAs identified as differentially expressed between the two etiologies were validated by quantitative real-time polymerase chain reaction (qPCR). The receiver operating characteristic (ROC) curve analysis was performed to examine the ability of relevant miRNA to differentiate between calcification and rheumatism etiologies. RESULTS: Rheumatic and calcified AV samples were prepared for the NGS and were successfully sequenced. The expression was validated by the qPCR approach in 46 AVs, 13 rheumatic, and 33 calcified AVs, confirming that miR-145-5p, miR-199a-5p, and miR-5701 were significantly higher in rheumatic AVs as compared with calcified AVs. ROC curve analysis revealed that miR-145-5p had a sensitivity of 76.92% and a specificity of 94.12%, area under the curve (AUC) = 0.88 (P = .0001), and miR-5701 had a sensitivity of 84.62% and a specificity of 76.47%, AUC = 0.78 (P = .0001), whereas miR-199a-5p had a sensitivity of 84.62%, and a specificity of 57.58%, AUC = 0.73 (P = .0083). CONCLUSION: We documented differential miRNA expression between AV disease etiologies. The miRNAs identified in this study advance our understanding of the mechanisms underlining AV disease.

A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes.

Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity).

Vascular histone deacetylation by pharmacological HDAC inhibition.

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.

Comparative microRNA profiling of sporadic and BRCA1 associated basal-like breast cancers.

BACKGROUND: While a number of studies have examined miRNA profiles across the molecular subtypes of breast cancer, it is unclear whether BRCA1 basal-like cancers have a specific miRNA profile. This study aims to compare grade independent miRNA expression in luminal cancers, sporadic and BRCA1 basal-type breast cancers. It also aims to ascertain an immunohistochemical profile regulated by BRCA1 specific miRNAs for potential diagnostic use. METHODS: miRNA expression was assessed in 11 BRCA1 basal, 16 sporadic basal, 17 luminal grade 3 cancers via microarrays. The expression of Cyclin D1, FOXP1, FIH-1, pan-ERß, NRP1 and CD99, predicted to be regulated by BRCA1 specific miRNAs by computer prediction algorithms, was assessed via immunohistochemistry in a cohort of 35 BRCA1 and 52 sporadic basal-like cancers. Assessment of cyclin D1, FOXP1, NRP1 and CD99 expression was repeated on a validation cohort of 82 BRCA1 and 65 sporadic basal-like breast cancers. RESULTS: Unsupervised clustering of basal cancers resulted in a "sporadic" cluster of 11 cancers, and a "BRCA1" cluster of 16 cancers, including a subgroup composed entirely of 10 BRCA1 cancers. Compared with sporadic basal cancers, BRCA1 cancers showed reduced positivity for proteins predicted to be regulated by miRNAs: FOXP1 (6/20[30 %] vs. 37/49[76 %], p < 0.001), cyclin D1 (8/22[36 %] vs. 30/46[65 %], p = 0.025), NRP1 (2/20[10 %] vs. 23/46[50 %], p = 0.002). This was confirmed in the validation cohort (all p < 0.001). Negative staining for 2 or more out of FOXP1, cyclin D1 and NRP1 predicts germline BRCA1 mutation with a sensitivity of 92 %, specificity of 44 %, positive predictive value of 38 % and a negative predictive value of 94 %. CONCLUSION: Sporadic and BRCA1 basal-like cancers have grade independent miRNA expression profiles. Furthermore miRNA driven differences in the expression of proteins in BRCA1 basal cancers may be detected via immunohistochemistry. These findings may have important diagnostic implications, as immunohistochemical assessment of basal cancers, in addition to the patient's family and clinical history, may potentially identify patients who may benefit from BRCA1 gene testing.

A novel mouse model of atherosclerotic plaque instability for drug testing and mechanistic/therapeutic discoveries using gene and microRNA expression profiling.

