Publications by authors named "Frank Lyko"

126 Publications

DAZAP2 acts as specifier of the p53 response to DNA damage.

Nucleic Acids Res 2021 Feb 16. Epub 2021 Feb 16.

Institute of Toxicology, University Medical Center Mainz, Johannes Gutenberg University, Mainz, Germany.

The DNA damage-responsive tumor suppressors p53 and HIPK2 are well established regulators of cell fate decision-making and regulate the cellular sensitivity to DNA-damaging drugs. Here, we identify Deleted in Azoospermia-associated protein 2 (DAZAP2), a small adaptor protein, as a novel regulator of HIPK2 and specifier of the DNA damage-induced p53 response. Knock-down or genetic deletion of DAZAP2 strongly potentiates cancer cell chemosensitivity both in cells and in vivo using a mouse tumour xenograft model. In unstressed cells, DAZAP2 stimulates HIPK2 polyubiquitination and degradation through interplay with the ubiquitin ligase SIAH1. Upon DNA damage, HIPK2 site-specifically phosphorylates DAZAP2, which terminates its HIPK2-degrading function and triggers its re-localization to the cell nucleus. Interestingly, nuclear DAZAP2 interacts with p53 and specifies target gene expression through modulating a defined subset of p53 target genes. Furthermore, our results suggest that DAZAP2 co-occupies p53 response elements to specify target gene expression. Collectively, our findings propose DAZAP2 as novel regulator of the DNA damage-induced p53 response that controls cancer cell chemosensitivity.
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http://dx.doi.org/10.1093/nar/gkab084DOI Listing
February 2021

Genome analysis of the monoclonal marbled crayfish reveals genetic separation over a short evolutionary timescale.

Commun Biol 2021 Jan 18;4(1):74. Epub 2021 Jan 18.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.

The marbled crayfish (Procambarus virginalis) represents a very recently evolved parthenogenetic freshwater crayfish species that has invaded diverse habitats in Europe and in Madagascar. However, population genetic analyses have been hindered by the homogeneous genetic structure of the population and the lack of suitable tools for data analysis. We have used whole-genome sequencing to characterize reference specimens from various known wild populations. In parallel, we established a whole-genome sequencing data analysis pipeline for the population genetic analysis of nearly monoclonal genomes. Our results provide evidence for systematic genetic differences between geographically separated populations and illustrate the emerging differentiation of the marbled crayfish genome. We also used mark-recapture population size estimation in combination with genetic data to model the growth pattern of marbled crayfish populations. Our findings uncover evolutionary dynamics in the marbled crayfish genome over a very short evolutionary timescale and identify the rapid growth of marbled crayfish populations as an important factor for ecological monitoring.
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http://dx.doi.org/10.1038/s42003-020-01588-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814009PMC
January 2021

A chicken DNA methylation clock for the prediction of broiler health.

Commun Biol 2021 Jan 18;4(1):76. Epub 2021 Jan 18.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.

The domestic chicken (Gallus gallus domesticus) is the globally most important source of commercially produced meat. While genetic approaches have played an important role in the development of chicken stocks, little is known about chicken epigenetics. We have systematically analyzed the chicken DNA methylation machinery and DNA methylation landscape. While overall DNA methylation distribution was similar to mammals, sperm DNA appeared hypomethylated, which correlates with the absence of the DNMT3L cofactor in the chicken genome. Additional analysis revealed the presence of low-methylated regions, which are conserved gene regulatory elements that show tissue-specific methylation patterns. We also used whole-genome bisulfite sequencing to generate 56 single-base resolution methylomes from various tissues and developmental time points to establish an LMR-based DNA methylation clock for broiler chicken age prediction. This clock was used to demonstrate epigenetic age acceleration in animals with experimentally induced inflammation. Our study provides detailed insights into the chicken methylome and suggests a novel application of the DNA methylation clock as a marker for livestock health.
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http://dx.doi.org/10.1038/s42003-020-01608-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814119PMC
January 2021

Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans.

EMBO J 2020 Dec 7:e105496. Epub 2020 Dec 7.

