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International audience; Glioblastomas represent approximatively half of all gliomas and are the most deadly and aggressive form. Their therapeutic resistance and tumor relapse rely on a subpopulation of cells that are called Glioma Stem Cells (GSCs). Here, we investigated the role of the long non-coding RNA HOXA-AS2 in GSC biology using descriptive and functional analyses of glioma samples classified according to their isocitrate dehydrogenase (IDH) gene mutation status, and of GSC lines. We found that HOXA-AS2 is overexpressed only in aggressive (IDHwt) glioma and GSC lines. ShRNA-based depletion of HOXA-AS2 in GSCs decreased cell proliferation and altered the expression of several hundreds of genes. Integrative analysis revealed that these expression changes were not associated with changes in DNA methylation or chromatin signatures at the promoter of the majority of genes deregulated following HOXA-AS2 silencing in GSCs, suggesting a post-transcriptional regulation. In addition, transcription factor binding motif enrichment and correlation analyses indicated that HOXA-AS2 affects, directly or indirectly, the expression of key transcription factors implicated in GCS biology, including E2F8, E2F1, STAT1, and ATF3, thus contributing to GCS aggressiveness by promoting their proliferation and modulating the inflammation pathway.

In human, the 39 coding HOX genes and 18 referenced noncoding antisense transcripts are arranged in four genomic clusters named HOXA, B, C, and D. This highly conserved family belongs to the homeobox class of genes that encode transcription factors required for normal development. Therefore, HOX gene deregulation might contribute to the development of many cancer types. Here, we study HOX gene deregulation in adult glioma, a common type of primary brain tumor. We performed extensive molecular analysis of tumor samples, classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found widespread expression of sense and antisense HOX transcripts only in aggressive (IDHwt) glioma samples, although the four HOX clusters displayed DNA hypermethylation. Integrative analysis of expression, DNA methylation, and histone modification signatures along the clusters revealed that HOX gene upregulation relies on canonical and alternative bivalent CpG island promoters that escape hypermethylation. H3K27me3 loss at these promoters emerges as the main cause of widespread HOX gene upregulation in IDHwt glioma cell lines and tumors. Our study provides the first comprehensive description of the epigenetic changes at HOX clusters and their contribution to the transcriptional changes observed in adult glioma. It also identified putative ‘master’ HOX proteins that might contribute to the tumorigenic potential of glioma stem cells., This study describes the epigenetic and transcriptional changes at HOX clusters in aggressive glioma. It shows how DNA hypermethylation and gene overexpression can coexist and suggest that loss of H3K27me3 along the HOX clusters is the main driving force of HOX widespread transcriptional alteration in aggressive glioma samples. It highlights the complexity of HOX gene expression pattern in patients where the usage of alternative promoters contributes to splice variant variability among samples.

// Franck Court 1, 2, 3 , Philippe Arnaud 1, 2, 3 1 CNRS-UMR 6293, Clermont-Ferrand, 63001, France 2 INSERM-U1103, Clermont-Ferrand, 63001, France 3 Universite Clermont Auvergne, GReD Laboratory, Clermont-Ferrand, 63000, France Correspondence to: Philippe Arnaud, email: philippe.arnaud@udamail.fr Franck Court, email: franck.court1@udamail.fr Keywords: cancer epigenetics, bivalent chromatin, DNA methylation, human stem cell chromatin signature Received: September 13, 2016 Accepted: November 23, 2016 Published: December 01, 2016 ABSTRACT CpG islands (CGI) marked by bivalent chromatin in stem cells are believed to be more prone to aberrant DNA methylation in tumor cells. The robustness and genome-wide extent of this instructive program in different cancer types remain to be determined. To address this issue we developed a user-friendly approach to integrate the stem cell chromatin signature in customized DNA methylation analyses. We used publicly available ChIP-sequencing datasets of several human embryonic stem cell (hESC) lines to determine the extent of bivalent chromatin genome-wide. We then created annotated lists of high-confidence bivalent, H3K4me3-only and H3K27me3-only chromatin regions. The main features of bivalent regions included localization in CGI/promoters, depletion in retroelements and enrichment in specific histone modifications, including the poorly characterized H3K23me2 mark. Moreover, bivalent promoters could be classified in three clusters based on PRC2 and PolII complexes occupancy. Genes with bivalent promoters of the PRC2-defined cluster displayed the lowest expression upon differentiation. As proof-of-concept, we assessed the DNA methylation pattern of eight types of tumors and confirmed that aberrant cancer-associated DNA hypermethylation preferentially targets CGI characterized by bivalent chromatin in hESCs. We also found that such aberrant DNA hypermethylation affected particularly bivalent CGI/promoters associated with genes that tend to remain repressed upon differentiation. Strikingly, bivalent CGI were the most affected by aberrant DNA hypermethylation in both CpG Island Methylator Phenotype-positive (CIMP+) and CIMP-negative tumors, suggesting that, besides transcriptional silencing in the pre-tumorigenic cells, the bivalent chromatin signature in hESCs is a key determinant of the instructive program for aberrant DNA methylation.

