Publications by authors named "Gilles Gasparoni"

46 Publications

Transmission of trained immunity and heterologous resistance to infections across generations.

Nat Immunol 2021 11 18;22(11):1382-1390. Epub 2021 Oct 18.

Department of Internal Medicine and Radboud Center for Infectious diseases, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.

Intergenerational inheritance of immune traits linked to epigenetic modifications has been demonstrated in plants and invertebrates. Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. The progeny of trained mice exhibited cellular, developmental, transcriptional and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment. Moreover, the progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous Escherichia coli and Listeria monocytogenes infections. Sperm DNA of parental male mice intravenously infected with the fungus C. albicans showed DNA methylation differences linked to immune gene loci. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections.
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http://dx.doi.org/10.1038/s41590-021-01052-7DOI Listing
November 2021

Two Piwis with Ago-like functions silence somatic genes at the chromatin level.

RNA Biol 2021 Oct 18:1-13. Epub 2021 Oct 18.

Molecular Cell Biology and Microbiology, Wuppertal University, Wuppertal, Germany.

Most sRNA biogenesis mechanisms involve either RNAse III cleavage or ping-pong amplification by different Piwi proteins harbouring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from targeted endogenous loci. Our data does not indicate any signatures from ping-pong amplification but Dicer cleavage of long dsRNA. Ptiwi13 and 14 prefer different sub-cellular localizations and different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and show a general preference for uridine-rich sRNAs along the entire sRNA length. In addition, Ptiwi14-loaded siRNAs show a 5´-U signature. Our data indicates both Ptiwis and 2´-O-methylation contributing to strand selection of Dicer cleaved siRNAs. This unexpected function of the two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. We describe an unusual mode of action of Piwi proteins extending not only the great variety of Piwi-associated RNAi pathways but moreover raising the question whether this could have been the primordial one.
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http://dx.doi.org/10.1080/15476286.2021.1991114DOI Listing
October 2021

Dysregulation of cholesterol homeostasis in human lung cancer tissue and tumour-associated macrophages.

EBioMedicine 2021 Oct 25;72:103578. Epub 2021 Sep 25.

Department of Pharmacy, Pharmaceutical Biology, Saarland University, Saarbrücken, Germany. Electronic address:

Background: Based on reports on elevated cholesterol levels in cancer cells, strategies to lower cholesterol synthesis have been suggested as an antitumour strategy. However, cholesterol depletion has also been shown to induce tumour-promoting actions in tumour-associated macrophages (TAMs).

Methods: We performed lipidomic and transcriptomic analyses of human lung cancer material. To assess whether the TAM phenotype is shaped by secreted factors produced by tumour cells, primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype using tumour cell-conditioned medium.

Findings: Lipidomic analysis of lung adenocarcinoma (n=29) and adjacent non-tumour tissues (n=22) revealed a significant accumulation of free cholesterol and cholesteryl esters within the tumour tissue. In contrast, cholesterol levels were reduced in TAMs isolated from lung adenocarcinoma tissues when compared with alveolar macrophages (AMs) obtained from adjacent non-tumour tissues. Bulk-RNA-Seq revealed that genes involved in cholesterol biosynthesis and metabolism were downregulated in TAMs, while cholesterol efflux transporters were upregulated. In vitro polarized TAM-like macrophages showed an attenuated lipogenic gene expression signature and exhibited lower cholesterol levels compared with non-polarized macrophages. A genome-wide comparison by bulk RNA-Seq confirmed a high similarity of ex vivo TAMs and in vitro TAM-like macrophages. Modulation of intracellular cholesterol levels by either starving, cholesterol depletion, or efflux transporter inhibition indicated that cholesterol distinctly shapes macrophage gene expression.

Interpretation: Our data show an opposite dysregulation of cholesterol homeostasis in tumour tissue vs. TAMs. Polarization of in vitro differentiated macrophages by tumour cell-conditioned medium recapitulates key features of ex vivo TAMs.

Funding: Deutsche Forschungsgemeinschaft (DFG), Landesforschungsf €orderungsprogramm Saarland (LFPP).
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http://dx.doi.org/10.1016/j.ebiom.2021.103578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8479395PMC
October 2021

Identification of tissue-specific and common methylation quantitative trait loci in healthy individuals using MAGAR.

Epigenetics Chromatin 2021 Sep 16;14(1):44. Epub 2021 Sep 16.

Department of Genetics/Epigenetics, Saarland University, Saarbrücken, Germany.

Background: Understanding the influence of genetic variants on DNA methylation is fundamental for the interpretation of epigenomic data in the context of disease. There is a need for systematic approaches not only for determining methylation quantitative trait loci (methQTL), but also for discriminating general from cell type-specific effects.

