Biology subjects

Simon, M.

Publications and source records attributed to Simon, M..

88 records · Page 5Linked to original sources

bioRxiv2020

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

Most sRNA biogenesis mechanisms involve either RNAseIII cleavage or ping-pong amplification by different Piwi proteins harboring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from endogenous remote loci. Our data does not indicate any signatures from ping-pong amplification but Dicer cleavage of long dsRNA. We show that Ptiwi13 and 14 have different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and Ptiwi14 loaded siRNAs show a 5'-U signature. Both Ptiwis show in addition a general preference for Uridine-rich sRNAs along the entire sRNA length. Our data indicates both Ptiwis and 2-O-methylation to contribute to strand selection of Dicer cleaved siRNAs. This unexpected function of two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. As both Ptiwis show differential subcellular localisation, Ptiwi13 in the cytoplasm and Ptiwi14 in the vegetative macronucleus, we conclude that cytosolic and nuclear silencing factors are necessary for efficient chromatin silencing.

genetics↗

bioRxiv2020

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-{beta} signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.

genetics↗

bioRxiv2019

Differential degradation of RNA species by autophagy related pathways in plants

An important function of the plant vacuole is the recycling of the delivered proteins and RNA by autophagy. We provide the first plant vacuolar small RNome by isolation of intact vacuoles from Barley and Arabidopsis, subsequent RNA purification and Next Generation Sequencing. In these vacuolar sRNomes, all types of cellular RNAs were found including those of chloroplast origin, suggesting a bulk-type of RNA transfer to, and breakdown in vacuoles. ATG5 is a major representative of autophagy genes and the vacuolar RNA composition in corresponding knockout plants differed clearly from controls as most chloroplast derived RNA species were missing. Moreover, the read length distribution of RNAs found in ATG5 mutants differed to control samples, indicating altered RNA processing. In contrast, vacuolar RNA length and composition of plants lacking the vacuolar RNase2 (rns2-2), involved in cellular RNA homeostasis, showed minor alterations, only. Our data therefore suggests that mainly autophagy components are responsible for selective transport and targeting of different RNA species into the vacuole for degradation. In addition, mature miRNAs were detected in all vacuolar preparations, however in ATG5 mutants at much lower frequency, indicating a new biological role for vacuolar miRNAs apart from becoming degraded.

plant biology↗

bioRxiv2019

High throughput competitive fluorescence polarization assay reveals functionalredundancy in the S100 protein family

The calcium-binding, vertebrate-specific S100 protein family consists of 20 paralogs in humans (referred as the S100ome), with several clinically important members. To assess their interactome, high-throughput, systematic analysis is indispensable, which allows one to get not only qualitative but quantitative insight into their protein-protein interactions (PPIs). We have chosen an unbiased assay, fluorescence polarization (FP) that revealed a partial functional redundancy when the complete S100ome (n=20) was tested against numerous model partners (n=13). Based on their specificity, the S100ome can be grouped into two distinct classes: promiscuous and orphan. In the first group, members bound to several ligands (>4-5) with comparable high affinity, while in the second one, the paralogs bound only one partner weakly, or no ligand was identified (orphan). Our results demonstrate that in vitro FP assays are highly suitable for quantitative ligand binding studies of selected protein families. Moreover, we provide evidence that PPI-based phenotypic characterization can complement the information obtained from the sequence-based phylogenetic analysis of the S100ome, an evolutionary young protein family.\n\nAuthor summaryFunctional similarity among a protein family can be essential in order to understand proteomic data, to find biomarkers, or in inhibitor design. Proteins with similar functions can compensate the loss-of-function of the others, their expression can co-vary under pathological conditions, and simultaneous targeting can lead to better results in the clinics. To investigate this property one can use sequence-based approaches. However, this path can be difficult. In the case of the vertebrate specific, evolutionary young, S100 family, phylogenetic approaches lead to ambiguous results. To overcome this problem, we applied a high-throughput biochemical approach to experimentally measure the binding affinities of a large number of S100 interactions. We performed unbiased fluorescence polarization assay, involving the complete human S100ome (20 paralogs) and 13 known interaction partners. We used this measured 20x13 (260) protein-protein interaction array to reveal the functional relationships within the family. Our work provide a general framework for studies focusing on phenotype-based domain classification.