RATIONALE: The high morbidity/mortality of atherosclerosis is typically precipitated by plaque rupture and consequent thrombosis. However, research on underlying mechanisms and therapeutic approaches is limited by the lack of animal models that reproduce plaque instability observed in humans. OBJECTIVE: Development and use of a mouse model of plaque rupture that reflects the end stage of human atherosclerosis. METHODS AND RESULTS: On the basis of flow measurements and computational fluid dynamics, we applied a tandem stenosis to the carotid artery of apolipoprotein E-deficient mice on high-fat diet. At 7 weeks postoperatively, we observed intraplaque hemorrhage in ≈50% of mice, as well as disruption of fibrous caps, intraluminal thrombosis, neovascularization, and further characteristics typically seen in human unstable plaques. Administration of atorvastatin was associated with plaque stabilization and downregulation of monocyte chemoattractant protein-1 and ubiquitin. Microarray profiling of mRNA and microRNA (miR) and, in particular, its combined analysis demonstrated major differences in the hierarchical clustering of genes and miRs among nonatherosclerotic arteries, stable, and unstable plaques and allows the identification of distinct genes/miRs, potentially representing novel therapeutic targets for plaque stabilization. The feasibility of the described animal model as a discovery tool was established in a pilot approach, identifying a disintegrin and metalloprotease with thrombospondin motifs 4 (ADAMTS4) and miR-322 as potential pathogenic factors of plaque instability in mice and validated in human plaques. CONCLUSIONS: The newly described mouse model reflects human atherosclerotic plaque instability and represents a discovery tool toward the development and testing of therapeutic strategies aimed at preventing plaque rupture. Distinctly expressed genes and miRs can be linked to plaque instability.

Genome-wide analysis distinguishes hyperglycemia regulated epigenetic signatures of primary vascular cells.

Emerging evidence suggests that poor glycemic control mediates post-translational modifications to the H3 histone tail. We are only beginning to understand the dynamic role of some of the diverse epigenetic changes mediated by hyperglycemia at single loci, yet elevated glucose levels are thought to regulate genome-wide changes, and this still remains poorly understood. In this article we describe genome-wide histone H3K9/K14 hyperacetylation and DNA methylation maps conferred by hyperglycemia in primary human vascular cells. Chromatin immunoprecipitation (ChIP) as well as CpG methylation (CpG) assays, followed by massive parallel sequencing (ChIP-seq and CpG-seq) identified unique hyperacetylation and CpG methylation signatures with proximal and distal patterns of regionalization associative with gene expression. Ingenuity knowledge-based pathway and gene ontology analyses indicate that hyperglycemia significantly affects human vascular chromatin with the transcriptional up-regulation of genes involved in metabolic and cardiovascular disease. We have generated the first installment of a reference collection of hyperglycemia-induced chromatin modifications using robust and reproducible platforms that allow parallel sequencing-by-synthesis of immunopurified content. We uncover that hyperglycemia-mediated induction of genes and pathways associated with endothelial dysfunction occur through modulation of acetylated H3K9/K14 inversely correlated with methyl-CpG content.

Epigenetic regulation of cell type-specific expression patterns in the human mammary epithelium.

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type-specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type-specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation.

Discovery and analysis of consistent active sub-networks in cancers.

Gene expression profiles can show significant changes when genetically diseased cells are compared with non-diseased cells. Biological networks are often used to identify active subnetworks (ASNs) of the diseases from the expression profiles to understand the reason behind the observed changes. Current methodologies for discovering ASNs mostly use undirected PPI networks and node centric approaches. This can limit their ability to find the meaningful ASNs when using integrated networks having comprehensive information than the traditional protein-protein interaction networks. Using appropriate scoring functions to assess both genes and their interactions may allow the discovery of better ASNs. In this paper, we present CASNet, which aims to identify better ASNs using (i) integrated interaction networks (mixed graphs), (ii) directions of regulations of genes, and (iii) combined node and edge scores. We simplify and extend previous methodologies to incorporate edge evaluations and lessen their sensitivity to significance thresholds. We formulate our objective functions using mixed integer programming (MIP) and show that optimal solutions may be obtained. We compare the ASNs obtained by CASNet and similar other approaches to show that CASNet can often discover more meaningful and stable regulatory ASNs. Our analysis of a breast cancer dataset finds that the positive feedback loops across 7 genes, AR, ESR1, MYC, E2F2, PGR, BCL2 and CCND1 are conserved across the basal/triple negative subtypes in multiple datasets that could potentially explain the aggressive nature of this cancer subtype. Furthermore, comparison of the basal subtype of breast cancer and the mesenchymal subtype of glioblastoma ASNs shows that an ASN in the vicinity of IL6 is conserved across the two subtypes. This result suggests that subtypes of different cancers can show molecular similarities indicating that the therapeutic approaches in different types of cancers may be shared.