Gurdon Institute, University of Cambridge, Cambridge, UK.

Methylation of carbon-5 of cytosines (m C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m C tRNA wobble methylation in the adaptation to higher temperatures.
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http://dx.doi.org/10.15252/embj.2020105496DOI Listing
December 2020

Epigenetic deregulation of lamina-associated domains in Hutchinson-Gilford progeria syndrome.

Genome Med 2020 05 25;12(1):46. Epub 2020 May 25.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany.

Background: Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid disease characterized by the early onset of age-related phenotypes including arthritis, loss of body fat and hair, and atherosclerosis. Cells from affected individuals express a mutant version of the nuclear envelope protein lamin A (termed progerin) and have previously been shown to exhibit prominent histone modification changes.

Methods: Here, we analyze the possibility that epigenetic deregulation of lamina-associated domains (LADs) is involved in the molecular pathology of HGPS. To do so, we studied chromatin accessibility (Assay for Transposase-accessible Chromatin (ATAC)-see/-seq), DNA methylation profiles (Infinium MethylationEPIC BeadChips), and transcriptomes (RNA-seq) of nine primary HGPS fibroblast cell lines and six additional controls, two parental and four age-matched healthy fibroblast cell lines.

Results: Our ATAC-see/-seq data demonstrate that primary dermal fibroblasts from HGPS patients exhibit chromatin accessibility changes that are enriched in LADs. Infinium MethylationEPIC BeadChip profiling further reveals that DNA methylation alterations observed in HGPS fibroblasts are similarly enriched in LADs and different from those occurring during healthy aging and Werner syndrome (WS), another premature aging disease. Moreover, HGPS patients can be stratified into two different subgroups according to their DNA methylation profiles. Finally, we show that the epigenetic deregulation of LADs is associated with HGPS-specific gene expression changes.

Conclusions: Taken together, our results strongly implicate epigenetic deregulation of LADs as an important and previously unrecognized feature of HGPS, which contributes to disease-specific gene expression. Therefore, they not only add a new layer to the study of epigenetic changes in the progeroid syndrome, but also advance our understanding of the disease's pathology at the cellular level.
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http://dx.doi.org/10.1186/s13073-020-00749-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249329PMC
May 2020

Rapid Epigenetic Adaptation in Animals and Its Role in Invasiveness.

Integr Comp Biol 2020 08;60(2):267-274

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.

Invasive species represent a serious ecological threat for many ecosystems worldwide and provide a unique opportunity to investigate rapid adaptation and evolution. Genetic variation allows populations of organisms to be both robust and adaptable to different environmental conditions over evolutionary timeframes. In contrast, invasive animals can rapidly adapt to new environments, with minimal genetic diversity. Thus, the extent to which environmental effects can trigger epigenetic responses is particularly interesting for understanding the role of epigenetics in rapid adaptation. In this review, we provide a brief overview of the different epigenetic mechanisms that control gene expression, and emphasize the importance of epigenetics for environmental adaptation. We also discuss recent publications that provide important examples for the role of epigenetic mechanisms in environmental adaptation. Furthermore, we present an overview of the current knowledge about epigenetic modulation as an adaptive strategy for invasive species. A particularly interesting example is provided by the marbled crayfish, a novel, monoclonal freshwater crayfish species that has colonized diverse habitats within a few years. Finally, we address important limitations of current approaches and highlight the potential importance of less well-known mechanisms for non-genetic organismal adaptation.
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http://dx.doi.org/10.1093/icb/icaa023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526798PMC
August 2020

Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming.

Commun Biol 2020 04 23;3(1):188. Epub 2020 Apr 23.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, 69120, Heidelberg, Germany.

