Team
Team manager: Cuvier Olivier
Presentation
We study how chromatin dynamics govern stem cell fate/proliferation through:
a) the control of the accessibility of key genomic features such as gene promoters/bodies or enhancers, notably through nucleosome positioning.
b) the 3D folding of chromatin that plays key roles in the regulation of transcription, DNA replication and repair, which involves 3D clustering or hubs and DNA loops among distinct functional elements.
c) the regulation of noncoding RNAs by specific co-factors like RNA helicases.
d) epigenetic demarcation by chromatin barriers and transcription coupled histone modifications histone methyltransferases of H3K36 (dNSD/Mes-4, dSet2/Hypb etc.), which participate to regulate oncogenes depending on nucleosome positioning and chromatin hubs.
To this end, we employ biochemistry, molecular genetics, cellular biology combined with computational biology of high throughput sequencing data to tackle key aspects of chromatin organization.
Project 1
Enhancer-promoter interactions bring distant enhancers and target promoters in spatial proximity, involving dynamic long-range contacts. We have developed biochemical purification schemes of chromatin insulator complexes allowing to identify co-factors of insulator proteins including the factors participating to form 3D clusters, involving CP190, Cohesin, and Chromator. We also find epigenetic regulators such as NSD/dMes-4 that participate to define euchromatic hubs in 3D, similar to chromatin islands in tumor cells. Finally, we identified new co-factors that regulate the stability of noncoding RNAs including RNA helicases.
In parallel, we develop computational biology approaches including the method entitled Aggregation, allowing to detect long-range contacts even when they are less stable than those formed at TAD borders e.g. as insulator loops and have now made available a package resource (Bioconductor package: HiCAggR). Aggregation allows to probe long-range contacts at very high resolution (< 1 kb) and so to identify the proper gene targets. Furthermore, we can now tackle systematically how transient long-range contacts may impede on fortuitous deregulation of genes beyond TAD borders or across insulators by using and integrating data from our single-cell omics-, microscopic-, and CRISPR- based approaches. Notably, enhancer-promoter transient contacts are under the influence of factors like cohesin that mediate loop extrusion, and insulators and non-coding RNAs. Accumulation of ncRNAs by several means including RNA helicase depletion, may impede 3D looping by regulating the tethering or stability of RNA-binding domain containing insulator proteins and co-factors. Aided by a large set of preliminary data, we investigate these mechanisms combining genomic engineering CRIPR/dCas9 approaches and computational approaches. We analyze how accumulation of ncRNAs regulate insulator and co-factors dynamics and the associated long-range contacts between enhancer and distant target promoters, establishing a linkage between structural and functional regulations.
Project 2
Innate immunity is an ancient form of host defence that has been conserved throughout evolution. Like mammals, flies encode a number of pattern recognition receptors (PRRs) that recognize conserved pathogen motifs called pathogen-associated molecular patterns (PAMPs). In response to a microbial infection and following the activation of Toll or IMD pathways, fat body cells produce antimicrobial peptides that are release into the hemolymph. Thanks to the Sting pathway, drosophila is also able to recognize cyclic dinucleotides, another class of PAMPs. This allows the recognition of both bacteria and virus leading to activate Relish, the Drosophila NF-κB homolog, and the production of antimicrobial peptides. To date, although the cGAS/Sting pathway is involved in the cytosolic recognition of DNA fragments or DNA/RNA hybrids from DNA breaks in humans, and cGLR1, an orthologue of cGAS, is able to detect dsRNA to induce an immune response via dSTING, the function of this pathway in the detection of DNA damage has not yet been studied in Drosophila. After characterizing the presence of DNA breaks and/or R-loops in several tumorigenic contexts, we study the function of the cGAS/Sting pathway, first in detecting DNA damage and then in its involvement in tumor progression.
In humans: In collaboration with the group of M. Benkirane, we sought at deciphering the nuclear function of cGAS/Sting in signaling of foreign viral DNA or in illegitimate DNA to trigger a proper cellular response towards innate immunity. We performed the bioinformatic analyses of such data, re-visiting the chromatin dimensions of cGAS signaling from endogenous sites, up to the induction of IRF3 and the associated innate immunity pathway. Our work aims at the understanding of how host genomes structurally and functionally recognize foreign DNA and possibly interact with viruses, or for an efficient fight by immunological means.
In Drosophila: we study such pathways to characterize the endogenous sensing of RNA-DNA upon oncogenic stress, upon production of ncRNAs and Rloops, in context of the RNA helicases identified in our team, which also serves to process viral DNA. We characterize how their dysfunctions impact transforming adult stem cells, to better understand how the innate immunity pathway protects the genome in such context.
