Publications by authors named "Vyacheslav Amstislavskiy"

23 Publications

  • Page 1 of 1

RNA Sequencing of Human Peripheral Blood Cells Indicates Upregulation of Immune-Related Genes in Huntington's Disease.

Front Neurol 2020 27;11:573560. Epub 2020 Nov 27.

Department of Neuropsychiatry, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the gene. As disease-modifying therapies for HD are being developed, peripheral blood cells may be used to indicate disease progression and to monitor treatment response. In order to investigate whether gene expression changes can be found in the blood of individuals with HD that distinguish them from healthy controls, we performed transcriptome analysis by next-generation sequencing (RNA-seq). We detected a gene expression signature consistent with dysregulation of immune-related functions and inflammatory response in peripheral blood from HD cases vs. controls, including induction of the interferon response genes, and . Our results suggest that it is possible to detect gene expression changes in blood samples from individuals with HD, which may reflect the immune pathology associated with the disease.
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http://dx.doi.org/10.3389/fneur.2020.573560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731869PMC
November 2020

The Leukemogenic TCF3-HLF Complex Rewires Enhancers Driving Cellular Identity and Self-Renewal Conferring EP300 Vulnerability.

Cancer Cell 2019 12 14;36(6):630-644.e9. Epub 2019 Nov 14.

Division of Oncology and Children's Research Centre, University Children's Hospital Zurich, 8032 Zurich, Switzerland. Electronic address:

The chimeric transcription factor TCF3-HLF defines an incurable acute lymphoblastic leukemia subtype. Here we decipher the regulome of endogenous TCF3-HLF and dissect its essential transcriptional components and targets by functional genomics. We demonstrate that TCF3-HLF recruits HLF binding sites at hematopoietic stem cell/myeloid lineage associated (super-) enhancers to drive lineage identity and self-renewal. Among direct targets, hijacking an HLF binding site in a MYC enhancer cluster by TCF3-HLF activates a conserved MYC-driven transformation program crucial for leukemia propagation in vivo. TCF3-HLF pioneers the cooperation with ERG and recruits histone acetyltransferase p300 (EP300), conferring susceptibility to EP300 inhibition. Our study provides a framework for targeting driving transcriptional dependencies in this fatal leukemia.
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http://dx.doi.org/10.1016/j.ccell.2019.10.004DOI Listing
December 2019

Mechanisms of Progression of Myeloid Preleukemia to Transformed Myeloid Leukemia in Children with Down Syndrome.

Cancer Cell 2019 08 11;36(2):123-138.e10. Epub 2019 Jul 11.

MRC MHU, BRC Hematology Theme, Oxford Biomedical Research Centre, Oxford Centre for Haematology, WIMM, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DU, UK; Department of Paediatrics, University of Oxford, Oxford OX3 9DS, UK.

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.
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http://dx.doi.org/10.1016/j.ccell.2019.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863161PMC
August 2019

Molecular Evolution of Early-Onset Prostate Cancer Identifies Molecular Risk Markers and Clinical Trajectories.

Cancer Cell 2018 12;34(6):996-1011.e8

Department of Pathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ≤55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.
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http://dx.doi.org/10.1016/j.ccell.2018.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444093PMC
December 2018

Impact of congenital cytomegalovirus infection on transcriptomes from archived dried blood spots in relation to long-term clinical outcome.

PLoS One 2018 19;13(7):e0200652. Epub 2018 Jul 19.

Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.

Congenital Cytomegalovirus infection (cCMV) is the leading infection in determining permanent long-term impairments (LTI), and its pathogenesis is largely unknown due to the complex interplay between viral, maternal, placental, and child factors. The cellular activity, considered to be the result of the response to exogenous and endogenous factors, is captured by the determination of gene expression profiles. In this study, we determined whole blood transcriptomes in relation to cCMV, CMV viral load and LTI development at 6 years of age by using RNA isolated from neonatal dried blood spots (DBS) stored at room temperature for 8 years. As DBS were assumed to mainly reflect the neonatal immune system, particular attention was given to the immune pathways using the global test. Additionally, differential expression of individual genes was performed using the voom/limma function packages. We demonstrated feasibility of RNA sequencing from archived neonatal DBS of children with cCMV, and non-infected controls, in relation to LTI and CMV viral load. Despite the lack of statistical power to detect individual genes differences, pathway analysis suggested the involvement of innate immune response with higher CMV viral loads, and of anti-inflammatory markers in infected children that did not develop LTI. Finally, the T cell exhaustion observed in infected neonates, in particular with higher viral load, did not correlate with LTI, therefore other mechanisms are likely to be involved in the long-term immune dysfunction. Despite these data demonstrate limitation in determining prognostic markers for LTI by means of transcriptome analysis, this exploratory study represents a first step in unraveling the pathogenesis of cCMV, and the aforementioned pathways certainly merit further evaluation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200652PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053152PMC
January 2019

The whole-genome landscape of medulloblastoma subtypes.