Fibroblasts are an essential cell population for human skin architecture and function. While fibroblast heterogeneity is well established, this phenomenon has not been analyzed systematically yet. We have used single-cell RNA sequencing to analyze the transcriptomes of more than 5,000 fibroblasts from a sun-protected area in healthy human donors. Our results define four main subpopulations that can be spatially localized and show differential secretory, mesenchymal and pro-inflammatory functional annotations. Importantly, we found that this fibroblast 'priming' becomes reduced with age. We also show that aging causes a substantial reduction in the predicted interactions between dermal fibroblasts and other skin cells, including undifferentiated keratinocytes at the dermal-epidermal junction. Our work thus provides evidence for a functional specialization of human dermal fibroblasts and identifies the partial loss of cellular identity as an important age-related change in the human dermis. These findings have important implications for understanding human skin aging and its associated phenotypes.
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http://dx.doi.org/10.1038/s42003-020-0922-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181753PMC
April 2020

Perceived socio-economic impacts of the marbled crayfish invasion in Madagascar.

PLoS One 2020 15;15(4):e0231773. Epub 2020 Apr 15.

Division of Epigenetics, DKFZ-ZMBH Alliance German Cancer Research Center (DKFZ), Heidelberg, Germany.

The negative environmental and economic impacts of many invasive species are well known. However, given the increased homogenization of global biota, and the difficulty of eradicating species once established, a balanced approach to considering the impacts of invasive species is needed. The marbled crayfish (Procambarus virginalis) is a parthenogenetic freshwater crayfish that was first observed in Madagascar around 2005 and has spread rapidly. We present the results of a socio-economic survey (n = 385) in three regions of Madagascar that vary in terms of when the marbled crayfish first arrived. Respondents generally considered marbled crayfish to have a negative impact on rice agriculture and fishing, however the animals were seen as making a positive contribution to household economy and food security. Regression modeling showed that respondents in regions with longer experience of marbled crayfish have more positive perceptions. Unsurprisingly, considering the perception that crayfish negatively impact rice agriculture, those not involved in crayfish harvesting and trading had more negative views towards the crayfish than those involved in crayfish-related activities. Food preference ranking and market surveys revealed the acceptance of marbled crayfish as a cheap source of animal protein; a clear positive in a country with widespread malnutrition. While data on biodiversity impacts of the marbled crayfish invasion in Madagascar are still completely lacking, this study provides insight into the socio-economic impacts of the dramatic spread of this unique invasive species. "Biby kely tsy fantam-piaviana, mahavelona fianakaviana" (a small animal coming from who knows where which supports the needs of the family). Government worker Analamanga, Madagascar.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231773PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159205PMC
July 2020

The microbiota programs DNA methylation to control intestinal homeostasis and inflammation.

Nat Microbiol 2020 04 3;5(4):610-619. Epub 2020 Feb 3.

Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew University Medical School, Jerusalem, Israel.

Although much research has been done on the diversity of the gut microbiome, little is known about how it influences intestinal homeostasis under normal and pathogenic conditions. Epigenetic mechanisms have recently been suggested to operate at the interface between the microbiota and the intestinal epithelium. We performed whole-genome bisulfite sequencing on conventionally raised and germ-free mice, and discovered that exposure to commensal microbiota induced localized DNA methylation changes at regulatory elements, which are TET2/3-dependent. This culminated in the activation of a set of 'early sentinel' response genes to maintain intestinal homeostasis. Furthermore, we demonstrated that exposure to the microbiota in dextran sodium sulfate-induced acute inflammation results in profound DNA methylation and chromatin accessibility changes at regulatory elements, leading to alterations in gene expression programs enriched in colitis- and colon-cancer-associated functions. Finally, by employing genetic interventions, we show that microbiota-induced epigenetic programming is necessary for proper intestinal homeostasis in vivo.
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http://dx.doi.org/10.1038/s41564-019-0659-3DOI Listing
April 2020

Limited antibody specificity compromises epitranscriptomic analyses.

Nat Commun 2019 12 11;10(1):5669. Epub 2019 Dec 11.

IMoPA, UMR7365 CNRS-University of Lorraine, Biopôle, 9 Avenue de la Forêt de Haye, 54505, Vandœuvre-lès-Nancy, France.

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http://dx.doi.org/10.1038/s41467-019-13684-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906430PMC
December 2019

The importance of non-histone protein methylation in cancer therapy.