Results: Here, we present a two-step computational framework MAGAR ( https://bioconductor.org/packages/MAGAR ), which fully supports the identification of methQTLs from matched genotyping and DNA methylation data, and additionally allows for illuminating cell type-specific methQTL effects. In a pilot analysis, we apply MAGAR on data in four tissues (ileum, rectum, T cells, B cells) from healthy individuals and demonstrate the discrimination of common from cell type-specific methQTLs. We experimentally validate both types of methQTLs in an independent data set comprising additional cell types and tissues. Finally, we validate selected methQTLs located in the PON1, ZNF155, and NRG2 genes by ultra-deep local sequencing. In line with previous reports, we find cell type-specific methQTLs to be preferentially located in enhancer elements.

Conclusions: Our analysis demonstrates that a systematic analysis of methQTLs provides important new insights on the influences of genetic variants to cell type-specific epigenomic variation.
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http://dx.doi.org/10.1186/s13072-021-00415-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8444396PMC
September 2021

Epigenomic and transcriptional profiling identifies impaired glyoxylate detoxification in NAFLD as a risk factor for hyperoxaluria.

Cell Rep 2021 Aug;36(8):109526

Department of Toxicology, Leibniz-Research Centre for Working Environment and Human Factors at the TU Dortmund (IfADo), 44139 Dortmund, Germany. Electronic address:

Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release-a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.
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http://dx.doi.org/10.1016/j.celrep.2021.109526DOI Listing
August 2021

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

Nat Commun 2021 06 15;12(1):3624. Epub 2021 Jun 15.

Max Planck Institute for Molecular Genetics, Otto-Warburg-Laboratories, Epigenomics, Ihnestraße 63-73, Berlin, Germany.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/β-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.
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http://dx.doi.org/10.1038/s41467-021-23706-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206096PMC
June 2021

A meta-analysis of epigenome-wide association studies in Alzheimer's disease highlights novel differentially methylated loci across cortex.

Nat Commun 2021 06 10;12(1):3517. Epub 2021 Jun 10.

University of Exeter Medical School, College of Medicine and Health, University of Exeter, Exeter, UK.

Epigenome-wide association studies of Alzheimer's disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimer's disease (N = 1453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N = 1408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further >600 unique donors. The meta-analysis summary statistics are available in our online data resource ( www.epigenomicslab.com/ad-meta-analysis/ ).
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http://dx.doi.org/10.1038/s41467-021-23243-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192929PMC
June 2021

Epigenetic control of region-specific transcriptional programs in mouse cerebellar and cortical astrocytes.

Glia 2021 Sep 24;69(9):2160-2177. Epub 2021 May 24.

Department of Genetics and EpiGenetics, University of Saarland, Saarbrücken, Germany.

Astrocytes from the cerebral cortex (CTX) and cerebellum (CB) share basic molecular programs, but also form distinct spatial and functional subtypes. The regulatory epigenetic layers controlling such regional diversity have not been comprehensively investigated so far. Here, we present an integrated epigenome analysis of methylomes, open chromatin, and transcriptomes of astroglia populations isolated from the cortex or cerebellum of young adult mice. Besides a basic overall similarity in their epigenomic programs, cortical astrocytes and cerebellar astrocytes exhibit substantial differences in their overall open chromatin structure and in gene-specific DNA methylation. Regional epigenetic differences are linked to differences in transcriptional programs encompassing genes of region-specific transcription factor networks centered around Lhx2/Foxg1 in CTX astrocytes and the Zic/Irx families in CB astrocytes. The distinct epigenetic signatures around these transcription factor networks point to a complex interconnected and combinatorial regulation of region-specific transcriptomes. These findings suggest that key transcription factors, previously linked to temporal, regional, and spatial control of neurogenesis, also form combinatorial networks important for astrocytes. Our study provides a valuable resource for the molecular basis of regional astrocyte identity and physiology.
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http://dx.doi.org/10.1002/glia.24016DOI Listing
September 2021

Human IgA-Expressing Bone Marrow Plasma Cells Characteristically Upregulate Programmed Cell Death Protein-1 Upon B Cell Receptor Stimulation.

Front Immunol 2020 12;11:628923. Epub 2021 Feb 12.

Department of Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin, Berlin, Germany.