High-Resolution Genomic Profiling of Liver Cancer Links Etiology With Mutation and Epigenetic Signatures.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization. METHODS: To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection. RESULTS: Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway. CONCLUSIONS: We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general.

Enhanced RAD21 cohesin expression confers poor prognosis in BRCA2 and BRCAX, but not BRCA1 familial breast cancers.

INTRODUCTION: The RAD21 gene encodes a key component of the cohesin complex, which is essential for chromosome segregation, and together with BRCA1 and BRCA2, for high-fidelity DNA repair by homologous recombination. Although its expression correlates with early relapse and treatment resistance in sporadic breast cancers, it is unclear whether familial breast cancers behave in a similar manner. METHODS: We performed an immunohistochemical analysis of RAD21 expression in a cohort of 94 familial breast cancers (28 BRCA1, 27 BRCA2, and 39 BRCAX) and correlated these data with genotype and clinicopathologic parameters, including survival. In these cancers, we also correlated RAD21 expression with genomic expression profiling and gene copy-number changes and miRNAs predicted to target RAD21. RESULTS: No significant differences in nuclear RAD21 expression were observed between BRCA1 (12 (43%) of 28), BRCA2 (12 (44%) of 27), and BRCAX cancers (12 (33%) of 39 (p = 0.598). No correlation was found between RAD21 expression and grade, size, or lymph node, ER, or HER2 status (all P > 0.05). As for sporadic breast cancers, RAD21 expression correlated with shorter survival in grade 3 (P = 0.009) and but not in grade 1 (P = 0.065) or 2 cancers (P = 0.090). Expression of RAD21 correlated with poorer survival in patients treated with chemotherapy (P = 0.036) but not with hormonal therapy (P = 0.881). RAD21 expression correlated with shorter survival in BRCA2 (P = 0.006) and BRCAX (P = 0.008), but not BRCA1 cancers (P = 0.713). Changes in RAD21 mRNA were reflected by genomic changes in DNA copy number (P < 0.001) and by RAD21 protein expression, as assessed with immunohistochemistry (P = 0.047). High RAD21 expression was associated with genomic instability, as assessed by the total number of base pairs affected by genomic change (P = 0.048). Of 15 miRNAs predicted to target RAD21, mir-299-5p inversely correlated with RAD21 expression (P = 0.002). CONCLUSIONS: Potential use of RAD21 as a predictive and prognostic marker in familial breast cancers is hence feasible and may therefore take into account the patient's BRCA1/2 mutation status.

Co-evolution of tumor cells and their microenvironment.

Increasing evidence indicates that tumor-stromal cell interactions have a crucial role in tumor initiation and progression. These interactions modify cellular compartments, leading to the co-evolution of tumor cells and their microenvironment. Although the importance of microenvironmental alterations in tumor development is recognized, the molecular mechanisms underlying these changes are only now beginning to be understood. Epigenetic and gene expression changes have consistently been reported in cancer-associated stromal cells and the influence of the host genotype on tumorigenesis is also well documented. However, the presence of clonally selected somatic genetic alterations within the tumor microenvironment has been controversial. A thorough understanding of the co-evolution of these two cellular compartments will require carefully executed molecular studies combined with mathematical modeling.

