Florian Erhard

Florian Erhard
Ludwig-Maximilians-University of Munich | LMU · Institut für Informatik

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84
Publications
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Publications

Publications (84)
Preprint
Activation of intronic polyadenylation signals results in premature cleavage and polyadenylation (PCPA). The majority of mammalian miRNAs are also located within intronic regions of protein-coding genes and are transcriptionally co-expressed with their host genes. Here we show that U1-dependent PCPA by telescripting dysregulates miRNA biogenesis. W...
Article
Full-text available
Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic–latent switch and viral non-coding RNAs3,4. Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cell...
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Full-text available
Viruses play a key role in explaining the pathogenesis of various autoimmune disorders, whose underlying principle is defined by the activation of autoreactive T-cells. In many cases, T-cells escape self-tolerance due to the failure in encountering certain MHC-I self-peptide complexes at substantial levels, whose peptides remain invisible from the...
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Our understanding of the composition and functions of splenic stromal cells remains incomplete. Here, based on analysis of over 20,000 single cell transcriptomes of splenic fibroblasts, we characterized the phenotypic and functional heterogeneity of these cells in healthy state and during virus infection. We describe eleven transcriptionally distin...
Article
Background Despite recent advances in exome and RNA sequencing to identify tumor-rejection antigens including neoantigens, the existing techniques fail to identify the vast majority of antigens targeted by tumor-reactive cells. A growing number of studies suggest that HLA-I peptides derived from non-canonical (nonC) open reading frames or derived f...
Article
COVID-19-induced ‘acute respiratory distress syndrome’ (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyzed pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single cell genomi...
Preprint
Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary an...
Preprint
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Human cytomegalovirus (HCMV) is a widespread obligatory human pathogen causing life-threatening disease in immunocompromised hosts. Myeloid cells such as monocyte-derived dendritic cells (moDCs) are targets of HCMV. Here, we performed single-cell RNA sequencing, which revealed infection of most moDCs upon in vitro HCMV exposure, whereas only a frac...
Preprint
Full-text available
Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a novel c...
Article
Full-text available
Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegal...
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Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Skłodowska-Curie Initiative International Traini...
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B cell maturation antigen (BCMA) is a target for various immunotherapies and a biomarker for tumor load in multiple myeloma (MM). We report a case of irreversible BCMA loss in a patient with MM who was enrolled in the KarMMa trial (NCT03361748) and progressed after anti-BCMA CAR T cell therapy. We identified selection of a clone with homozygous del...
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Background The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign ti...
Article
Many experimental approaches have been developed to identify transcription start sites (TSS) from genomic scale data. However, experiment specific biases lead to large numbers of false positive calls. Here, we present our integrative approach iTiSS, which is an accurate and generic TSS caller for any TSS profiling experiment in eukaryotes, and subs...
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Full-text available
Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays a...
Preprint
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The conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal de-ubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo by acting as a potent viral immune evasin. Here, we show that the pUL36 DUB activity was required t...
Preprint
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Rationale Accumulation of mononuclear phagocytes (monocytes, macrophages and dendritic cells) in the vessel wall is a hallmark of atherosclerosis. Although single-cell RNA-sequencing (scRNA-seq) has shed new light on immune cell transcriptional diversity in atherosclerosis, it is still unknown whether the transcriptional states of mononuclear phago...
Preprint
Full-text available
Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays a...
Article
Full-text available
The HSV-1 virion host shutoff ( vhs ) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA leve...
Preprint
Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonises several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is largely unknown. Here, we decipher the role of ZAP in the context of human cytomegaloviru...
Article
The success of cancer immunotherapy relies on the ability of cytotoxic T cells to specifically recognize and eliminate tumor cells based on peptides presented by HLA-I. Although the peptide epitopes that elicit the corresponding immune response often remain unidentified, it is generally assumed that neoantigens, due to tumor-specific mutations, are...
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Full-text available
Success of DC vaccines relies on the quality of antigen presentation, costimulation, lymph node migration, and the release of IL-12, in case of Th1 priming. Here, we provide evidence for interaction between the injected vaccine DCs with endogenous lymph node-resident DCs for Th1 induction. While migration of the injected DCs was essential for antig...
Preprint
Full-text available
Herpes simplex virus 1 (HSV-1) installs a profound host shut-off during lytic infection. The virion host shut-off ( vhs ) protein plays a key role in this process by efficiently cleaving both host and viral mRNAs in a translation-initiation-dependent manner. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in transc...
Article
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The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including al...
Article
Full-text available
Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to th...