The functions of bone marrow plasma cells (BMPC) beyond antibody production are not fully elucidated and distinct subsets of BMPC suggest potential different functions. Phenotypic differences were identified for human BMPC depending on CD19 expression. Since CD19 is a co-stimulatory molecule of the B-cell-receptor (BCR), and IgA and IgM BMPC express the BCR on their surface, we here studied whether CD19 expression affects cellular responses, such as BCR signaling and the expression of checkpoint molecules. We analyzed 132 BM samples from individuals undergoing routine total hip arthroplasty. We found that both CD19 and CD19 BMPC expressed BCR signaling molecules. Notably, the BCR-associated kinase spleen tyrosine kinase (SYK) including pSYK was higher expressed in CD19 BMPC compared to CD19 BMPC. BCR stimulation also resulted in increased kinase phosphorylation downstream of the BCR while expression of CD19 remained stable afterwards. Interestingly, the BCR response was restricted to IgA BMPC independently of CD19 expression. With regard to the expression of checkpoint molecules, CD19 BMPC expressed higher levels of co-inhibitory molecule programmed cell death protein-1 (PD-1) than CD19 BMPC. IgA BMPC characteristically upregulated PD-1 upon BCR stimulation in contrast to other PC subsets and inhibition of the kinase SYK abrogated PD-1 upregulation. In contrast, expression of PD-1 ligand, B and T lymphocyte attenuator (BTLA) and CD28 did not change upon BCR activation of IgA BMPC. Here, we identify a distinct characteristic of IgA BMPC that is independent of the phenotypic heterogeneity of the subsets according to their CD19 expression. The data suggest that IgA BMPC underlie different regulatory principles and/or exert distinct regulatory functions.
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http://dx.doi.org/10.3389/fimmu.2020.628923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907642PMC
June 2021

Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Treg Phenotype.

Front Immunol 2020 7;11:609891. Epub 2021 Jan 7.

Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.

CD4+ regulatory T cells (Tregs) are key mediators of immunological tolerance and promising effector cells for immuno-suppressive adoptive cellular therapy to fight autoimmunity and chronic inflammation. Their functional stability is critical for their clinical utility and has been correlated to the demethylated state of the TSDR/CNS2 enhancer element in the Treg lineage transcription factor FOXP3. However, proof for a causal contribution of the TSDR de-methylation to FOXP3 stability and Treg induction is so far lacking. We here established a powerful transient-transfection CRISPR-Cas9-based epigenetic editing method for the selective de-methylation of the TSDR within the endogenous chromatin environment of a living cell. The induced de-methylated state was stable over weeks in clonal T cell proliferation cultures even after expression of the editing complex had ceased. Epigenetic editing of the TSDR resulted in FOXP3 expression, even in its physiological isoform distribution, proving a causal role for the de-methylated TSDR in FOXP3 regulation. However, successful FOXP3 induction was not associated with a switch towards a functional Treg phenotype, in contrast to what has been reported from FOXP3 overexpression approaches. Thus, TSDR de-methylation is required, but not sufficient for a stable Treg phenotype induction. Therefore, targeted demethylation of the TSDR may be a critical addition to published Treg induction protocols which so far lack FOXP3 stability.
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http://dx.doi.org/10.3389/fimmu.2020.609891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817622PMC
June 2021

The HPV and p63 Status in Penile Cancer Are Linked with the Infiltration and Therapeutic Availability of Neutrophils.

Mol Cancer Ther 2021 02 3;20(2):423-437. Epub 2020 Dec 3.

Institute of Virology, University of Saarland, Homburg, Germany.

Squamous penile cancer displays a rare human papillomavirus (HPV)-associated tumor entity. Investigations on the molecular pathogenesis of HPV-driven penile cancer are impaired by the rareness of clinical specimens and, in particular, are missing relevant cell culture models. Here, we identified in HPV-positive penile cancer cell lines that HPV16 oncoproteins control expression by modulating critical regulators, while integration into the open reading frame facilitates oncogene expression. The resulting feed-forward loop leads to elevated p63 levels that in turn enhance the release of the neutrophil-recruiting chemokine CXCL8. Remarkably, elevated CXCL8 amounts lead to the increased surface exposition of the Fc receptor of human IgA antibodies, FcαRI, on neutrophils and correlated with a higher susceptibility to antibody-dependent neutrophil-mediated cytotoxicity (ADCC) using an EGFR-specific IgA2 antibody. IHC staining of tissue microarrays proved that elevated expression of p63 together with neutrophil infiltration were significantly more frequent in HPV-positive penile cancer displaying a higher tumor grade. In summary, we identified a promising marker profile of patients with penile cancer at higher risk for worse prognosis. However, these patients may benefit from immunotherapeutic approaches efficiently engaging neutrophils for tumor cell killing.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0173DOI Listing
February 2021

Prediction of single-cell gene expression for transcription factor analysis.

Gigascience 2020 10;9(11)

Institute for Cardiovascular Regeneration, Goethe University, 60590 Frankfurt am Main, Germany; Theodor-Stern-Kai 7.

Background: Single-cell RNA sequencing is a powerful technology to discover new cell types and study biological processes in complex biological samples. A current challenge is to predict transcription factor (TF) regulation from single-cell RNA data.