Nature 2017 07;547(7663):311-317

Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.
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http://dx.doi.org/10.1038/nature22973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905700PMC
July 2017

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors.

Nat Commun 2017 02 10;8:14262. Epub 2017 Feb 10.

Max Planck Institute for Molecular Genetics, Department of Computational Molecular Biology, Ihnestrasse 73, D-14195 Berlin, Germany.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.
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http://dx.doi.org/10.1038/ncomms14262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309787PMC
February 2017

Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.

Cell 2016 11;167(5):1398-1414.e24

Human Genetics, McGill University, 740 Dr. Penfield, Montreal, QC H3A 0G1, Canada.

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14 monocytes, CD16 neutrophils, and naive CD4 T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.
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http://dx.doi.org/10.1016/j.cell.2016.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5119954PMC
November 2016

Active medulloblastoma enhancers reveal subgroup-specific cellular origins.

Nature 2016 Feb 27;530(7588):57-62. Epub 2016 Jan 27.

Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, Washington 98105, USA.

Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.
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http://dx.doi.org/10.1038/nature16546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5168934PMC
February 2016

Erratum to: 'The direction of cross affects obesity after puberty in male but not female offspring'.

BMC Genomics 2015 Nov 30;16(1):1018. Epub 2015 Nov 30.

Albrecht Daniel Thaer-Institut für Agrar- und Gartenbauwissenschaften, Humboldt-Universität zu Berlin, Invalidenstraße 42, Berlin, D-10115, Germany.

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http://dx.doi.org/10.1186/s12864-015-2204-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665910PMC
November 2015

The direction of cross affects [corrected] obesity after puberty in male but not female offspring.

BMC Genomics 2015 Nov 6;16:904. Epub 2015 Nov 6.

Albrecht Daniel Thaer-Institut für Agrar- und Gartenbauwissenschaften, Humboldt-Universität zu Berlin, Invalidenstraße 42, D-10115, Berlin, Germany.

Background: We investigated parent-of-origin and allele-specific expression effects on obesity and hepatic gene expression in reciprocal crosses between the Berlin Fat Mouse Inbred line (BFMI) and C57Bl/6NCrl (B6N).

Results: We found that F1-males with a BFMI mother developed 1.8 times more fat mass on a high fat diet at 10 weeks than F1-males of a BFMI father. The phenotype was detectable from six weeks on and was preserved after cross-fostering. RNA-seq data of liver provided evidence for higher biosynthesis and elongation of fatty acids (p = 0.00635) in obese male offspring of a BFMI mother versus lean offspring of a BFMI father. Furthermore, fatty acid degradation (p = 0.00198) and the peroxisome pathway were impaired (p = 0.00094). The circadian rhythm was affected as well (p = 0.00087). Among the highest up-regulated protein coding genes in obese males were Acot4 (1.82 fold, p = 0.022), Cyp4a10 (1.35 fold, p = 0.026) and Cyp4a14 (1.32 fold, p = 0.012), which hydroxylize fatty acids and which are known to be increased in liver steatosis. Obese males showed lower expression of the genetically imprinted and paternally expressed 3 (Peg3) gene (0.31 fold, p = 0.046) and higher expression of the androgen receptor (Ar) gene (2.38 fold, p = 0.068). Allelic imbalance was found for expression of ATP-binding cassette transporter gene Abca8b. Several of the differentially expressed genes contain estrogen response elements.

Conclusions: Parent-of-origin effects during gametogenesis and/or fetal development in an obese mother epigenetically modify the transcription of genes that lead to enhanced fatty acid synthesis and impair β-oxidation in the liver of male, but not female F1 offspring. Down-regulation of Peg3 could contribute to trigger this metabolic setting. At puberty, higher amounts of the androgen receptor and altered access to estrogen response elements in affected genes are likely responsible for male specific expression of genes that were epigenetically triggered. A suggestive lack of estrogen binding motifs was found for highly down-regulated genes in adult hepatocytes of obese F1 males (p = 0.074).
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http://dx.doi.org/10.1186/s12864-015-2164-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636810PMC
November 2015

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

Nat Genet 2015 Sep 27;47(9):1020-1029. Epub 2015 Jul 27.

Children's Cancer Research Institute, Vienna, Austria.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.
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http://dx.doi.org/10.1038/ng.3362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603357PMC
September 2015

Comparative analysis and modeling of the severity of steatohepatitis in DDC-treated mouse strains.

PLoS One 2014 27;9(10):e111006. Epub 2014 Oct 27.

Max Planck Institute for Molecular Genetics, Department Vertebrate Genomics, Berlin, Germany.