biochemistry↗

bioRxiv2019

Tolerance of nonsynonymous variation is closely correlated between human and mouse orthologues

Genic constraint describes how tolerant a gene is of nonsynonymous variation before it is removed from the population by negative selection. Here, we provide the first estimates of intraspecific constraint for mouse genes genome-wide, and show constraint is positively correlated between human and mouse orthologues (r = 0.806). We assess the relationships between mouse gene constraint and knockout phenotypes, showing gene constraint is positively associated with pleiotropy (ie an increased number of phenotype associations (R2 = 0.65)), in addition to an enrichment in lethal, developmental, and craniofacial knockout phenotypes amongst the most constrained genes. Finally, we show mouse constraint can be used to predict human genes associated with Mendelian disease, and is positively correlated with an increase in the number of known pathogenic variants in the human orthologue (R2 = 0.23). Our metrics of mouse and human constraint are available to inform future research using mouse models.

genomics↗

bioRxiv2018

Exploration Of The Timeliness Of ICD Codes In Administrative Databases: A Nationwide Study

INTRODUCTIONThe ICD codes are ubiquitously available in hospital information systems and have been used in a number of areas such as epidemiology, phenotype-genotype association mining, surveillance of the use of drugs and medical devices and health care evaluation. We aimed to analyze the timeliness of the 3-character ICD-10 codes collected in the French national hospital discharge summary database between 2008 and 2017 and we classified the codes according to their evolution.\n\nMATERIAL AND METHODSWe extracted all 3-character ICD-10 codes from all French hospital discharge summaries between 2008 and 2017. For each code and by the month, we computed a relative frequency; we also computed the overall amplitude of the study period. Temporal clustering, according to the SAX representation, was performed to classify the main evolution patterns.\n\nRESULTSWe extracted 238,334,751 encounters corresponding to 56,621,773 distinct patients. 1,006 ICD codes presented a variation of the relative amplitude of frequencies lower than 50%, 510 codes between 50% and 100% and 521 greater than 100%. Out of the 2,037 codes included in the study, we kept the 1,758 for the temporal clustering. Four clusters were identified, including a global increase and a global decrease patterns.\n\nDISCUSSIONThe overall results showed a strong instability (i.e. large variation of frequency over time) of the use of ICD codes over time, with an important variation of the relative amplitude of the frequencies. We distinguished between external factors due to changes in billing, organization, policy or regulation and intrinsic factors due to epidemiological phenomenon. The detailed analysis of profiles show that the same cluster can contain profiles influenced by intrinsic epidemiological or external factors or both. Additional knowledge and sources are probably required to determine automatically the origin of the profile.

epidemiology↗

bioRxiv2018

B cells sustain inflammation and improve survival in human melanoma

Tumor associated inflammation is one of important predictors of response to immune checkpoint blockade. Understanding molecular processes that regulate tumor inflammation is key to improve the efficacy of checkpoint blockade. Established mechanisms that underlie therapy response and resistance have centered on anti-tumor T cell responses.\n\nWe show that tumor-associated B cells are vital to T cell functions. They promote recruitment of CD8+ T cells through plasmablast-like cells with expression of pro- and anti-inflammatory factors. Plasmablast-like cells are associated with improved survival of patients with metastatic melanoma and their response to checkpoint blockade. Plasmablast-like B cells express chemokines for T cell-attraction. Depletion of tumor-associated B cells by anti-CD20 immunotherapy of metastatic melanoma patients causes a remarkable decrease in tumor CD8+ T cells. These findings indicate that tumor-associated B cells orchestrate and sustain tumor inflammation, recruit CD8+ T effector cells and are key to therapeutic response and patients overall survival.

cancer biology↗

bioRxiv2018

Inhibition of retrotransposition improves health and extends lifespan of SIRT6 knockout mice