Summary L1 Chimeric Transcripts (LCTs) are initiated by repeated LINE-1 element antisense promoters and include the L1 5′UTR sequence in antisense orientation followed by the adjacent genomic region. LCTs have been characterized mainly using bioinformatics approaches to query dbEST. To take advantage of NGS data to unravel the transcriptome composition, we developed Chimeric LIne Finder (CLIFinder), a new bioinformatics tool. Using stranded paired-end RNA-seq data, we demonstrated that CLIFinder can identify genome-wide transcribed chimera sequences corresponding to potential LCTs. Moreover, CLIFinder can be adapted to study transcription from other repeat types. Availability and implementation The code is available at: https://github.com/GReD-Clermont/CLIFinder; and for Galaxy users, it is directly accessible in the tool shed at: https://toolshed.g2.bx.psu.edu/view/clifinder/clifinder/. Supplementary information Supplementary data are available at Bioinformatics online.

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation., [Author Summary] Differences in gamete DNA methylation is subject to genome-wide reprogramming during preimplantation development to establish an embryo with an epigenetic state compatible with totipotency. DNA sequences associated with imprinted differentially methylated regions (DMRs) are largely protected from this process, retaining their parent-of-origin epigenetic marks. By comparing the methylation profiles of human oocytes, sperm, blastocysts and various somatic tissues including placenta, we observe hundreds of CpG island sequences that maintain methylation on their maternal allele in blastocysts and placenta indicative of incomplete reprogramming. In some cases this maternal methylation influence transcription of nearby genes, revealing transient imprinting in embryos after genome-activation and in placenta. Strikingly, these placenta-specific DMRs are polymorphic between placenta samples with a minority of samples being robustly unmethylated on both alleles., This work was supported by Spanish Ministerio de Educación y Competitividad (MINECO) (BFU2014-53093 to DM) co-funded with the European Union Regional Development Fund (FEDER). AMS is a recipient of a FPI PhD studentship from MINECO. TMB is supported by ICREA, EMBO YIP 2013, MINECO BFU2014-55090-P (FEDER), BFU2015-7116-ERC and BFU2015-6215-ERCU01 MH106874 grant, Fundacio Zoo Barcelona and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya.