Nat Rev Mol Cell Biol 2019 10;20(10):569-570

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany.

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http://dx.doi.org/10.1038/s41580-019-0147-xDOI Listing
October 2019

DNA (de)methylation in embryonic stem cells controls CTCF-dependent chromatin boundaries.

Genome Res 2019 05 4;29(5):750-761. Epub 2019 Apr 4.

School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.

Coordinated changes of DNA (de)methylation, nucleosome positioning, and chromatin binding of the architectural protein CTCF play an important role for establishing cell-type-specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes, we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the and dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC), or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation, and gene expression in DKO ESCs and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, whereas unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild-type (WT) cells but not from 5fC-enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, for example, at promoters, boundaries of chromatin loops, and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to down-regulation of neighboring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions, and DNA sequence that determines differential CTCF binding and regulates gene expression.
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http://dx.doi.org/10.1101/gr.239707.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499307PMC
May 2019

Ecological plasticity and commercial impact of invasive marbled crayfish populations in Madagascar.

BMC Ecol 2019 02 6;19(1). Epub 2019 Feb 6.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.

Background: The marbled crayfish (Procambarus virginalis) is a monoclonal, parthenogenetically reproducing freshwater crayfish species that has formed multiple stable populations worldwide. Madagascar hosts a particularly large and rapidly expanding colony of marbled crayfish in a unique environment characterized by a very high degree of ecological diversity.

Results: Here we provide a detailed characterization of five marbled crayfish populations in Madagascar and their habitats. Our data show that the animals can tolerate a wide range of ecological parameters, consistent with their invasive potential. While we detected marbled crayfish in sympatry with endemic crayfish species, we found no evidence for the transmission of the crayfish plague pathogen, a potentially devastating oomycete. Furthermore, our results also suggest that marbled crayfish are active predators of the freshwater snails that function as intermediate hosts for human schistosomiasis. Finally, we document fishing, farming and market sales of marbled crayfish in Madagascar.

Conclusions: Our results provide a paradigm for the complex network of factors that promotes the invasive spread of marbled crayfish. The commercial value of the animals is likely to result in further anthropogenic distribution.
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http://dx.doi.org/10.1186/s12898-019-0224-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366054PMC
February 2019

Queuine links translational control in eukaryotes to a micronutrient from bacteria.

Nucleic Acids Res 2019 04;47(7):3711-3727

Institut für Biologie, Molekulare Zellbiologie, Humboldt-Universität zu Berlin, Berlin, Germany.

In eukaryotes, the wobble position of tRNA with a GUN anticodon is modified to the 7-deaza-guanosine derivative queuosine (Q34), but the original source of Q is bacterial, since Q is synthesized by eubacteria and salvaged by eukaryotes for incorporation into tRNA. Q34 modification stimulates Dnmt2/Pmt1-dependent C38 methylation (m5C38) in the tRNAAsp anticodon loop in Schizosaccharomyces pombe. Here, we show by ribosome profiling in S. pombe that Q modification enhances the translational speed of the C-ending codons for aspartate (GAC) and histidine (CAC) and reduces that of U-ending codons for asparagine (AAU) and tyrosine (UAU), thus equilibrating the genome-wide translation of synonymous Q codons. Furthermore, Q prevents translation errors by suppressing second-position misreading of the glycine codon GGC, but not of wobble misreading. The absence of Q causes reduced translation of mRNAs involved in mitochondrial functions, and accordingly, lack of Q modification causes a mitochondrial defect in S. pombe. We also show that Q-dependent stimulation of Dnmt2 is conserved in mice. Our findings reveal a direct mechanism for the regulation of translational speed and fidelity in eukaryotes by a nutrient originating from bacteria.
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http://dx.doi.org/10.1093/nar/gkz063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468285PMC
April 2019

Division of labour: tRNA methylation by the NSun2 tRNA methyltransferases Trm4a and Trm4b in fission yeast.

RNA Biol 2019 03 1;16(3):249-256. Epub 2019 Feb 1.

a Institut für Biologie, Molekulare Zellbiologie , Humboldt-Universität zu Berlin , Berlin , Germany.