Results: Here, we propose a novel approach for predicting gene expression at the single-cell level using cis-regulatory motifs, as well as epigenetic features. We designed a tree-guided multi-task learning framework that considers each cell as a task. Through this framework we were able to explain the single-cell gene expression values using either TF binding affinities or TF ChIP-seq data measured at specific genomic regions. TFs identified using these models could be validated by the literature.

Conclusion: Our proposed method allows us to identify distinct TFs that show cell type-specific regulation. This approach is not limited to TFs but can use any type of data that can potentially be used in explaining gene expression at the single-cell level to study factors that drive differentiation or show abnormal regulation in disease. The implementation of our workflow can be accessed under an MIT license via https://github.com/SchulzLab/Triangulate.
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http://dx.doi.org/10.1093/gigascience/giaa113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596801PMC
October 2020

A cell-of-origin epigenetic tracer reveals clinically distinct subtypes of high-grade serous ovarian cancer.

Genome Med 2020 10 30;12(1):94. Epub 2020 Oct 30.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCSS, Milan, Italy.

Background: High-grade serous ovarian cancer (HGSOC) is a major unmet need in oncology. The remaining uncertainty on its originating tissue has hampered the discovery of molecular oncogenic pathways and the development of effective therapies.

Methods: We used an approach based on the retention in tumors of a DNA methylation trace (OriPrint) that distinguishes the two putative tissues of origin of HGSOC, the fimbrial (FI) and ovarian surface epithelia (OSE), to stratify HGSOC by several clustering methods, both linear and non-linear. The identified tumor subtypes (FI-like and OSE-like HGSOC) were investigated at the RNAseq level to stratify an in-house cohort of macrodissected HGSOC FFPE samples to derive overall and disease-free survival and identify specific transcriptional alterations of the two tumor subtypes, both by classical differential expression and weighted correlation network analysis. We translated our strategy to published datasets and verified the co-occurrence of previously described molecular classification of HGSOC. We performed cytokine analysis coupled to immune phenotyping to verify alterations in the immune compartment associated with HGSOC. We identified genes that are both differentially expressed and methylated in the two tumor subtypes, concentrating on PAX8 as a bona fide marker of FI-like HGSOC.

Results: We show that: - OriPrint is a robust DNA methylation tracer that exposes the tissue of origin of HGSOC. - The tissue of origin of HGSOC is the main determinant of DNA methylation variance in HGSOC. - The tissue of origin is a prognostic factor for HGSOC patients. - FI-like and OSE-like HGSOC are endowed with specific transcriptional alterations that impact patients' prognosis. - OSE-like tumors present a more invasive and immunomodulatory phenotype, compatible with its worse prognostic impact. - Among genes that are differentially expressed and regulated in FI-like and OSE-like HGSOC, PAX8 is a bona fide marker of FI-like tumors.

Conclusions: Through an integrated approach, our work demonstrates that both FI and OSE are possible origins for human HGSOC, whose derived subtypes are both molecularly and clinically distinct. These results will help define a new roadmap towards rational, subtype-specific therapeutic inroads and improved patients' care.
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http://dx.doi.org/10.1186/s13073-020-00786-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7597028PMC
October 2020

A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation.

Sci Adv 2020 Aug 19;6(34):eaba5614. Epub 2020 Aug 19.

Center for Integrated Protein Science Munich CIPSM, Munich, Germany.

Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin () in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR-mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.
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http://dx.doi.org/10.1126/sciadv.aba5614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438099PMC
August 2020

Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation.

Mol Ther Nucleic Acids 2020 Sep 31;21:1050-1061. Epub 2020 Jul 31.

Center for Integrated Protein Science Munich CIPSM, Munich, Germany; Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany. Electronic address:

Mutations in CLRN1 cause Usher syndrome (USH) type III (USH3A), a disease characterized by progressive hearing impairment, retinitis pigmentosa, and vestibular dysfunction. Due to the lack of appropriate disease models, no efficient therapy for retinitis pigmentosa in USH patients exists so far. In addition, given the yet undefined functional role and expression of the different CLRN1 splice isoforms in the retina, non-causative therapies such as gene supplementation are unsuitable at this stage. In this study, we focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect. Surprisingly, however, no synergistic effect was detected when combining both methods. Finally, the injection of naked AONs into mice expressing the mutant CLRN1 minigene in the retina also led to a significant splice rescue. We propose that both AONs and CRISPR-Cas9 are suitable strategies to initiate advanced preclinical studies for treatment of USH3A patients.
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http://dx.doi.org/10.1016/j.omtn.2020.07.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452116PMC
September 2020

Dual-Seq reveals genome and transcriptome of Caedibacter taeniospiralis, obligate endosymbiont of Paramecium.

