ROCK: digital normalization of whole genome sequencing data

International audienceDue to advances in high-throughput sequencing technologies, generating whole genome sequencing (WGS) data with high coverage depth (e.g. ≥ 500×) is now becoming common, especially when dealing with non-eukaryotic genomes. Such high coverage WGS data often fulfills the expectation that most nucleotide positions of the genome are sequenced a sufficient number of times without error. However, performing bioinformatic analyses (e.g. sequencing error correction, whole genome de novo assembly) on such highly redundant data requires substantial running times and memory footprint.To reduce redundancy within a WGS dataset, randomly downsampling high-throughput sequencing reads (HTSR) is trivial. Nevertheless, this first-in-mind strategy is not efficient as it does not minimize variation in sequencing depth, thereby eroding the coverage depth of genome regions that are under-covered (if any). To cope with this problem, a simple greedy algorithm, named digital normalization, was designed to efficiently downsample HTSRs over genome regions that are over-covered (Brown et al., 2012). Given an upper-bound threshold κ > 1, it returns a subset Sκ such that the coverage depth induced by the HTSRs in Sκ is expected to be at most εκ across genome (where ε > 1 is a constant). By discarding highly redundant HTSRs while retaining sufficient and homogeneous coverage depth (≈ εκ), this algorithm strongly decreases both running times and memory required to subsequently analyze WGS data, with often little impact on the expected results (Crusoe et al., 2015). Interestingly, the digital normalization algorithm can be easily enhanced in several ways, so that the final subset contains fewer but more qualitative HTSRs. Unfortunately, these different improvements are scattered in distinct program tools. ROCK (Reducing Over-Covering K-mers) was therefore developed with the key purpose of implementing a fast, accurate and easy-to-use digital normalization procedure. The C++ source code is available under GNU Affero GeneralPublic License v3.0 at https://gitlab.pasteur.fr/vlegrand/ROC

Antisense transcriptional interference mediates condition-specific gene repression in budding yeast

International audiencePervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular an-tisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilized pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asR-NAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chro-matin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption

Loss of Apc Rapidly Impairs DNA Methylation Programs and Cell Fate Decisions in Lgr5 + Intestinal Stem Cells

International audienceColorectal cancer initiation and progression result from the accumulation of genetic and epigenetic alterations. Although aberrant gene expression and DNA methylation profiles are considered hallmarks of colorectal cancer development, the precise timing at which these are produced during tumor establishment remains elusive. Here we investigated the early transcriptional and epigenetic changes induced by adenomatous polyposis coli (Apc) inactivation in intestinal crypts. Hyperactivation of the Wnt pathway via Apc inactivation in crypt base columnar intestinal stem cells (ISC) led to their rapid accumulation driven by an impaired molecular commitment to differentiation, which was associated with discrete alterations in DNA methylation. Importantly, inhibiting the enzymes responsible for de novo DNA methylation restored the responsiveness of Apc-deficient intestinal organoids to stimuli regulating the proliferation-to-differentiation transition in ISC. This work reveals that early DNA methylation changes play critical roles in the establishment of the impaired fate decision program consecutive to Apc loss of function. SIGNIFICANCE: This study demonstrates the functional impact of changes in DNA methylation to determine the colorectal cancer cell phenotype following loss of Apc function

VRK3 depletion induces cell cycle arrest and metabolic reprogramming of pontine diffuse midline glioma - H3K27 altered cells

International audienceWe previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-H3K27M cell fitness. Gene expression studies after VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knockdown. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201

DIPG-43. CLINICAL AND MOLECULAR CHARACTERISTIC OF A NEW SUBTYPE OF DMG, H3K27-ALTERED WITH MAPK-ACTIVATING CO-DRIVER MUTATIONS

International audienceDiffuse midline gliomas (DMG) represent a big challenge in neuro-oncology. These tumors occur more frequently in children and are presently incurable. They are characterized by a K27M substitution in H3.1 or H3.3 histone tail or the overexpression of EZHIP (EZH Inhibitory Protein). These three alterations induce a global loss of trimethylation in H3K27 with a specific epigenic and transcriptomic remodeling. The additional oncogenic events and the clinical behavior are also distinct according to the driver event. Based on these differences, the H3K27-altered DMG is now classified in 4 subtypes by the latest edition of the WHO Classification of CNS tumors. Even with this new subclassification, the H3.3K27M subgroup still appears heterogenous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tend to have a better prognosis. To better study the role of these co-driver alterations that activate the mitogen activated protein kinase (MAPK) signaling, we assembled a large pediatric and adult cohort of H3K27-altered DMG comprising 25 new DMG patients mutated in FGFR1 or BRAFV600E and 37 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylome analysis on this extended cohort. Interestingly, the results show clear differences with other DMG subtypes, including: specific DNA methylation profile, senescence signature, better overall survival (median around 3 years), older age at diagnosis, specific histological and radiological presentations with calcifications or more circumscribed tumors. Additionally, in specific cases, we show that the MAPK-activating mutation occurred subsequently to the histone H3K27M mutation. In conclusion, DMG, H3K27-altered harboring MAPK activating mutations represent a new subtype of DMG also frequent in adults, and deserve further attention with respect to specific therapeutic challenges