Enzymes of the cytosine-5 RNA methyltransferase Trm4/NSun2 family methylate tRNAs at C48 and C49 in multiple tRNAs, as well as C34 and C40 in selected tRNAs. In contrast to most other organisms, fission yeast Schizosaccharomyces pombe carries two Trm4/NSun2 homologs, Trm4a (SPAC17D4.04) and Trm4b (SPAC23C4.17). Here, we have employed tRNA methylome analysis to determine the dependence of cytosine-5 methylation (mC) tRNA methylation in vivo on the two enzymes. Remarkably, Trm4a is responsible for all C48 methylation, which lies in the tRNA variable loop, as well as for C34 in tRNA and tRNA, which are at the anticodon wobble position. Conversely, Trm4b methylates C49 and C50, which both lie in the TΨC-stem. Thus, S. pombe show an unusual separation of activities of the NSun2/Trm4 enzymes that are united in a single enzyme in other eukaryotes like humans, mice and Saccharomyces cerevisiae. Furthermore, in vitro activity assays showed that Trm4a displays intron-dependent methylation of C34, whereas Trm4b activity is independent of the intron. The absence of Trm4a, but not Trm4b, causes a mild resistance of S. pombe to calcium chloride.
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http://dx.doi.org/10.1080/15476286.2019.1568819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380293PMC
March 2019

Whole-Genome Bisulfite Sequencing for the Methylation Analysis of Insect Genomes.

Methods Mol Biol 2019 ;1858:141-156

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany.

DNA methylation is a conserved epigenetic modification of animal genomes, but genome methylation patterns appear surprisingly diverse in insects. Whole-genome bisulfite sequencing (WGBS) represents a sensitive and robust method for the characterization of genome-wide methylation patterns at single-base resolution. Here, we describe a step-by-step protocol for the generation and analysis of WGBS datasets using standard Illumina sequencing platforms. In comparison to whole-genome sequencing, WGBS has additional caveats that require particular attention and are highlighted in this chapter.
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http://dx.doi.org/10.1007/978-1-4939-8775-7_11DOI Listing
July 2019

Dnmt1 has an essential function despite the absence of CpG DNA methylation in the red flour beetle Tribolium castaneum.

Sci Rep 2018 11 7;8(1):16462. Epub 2018 Nov 7.

Institute for Evolution and Biodiversity, University of Münster, Münster, Germany.

Epigenetic mechanisms, such as CpG DNA methylation enable phenotypic plasticity and rapid adaptation to changing environments. CpG DNA methylation is established by DNA methyltransferases (DNMTs), which are well conserved across vertebrates and invertebrates. There are insects with functional DNA methylation despite lacking a complete set of Dnmts. But at least one of the enzymes, DNMT1, appears to be required to maintain an active DNA methylation system. The red flour beetle, Tribolium castaneum, lacks Dnmt3 but possesses Dnmt1 and it has been controversial whether it has a functional DNA methylation system. Using whole genome bisulfite sequencing, we did not find any defined patterns of CpG DNA methylation in embryos. Nevertheless, we found Dnmt1 expressed throughout the entire life cycle of the beetle, with mRNA transcripts significantly more abundant in eggs and ovaries. A maternal knockdown of Dnmt1 caused a developmental arrest in offspring embryos. We show that Dnmt1 plays an essential role in T. castaneum embryos and that its downregulation leads to an early developmental arrest. This function appears to be unrelated to DNA methylation, since we did not find any evidence for this modification. This strongly suggests an alternative role of this protein.
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http://dx.doi.org/10.1038/s41598-018-34701-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220294PMC
November 2018

BisAMP: A web-based pipeline for targeted RNA cytosine-5 methylation analysis.