Article
Full-text available
CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral pro...
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Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease¹. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further...
Preprint
Full-text available
Since the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral t...
Preprint
Full-text available
Current single-cell RNA sequencing approaches gives a snapshot of a cellular phenotype but convey no information on the temporal dynamics of transcription. Moreover, the stochastic nature of transcription at molecular level is not recovered. Here, we present single-cell SLAM-seq (scSLAM-seq), which integrates metabolic RNA labeling, biochemical nuc...
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Global quantification of total RNA is used to investigate steady state levels of gene expression. However, being able to differentiate pre-existing RNA (that has been synthesized prior to a defined point in time) and newly transcribed RNA can provide invaluable information e.g. to estimate RNA half-lives or identify fast and complex regulatory proc...
Article
Motivation: Fold changes from count based high-throughput experiments such as RNA-seq suffer from a zero-frequency problem. To circumvent division by zero, so-called pseudocounts are added to make all observed counts strictly positive. The magnitude of pseudocounts for digital expression measurements and on which stage of the analysis they are int...
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Background -Alternative macrophage activation, which relies on mitochondrial oxidative metabolism, plays a central role in resolution of inflammation and prevents atherosclerosis. Moreover, macrophages handle large amounts of cholesterol and triglycerides derived from the engulfed modified lipoproteins during atherosclerosis. Although several micro...
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Ribosome profiling has been used to predict thousands of short open reading frames (sORFs) in eukaryotic cells, but it suffers from substantial levels of noise. PRICE (https://github.com/erhard-lab/price) is a computational method that models experimental noise to enable researchers to accurately resolve overlapping sORFs and noncanonical translati...
Article
Full-text available
Lytic herpes simplex virus 1 (HSV-1) infection triggers disruption of transcription termination (DoTT) of most cellular genes, resulting in extensive intergenic transcription. Similarly, cellular stress responses lead to gene-specific transcription downstream of genes (DoG). In this study, we performed a detailed comparison of DoTT/DoG transcriptio...
Data
Supplementary tables. Table A provides read-through values obtained from 4sU-seq data for HSV-1 infection, salt and heat stress. Table B provides read-through values for total, cytoplasmic, nucleoplasmic and chromatin-associated RNA at 8h p.i. HSV-1 infection. Table C lists genes for which read-through transcripts at 8h p.i. HSV-1 infection appear...
Data
Supplementary methods. Protocol for the separation of subcellular RNA fractions used in this study. (PDF)
Data
Supplementary figures. Contains Supplementary Figures A-R and legends. (PDF)
Article
Ribosome profiling has predicted thousands of short open reading frames (sORFs) in eukaryotic cells, but still suffers from substantial levels of noise. PRICE (https://github.com/erhard-lab/price) is a computational method modeling the experimental noise to accurately resolve overlapping sORFs and non-canonical translation initiation. We experiment...
Article
Full-text available
Dendritic cells (DCs) are key directors of tolerogenic and immunogenic immune responses. During the steady state, DCs maintain T cell tolerance to self-antigens by multiple mechanisms including inducing anergy, deletion, and Treg activity. All of these mechanisms help to prevent autoimmune diseases or other hyperreactivities. Different DC subsets c...
Article
Full-text available
Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular...
Data
Public resources used for this study. (PDF)
Data
Validation of gene array hybridization results by quantitative RT-PCR. (A) Relative transcript levels of EBNA2 target genes were quantified from total RNA samples of the indicated cell lines by RT-qPCR. All results were normalized to actin B transcript levels. (B) For comparison the expression levels measured by gene array hybridization are shown i...
Data
Heatmap showing microRNAs regulated at least 1.5-fold (p ≤ 0.05) by EBNA2 in DG75ER/EBNA2 CBF1 wt cells (for all details see S1 Fig). (TIF)
Data
Validation of efficient EBF1 knockdown by Western blot analysis. DG75doxHA-E2 CBF1 wt or CBF1 ko B cells were transfected with a mixture of scrambled non-targeting siRNAs (siCNTRL) or EBF1 specific siRNAs (siEBF1). 8 h post transfection, EBNA2 transcription was induced. 24 h post transfection, cells were harvested and analyzed by immunoblots and Ch...
Data
Control panels documenting estrogen responses in ER/EBNA2 expressing DG75 cells compared to estrogen treated untransfected parental cell lines. (A) DG75 parental cells (DG75 CBF1 wt), CBF1 deficient (DG75 CBF1 ko), ER/EBNA2 expressing (DG75ER/EBNA2 CBF1 wt), and CBF1 deficient ER/EBNA2 expressing DG75 cells (DG75ER/EBNA2 CBF1 ko) were treated with...
