Genome integration structures and genotype variants of oncogenic human papillomavirus types HPV16 and HPV68 in cervical carcinoma-derived cell lines, cervical precursor lesions and carcinomas

Persistent infection with high-risk human papillomavirus (hr-HPV) is essential for cervical carcinogenesis, and is frequently followed by integration of the viral DNA into the host genome. Upon integration, the viral E2 gene is usually disrupted or deleted leading to deregulated transcription of the E6/E7 oncogenes from the upstream regulatory region (URR). Integrated HPV DNA may also affect critical cellular genes through insertional mutagenesis, which can contribute to the multi-step process of cervical carcinogenesis. HPV16 is the most frequent and HPV68 is a rare hr-HPV type, present in about 55% and less than 1% of cervical carcinomas worldwide, respectively. In this work, HPV68 DNA structures in cervical carcinoma cell lines and clinical samples were analyzed. HPV16 integration and E1-E2 sequences were studied using the novel “amplification selection pyrosequencing of HPV16” (ASP16) strategy. HPV68 is divided into two subtypes, a and b. A hallmark of HPV68b is its presence as integrated DNA in the cervical carcinoma cell line ME180. In the mutant cell line ME180R, selected for resistance to growth inhibition by tumor-necrosis-factor alpha (TNFalpha), partial deletions in the integrated HPV68b DNA had been detected. In this study, the complete structures of the integrated HPV68b in ME180 and ME180R have been determined. ME180 cells contain two disrupted HPV68b copies, integrated in a unique head-to-head arrangement into chromosome 18q21. By selection of new TNFalpha-resistant ME180 sub-lines, it was found that the rearrangements and partial deletions of HPV68b in ME180R are unnecessary for the TNFalpha-resistance phenotype. In addition, a full-length and a mutant HPV68b genome were isolated from a cervical intraepithelial neoplasia grade 2 (CIN2) precursor lesion, cloned and completely sequenced. The mutant genome carrying a 1.2-kb deletion in the E1 gene is probably integrated. Based on partial URR sequences, ten HPV68b variants, nine of them new, and one HPV68a variant have been identified in eleven clinical samples, suggesting that HPV68b is more widely distributed than HPV68a and is present in a multitude of molecular variants. ASP16 was developed for simultaneous determination of HPV16 integration junctions in multiple clinical DNA samples. It consists of four main steps: GenomePlex whole genome amplification, HPV16 E1-E2 sequence enrichment, Roche/454 GS-FLX pyrosequencing, and data analysis. In this work, computer programs for ASP16 data analysis were developed and applied. The ASP16 strategy was further optimized and used for the analysis of 25 HPV16-positive samples. The optimized ASP16 delivered longer sequence read lengths and 89% average sequence coverage. HPV16 integration junctions were identified in 3 out of 4 cell lines, and 6 out of 21 clinical samples. The HPV16 integration sites identified in the clinical samples are all located near cellular proto-oncogenes or tumor suppressor genes, supporting the assumption that HPV integration contributes to cervical carcinogenesis by altering cancer-relevant cellular genes. The high E1-E2 sequence coverage also allowed HPV16 variant assignments. Altogether, the ASP16 strategy, which is the first method combining next generation sequencing technologies with HPV integration analysis in a multiplex format, shows the potential to identify HPV16 integration junctions in series of clinical samples in parallel and at the same time provides E1-E2 sequences suitable for mutation/variant analysis

Retrotransposon Alu Is Enriched In The Epichromatin Of HL-60 Cells

Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a “rim” staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells, \u3e95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither “classical” heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus

A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing.