Methods 2019 03 24;156:121-127. Epub 2018 Oct 24.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Electronic address:

RNA cytosine-5 methylation (mC) has emerged as a key epitranscriptomic mark, which fulfills multiple roles in structural modulation, stress signaling and the regulation of protein translation. Bisulfite sequencing is currently the most accurate and reliable method to detect mC marks at nucleotide resolution. Targeted bisulfite sequencing allows mC detection at single base resolution, by combining the use of tailored primers with bisulfite treatment. A number of computational tools currently exist to analyse mC marks in DNA bisulfite sequencing. However, these methods are not directly applicable to the analysis of RNA mC marks, because DNA analysis focuses on CpG methylation, and because artifactual unconversion and misamplification in RNA can obscure actual methylation signals. We describe a pipeline designed specifically for RNA cytosine-5 methylation analysis in targeted bisulfite sequencing experiments. The pipeline is directly applicable to Illumina MiSeq (or equivalent) sequencing datasets using a web interface (https://bisamp.dkfz.de), and is defined by optimized mapping parameters and the application of tailored filters for the removal of artifacts. We provide examples for the application of this pipeline in the unambiguous detection of mC marks in tRNAs from mouse embryonic stem cells and neuron-differentiated stem cells as well as in 28S rRNA from human fibroblasts. Finally, we also discuss the adaptability of BisAMP to the analysis of DNA methylation. Our pipeline provides an accurate, fast and user-friendly framework for the analysis of cytosine-5 methylation in amplicons from bisulfite-treated RNA.
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http://dx.doi.org/10.1016/j.ymeth.2018.10.013DOI Listing
March 2019

The methylome of the marbled crayfish links gene body methylation to stable expression of poorly accessible genes.

Epigenetics Chromatin 2018 10 4;11(1):57. Epub 2018 Oct 4.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.

Background: The parthenogenetic marbled crayfish (Procambarus virginalis) is a novel species that has rapidly invaded and colonized various different habitats. Adaptation to different environments appears to be independent of the selection of genetic variants, but epigenetic programming of the marbled crayfish genome remains to be understood.

Results: Here, we provide a comprehensive analysis of DNA methylation in marbled crayfish. Whole-genome bisulfite sequencing of multiple replicates and different tissues revealed a methylation pattern that is characterized by gene body methylation of housekeeping genes. Interestingly, this pattern was largely tissue invariant, suggesting a function that is unrelated to cell fate specification. Indeed, integrative analysis of DNA methylation, chromatin accessibility and mRNA expression patterns revealed that gene body methylation correlated with limited chromatin accessibility and stable gene expression, while low-methylated genes often resided in chromatin with higher accessibility and showed increased expression variation. Interestingly, marbled crayfish also showed reduced gene body methylation and higher gene expression variability when compared with their noninvasive mother species, Procambarus fallax.

Conclusions: Our results provide novel insights into invertebrate gene body methylation and its potential role in adaptive gene regulation.
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http://dx.doi.org/10.1186/s13072-018-0229-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172769PMC
October 2018

Queuosine-modified tRNAs confer nutritional control of protein translation.

EMBO J 2018 09 9;37(18). Epub 2018 Aug 9.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany.

Global protein translation as well as translation at the codon level can be regulated by tRNA modifications. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the diet and gut microbiota. We show here that nutritionally determined Q-tRNA levels promote Dnmt2-mediated methylation of tRNA Asp and control translational speed of Q-decoded codons as well as at near-cognate codons. Deregulation of translation upon queuine depletion results in unfolded proteins that trigger endoplasmic reticulum stress and activation of the unfolded protein response, both in cultured human cell lines and in germ-free mice fed with a queuosine-deficient diet. Taken together, our findings comprehensively resolve the role of this anticodon tRNA modification in the context of native protein translation and describe a novel mechanism that links nutritionally determined modification levels to effective polypeptide synthesis and cellular homeostasis.
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http://dx.doi.org/10.15252/embj.201899777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138434PMC
September 2018

Cell-of-Origin DNA Methylation Signatures Are Maintained during Colorectal Carcinogenesis.