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6 deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA which triggers massive type I interferon response via activation of cGAS. Remarkably, nucleoside reverse transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number and type I interferons were elevated in the wild type aged mice. As sterile inflammation is a hallmark of aging we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.\n\nHighlightsO_LISIRT6 KO mice accumulate L1 cDNA triggering type I interferon response via cGAS pathway\nC_LIO_LIWild type aged mice accumulate L1 cDNA and display type I interferon response\nC_LIO_LIReverse transcriptase inhibitors rescue type I interferon response and DNA damage\nC_LIO_LIReverse transcriptase inhibitors extend lifespan and improve health of SIRT6 KO mice\nC_LI

molecular biology↗

bioRxiv2018

MYCN-induced metabolic rewiring creates novel therapeutic vulnerabilities in neuroblastoma

MYCN is a transcription factor that is aberrantly expressed in many tumor types and is often correlated with poor patient prognosis. Recently, several lines of evidence pointed to the fact that oncogenic activation of MYC family proteins is concomitant with reprogramming of tumor cells to cope with an enhanced need for metabolites during cell growth. These adaptions are driven by the ability of MYC proteins to act as transcriptional amplifiers in a tissue-of-origin specific manner. Here, we describe the effects of MYCN overexpression on metabolic reprogramming in neuroblastoma cells. Ectopic expression of MYCN induced a glycolytic switch that was concomitant with enhanced sensitivity towards 2-deoxyglucose, an inhibitor of glycolysis. Moreover, global metabolic profiling revealed extensive alterations in the cellular metabolome resulting from overexpression of MYCN. Limited supply with either of the two main carbon sources, glucose or glutamine, resulted in distinct shifts in steady-state metabolite levels and significant changes in glutathione metabolism. Interestingly, interference with glutamine-glutamate conversion preferentially blocked proliferation of MYCN overexpressing cells, when glutamine levels were reduced. Thus, our study uncovered MYCN induction and nutrient levels as important metabolic master switches in neuroblastoma cells and identified critical nodes that restrict tumor cell proliferation.

cancer biology↗

bioRxiv2018

Rewiring of RSK-PDZ interactions by linear motif phosphorylation

Protein phosphorylation is a key regulator of protein-protein interactions. How does the interactome of a protein change during extracellular stimulations? While many individual examples of phosphorylation-regulated interactions were described previously, studies addressing the interactome changes induced by a particular phosphorylation event remain scarce. Here, we try to answer this question, by focusing on interactions between a phosphorylable PDZ-binding linear motif and the entire complement of human PDZ domains. Using a combination of in vitro quantitative techniques and cell-based approaches, we demonstrate that the activation of the mitotic effector kinase RSK1 causes dramatic changes in its connectivity with PDZ domain containing proteins. These changes consist of modulations of the binding affinity of numerous interactions, rather than on/off switching of a few interactions. Our results highlight the previously unappreciated role of phosphorylation in the complex and subtle rewiring of large numbers of protein-protein interactions.

systems biology↗

bioRxiv2018

Objective Evaluation of Multiple Sclerosis Lesion Segmentation using a Data Management and Processing Infrastructure

We present a study of multiple sclerosis segmentation algorithms conducted at the international MICCAI 2016 challenge. This challenge was operated using a new open-science computing infrastructure. This allowed for the automatic and independent evaluation of a large range of algorithms in a fair and completely automatic manner. This computing infrastructure was used to evaluate thirteen methods of MS lesions segmentation, exploring a broad range of state-of-the-art algorithms, against a high-quality database of 53 MS cases coming from four centers following a common definition of the acquisition protocol. Each case was annotated manually by an unprecedented number of seven different experts. Results of the challenge highlighted that automatic algorithms, including the recent machine learning methods (random forests, deep learning, ...), are still trailing human expertise on both detection and delineation criteria. In addition, we demonstrate that computing a statistically robust consensus of the algorithms performs closer to human expertise on one score (segmentation) although still trailing on detection scores.