Malignant gliomas are the most common primary brain tumors. Grade III and IV gliomas harboring wild-type IDH1/2 are the most aggressive. In addition to surgery and radiotherapy, concomitant and adjuvant chemotherapy with temozolomide (TMZ) significantly improves overall survival (OS). The methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter is predictive of TMZ response and a prognostic marker of cancer outcome. However, the promoter regions the methylation of which correlates best with survival in aggressive glioma and whether the promoter methylation status predictive value could be refined or improved by other MGMT-associated molecular markers are not precisely known. In a cohort of 87 malignant gliomas treated with radiotherapy and TMZ-based chemotherapy, we retrospectively determined the MGMT promoter methylation status, genotyped single nucleotide polymorphisms (SNPs) in the promoter region and quantified MGMT mRNA expression level. Each of these variables was correlated with each other and with the patients' OS. We found that methylation of the CpG sites within MGMT exon 1 best correlated with OS and MGMT expression levels, and confirmed MGMT methylation as a stronger independent prognostic factor compared to MGMT transcription levels. Our main finding is that the presence of only the A allele at the rs34180180 SNP in the tumor was significantly associated with shorter OS, independently of the MGMT methylation status. In conclusion, in the clinic, rs34180180 SNP genotyping could improve the prognostic value of the MGMT promoter methylation assay in patients with aggressive glioma treated with TMZ., ARC -Fondation ARC pour la Recherche sur le Cancer(EML20120904843)

Chantier qualité GA; International audience; The myogenic regulatory factor Myod and insulin-like growth factor 2 (Igf2) have been shown to interact in vitro during myogenic differentiation. In order to understand how they interact in vivo, we produced double-mutant mice lacking both the Myod and Igf2 genes. Surprisingly, these mice display neonatal lethality due to severe diaphragm atrophy. Alteration of diaphragm muscle development occurs as early as 15.5 days post-coitum in the double-mutant embryos and leads to a defect in the terminal differentiation of muscle progenitor cells. A negative-feedback loop was detected between Myod and Igf2 in embryonic muscles. Igf2 belongs to the imprinted H19-Igf2 locus. Molecular analyses show binding of Myod on a mesodermal enhancer (CS9) of the H19 gene. Chromatin conformation capture experiments reveal direct interaction of CS9 with the H19 promoter, leading to increased H19 expression in the presence of Myod. In turn, the non-coding H19 RNA represses Igf2 expression in trans. In addition, Igf2 also negatively regulates Myod expression, possibly by reducing the expression of the Srf transcription factor, a known Myod activator. In conclusion, Igf2 and Myod are tightly co-regulated in skeletal muscles and act in parallel pathways in the diaphragm, where they affect the progression of myogenic differentiation. Igf2 is therefore an essential player in the formation of a functional diaphragm in the absence of Myod.

The junb gene behaves as an immediate early gene in bacterial lipopolysaccharide (LPS)-stimulated dendritic cells (DCs), where its transient transcriptional activation is necessary for the induction of inflammatory cytokines. junb is a short gene and its transcriptional activation by LPS depends on the binding of NF-kappaB to an enhancer located just downstream of its 3' UTR. Here, we have addressed the mechanisms underlying the transcriptional hyper-reactivity of junb. Using transfection and pharmacological assays to complement chromatin immunoprecipitation analyses addressing the localization of histones, polymerase II, negative elongation factor (NELF)-, DRB sensitivity-inducing factor (DSIF)- and Positive Transcription Factor b complexes, we demonstrate that junb is a RNA Pol II-paused gene where Pol II is loaded in the transcription start site domain but poorly active. Moreover, High salt-Recovered Sequence, chromosome conformation capture (3C)- and gene transfer experiments show that (i) junb is organized in a nuclear chromatin loop bringing into close spatial proximity the upstream promoter region and the downstream enhancer and (ii) this configuration permits immediate Pol II release on the junb body on binding of LPS-activated NF-kappaB to the enhancer. Thus, our work unveils a novel topological framework underlying fast junb transcriptional response in DCs. Moreover, it also points to a novel layer of complexity in the modes of action of NF-kappaB.

