Accurate reconstruction of viral genomes in human cells from short reads using iterative refinement.

BACKGROUND: After an infection, human cells may contain viral genomes in the form of episomes or integrated DNA. Comparing the genomic sequences of different strains of a virus in human cells can often provide useful insights into its behaviour, activity and pathology, and may help develop methods for disease prevention and treatment. To support such comparative analyses, the viral genomes need to be accurately reconstructed from a large number of samples. Previous efforts either rely on customized experimental protocols or require high similarity between the sequenced genomes and a reference, both of which limit the general applicability of these approaches. In this study, we propose a pipeline, named ASPIRE, for reconstructing viral genomes accurately from short reads data of human samples, which are increasingly available from genome projects and personal genomics. ASPIRE contains a basic part that involves de novo assembly, tiling and gap filling, and additional components for iterative refinement, sequence corrections and wrapping. RESULTS: Evaluated by the alignment quality of sequencing reads to the reconstructed genomes, these additional components improve the assembly quality in general, and in some particular samples quite substantially, especially when the sequenced genome is significantly different from the reference. We use ASPIRE to reconstruct the genomes of Epstein Barr Virus (EBV) from the whole-genome sequencing data of 61 nasopharyngeal carcinoma (NPC) samples and provide these sequences as a resource for EBV research. CONCLUSIONS: ASPIRE improves the quality of the reconstructed EBV genomes in published studies and outperforms TRACESPipe in some samples considered.

Loss of tumor suppressor IGFBP4 drives epigenetic reprogramming in hepatic carcinogenesis.

Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition.

Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.

ECplot: an online tool for making standardized plots from large datasets for bioinformatics publications.

MOTIVATION AND RESULTS: We have implemented ECplot, an online tool for plotting charts from large datasets. This tool supports a variety of chart types commonly used in bioinformatics publications. In our benchmarking, it was able to create a Box-and-Whisker plot with about 67 000 data points and 8 MB total file size within several seconds. The design of the tool makes common formatting operations easy to perform. It also allows more complex operations to be achieved by advanced XML (Extensible Markup Language) and programming options. Data and formatting styles are stored in separate files, such that style templates can be made and applied to new datasets. The text-based file formats based on XML facilitate efficient manipulation of formatting styles for a large number of data series. These file formats also provide a means to reproduce published figures from raw data, which complement parallel efforts in making the data and software involved in published analysis results accessible. We demonstrate this idea by using ECplot to replicate some complex figures from a previous publication. AVAILABILITY AND IMPLEMENTATION: ECplot and its source code (under MIT license) are available at https://yiplab.cse.cuhk.edu.hk/ecplot/. CONTACT: kevinyip@cse.cuhk.edu.hk.

VAS: a convenient web portal for efficient integration of genomic features with millions of genetic variants.

BACKGROUND: High-throughput experimental methods have fostered the systematic detection of millions of genetic variants from any human genome. To help explore the potential biological implications of these genetic variants, software tools have been previously developed for integrating various types of information about these genomic regions from multiple data sources. Most of these tools were designed either for studying a small number of variants at a time, or for local execution on powerful machines. RESULTS: To make exploration of whole lists of genetic variants simple and accessible, we have developed a new Web-based system called VAS (Variant Annotation System, available at https://yiplab.cse.cuhk.edu.hk/vas/). It provides a large variety of information useful for studying both coding and non-coding variants, including whole-genome transcription factor binding, open chromatin and transcription data from the ENCODE consortium. By means of data compression, millions of variants can be uploaded from a client machine to the server in less than 50 megabytes of data. On the server side, our customized data integration algorithms can efficiently link millions of variants with tens of whole-genome datasets. These two enabling technologies make VAS a practical tool for annotating genetic variants from large genomic studies. We demonstrate the use of VAS in annotating genetic variants obtained from a migraine meta-analysis study and multiple data sets from the Personal Genomes Project. We also compare the running time of annotating 6.4 million SNPs of the CEU trio by VAS and another tool, showing that VAS is efficient in handling new variant lists without requiring any pre-computations. CONCLUSIONS: VAS is specially designed to handle annotation tasks with long lists of genetic variants and large numbers of annotating features efficiently. It is complementary to other existing tools with more specific aims such as evaluating the potential impacts of genetic variants in terms of disease risk. We recommend using VAS for a quick first-pass identification of potentially interesting genetic variants, to minimize the time required for other more in-depth downstream analyses.

Are special read alignment strategies necessary and cost-effective when handling sequencing reads from patient-derived tumor xenografts?

BACKGROUND: Patient-derived tumor xenografts in mice are widely used in cancer research and have become important in developing personalized therapies. When these xenografts are subject to DNA sequencing, the samples could contain various amounts of mouse DNA. It has been unclear how the mouse reads would affect data analyses. We conducted comprehensive simulations to compare three alignment strategies at different mutation rates, read lengths, sequencing error rates, human-mouse mixing ratios and sequenced regions. We also sequenced a nasopharyngeal carcinoma xenograft and a cell line to test how the strategies work on real data. RESULTS: We found the "filtering" and "combined reference" strategies performed better than aligning reads directly to human reference in terms of alignment and variant calling accuracies. The combined reference strategy was particularly good at reducing false negative variants calls without significantly increasing the false positive rate. In some scenarios the performance gain of these two special handling strategies was too small for special handling to be cost-effective, but it was found crucial when false non-synonymous SNVs should be minimized, especially in exome sequencing. CONCLUSIONS: Our study systematically analyzes the effects of mouse contamination in the sequencing data of human-in-mouse xenografts. Our findings provide information for designing data analysis pipelines for these data.