Sci Rep 2020 06 16;10(1):9727. Epub 2020 Jun 16.

Microbiology, Institute of Biology II, Albert Ludwig University of Freiburg, Freiburg, Germany.

Interest in host-symbiont interactions is continuously increasing, not only due to the growing recognition of the importance of microbiomes. Starting with the detection and description of novel symbionts, attention moves to the molecular consequences and innovations of symbioses. However, molecular analysis requires genomic data which is difficult to obtain from obligate intracellular and uncultivated bacteria. We report the identification of the Caedibacter genome, an obligate symbiont of the ciliate Paramecium. The infection does not only confer the host with the ability to kill other cells but also renders them immune against this effect. We obtained the C. taeniospiralis genome and transcriptome by dual-Seq of DNA and RNA from infected paramecia. Comparison of codon usage and expression level indicates that genes necessary for a specific trait of this symbiosis, i.e. the delivery of an unknown toxin, result from horizontal gene transfer hinting to the relevance of DNA transfer for acquiring new characters. Prediction of secreted proteins of Caedibacter as major agents of contact with the host implies, next to several toxin candidates, a rather uncharacterized secretome which appears to be highly adapted to this symbiosis. Our data provides new insights into the molecular establishment and evolution of this obligate symbiosis and for the pathway characterization of toxicity and immunity.
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http://dx.doi.org/10.1038/s41598-020-65894-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297999PMC
June 2020

Neuronal deficiency of p38α-MAPK ameliorates symptoms and pathology of APP or Tau-transgenic Alzheimer's mouse models.

FASEB J 2020 07 31;34(7):9628-9649. Epub 2020 May 31.

Department of Neurology, Saarland University, Homburg, Germany.

Alzheimer's disease (AD) is the leading cause of dementia with very limited therapeutic options. Amyloid β (Aβ) and phosphorylated Tau (p-Tau) are key pathogenic molecules in AD. P38α-MAPK is specifically activated in AD lesion sites. However, its effects on AD pathogenesis, especially on p-Tau-associated brain pathology, and the underlying molecular mechanisms remain unclear. We mated human APP-transgenic mice and human P301S Tau-transgenic mice with mapk14-floxed and neuron-specific Cre-knock-in mice. We observed that deletion of p38α-MAPK specifically in neurons improves the cognitive function of both 9-month-old APP and Tau-transgenic AD mice, which is associated with decreased Aβ and p-Tau load in the brain. We further used next-generation sequencing to analyze the gene transcription in brains of p38α-MAPK deficient and wild-type APP-transgenic mice, which indicated that deletion of p38α-MAPK regulates the transcription of calcium homeostasis-related genes, especially downregulates the expression of grin2a, a gene encoding NMDAR subunit NR2A. Cell culture experiments further verified that deletion of p38α-MAPK inhibits NMDA-triggered calcium influx and neuronal apoptosis. Our systemic studies of AD pathogenic mechanisms using both APP- and Tau-transgenic mice suggested that deletion of neuronal p38α-MAPK attenuates AD-associated brain pathology and protects neurons in AD pathogenesis. This study supports p38α-MAPK as a novel target for AD therapy.
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http://dx.doi.org/10.1096/fj.201902731RRDOI Listing
July 2020

Yeast Viral Killer Toxin K1 Induces Specific Host Cell Adaptions via Intrinsic Selection Pressure.

Appl Environ Microbiol 2020 02 3;86(4). Epub 2020 Feb 3.

Molecular and Cell Biology, Saarland University and Max Planck Institute for Informatics, Saarbrücken, Germany

The killer phenomenon in yeast () not only provides the opportunity to study host-virus interactions in a eukaryotic model but also represents a powerful tool to analyze potential coadaptional events and the role of killer yeast in biological diversity. Although undoubtedly having a crucial impact on the abundance and expression of the killer phenotype in killer-yeast harboring communities, the influence of a particular toxin on its producing host cell has not been addressed sufficiently. In this study, we describe a model system of two K1 killer yeast strains with distinct phenotypical differences pointing to substantial selection pressure in response to the toxin secretion level. Transcriptome and lipidome analyses revealed specific and intrinsic host cell adaptions dependent on the amount of K1 toxin produced. High basal expression of genes coding for osmoprotectants and stress-responsive proteins in a killer yeast strain secreting larger amounts of active K1 toxin implies a generally increased stress tolerance. Moreover, the data suggest that immunity of the host cell against its own toxin is essential for the balanced virus-host interplay providing valuable hints to elucidate the molecular mechanisms underlying K1 immunity and implicating an evolutionarily conserved role for toxin immunity in natural yeast populations. The killer phenotype in relies on the cytoplasmic persistence of two RNA viruses. In contrast to bacterial toxin producers, killer yeasts necessitate a specific immunity mechanism against their own toxin because they bear the same receptor populations as sensitive cells. Although the killer phenomenon is highly abundant and has a crucial impact on the structure of yeast communities, the influence of a particular toxin on its host cell has been barely addressed. In our study, we used two derivatives secreting different amount of the killer toxin K1 to analyze potential coadaptional events in this particular host/virus system. Our data underline the dependency of the host cell's ability to cope with extracellular toxin molecules and intracellular K1 molecules provided by the virus. Therefore, this research significantly advances the current understanding of the evolutionarily conserved role of this molecular machinery as an intrinsic selection pressure in yeast populations.
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http://dx.doi.org/10.1128/AEM.02446-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997729PMC
February 2020