Genomic imprinting is a complex epigenetic mechanism of transcriptional control that utilizes DNA methylation and histone modifications to bring about parent-of-origin specific monoallelic expression in mammals. Genes subject to imprinting are often organised in clusters associated with large non-coding RNAs (ncRNAs), some of which have cis-regulatory functions. Here we have undertaken a detailed allelic expression analysis of an imprinted domain on mouse proximal chromosome 10 comprising the paternally expressed Plagl1 gene. We identified three novel Plagl1 transcripts, only one of which contains protein-coding exons. In addition, we characterised two unspliced ncRNAs, Hymai, the mouse orthologue of HYMAI, and Plagl1it (Plagl1 intronic transcript), a transcript located in intron 5 of Plagl1. Imprinted expression of these novel ncRNAs requires DNMT3L-mediated maternal DNA methylation, which is also indispensable for establishing the correct chromatin profile at the Plagl1 DMR. Significantly, the two ncRNAs are retained in the nucleus, consistent with a potential regulatory function at the imprinted domain. Analysis with catRAPID, a protein-ncRNA association prediction algorithm, suggests that Hymai and Plagl1it RNAs both have potentially high affinity for Trithorax chromatin regulators. The two ncRNAs could therefore help to protect the paternal allele from DNA methylation by attracting Trithorax proteins that mediate H3 lysine-4 methylation. This work was supported by Ayuda Merck Serono- Fundación Salud 2000 de Investigación en Endocrinología 2009 (to DM and IIP); Spanish Ministerio de Educación y Ciencia (grant number SAF2008-1578 to DM); Centre National de la Recherche Scientifique ‘‘Projects for International Scientific Cooperation’’ 34622 (to PA and DM); Agence National de la Recherche (to RF); ‘Ligue Contre le Cancer’ (to RF); Ligue contre le Cancer comité Hérault and Association pour la Recherche sur la cancer- ARC nu 4980- (for PA). DM is a Ramon y Cajal research fellow and AGA is funded by a FPU studentship. Research in the Neonatal Unit in Hospital Sant Joan de Déu is partially funded by an unrestricted grant from BebéDue Spain

International audience; The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea.

Parental genomic imprinting at the Igf2/H19 locus is controlled by a methylation-sensitive CTCF insulator that prevents the access of downstream enhancers to the Igf2 gene on the maternal chromosome. However, on the paternal chromosome, it remains unclear whether long-range interactions with the enhancers are restricted to the Igf2 promoters or whether they encompass the entire gene body. Here, using the quantitative chromosome conformation capture assay, we show that, in the mouse liver, the endodermal enhancers have low contact frequencies with the Igf2 promoters but display, on the paternal chromosome, strong interactions with the intragenic differentially methylated regions 1 and 2. Interestingly, we found that enhancers also interact with a so-far poorly characterized intergenic region of the locus that produces a novel imprinted long non-coding transcript that we named the paternally expressed Igf2/H19 intergenic transcript (PIHit) RNA. PIHit is expressed exclusively from the paternal chromosome, contains a novel discrete differentially methylated region in a highly conserved sequence and, surprisingly, does not require an intact ICR/H19 gene region for its imprinting. Altogether, our data reveal a novel imprinted domain in the Igf2/H19 locus and lead us to propose a model for chromatin folding of this locus on the paternal chromosome.

International audience; ABSTRACT: BACKGROUND: Despite its critical role for mammalian gene regulation, the basic structural landscape of chromatin in living cells remains largely unknown within chromosomal territories below the megabase scale. RESULTS: Here, using the 3C-qPCR method, we investigate contact frequencies at high resolution within the interphase chromatin at several mouse loci. We find that, at several gene-rich loci, contact frequencies undergo a periodical modulation (every 90-100 kb) that affects chromatin dynamics over large genomic distances (few hundred kb). Interestingly, this modulation appears to be conserved in human cells and bioinformatic analyses of locus-specific, long-range cis-interactions suggest that it may underlie the dynamics of a significant number of gene-rich domains in mammals, thus contributing to genome evolution. Finally, using an original model derived from polymer physics, we show that this modulation can be understood as a fundamental helix shape that chromatin tends to adopt in gene-rich domains when no significant locus-specific interaction takes place. CONCLUSIONS: Altogether, our work unveils a fundamental aspect of chromatin dynamics in mammals and contributes to a better understanding of genome organization within chromosomal territories.

International audience