neuroscience↗

bioRxiv2018

Antisense lncRNA transcription drives stochastic Protocadherin α promoter choice

Stochastic and combinatorial activation of clustered Protocadherin (Pcdh) , {beta}, and {gamma} gene promoters generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here we show that Pcdh promoter choice requires transcription of a long noncoding RNA (lncRNA) initiated from newly identified promoters located in the protein coding sequence of each Pcdh exon. Antisense transcription of the lncRNA through the sense promoter results in its activation and in DNA demethylation of the binding sites for the CCCTC-binding protein, CTCF, located in close proximity to both sense and antisense promoters. Increased CTCF binding promotes the assembly of long-range DNA contacts between the activated promoter and a neuron-specific enhancer, thus locking in the epigenetic state of the stochastically chosen Pcdh promoter. Examination of this hierarchical molecular mechanism in differentiating olfactory sensory neurons, suggests that antisense Pcdh transcription is a key prerequisite for stochastic Pcdh promoter choice in vivo.

genomics↗

bioRxiv2018

DAF-16/Foxo suppresses the transgenerational sterility of prg-1 piRNA mutants via a systemic small RNA pathway

Mutation of the daf-2 insulin/IGF-1 receptor activates the DAF-16/Foxo transcription factor to suppress the transgenerational sterility phenotype of prg-1/piRNA mutants that are deficient for piRNA-mediated genome silencing. As with PRG-1/piRNAs, mutations in the nuclear RNA interference gene nrde-1 compromised germ cell immortality, but deficiency for daf-2 did not suppress the transgenerational sterility of nrde-1 or nrde-4 single mutants or of prg-1; nrde-4 or prg-1; hrde-1 double mutants. NRDE-1 and NRDE-4 promote transcriptional silencing in somatic cells via the nuclear Argonaute protein NRDE-3, which was dispensable for germ cell immortality. However, daf-2 deficiency failed to promote germ cell immortality in prg-1; nrde-3 mutants. Consistently, we found that DAF-16 activity in somatic cells suppressed the transgenerational sterility of prg-1 mutants via the SID-1 dsRNA transmembrane channel that promotes systemic RNAi as well as Dicer, the dsRNA binding protein RDE-4 and the RDRP RRF-3. We conclude that DAF-16 activates a cell-non-autonomous systemic RNAi pathway that promotes small RNA-mediated genome silencing in germ cells to suppress loss of the genomic immune surveillance factor Piwi/PRG-1. Author SummarySmall RNAs can promote genome silencing. The Argonaute protein Piwi interacts with thousands of small RNAs termed piRNAs in germ cells to suppress expression of transposons and foreign genetic elements. However, the Piwi silencing system may be commonly targeted by viral or transposon genomic parasites that seek to suppress the endogenous defences against their expression and replication. Activation of the DAF-16 stress response pathway promotes adult longevity and can also abolish the transgenerational sterility of C. elegans Piwi mutants. We found that DAF-16 accomplishes this by activating a somatic small RNA pathway where small RNAs are initially produced in the soma and are then transported into the germline to suppress expression of a toxic genetic locus in Piwi mutants. Thus, the DAF-16 stress response pathway activates a systemic small RNA cascade to suppress defects in the Piwi/piRNA genome silencing system.

genetics↗

bioRxiv2018

Deficiency for Piwi results in transmission of a heritable stress that promotes longevity via DAF-16/Foxo

The C. elegans Argonaute protein PRG-1/Piwi and associated piRNAs protect metazoan genomes by silencing transposons and other types of foreign DNA. As prg-1 mutants are propagated, their fertility deteriorates prior to the onset of a reproductive arrest phenotype that resembles a starvation-induced stress response. We found that late-generation prg-1 mutants with substantially reduced fertility were long-lived, whereas early- or mid-generation prg-1 mutants had normal lifespans. Loss of the stress response transcription factor DAF-16 caused mid- or late-generation prg-1 mutants to live very short lives, whereas overexpression of DAF-16 enabled both mid- and late-generation prg-1 mutants to live long. Cytoplasmic P-bodies that respond to stress increased in long-lived late-generation prg-1 mutants and were transmitted to F1 but not F2 cross-progeny. Moreover, moderate levels of heritable stress shorten late-generation prg-1 mutant longevity when DAF-16 or P bodies are deficient. Together, these results suggest that the longevity of late-generation prg-1 mutants is a hormetic stress response. However, dauer larvae that occur in response to stress were not observed in late-generation prg-1 mutants. Small germ cell nucleoli that depended on germline DAF-16 were present in late-generation prg-1 mutants but were not necessary for their longevity. We propose that prg-1 mutant germ cells transmit a form of heritable stress, high levels of which promote longevity and strongly reduce fertility. The heritable stress transmitted by PRG-1/Piwi mutant germ cells may be generally relevant to epigenetic inheritance of longevity. Core message of paperprg-1/Piwi mutants with strongly reduced fertility live long and longevity is transmitted for one generation to F1 cross progeny. Stress granules are increased and germ cell nucleoli are small for long-lived Piwi mutants and their F1 progeny. Loss of daf-16 stress response transcription factor or dcap-1 P body protein causes very short life for worms when prg-1 mutant fertility is moderately reduced, whereas moderate fertility is sufficient to extend lifespan when somatic DAF-16 is overexpressed. We propose that prg-1 mutant germ cells transmit a heritable epigenetic factor that is stressful and elicits two hormetic stress responses: reproductive arrest and longevity.