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼ 100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.We thank the DKFZ Genomics and Proteomics Core Facility and the OICR Genome Technologies Platform for provision of sequencing services. Financial support was provided by the consortium projects READNA under grant agreement FP7 Health-F4-2008-201418, ESGI under grant agreement 262055, GEUVADIS under grant agreement 261123 of the European Commission Framework Programme 7, ICGC-CLL through the Spanish Ministry of Science and Innovation (MICINN), the Instituto de Salud Carlos III (ISCIII) and the Generalitat de Catalunya. Additional financial support was provided by the PedBrain Tumor Project contributing to the International Cancer Genome Consortium, funded by German Cancer Aid (109252) and by the German Federal Ministry of Education and Research (BMBF, grants #01KU1201A, MedSys #0315416C and NGFNplus #01GS0883; the Ontario Institute for Cancer Research to PCB and JDM through funding provided by the Government of Ontario, Ministry of Research and Innovation; Genome Canada; the Canada Foundation for Innovation and Prostate Cancer Canada with funding from the Movember Foundation (PCB). PCB was also supported by a Terry Fox Research Institute New Investigator Award, a CIHR New Investigator Award and a Genome Canada Large-Scale Applied Project Contract. The Synergie Lyon Cancer platform has received support from the French National Institute of Cancer (INCa) and from the ABS4NGS ANR project (ANR-11-BINF-0001-06). The ICGC RIKEN study was supported partially by RIKEN President’s Fund 2011, and the supercomputing resource for the RIKEN study was provided by the Human Genome Center, University of Tokyo. MDE, LB, AGL and CLA were supported by Cancer Research UK, the University of Cambridge and Hutchison-Whampoa Limited. SD is supported by the Torres Quevedo subprogram (MI CINN) under grant agreement PTQ-12-05391. EH is supported by the Research Council of Norway under grant agreements 221580 and 218241 and by the Norwegian Cancer Society under grant agreement 71220-PR-2006-0433. Very special thanks go to Jennifer Jennings for administrating the activity of the ICGC Verification Working Group and Anna Borrell for administrative support.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1000

Retrotransposon Alu is enriched in the epichromatin of HL-60 cells

Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a “rim” staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells, >95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither “classical” heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus

Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas

<div><p>Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3′-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3′-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.</p></div

HPV16 E6 variants.

<p>E-p = European prototype; E-T350G = European T>G at position 350; NA1 = North-American type 1; AA = Asian-American; As = Asian; Af1 = African type 1; Af2 = African type 2.</p>*<p>Underlined are those tumors which seem to contain episomal HPV16 DNA, because no DNA junctions of integrated HPV16 DNA could be identified by the TEN16 analysis.</p

Tumor T2548 with six HPV16 DNA integration sites.

<p>The six integration sites are distributed on the chromosomes 3, 19 and 20 (two DJs on each). All six integration sites are intragenic, but none of them is transcriptionally active. The cellular genes directly targeted by HPV16 DNA integration in T2548 include <i>FHIT</i> (transcript 002, Ensembl ID: ENST00000468189, minus strand, 9 exons), <i>ARID3A</i> (transcript 001, Ensembl ID: ENST00000263620, plus strand, 9 exons), <i>MOB3A</i> (transcript 001, Ensembl ID: ENST00000357066, minus strand, 5 exons), <i>MACROD2</i> (transcript 010, Ensembl ID: ENST00000217246, plus strand, 17 exons) and <i>CBFA2T2</i> (transcript 003, Ensembl ID: ENST00000375279, plus strand, 12 exons).</p

Comparison of HPV16-cellular DNA junctions and RNA junctions.