Both rare and common genetic variants contribute to autism in the Faroe Islands

Genetic correlates of autism in natives of remote islands A study of the genetic architecture of autism among people living on the remote Faroe Islands highlights the role of both common and rare gene variants to autism. Claire Leblond from the Institut Pasteur in Paris, France, and colleagues profiled the genetics of 357 Faroese individuals, including 36 with autism, 136 of their relatives and 185 non-autistic controls. Similar to the findings of genetic studies of autism from elsewhere, the researchers discovered rare structural variants in known autism-associated genes and a few new candidate genes linked to brain function. However, unlike studies from larger mainland populations, they also showed that inbreeding on these remote islands increased the likelihood of carrying two copies of the point mutations that contribute to autism

DIPG-10. TP53 PATHWAY ALTERATION IS DRIVING RADIORESISTANCE IN DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPG)

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Transcriptomic and epigenetic profiling of ‘diffuse midline gliomas, H3 K27M-mutant’ discriminate two subgroups based on the type of histone H3 mutated and not supratentorial or infratentorial location

Abstract Diffuse midline glioma (DMG), H3 K27M-mutant, is a new entity in the updated WHO classification grouping together diffuse intrinsic pontine gliomas and infiltrating glial neoplasms of the midline harboring the same canonical mutation at the Lysine 27 of the histones H3 tail. Two hundred and fifteen patients younger than 18 years old with centrally-reviewed pediatric high-grade gliomas (pHGG) were included in this study. Comprehensive transcriptomic (n = 140) and methylation (n = 80) profiling was performed depending on the material available, in order to assess the biological uniqueness of this new entity compared to other midline and hemispheric pHGG. Tumor classification based on gene expression (GE) data highlighted the similarity of K27M DMG independently of their location along the midline. T-distributed Stochastic Neighbor Embedding (tSNE) analysis of methylation profiling confirms the discrimination of DMG from other well defined supratentorial tumor subgroups. Patients with diffuse intrinsic pontine gliomas (DIPG) and thalamic DMG exhibited a similarly poor prognosis (11.1 and 10.8 months median overall survival, respectively). Interestingly, H3.1-K27M and H3.3-K27M primary tumor samples could be distinguished based both on their GE and DNA methylation profiles, suggesting that they might arise from a different precursor or from a different epigenetic reorganization. These differences in DNA methylation profiles were conserved in glioma stem-like cell culture models of DIPG which mimicked their corresponding primary tumor. ChIP-seq profiling of H3K27me3 in these models indicate that H3.3-K27M mutated DIPG stem cells exhibit higher levels of H3K27 trimethylation which are correlated with fewer genes expressed by RNAseq. When considering the global distribution of the H3K27me3 mark, we observed that intergenic regions were more trimethylated in the H3.3-K27M mutated cells compared to the H3.1-K27M mutated ones. H3 K27M-mutant DMG represent a homogenous group of neoplasms compared to other pediatric gliomas that could be further separated based on the type of histone H3 variant mutated and their respective epigenetic landscapes. As these characteristics drive different phenotypes, these findings may have important implication for the design of future trials in these specific types of neoplasms

PDTM-36. WHOLE EXOME SEQUENCING (WES) OF DIPG PATIENTS FROM THE BIOMEDE TRIAL REVEALS NEW PROGNOSTIC SUBGROUPS WITH SPECIFIC ONCOGENIC PROGRAMMES

International audienceAbstract BACKGROUND & METHODS. The BIOlogical MEdicines for DIPG Eradication (BIOMEDE) trial is a randomized multi-arm multi-stage program (drop-the-loser adaptive design). The first trial was an open-label phase-II trial comparing three drugs (everolimus, dasatinib, erlotinib) combined with irradiation, allocated according to the presence of their specific targets. All patients underwent a biopsy at diagnosis to confirm the DIPG molecularly and establish the expression of pre-specified biomarkers. Patient samples were explored by WES with a sequencing depth average >100X for the tumor and >60X for the blood. RESULTS. Unsupervised clustering of copy-number-variations (CNV) identified 4 groups corresponding to a new stratification of DIPG: cluster 1 (n=42) with high CNV, cluster 2 (n=23) with chromosome 1q + chromosome 2 gains, cluster 3 (n=9) with low CNV and cluster 4 (n=21) with isolated 1q gain. Clusters 1 had higher hazard-ratio for death than the others (mean HR 1.959, range 1.054–3.643, p< 0.0001, adjusted Cox model). Extensive structural rearrangements/chromotrypsis were significantly more frequent in TP53-mutated samples. Indeed, when stratiftying patients in 4 groups based on type of histone H3 mutated at K27 and the presence of a TP53 pathway alteration the subgroup with TP53 altered pathway had a significantly higher hazard-ratio for death than the others (mean HR 3.450, p=0.0017, adjusted Cox model). Additional drug targets were identified, especially in the DNA repair machinery that could be exploited for new targeted therapies. CONCLUSION. WES at diagnosis was feasible in most patients and brings new prognostic and theranostic informations. This allows a better patients stratification and the development of personalized medicine in DIPG

Table_3_VRK3 depletion induces cell cycle arrest and metabolic reprogramming of pontine diffuse midline glioma - H3K27 altered cells.xlsx

We previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-H3K27M cell fitness. Gene expression studies after VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knockdown. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201.</p