genetics↗

bioRxiv2018

Automated analysis of small RNA datasets with RAPID

Summary: Understanding the role of short-interfering RNA (siRNA) in diverse biological processes is of current interest and often approached through small RNA sequencing. However, analysis of these datasets is difficult due to the complexity of biological RNA processing pathways, which differ between species. Several properties like strand specificity, length distribution, and distribution of soft-clipped bases are few parameters known to guide researchers in understanding the role of siRNAs. We present RAPID, a generic eukaryotic siRNA analysis pipeline, which captures information inherent in the datasets and automatically produces numerous visualizations as user-friendly HTML reports, covering multiple categories required for siRNA analysis. RAPID also facilitates an automated comparison of multiple datasets, with one of the normalization techniques dedicated for siRNA knockdown analysis, and integrates differential expression analysis using DESeq2. RAPID is available under MIT license at https://github.com/SchulzLab/RAPID. We recommend using it as a conda environment available from https://anaconda.org/bioconda/rapid.

bioinformatics↗

bioRxiv2018

The conserved phosphatase GSP-2/PP1 promotes germline immortality via small RNA-mediated genome silencing during meiosis

Genomic silencing can promote germ cell immortality, or transgenerational maintenance of the germ line, via mechanisms that may occur during mitosis or meiosis. Here we report that the gsp-2 PP1/Glc7 phosphatase promotes germ cell immortality. We identified a separation-of-function allele of C. elegans GSP-2 that caused a meiosis-specific chromosome segregation defect and defects in transgenerational small RNA-induced genome silencing. GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. Sterile gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents and histone phosphorylation defects in oocytes, similar to small RNA genome silencing mutants. Epistasis and RNA analysis suggested that GSP-2 functions downstream of small RNAs. We conclude that a meiosis-specific function of GSP-2/LAB-1 ties small RNA-mediated silencing of the epigenome to germ cell immortality. Given that hemizygous genetic elements can drive transgenerational epigenomic silencing, and given that LAB-1 promotes pairing of homologous chromosomes and localizes to the interface between homologous chromosomes during pachytene, we suggest that discontinuities at this interface could promote nuclear silencing in a manner that depends on GSP-2.\n\nAuthor SummaryThe germ line of an organism is considered immortal in its capacity to give rise to an unlimited number of future generations. To protect the integrity of the germ line, mechanisms act to suppress the accumulation of transgenerational damage to the genome or epigenome. Loss of germ cell immortality can result from mutations that disrupt the small RNA-mediated silencing pathway that helps to protect the integrity of the epigenome. Here we report for the first time that the C. elegans protein phosphatase GSP-2 that promotes core chromosome biology functions during meiosis is also required for germ cell immortality. Specifically, we identified a partial loss of function allele of gsp-2 that exhibits defects in meiotic chromosome segregation and is also dysfunctional for transgenerational small RNA-mediated genome silencing. Our results are consistent with a known role of Drosophila Protein Phosphatase 1 in heterochromatin silencing, and point to a meiotic phosphatase function that is relevant to germ cell immortality, conceivably related to its roles in chromosome pairing or sister chromatid cohesion.

genetics↗