Background: Non-alcoholic fatty liver disease (NAFLD) has a broad spectrum of disease states ranging from mild steatosis characterized by an abnormal retention of lipids within liver cells to steatohepatitis (NASH) showing fat accumulation, inflammation, ballooning and degradation of hepatocytes, and fibrosis. Ultimately, steatohepatitis can result in liver cirrhosis and hepatocellular carcinoma.

Methodology And Results: In this study we have analyzed three different mouse strains, A/J, C57BL/6J, and PWD/PhJ, that show different degrees of steatohepatitis when administered a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) containing diet. RNA-Seq gene expression analysis, protein analysis and metabolic profiling were applied to identify differentially expressed genes/proteins and perturbed metabolite levels of mouse liver samples upon DDC-treatment. Pathway analysis revealed alteration of arachidonic acid (AA) and S-adenosylmethionine (SAMe) metabolism upon other pathways. To understand metabolic changes of arachidonic acid metabolism in the light of disease expression profiles a kinetic model of this pathway was developed and optimized according to metabolite levels. Subsequently, the model was used to study in silico effects of potential drug targets for steatohepatitis.

Conclusions: We identified AA/eicosanoid metabolism as highly perturbed in DDC-induced mice using a combination of an experimental and in silico approach. Our analysis of the AA/eicosanoid metabolic pathway suggests that 5-hydroxyeicosatetraenoic acid (5-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE) and prostaglandin D2 (PGD2) are perturbed in DDC mice. We further demonstrate that a dynamic model can be used for qualitative prediction of metabolic changes based on transcriptomics data in a disease-related context. Furthermore, SAMe metabolism was identified as being perturbed due to DDC treatment. Several genes as well as some metabolites of this module show differences between A/J and C57BL/6J on the one hand and PWD/PhJ on the other.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111006PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210132PMC
June 2015

Influence of RNA extraction methods and library selection schemes on RNA-seq data.

BMC Genomics 2014 Aug 11;15:675. Epub 2014 Aug 11.

Max Planck Institute for Molecular Genetics, Ihnestr, 63-73, Berlin 14195, Germany.

Background: Gene expression analysis by RNA sequencing is now widely used in a number of applications surveying the whole transcriptomes of cells and tissues. The recent introduction of ribosomal RNA depletion protocols, such as RiboZero, has extended the view of the polyadenylated transcriptome to the poly(A)- fraction of the RNA. However, substantial amounts of intronic transcriptional activity has been reported in RiboZero protocols, raising issues regarding their potential nuclear origin and the impact on the actual sequence depth in exonic regions.

Results: Using HEK293 human cells as source material, we assessed here the impact of the two commonly used RNA extraction methods and of the library construction protocols (rRNA depletion versus mRNA) on 1) the relative abundance of intronic reads and 2) on the estimation of gene expression values. We benchmarked the rRNA depletion-based sequencing with a specific analysis of the cytoplasmic and nuclear transcriptome fractions, suggesting that the large majority of the intronic reads correspond to unprocessed nuclear transcripts rather than to independent transcriptional units. We show that Qiagen or TRIzol extraction methods retain differentially nuclear RNA species, and that consequently, rRNA depletion-based RNA sequencing protocols are particularly sensitive to the extraction methods.

Conclusions: We could show that the combination of Trizol-based RNA extraction with rRNA depletion sequencing protocols led to the largest fraction of intronic reads, after the sequencing of the nuclear transcriptome. We discuss here the impact of the various strategies on gene expression and alternative splicing estimation measures. Further, we propose guidelines and a double selection strategy for minimizing the expression biases, without loss of information.
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http://dx.doi.org/10.1186/1471-2164-15-675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148917PMC
August 2014

Exome sequencing from nanogram amounts of starting DNA: comparing three approaches.

PLoS One 2014 3;9(7):e101154. Epub 2014 Jul 3.

Max-Planck Institute for Molecular Genetics, Berlin, Germany; AlacrisTheranostics GmbH, Berlin, Germany.

Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelectXT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input samples.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101154PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4081514PMC
October 2015

Parallel profiling of the transcriptome, cistrome, and epigenome in the cellular response to ionizing radiation.

Sci Signal 2014 May 13;7(325):rs3. Epub 2014 May 13.

The David and Inez Myers Laboratory for Cancer Research, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

The DNA damage response (DDR) is a vast signaling network that is robustly activated by DNA double-strand breaks, the critical lesion induced by ionizing radiation (IR). Although much of this response operates at the protein level, a critical component of the network sustains many DDR branches by modulating the cellular transcriptome. Using deep sequencing, we delineated three layers in the transcriptional response to IR in human breast cancer cells: changes in the expression of genes encoding proteins or long noncoding RNAs, alterations in genomic binding by key transcription factors, and dynamics of epigenetic markers of active promoters and enhancers. We identified protein-coding and previously unidentified noncoding genes that were responsive to IR, and demonstrated that IR-induced transcriptional dynamics was mediated largely by the transcription factors p53 and nuclear factor κB (NF-κB) and was primarily dependent on the kinase ataxia-telangiectasia mutated (ATM). The resultant data set provides a rich resource for understanding a basic, underlying component of a critical cellular stress response.
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http://dx.doi.org/10.1126/scisignal.2005032DOI Listing
May 2014

Transcriptome and genome sequencing uncovers functional variation in humans.