Data
Heatmap representing the 132 transcripts regulated at least 2-fold (p ≤ 0.001) by EBNA2 in CBF1 deficient DG75ER/EBNA2 cells. Total cellular RNA was isolated and submitted to gene expression analysis using the Human Gene 2.0 ST array. All probe sets represent single transcripts. For each condition 3 biological replicates were examined. Each vertica...
Data
Doxycycline inducible HA-EBNA2 expression in CBF1 proficient or deficient DG75 B cells. (A) pRTRdoxHA-E2 vector used to generate stable DG75 cell lines. The coding sequence for EBNA2 fused to a N-terminal HA-tag (HA-E2), plus a preceding intron of the beta-globin gene for enhanced expression, was cloned into the pRTR vector [69, 70] using SfiI rest...
Data
Intersection of EBNA2 binding sites identified in LCLs, DG75doxHA-E2 CBF1 wt or ko. The table lists (lower part) the called peaks and the fraction of peaks which also score in LCL EBNA2. (TIF)
Data
Based on the expression level changes of 950 transcripts which are regulated in DG75ER/EBNA2 CBF1 wt at least 2-fold (p ≤ 0.05) and expression levels of the same transcripts in DG75ER/EBNA2 CBF1 ko cells, 12 clusters of transcripts were defined. Number of transcripts contained in each cluster is indicated on the left. Unique ID and Gene Name are li...
Data
Identification of individual target gene subsets based on principle component analysis. Since on average target gene expression changes in CBF1 positive cells were stronger than in CBF1 negative cells, principle component analysis on EBNA2 regulated genes was used to identify specific subpopulations: The first principle component (green arrow) desc...
Data
Gene list k-means clustering (cluster 1–12). (XLSX)
Article
Full-text available
We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics of newly synthesized proteins (pulsed stable isotope labeling with amino acids in cell culture, pSILAC) in combination with mRNA and non-coding RNA expression analyses by next generation sequencing (RNA-, miR-Seq) in the colorectal cancer (CRC) cel...
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Various biases affect high-throughput sequencing read counts. Contrary to the general assumption, we show that bias does not always cancel out when fold changes are computed and that bias affects more than 20% of genes that are called differentially regulated in RNA-seq experiments with drastic effects on subsequent biological interpretation. Here,...
Article
Full-text available
Herpes simplex virus 1 (HSV-1) is an important human pathogen and a paradigm for virus-induced host shut-off. Here we show that global changes in transcription and RNA processing and their impact on translation can be analysed in a single experimental setting by applying 4sU-tagging of newly transcribed RNA and ribosome profiling to lytic HSV-1 inf...
Article
Full-text available
Bone fracture healing impairment related to mechanical problems has been largely corrected by advances in fracture management. Better protocols, more strict controls of time and function, hardware and surgical technique evolution have contributed to better prognosis, even in complex fractures. However, atrophic nonunion persists in clinical cases w...
Article
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Gene expression is regulated in a context-dependent, cell-type specific manner. Condition-specific transcription is dependent on the presence of transcription factors (TFs) that can activate or inhibit its target genes (global context). Additional factors such as chromatin structure, histone or DNA modifications also influence the activity of indiv...
Article
Full-text available
Epstein-Barr virus (EBV) causes a persistent infection in human B cells by establishing specific transcription programs to control B cell activation and differentiation. Transcriptional reprogramming of EBV infected B cells is predominantly driven by the action of EBV nuclear antigens, among them the transcriptional repressor EBNA3A. By comparing g...
Article
Full-text available
PARma is a complete data analysis software for AGO-PAR-CLIP experiments to identify target sites of microRNAs as well as the microRNA binding to these sites. It integrates specific characteristics of the experiments into a generative model. The model and a novel pattern discovery tool are iteratively applied to data to estimate seed activity probab...
Article
Motivation: RIP-chip is a high-throughput method to identify mRNAs that are targeted by RNA-binding proteins. The protein of interest is immunoprecipitated, and the identity and relative amount of mRNA associated with it is measured on microarrays. Even if a variety of methods is available to analyse microarray data, e.g. to detect differentially...
Article
Quantitative high-throughput mass spectrometry has become an established tool to measure relative gene expression proteome-wide. The output of such an experiment usually consists of a list of expression ratios (fold changes) for several thousand proteins between two conditions. However, we observed that individual peptide fold changes may show a si...