<p>TA = TEN16/APOT comparison; DJ = DNA junction; RJ = RNA junction; Chr. = chromosome; n.a. = not applicable; Do = splice donor; Ac = splice acceptor.</p>(1)<p>TA-group 1: samples with one DJ and a corresponding RJ.</p>(2)<p>TA-group 2: samples with one corresponding DJ/RJ pair and additional DJs without RJ counterpart.</p>(3)<p>TA-group 3: samples without corresponding DJ/RJ.</p>&<p>The 22 RNA junctions (APOT) are part of a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693-Schmitz2" target="_blank">[50]</a> in which information on chromosomal locations, cellular genes and splicing is given, but without the exact position numbers of the cellular breakpoints.</p>§<p>Genomic distance between the cellular breakpoints of DJ and RJ.</p>$<p>Distance from the HPV16 splice donor (position 880) to the cellular splice acceptor (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone-0066693-g001" target="_blank">Figure 1</a>).</p>#<p>Discovered by searching in the TEN16 sequence library for DJs located within 1 Mb upstream of the respective RJs.</p>*<p>For sample T186e, the cellular sequences of DJ1 and RJ were both mapped to chromosome 9, but in opposite orientation to each other.</p>a)<p>In the fusion transcript, the viral E6/E7 exon is spliced to the next downstream exon of the cellular gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone-0066693-g003" target="_blank">Figure 3</a>).</p

Frequency distribution of HPV16 3′-breakpoints in different segments of the HPV16 early region.

<p>The distribution of HPV16 3′-breakpoints of viral-cellular DNA junctions (n = 74) was analyzed within the five different segments E1, E2, E5, E1-Ac and Ac-PAE of the HPV16 early region. The positions of each segment in the HPV16 genome are given in parentheses. The E1-PAE segment of the HPV16 early region (pos. 865–4215, 3351 bp) was taken as reference. The relative length of each segment is shown by the white bars. The relative frequency of HPV16 3′-breakpoints within each segment is shown by the grey bars for all DNA junctions (DJ_all, n = 74, middle-grey bar), the transcribed DNA junctions (DJ_tr., n = 21, light-grey bar) and the non-transcribed DNA junctions (DJ_n.tr., n = 53, dark-grey bar). The exact two-tailed one-sample binomial test was used for statistical analysis by comparing the relative frequency of HPV16 3′-breakpoints in each segment to the relative segment length. Bars marked with asterisks indicate statistically significant results (P<0.05). Data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693.s005" target="_blank">Table S4</a>.</p

Examples of intragenic HPV16 DNA integration sites.

<p>(A) Tumor T182e has one HPV16 integration site 0182_DJ1, which is transcriptionally active (TA-group 1). The integrated HPV16 DNA is located in the intron (i) between exons (e) 5 and 6 of the cellular gene <i>LIPC</i> (transcript 003, Ensembl ID: ENST00000414170, plus strand, 10 exons), and has the same orientation as <i>LIPC</i>. APOT analysis identified an HPV16-cellular fusion transcript (0182_RJ) in which the viral exon is spliced to the downstream <i>LIPC</i> exon 6. (B) Tumor T892 (TA-group 2) has two HPV16 integration sites (0892_DJ1 and 0892_DJ2), which are both located in the intron between exons 12 and 13 of the cellular gene <i>GPN1</i> (transcript 001, Ensembl ID: ENST00000264718, plus strand, 14 exons). While 0892_DJ1 has the same orientation as <i>GPN1</i>, the transcriptionally active 0892_DJ2 has the opposite orientation. In the transcript 0892_RJ the viral exon is spliced to an alternative cellular exon. (C) In tumor T2319 (TA-group 2), all three identified HPV16 integration sites are located within the cellular gene <i>CASZ1</i> (transcript 003, Ensembl ID: ENST00000377022, 21 exons). Since the <i>CASZ1</i> gene is located on the minus strand, the sense orientation of the gene is from right to left. Junction 2319_DJ1 is located in an intron in opposite direction to <i>CASZ1</i>. Junctions 2319_DJ2 and 2319_DJ3 are located in the terminal exon 21 in the same direction as the <i>CASZ1</i> gene, DJ2 in the terminal part of the translated region and DJ3 in the 3′ untranslated region (3′-UTR). Both are possible templates for the HPV16-cellular fusion transcript 2319_RJ. – In the DJs and RJs, the open boxes denote the HPV16 part and the black boxes/arrows the fused cellular part. The arrow of the DJs indicates the sense orientation of the HPV16 oncogenes. Transcribed DJs and the RJs are shown in red, non-transcribed DJs in blue letters.</p