Cell Rep 2018 06;23(11):3407-3418

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, 69120 Heidelberg, Germany. Electronic address:

Colorectal adenomas are precursor lesions of colorectal cancers and represent clonal amplifications of single cells from colonic crypts. DNA methylation patterns specify cell-type identity during cellular differentiation and, therefore, provide opportunities for the molecular analysis of tumors. We have now analyzed DNA methylation patterns in colorectal adenomas and identified three biologically defined subclasses that describe different intestinal crypt differentiation stages. Importantly, colorectal carcinomas could be classified into the same methylation subtypes, reflecting their shared cell types of origin with adenomas. Further data analysis also revealed significantly reduced overall survival for one of the subtypes. Our results provide a concept for understanding the methylation patterns observed in colorectal cancer and provide opportunities for tumor subclassification and patient stratification.
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http://dx.doi.org/10.1016/j.celrep.2018.05.045DOI Listing
June 2018

Transforming growth factor β1-mediated functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia.

Haematologica 2018 09 17;103(9):1462-1471. Epub 2018 May 17.

Department of Hematology, Oncology and Clinical Immunology, University of Duesseldorf, Medical Faculty, Germany

Mesenchymal stromal cells are involved in the pathogenesis of myelodysplastic syndromes and acute myeloid leukemia, but the underlying mechanisms are incompletely understood. To further characterize the pathological phenotype we performed RNA sequencing of mesenchymal stromal cells from patients with myelodysplastic syndromes and acute myeloid leukemia and found a specific molecular signature of genes commonly deregulated in these disorders. Pathway analysis showed a strong enrichment of genes related to osteogenesis, senescence, inflammation and inhibitory cytokines, thereby reflecting the structural and functional deficits of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia on a molecular level. Further analysis identified transforming growth factor β1 as the most probable extrinsic trigger factor for this altered gene expression. Following exposure to transforming growth factor β1, healthy mesenchymal stromal cells developed functional deficits and adopted a phenotype reminiscent of that observed in patient-derived stromal cells. These suppressive effects of transforming growth factor β1 on stromal cell functionality were abrogated by SD-208, an established inhibitor of transforming growth factor β receptor signaling. Blockade of transforming growth factor β signaling by SD-208 also restored the osteogenic differentiation capacity of patient-derived stromal cells, thus confirming the role of transforming growth factor β1 in the bone marrow microenvironment of patients with myelodysplastic syndromes and acute myeloid leukemia. Our findings establish transforming growth factor β1 as a relevant trigger causing functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia and identify SD-208 as a candidate to revert these effects.
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http://dx.doi.org/10.3324/haematol.2017.186734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119130PMC
September 2018

Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs.

Nat Cell Biol 2018 05 25;20(5):535-540. Epub 2018 Apr 25.

Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA.

The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA . Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress and metabolic disorders. How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat-diet-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (mC, mG) in sperm 30-40 nt RNA fractions that are induced by a high-fat diet. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs and rRNA-derived small RNAs, which might be essential in composing a sperm RNA 'coding signature' that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated mC contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.
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http://dx.doi.org/10.1038/s41556-018-0087-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5926820PMC
May 2018

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

RNA Biol 2018 9;15(6):829-831. Epub 2018 May 9.

ba University of Nottingham School of Biosceinces , Nottingham , United Kingdom.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.
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http://dx.doi.org/10.1080/15476286.2018.1460996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152430PMC
November 2018

Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats.

Cell Rep 2018 02;22(7):1861-1874

Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria. Electronic address:

The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5) RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.
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http://dx.doi.org/10.1016/j.celrep.2018.01.061DOI Listing
February 2018

Methylation profiling identifies two subclasses of squamous cell carcinoma related to distinct cells of origin.

Nat Commun 2018 02 8;9(1):577. Epub 2018 Feb 8.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, 69120, Heidelberg, Germany.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and usually progresses from a UV-induced precancerous lesion termed actinic keratosis (AK). Despite various efforts to characterize these lesions molecularly, the etiology of AK and its progression to cSCC remain partially understood. Here, we use Infinium MethylationEPIC BeadChips to interrogate the DNA methylation status in healthy, AK and cSCC epidermis samples. Importantly, we show that AK methylation patterns already display classical features of cancer methylomes and are highly similar to cSCC profiles. Further analysis identifies typical features of stem cell methylomes, such as reduced DNA methylation age, non-CpG methylation, and stem cell-related keratin and enhancer methylation patterns. Interestingly, this signature is detected only in half of the samples, while the other half shows patterns more closely related to healthy epidermis. These findings suggest the existence of two subclasses of AK and cSCC emerging from distinct keratinocyte differentiation stages.
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http://dx.doi.org/10.1038/s41467-018-03025-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805678PMC
February 2018

Clonal genome evolution and rapid invasive spread of the marbled crayfish.