Project 3
Our expertise in computational biology has been integrated in a network of collaborations with teams working on viruses (laboratories of M Benkirane; R. Kiernan; C. Neuveut; D. Durantel; S. Emiliani; E. Bertrand, etc.) along with the development of a novel GDR, the DynaVIR network. Our goal is to decipher if and how chromatin dynamics, either through local chromatin accessibility, or more globally through 3D interactions, can regulate viral integration and/or re-activation e.g. by local nucleosome re-positioning or by 3D looping, e.g. when the viral genome interacts with neighbor genes. We tackled the mechanisms involving viral latency and/or (re-) activation that are crucial to viral-induced cancer cells such as in hepatocarcinoma (Collab: D. Durantel, C. Neuveut). We analyze how virus integration of HBV enacts on the local chromatin interactions, on epigenetic marks, or on higher-order folding at the scales of 3D clusters, TADs or compartments within infected cells from patients. We score changes at the scale of long-range contacts, between the viral genome with the neighbor chromatin landscape, including oncogenes, within specific cellular contexts and of cancer development or not, which can be eventually tested through dCas9/CRAB approaches seeking to unravel causal linkages between virus integration and oncogene induction (Collab: Diogo-Dias, Neuveut, Durantel).
– Heurteau A, Perrois C, Depierre D, Fosseprez O, Humbert J, Schaak S, Cuvier O#. Insulator-based loops mediate the spreading of H3K27me3 over distant micro-domains repressing euchromatin genes. (2020) Genome Biol. doi: 10.1186/s13059-020-02106-z.PMID: 32746892
– Machida S, Depierre D*, Chen HC, Thenin-Houssier S, Petitjean G, Doyen CM, Takak M, Cuvier O#, Benkirane M*. Exploring histone loading on HIV DNA reveals a dynamic nucleosome positioning between unintegrated and integrated viral genome. (2020). Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1913754117.
– Salifou K*, Burnard C*, Basavarajaiah P, Grasso G, Helsmoortel M, Mac V, Depierre D, Franckhauser C, Beyne E, Contreras X, Dejardin J, Rouquier S, Cuvier O, Kiernan R. Chromatin-associated MRN complex protects highly transcribing genes from genomic instability. (2021) Sci Adv. doi: 10.1126/sciadv.abb2947.
– Thenin-Houssier S, Machida S, Jahan C, Bonnet-Madin L, Abbou S, Chen HC, Tesfaye R, Cuvier O, Benkirane M. POLE3 is a repressor of unintegrated HIV-1 DNA required for efficient virus integration and escape from innate immune sensing. (2023) Sci Adv. doi: 10.1126/SciAdv.adh3642.
– Recoules L, Heurteau A, Raynal F, Karasu N, Moutahir F, Bejjani F, Jariel-Encontre I, Cuvier O, Sexton T, Lavigne AC, Bystricky K. The histone variant macroH2A1.1 regulates RNA polymerase II-paused genes within defined interaction landscapes. (2022) J Cell Sci. doi: 10.1242/jcs.259456.
– Depierre D, Perrois C, Schickele N, Lhoumaud P, Abdi-Galab M, Fosseprez O, Heurteau A, Margueron R, Cuvier O#. Chromatin in 3D distinguishes dMes-4/NSD and Hypb/dSet2 in protecting genes from H3K27me3 silencing. (2023) Life Sci Alliance. doi:10.26508/lsa.202302038.
– Contreras X*, Depierre D*, Akkawi C, Srbic M, Helsmoortel M, Nogaret M, LeHars M, Salifou K, Heurteau A, Cuvier O, Kiernan R. PAPγ associates with PAXT nuclear exosome to control the abundance of PROMPT ncRNAs. Nat Commun. (2023). doi:10.1038/s41467-023-42620-9.
– Mancheno-Ferris A, Immarigeon C, Rivero A, Depierre D, Schickele N, Fosseprez O, Chanard N, Aughey G, Lhoumaud P, Anglade J, Southall T, Plaza S, , Payre F, Cuvier O#, Polesello C#. Crosstalk between chromatin and Shavenbaby defines transcriptional output along the Drosophila intestinal stem cell lineage. (2023). iScience. doi: 10.1016/j.isci.2023.108624.
– Schickele, N Tesfaye, Schaak, Martin P. and O. Cuvier. HiCAggR –Bioconductor package https://www.bioconductor.org/packages/release/bioc/html/HicAggR.html
– Fosseprez O, Cuvier O Uncovering the functions and mechanisms of regulatory elements-associated non-coding RNAs. Biochim Biophys Acta Gene Regul Mech. (2024). doi:10.1016/j.bbagrm.2024. 195059.
Affiliation