Plasma lipidomic analysis of stable and unstable coronary artery disease.

OBJECTIVE: Traditional risk factors for coronary artery disease (CAD) fail to adequately distinguish patients who have atherosclerotic plaques susceptible to instability from those who have more benign forms. Using plasma lipid profiling, this study aimed to provide insight into disease pathogenesis and evaluate the potential of lipid profiles to assess risk of future plaque instability. METHODS AND RESULTS: Plasma lipid profiles containing 305 lipids were measured on 220 individuals (matched healthy controls, n=80; stable angina, n=60; unstable coronary syndrome, n=80) using electrospray-ionisation tandem mass spectrometry. ReliefF feature selection coupled with an L2-regularized logistic regression based classifier was used to create multivariate classification models which were verified via 3-fold cross-validation (1000 repeats). Models incorporating both lipids and traditional risk factors provided improved classification of unstable CAD from stable CAD (C-statistic=0.875, 95% CI 0.874-0.877) compared with models containing only traditional risk factors (C-statistic=0.796, 95% CI 0.795-0.798). Many of the lipids identified as discriminatory for unstable CAD displayed an association with disease acuity (severity), suggesting that they are antecedents to the onset of acute coronary syndrome. CONCLUSION: Plasma lipid profiling may contribute to a new approach to risk stratification for unstable CAD.

Genetic changes in tumour microenvironments.

Numerous in vitro and in vivo studies have established that carcinoma-associated fibroblasts differ phenotypically from fibroblasts associated with normal tissue but the mechanisms underlying these differences are unclear. Since carcinoma-associated fibroblasts can be propagated in vitro for extended periods and still maintain their cancer-promoting phenotype, some investigators have proposed that they might have acquired somatic genetic alterations analogous to those observed in malignant epithelium. Early molecular genetic studies appeared to validate this hypothesis by demonstrating remarkably high frequencies of clonal somatic genetic alterations in carcinoma-associated fibroblasts, including loss of heterozygosity, gene amplification, and point mutations in tumour suppressor genes such as TP53 and PTEN. The initial excitement of these paradigm-changing studies overshadowed concerns that there may have been a more mundane explanation for these observations. In addition to the fact that the data would necessarily invoke an unlikely scenario of the simultaneous generation of two symbiotic malignancies, subsequent molecular genetic studies found no evidence of frequent genomic aberrations. One striking common trait of those studies reporting frequent clonal somatic alterations in carcinoma-associated fibroblasts is the use of tissues and techniques which are well known to be highly prone to generating artefacts such as limiting and poor quality DNA followed by highly multiplexed PCR-based analyses. It is now clear that clonal somatic mutations are not the biological basis of the cancer-promoting attributes of carcinoma-associated fibroblasts.

EBNA1 regulates cellular gene expression by binding cellular promoters.

Epstein-Barr virus (EBV) is associated with several types of lymphomas and epithelial tumors including Burkitt's lymphoma (BL), HIV-associated lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. EBV nuclear antigen 1 (EBNA1) is expressed in all EBV associated tumors and is required for latency and transformation. EBNA1 initiates latent viral replication in B cells, maintains the viral genome copy number, and regulates transcription of other EBV-encoded latent genes. These activities are mediated through the ability of EBNA1 to bind viral-DNA. To further elucidate the role of EBNA1 in the host cell, we have examined the effect of EBNA1 on cellular gene expression by microarray analysis using the B cell BJAB and the epithelial 293 cell lines transfected with EBNA1. Analysis of the data revealed distinct profiles of cellular gene changes in BJAB and 293 cell lines. Subsequently, chromatin immune-precipitation revealed a direct binding of EBNA1 to cellular promoters. We have correlated EBNA1 bound promoters with changes in gene expression. Sequence analysis of the 100 promoters most enriched revealed a DNA motif that differs from the EBNA1 binding site in the EBV genome.

Resolving the conflicts around Par2 opposing roles in regeneration by comparing immune-mediated and toxic-induced injuries.