Comparative Analysis of Biochemical Biases by Ligation- and Template-Switch-Based Small RNA Library Preparation Protocols.

Clin Chem 2019 12 23;65(12):1581-1591. Epub 2019 Oct 23.

Molecular Cell Dynamics, Centre for Human and Molecular Biology, Saarland University, Saarbruecken, Germany;

Background: Small RNAs are key players in the regulation of gene expression and differentiation. However, many different classes of small RNAs (sRNAs) have been described with distinct biogenesis pathways and, as a result, with different biochemical properties. To analyze sRNAs by deep sequencing, complementary DNA synthesis requires manipulation of the RNA molecule itself. Thus, enzymatic activities during library preparation bias the library content owing to biochemical criteria.

Methods: We compared 4 different manipulations of RNA for library preparation: () a ligation-based procedure allowing only 5'-mono-phosphorylated RNA to enter the library, () a ligation-based procedure allowing additional 5'-triphosphates and Cap structures, () a ligation-independent, template-switch-based library preparation, and () a template-switch-based library preparation allowing 3'-phosphorylated RNAs to enter the library.

Results: Our data show large differences between ligation-dependent and ligation-independent libraries in terms of their preference for individual sRNA classes such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and transfer RNA fragments. Moreover, the miRNA composition is different between both procedures, and more microRNA isoforms (isomiRs) can be identified after pyrophosphatase treatment. piRNAs are enriched in template-switch libraries, and this procedure apparently includes more different RNA species.

Conclusions: Our data indicate that miRNAomics from both methods will hardly be comparable. Ligation-based libraries enrich for canonical miRNAs, which thus may be suitable methods for miRNAomics. Template-switch libraries contain increased numbers and different compositions of fragments and long RNAs. Following different interests for other small RNA species, ligation-independent libraries appear to show a more realistic sRNA landscape with lower bias against biochemical modifications.
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http://dx.doi.org/10.1373/clinchem.2019.305045DOI Listing
December 2019

Unique and assay specific features of NOMe-, ATAC- and DNase I-seq data.

Nucleic Acids Res 2019 11;47(20):10580-10596

Department of Genetics, Saarland University, 66123 Saarbrücken, Germany.

Chromatin accessibility maps are important for the functional interpretation of the genome. Here, we systematically analysed assay specific differences between DNase I-seq, ATAC-seq and NOMe-seq in a side by side experimental and bioinformatic setup. We observe that most prominent nucleosome depleted regions (NDRs, e.g. in promoters) are roboustly called by all three or at least two assays. However, we also find a high proportion of assay specific NDRs that are often 'called' by only one of the assays. We show evidence that these assay specific NDRs are indeed genuine open chromatin sites and contribute important information for accurate gene expression prediction. While technically ATAC-seq and DNase I-seq provide a superb high NDR calling rate for relatively low sequencing costs in comparison to NOMe-seq, NOMe-seq singles out for its genome-wide coverage allowing to not only detect NDRs but also endogenous DNA methylation and as we show here genome wide segmentation into heterochromatic B domains and local phasing of nucleosomes outside of NDRs. In summary, our comparisons strongly suggest to consider assay specific differences for the experimental design and for generalized and comparative functional interpretations.
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http://dx.doi.org/10.1093/nar/gkz799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6847574PMC
November 2019

DNA methylation signatures of monozygotic twins clinically discordant for multiple sclerosis.

Nat Commun 2019 05 7;10(1):2094. Epub 2019 May 7.

Department of Genetics/Epigenetics, Saarland University, 66123, Saarbrücken, Germany.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with a modest concordance rate in monozygotic twins, which strongly argues for involvement of epigenetic factors. We observe highly similar peripheral blood mononuclear cell-based methylomes in 45 MS-discordant monozygotic twins. Nevertheless, we identify seven MS-associated differentially methylated positions (DMPs) of which we validate two, including a region in the TMEM232 promoter and ZBTB16 enhancer. In CD4 + T cells we find an MS-associated differentially methylated region in FIRRE. Additionally, 45 regions show large methylation differences in individual pairs, but they do not clearly associate with MS. Furthermore, we present epigenetic biomarkers for current interferon-beta treatment, and extensive validation shows that the ZBTB16 DMP is a signature for prior glucocorticoid treatment. Taken together, this study represents an important reference for epigenomic MS studies, identifies new candidate epigenetic markers, and highlights treatment effects and genetic background as major confounders.
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http://dx.doi.org/10.1038/s41467-019-09984-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504952PMC
May 2019

The sncRNA Zoo: a repository for circulating small noncoding RNAs in animals.