Nature 2013 Sep 15;501(7468):506-11. Epub 2013 Sep 15.

Department of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, Switzerland.

Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project--the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.
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http://dx.doi.org/10.1038/nature12531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918453PMC
September 2013

Janus--a comprehensive tool investigating the two faces of transcription.

Bioinformatics 2013 Jul 24;29(13):1600-6. Epub 2013 Apr 24.

Institute for Clinical Molecular Biology, Christian-Albrechts-University, 24105 Kiel, Germany.

Motivation: Protocols to generate strand-specific transcriptomes with next-generation sequencing platforms have been used by the scientific community roughly since 2008. Strand-specific reads allow for detection of antisense events and a higher resolution of expression profiles enabling extension of current transcript annotations. However, applications making use of this strandedness information are still scarce.

Results: Here we present a tool (Janus), which focuses on the identification of transcriptional active regions in antisense orientation to known and novel transcribed elements of the genome. Janus can compare the antisense events of multiple samples and assigns scores to identify mutual expression of either transcript in a sense/antisense pair, which could hint to regulatory mechanisms. Janus is able to make use of single-nucleotide variant (SNV) and methylation data, if available, and reports the sense to antisense ratio of regions in the vicinity of the identified genetic and epigenetic variation. Janus interrogates positions of heterozygous SNVs to identify strand-specific allelic imbalance.

Availability: Janus is written in C/C++ and freely available at http://www.ikmb.uni-kiel.de/janus/janus.html under terms of GNU General Public License, for both, Linux and Windows 64×. Although the binaries will work without additional downloads, the software depends on bamtools (https://github.com/pezmaster31/bamtools) for compilation. A detailed tutorial section is included in the first section of the supplemental material and included as brief readme.txt in the tutorial archive.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btt185DOI Listing
July 2013

Integrative genomic analyses reveal an androgen-driven somatic alteration landscape in early-onset prostate cancer.

Cancer Cell 2013 Feb;23(2):159-70

Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany.

Early-onset prostate cancer (EO-PCA) represents the earliest clinical manifestation of prostate cancer. To compare the genomic alteration landscapes of EO-PCA with "classical" (elderly-onset) PCA, we performed deep sequencing-based genomics analyses in 11 tumors diagnosed at young age, and pursued comparative assessments with seven elderly-onset PCA genomes. Remarkable age-related differences in structural rearrangement (SR) formation became evident, suggesting distinct disease pathomechanisms. Whereas EO-PCAs harbored a prevalence of balanced SRs, with a specific abundance of androgen-regulated ETS gene fusions including TMPRSS2:ERG, elderly-onset PCAs displayed primarily non-androgen-associated SRs. Data from a validation cohort of > 10,000 patients showed age-dependent androgen receptor levels and a prevalence of SRs affecting androgen-regulated genes, further substantiating the activity of a characteristic "androgen-type" pathomechanism in EO-PCA.
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http://dx.doi.org/10.1016/j.ccr.2013.01.002DOI Listing
February 2013

Dissecting the genomic complexity underlying medulloblastoma.

Nature 2012 Aug;488(7409):100-5

Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany.

Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
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http://dx.doi.org/10.1038/nature11284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3662966PMC
August 2012

A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods.

Biochem Biophys Res Commun 2012 Jun 15;422(4):643-6. Epub 2012 May 15.

Max Planck Institute for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany.

Preserving the original RNA orientation information in RNA-Sequencing (RNA-Seq) experiment is essential to the analysis and understanding of the complexity of mammalian transcriptomes. We describe herein a simple, robust, and time-effective protocol for generating strand-specific RNA-seq libraries suited for the Illumina sequencing platform. We modified the Illumina TruSeq RNA sample preparation by implementing the strand specificity feature using the dUTP method. This protocol uses low amounts of starting material and allows a fast processing within two days. It can be easily implemented and requires only few additional reagents to the original Illumina kit.
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http://dx.doi.org/10.1016/j.bbrc.2012.05.043DOI Listing
June 2012

Transcriptome analysis by strand-specific sequencing of complementary DNA.

Nucleic Acids Res 2009 Oct 20;37(18):e123. Epub 2009 Jul 20.

Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Ihnestr. 73, 14195 Berlin, Germany.

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.
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http://dx.doi.org/10.1093/nar/gkp596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764448PMC
October 2009