Nat Ecol Evol 2018 03 5;2(3):567-573. Epub 2018 Feb 5.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.

The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarus fallax, and also established a triploid AA'B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.
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http://dx.doi.org/10.1038/s41559-018-0467-9DOI Listing
March 2018

The marbled crayfish (Decapoda: Cambaridae) represents an independent new species.

Authors:
Frank Lyko

Zootaxa 2017 Dec 12;4363(4):544-552. Epub 2017 Dec 12.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, 69120 Heidelberg, Germany.

Marbled crayfish are a globally expanding population of parthenogenetically reproducing freshwater decapods. They are closely related to the sexually reproducing slough crayfish, Procambarus fallax, which is native to the southeastern United States. Previous studies have shown that marbled crayfish are morphologically very similar to P. fallax. However, different fitness traits, reproductive incompatibility and substantial genetic differences suggest that the marbled crayfish should be considered an independent species. This article provides its formal description and scientific name, Procambarus virginalis sp. nov.
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http://dx.doi.org/10.11646/zootaxa.4363.4.6DOI Listing
December 2017

The DNA methyltransferase family: a versatile toolkit for epigenetic regulation.

Authors:
Frank Lyko

Nat Rev Genet 2018 02 16;19(2):81-92. Epub 2017 Oct 16.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.

The DNA methyltransferase (DNMT) family comprises a conserved set of DNA-modifying enzymes that have a central role in epigenetic gene regulation. Recent studies have shown that the functions of the canonical DNMT enzymes - DNMT1, DNMT3A and DNMT3B - go beyond their traditional roles of establishing and maintaining DNA methylation patterns. This Review analyses how molecular interactions and changes in gene copy numbers modulate the activity of DNMTs in diverse gene regulatory functions, including transcriptional silencing, transcriptional activation and post-transcriptional regulation by DNMT2-dependent tRNA methylation. This mechanistic diversity enables the DNMT family to function as a versatile toolkit for epigenetic regulation.
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http://dx.doi.org/10.1038/nrg.2017.80DOI Listing
February 2018

-associated DNA hypermethylation in acute myeloid leukemia reflects differentiation blockage rather than inhibition of TET-mediated demethylation.

Cell Stress 2017 Sep 20;1(1):55-67. Epub 2017 Sep 20.

Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center; 69120 Heidelberg, Germany.

Isocitrate dehydrogenases 1 and 2 () are recurrently mutated in acute myeloid leukemia (AML), but their mechanistic role in leukemogenesis is poorly understood. The inhibition of TET enzymes by D-2-hydroxyglutarate (D-2-HG), which is produced by mutant (), has been suggested to promote epigenetic deregulation during tumorigenesis. In addition, also induces a differentiation block in various cell culture and mouse models. Here we analyze the genomic methylation patterns of AML patients with using Infinium 450K data from a large AML cohort and found that is associated with pronounced DNA hypermethylation at tens of thousands of CpGs. Interestingly, however, myeloid leukemia cells overexpressing , cells that were cultured in the presence of D-2-HG or TET2 mutant AML patients did not show similar methylation changes. In further analyses, we also characterized the methylation landscapes of myeloid progenitor cells and analyzed their relationship to -associated hypermethylation. Our findings identify the differentiation state of myeloid cells, rather than inhibition of TET-mediated DNA demethylation, as a major factor of -associated hypermethylation in AML. Furthermore, our results are also important for understanding the mode of action of currently developed inhibitors.
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http://dx.doi.org/10.15698/cst2017.10.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551656PMC
September 2017