Identification of a recurrent transforming UBR5-ZNF423 fusion gene in EBV-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is prevalent in southern China, south-east Asia and northern Africa. The development and stepwise progression of NPC involves accumulation of multiple gross genetic changes during the clonal expansion of Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cell population. Here, using paired-end whole-transcriptome sequencing, we discovered a number of chimeric fusion transcripts in a panel of EBV-positive tumour lines. Among these transcripts, a novel fusion of ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and zinc finger protein 423 (ZNF423) on 16q12.1, identified from the NPC cell line C666-1, was recurrently detected in 12/144 (8.3%) of primary tumours. The fusion gene contains exon 1 of UBR5 and exons 7-9 of ZNF423 and produces a 94 amino acid chimeric protein including the original C-terminal EBF binding domain (ZF29-30) of ZNF423. Notably, the growth of NPC cells with UBR5-ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5-ZNF423 by fusion-specific siRNA significantly inhibited the cell proliferation and colony-forming ability of C666-1 cells. The transforming ability of UBR5-ZNF423 fusion was also confirmed in NIH3T3 fibroblasts. Constitutive expression of UBR5-ZNF423 in NIH3T3 fibroblasts significantly enhanced its anchorage-independent growth in soft agar and induced tumour formation in a nude mouse model. These findings suggest that expression of UBR5-ZNF423 protein might contribute to the transformation of a subset of NPCs, possibly by altering the activity of EBFs (early B cell factors). Identification of the oncogenic UBR5-ZNF423 provides new potential opportunities for therapeutic intervention in NPC.

Whole-genome profiling of nasopharyngeal carcinoma reveals viral-host co-operation in inflammatory NF-κB activation and immune escape.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer.

Aberrant enhancer hypomethylation contributes to hepatic carcinogenesis through global transcriptional reprogramming.

Hepatocellular carcinomas (HCC) exhibit distinct promoter hypermethylation patterns, but the epigenetic regulation and function of transcriptional enhancers remain unclear. Here, our affinity- and bisulfite-based whole-genome sequencing analyses reveal global enhancer hypomethylation in human HCCs. Integrative epigenomic characterization further pinpoints a recurrent hypomethylated enhancer of CCAAT/enhancer-binding protein-beta (C/EBPß) which correlates with C/EBPß over-expression and poorer prognosis of patients. Demethylation of C/EBPß enhancer reactivates a self-reinforcing enhancer-target loop via direct transcriptional up-regulation of enhancer RNA. Conversely, deletion of this enhancer via CRISPR/Cas9 reduces C/EBPß expression and its genome-wide co-occupancy with BRD4 at H3K27ac-marked enhancers and super-enhancers, leading to drastic suppression of driver oncogenes and HCC tumorigenicity. Hepatitis B X protein transgenic mouse model of HCC recapitulates this paradigm, as C/ebpß enhancer hypomethylation associates with oncogenic activation in early tumorigenesis. These results support a causal link between aberrant enhancer hypomethylation and C/EBPß over-expression, thereby contributing to hepatocarcinogenesis through global transcriptional reprogramming.

Establishment and characterization of new tumor xenografts and cancer cell lines from EBV-positive nasopharyngeal carcinoma.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.

Exome and genome sequencing of nasopharynx cancer identifies NF-κB pathway activating mutations.

Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression and rational therapy design. Here we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-κB pathway, including CYLD, TRAF3, NFKBIA and NLRC5, in a total of 41% of cases. Functional analysis confirmed inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent membrane protein 1 (LMP1) functions to constitutively activate NF-κB signalling, and we observed mutual exclusivity among tumours with somatic NF-κB pathway aberrations and LMP1-overexpression, suggesting that NF-κB activation is selected for by both somatic and viral events during NPC pathogenesis.

Complete genomic sequence of Epstein-Barr virus in nasopharyngeal carcinoma cell line C666-1.

BACKGROUND: Nasopharyngeal carcinoma is a distinct type of head and neck cancer which is consistently associated with Epstein-Barr virus (EBV). The C666-1 cell line is the only in vitro native EBV-infected NPC cell model commonly used for study of the viral-host interaction. Nevertheless, the complete EBV genome sequence in this in vitro EBV-infected NPC model has not been characterized. OBJECTIVE: To determine the complete EBV genome sequence in C666-1 cells. METHODS: The C666-1 genome was sequenced by 100-bases pair-end massive parallel sequencing. Bioinformatics analysis was performed to extract the EBV sequences and construct an EBV consensus sequence map. PCR amplification and Sanger DNA sequencing were used for sequence validation and gap filling. A phylogenetic analysis of EBV strain in C666-1 cells and other reported EBV strains was performed. RESULTS: A 171,317 bp complete EBV genome of C666-1 was successfully constructed (GenBank accession number: KC617875). Phylogenetic analysis of EBV genome in C666-1 revealed that the C666-1 EBV strain is closely related to the reported strains in NPC primary tumors. CONCLUSION: C666-1 contains a representative NPC-associated EBV genome and might serve as an important model for studying the roles or function of viral proteins in NPC tumorigenesis.