Nucleic Acids Res 2019 05;47(9):4431-4441

Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrücken, Germany.

The repertoire of small noncoding RNAs (sncRNAs), particularly miRNAs, in animals is considered to be evolutionarily conserved. Studies on sncRNAs are often largely based on homology-based information, relying on genomic sequence similarity and excluding actual expression data. To obtain information on sncRNA expression (including miRNAs, snoRNAs, YRNAs and tRNAs), we performed low-input-volume next-generation sequencing of 500 pg of RNA from 21 animals at two German zoological gardens. Notably, none of the species under investigation were previously annotated in any miRNA reference database. Sequencing was performed on blood cells as they are amongst the most accessible, stable and abundant sources of the different sncRNA classes. We evaluated and compared the composition and nature of sncRNAs across the different species by computational approaches. While the distribution of sncRNAs in the different RNA classes varied significantly, general evolutionary patterns were maintained. In particular, miRNA sequences and expression were found to be even more conserved than previously assumed. To make the results available for other researchers, all data, including expression profiles at the species and family levels, and different tools for viewing, filtering and searching the data are freely available in the online resource ASRA (Animal sncRNA Atlas) at https://www.ccb.uni-saarland.de/asra/.
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http://dx.doi.org/10.1093/nar/gkz227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511844PMC
May 2019

Sodium chloride is an ionic checkpoint for human T2 cells and shapes the atopic skin microenvironment.

Sci Transl Med 2019 02;11(480)

Institute of Virology, Technical University of Munich, 81675 Munich, Germany.

The incidence of allergic diseases has increased over the past 50 years, likely due to environmental factors. However, the nature of these factors and the mode of action by which they induce the type 2 immune deviation characteristic of atopic diseases remain unclear. It has previously been reported that dietary sodium chloride promotes the polarization of T helper 17 (T17) cells with implications for autoimmune diseases such as multiple sclerosis. Here, we demonstrate that sodium chloride also potently promotes T2 cell responses on multiple regulatory levels. Sodium chloride enhanced interleukin-4 (IL-4) and IL-13 production while suppressing interferon-γ (IFN-γ) production in memory T cells. It diverted alternative T cell fates into the T2 cell phenotype and also induced de novo T2 cell polarization from naïve T cell precursors. Mechanistically, sodium chloride exerted its effects via the osmosensitive transcription factor NFAT5 and the kinase SGK-1, which regulated T2 signature cytokines and master transcription factors in hyperosmolar salt conditions. The skin of patients suffering from atopic dermatitis contained elevated sodium compared to nonlesional atopic and healthy skin. These results suggest that sodium chloride represents a so far overlooked cutaneous microenvironmental checkpoint in atopic dermatitis that can induce T2 cell responses, the orchestrators of atopic diseases.
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http://dx.doi.org/10.1126/scitranslmed.aau0683DOI Listing
February 2019

Integrative analysis of single-cell expression data reveals distinct regulatory states in bidirectional promoters.

Epigenetics Chromatin 2018 11 10;11(1):66. Epub 2018 Nov 10.

Excellence Cluster for Multimodal Computing and Interaction, Saarland Informatics Campus, Saarland University, Campus E1 7, Saarbrücken, 66123, Germany.

Background: Bidirectional promoters (BPs) are prevalent in eukaryotic genomes. However, it is poorly understood how the cell integrates different epigenomic information, such as transcription factor (TF) binding and chromatin marks, to drive gene expression at BPs. Single-cell sequencing technologies are revolutionizing the field of genome biology. Therefore, this study focuses on the integration of single-cell RNA-seq data with bulk ChIP-seq and other epigenetics data, for which single-cell technologies are not yet established, in the context of BPs.

Results: We performed integrative analyses of novel human single-cell RNA-seq (scRNA-seq) data with bulk ChIP-seq and other epigenetics data. scRNA-seq data revealed distinct transcription states of BPs that were previously not recognized. We find associations between these transcription states to distinct patterns in structural gene features, DNA accessibility, histone modification, DNA methylation and TF binding profiles.