BACKGROUND: Different factors may lead to hepatitis. Among which are liver inflammation and poisoning. We chose two hepatitis models, typical for these two underlying causes. Thus, we aimed to characterize the role of protease-activated receptor 2 (Par2) in liver regeneration and inflammation to reconcile Par2 conflicting role in many damage models, which sometimes aggravates the induced damage and sometimes alleviates it. METHODS: WT and knockout (Par2KO) mice were injected with concanavalin A (ConA) to induce immune-mediated hepatitis or with carbon tetrachloride (CCl4) to elicit direct hepatic damage. To distinguish the immune component from the liver regenerative response, we conducted bone marrow (BM) replacements of WT and Par2KO mice and repeated the damage models. RESULTS: ConA injection caused limited damage in Par2KO mice livers, while in the WT mice severe damage followed by leukocyte infiltration was evident. Reciprocal BM replacement of WT and Par2KO showed that WT BM-reconstituted Par2KO mice displayed marked liver damage, while in Par2KO BM-reconstituted WT mice, the tissue was generally protected. In the CCl4 direct damage model, hepatocytes regenerated in WT mice, whereas Par2KO mice failed to recover. Reciprocal BM replacement did not show significant differences in hepatic regeneration. In Par2KO mice, hepatitis was more apparent, while WT recovered regardless of the BM origin. CONCLUSIONS: We conclude that Par2 activation in the immune system aggravates hepatitis and that Par2 activation in the damaged tissue promotes liver regeneration. When we incorporate this finding and revisit the literature reports, we reconciled the conflicts surrounding Par2's role in injury, recovery, and inflammation.

Meta-analysis of gene expression microarrays with missing replicates.

BACKGROUND: Many different microarray experiments are publicly available today. It is natural to ask whether different experiments for the same phenotypic conditions can be combined using meta-analysis, in order to increase the overall sample size. However, some genes are not measured in all experiments, hence they cannot be included or their statistical significance cannot be appropriately estimated in traditional meta-analysis. Nonetheless, these genes, which we refer to as incomplete genes, may also be informative and useful. RESULTS: We propose a meta-analysis framework, called "Incomplete Gene Meta-analysis", which can include incomplete genes by imputing the significance of missing replicates, and computing a meta-score for every gene across all datasets. We demonstrate that the incomplete genes are worthy of being included and our method is able to appropriately estimate their significance in two groups of experiments. We first apply the Incomplete Gene Meta-analysis and several comparable methods to five breast cancer datasets with an identical set of probes. We simulate incomplete genes by randomly removing a subset of probes from each dataset and demonstrate that our method consistently outperforms two other methods in terms of their false discovery rate. We also apply the methods to three gastric cancer datasets for the purpose of discriminating diffuse and intestinal subtypes. CONCLUSIONS: Meta-analysis is an effective approach that identifies more robust sets of differentially expressed genes from multiple studies. The incomplete genes that mainly arise from the use of different platforms may also have statistical and biological importance but are ignored or are not appropriately involved by previous studies. Our Incomplete Gene Meta-analysis is able to incorporate the incomplete genes by estimating their significance. The results on both breast and gastric cancer datasets suggest that the highly ranked genes and associated GO terms produced by our method are more significant and biologically meaningful according to the previous literature.

Appraisal of progenitor markers in the context of molecular classification of breast cancers.

Clinical management of breast cancer relies on case stratification, which increasingly employs molecular markers. The motivation behind delineating breast epithelial differentiation is to better target cancer cases through innate sensitivities bequeathed to the cancer from its normal progenitor state. A combination of histopathological and molecular classification of breast cancer cases suggests a role for progenitors in particular breast cancer cases. Although a remarkable fraction of the real tissue repertoire is maintained within a population of independent cell line cultures, some steps that are closer to the terminal differentiation state and that form a majority of primary human breast tissues are missing in the cell line cultures. This raises concerns about current breast cancer models.