Conclusions: Our results suggest that a complex interplay of all of these elements is required to achieve BP-specific transcriptional output in this specialized promoter configuration. Further, our study implies that novel statistical methods can be developed to deconvolute masked subpopulations of cells measured with different bulk epigenomic assays using scRNA-seq data.
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http://dx.doi.org/10.1186/s13072-018-0236-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230222PMC
November 2018

Epigenomic map of human liver reveals principles of zonated morphogenic and metabolic control.

Nat Commun 2018 10 8;9(1):4150. Epub 2018 Oct 8.

Medical Department 1, University Hospital Dresden, Technische Universität Dresden (TU Dresden), Dresden, Germany.

A deeper epigenomic understanding of spatial organization of cells in human tissues is an important challenge. Here we report the first combined positional analysis of transcriptomes and methylomes across three micro-dissected zones (pericentral, intermediate and periportal) of human liver. We identify pronounced anti-correlated transcriptional and methylation gradients including a core of 271 genes controlling zonated metabolic and morphogen networks and observe a prominent porto-central gradient of DNA methylation at binding sites of 46 transcription factors. The gradient includes an epigenetic and transcriptional Wnt signature supporting the concept of a pericentral hepatocyte regeneration pathway under steady-state conditions. While donors with non-alcoholic fatty liver disease show consistent gene expression differences corresponding to the severity of the disease across all zones, the relative zonated gene expression and DNA methylation patterns remain unchanged. Overall our data provide a wealth of new positional insights into zonal networks controlled by epigenetic and transcriptional gradients in human liver.
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http://dx.doi.org/10.1038/s41467-018-06611-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175862PMC
October 2018

A comprehensive analysis of 195 DNA methylomes reveals shared and cell-specific features of partially methylated domains.

Genome Biol 2018 09 28;19(1):150. Epub 2018 Sep 28.

Department of Genetics, Saarland University, Campus Saarbrücken, Saarbrücken, 66123, Germany.

Background: Partially methylated domains are extended regions in the genome exhibiting a reduced average DNA methylation level. They cover gene-poor and transcriptionally inactive regions and tend to be heterochromatic. We present a comprehensive comparative analysis of partially methylated domains in human and mouse cells, to identify structural and functional features associated with them.

Results: Partially methylated domains are present in up to 75% of the genome in human and mouse cells irrespective of their tissue or cell origin. Each cell type has a distinct set of partially methylated domains, and genes expressed in such domains show a strong cell type effect. The methylation level varies between cell types with a more pronounced effect in differentiating and replicating cells. The lowest level of methylation is observed in highly proliferating and immortal cancer cell lines. A decrease of DNA methylation within partially methylated domains tends to be linked to an increase in heterochromatic histone marks and a decrease of gene expression. Characteristic combinations of heterochromatic signatures in partially methylated domains are linked to domains of early and middle S-phase and late S-G2 phases of DNA replication.

Conclusions: Partially methylated domains are prominent signatures of long-range epigenomic organization. Integrative analysis identifies them as important general, lineage- and cell type-specific topological features. Changes in partially methylated domains are hallmarks of cell differentiation, with decreased methylation levels and increased heterochromatic marks being linked to enhanced cell proliferation. In combination with broad histone marks, partially methylated domains demarcate distinct domains of late DNA replication.
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http://dx.doi.org/10.1186/s13059-018-1510-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161375PMC
September 2018

Next Generation Sequencing Analysis of Total Small Noncoding RNAs from Low Input RNA from Dried Blood Sampling.

Anal Chem 2018 10 25;90(20):11791-11796. Epub 2018 Sep 25.

Molecular Cell Dynamics, Centre for Human and Molecular Biology , Campus A2.4, Saarland University , 66123 Saarbrücken , Germany.

Circulating miRNAs are favored for biomarker candidates as they can reflect tissue specific miRNA dysregulation in disease contexts. Moreover, they have the additional advantage that they can be monitored in a minimally invasive manner. Blood-borne miRNAs are therefore currently characterized to identify, describe, and validate their potential suitability as biomarkers; however, sampling and as well miRNA detection methods limit these studies in terms of sensitivity but also practicability in clinical, at-home, or low-resource sampling of high-quality circulating RNA samples. We describe here a novel and innovative method of circulating RNA microsampling from minimal volume dried blood samples with direct enrichment for small RNA fractions in combination with ligation free library preparation. We evaluated crucial parameters for efficient library preparation from low RNA inputs of 50 pg for efficient dissection not only of miRNAs but also isomiRs, piRNAs, and lincRNAs. We compared these data to classical microarrays and characterize the technical reproducibility and its sensitivity. We demonstrate and evaluate a method for easy low resource sampling and NGS analysis of circulating RNAs providing a powerful tool for massive cohort and remote patient monitoring.
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http://dx.doi.org/10.1021/acs.analchem.8b03557DOI Listing
October 2018
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