Biology subjects

Watrin, E.

Publications and source records attributed to Watrin, E..

8 recordsLinked to original sources

bioRxiv2024

TDG orchestrates ATF4-dependent gene transcription during retinoic acid-induced cell fate acquisition

Acquisition of cell identity is associated with a remodeling of the epigenome in part through active DNA demethylation. The T:G mismatch DNA glycosylase (TDG) participates to this process by removing 5-methylcytosines that have been oxidized by Ten-Eleven-Translocation (TET) enzymes. Despite this well-defined molecular function, a comprehensive view of the biological function of TDG is still lacking, especially during cell differentiation. Here, we combined transcriptomic and epigenomic approaches in a Tdg knock-out epiblast stem-like cell model to decipher TDG function in pluripotent cells and their retinoic acid-induced progeny. We determined that TDG occupies a majority of active promoters, a large fraction of which are also engaged by the transcription factor ATF4. Consistently, neural fate commitment upon retinoic acid treatment is associated with a TDG-dependent sustained expression of ATF4-dependent genes, in relation with a TDG-associated nucleosome positioning at promoters. We further evidenced that TDG maintains ATF4 pathway activity by positively regulating the mammalian target of rapamycin complex 1 (mTORC1), favoring neural cell fate commitment. These observations highlight the central role of TDG in cell differentiation and support a model linking metabolic reprogramming to cell fate acquisition.

genomics↗

bioRxiv2022

Inhibition of TGFB pathway prevents short body size and cardiac defects in Nipbl-deficient mice, a mouse model of Cornelia de Lange syndrome

Cornelia de Lange Syndrome (CdLS) largely caused by mutation of the cohesin loader NIPBL is a rare developmental disorder affecting the formation of many organs. Besides a short body size and neurological defects, more than half of CdLS children feature various cardiac malformations. To mimic the disease and test a therapeutic strategy, we generated a C57/Bl6 Nipbl+/- mouse model of the disease. These mice featured a severe delay in both embryonic and postnatal growth. The Nipbl-deficient embryonic and neonatal hearts developed ventricular hypertrophy, aortic and valve defects associated with a persistent truncus arteriosus and a ventricular septal defect. Muscles derived from the second heart field were deficient in the Nipbl haplo-insufficient mouse embryos. The adult hearts then featured a severe aortic senescence phenotype and a stenosis resulting in an increase in aortic flux velocity and persistent left ventricular hypertrophy. Using proteomics and RNA-sequencing in embryos, we identified a dysregulated TGF{beta} pathway in the outflow tract of embryonic hearts as well as the presence of senescent cells as early as in E13.5 Nipbl+/- embryonic hearts, limb primordium cartilage as well as in different post-natal tissues including muscle and brain cortex. Treatment of pregnant Nipbl+/- mice with a TGF{beta}R (ALK5) inhibitor from E9.5 to E13.5 prevented cell -senescence and rescued the cardiac phenotype as well as the body size of mice at birth. Altogether our data revealed that an exacerbated TGF{beta} pathway associated with cell senescence is at the origin of many defects in a CdL mouse model. This druggable pathway opens the path toward a potential preventive and/or therapeutic strategy for post-natal CdLS patients.

developmental biology↗

bioRxiv2022

Specific conformational dynamics of the ATPase head domains and DNA exit gate mediate the Cohesin ATPase cycle

Cohesin is key to eukaryotic genome organization and acts throughout the cell cycle in an ATP- dependent manner. The molecular mechanisms underlying the Cohesin ATPase activity are poorly understood. Here, we have characterized distinct steps of the human Cohesin ATPase cycle and show that the SMC1A and SMC3 ATPase domains undergo specific but concerted structural rearrangements along this cycle. Specifically, while the proximal coiled coil of the SMC1A ATPase domain remains conformationally stable, that of SMC3 displays an intrinsic flexibility. The ATP-dependent formation of the heterodimeric SMC1A/SMC3 ATPase module (engaged state) favours this flexibility, while it is counteracted by binding of NIPBL and DNA (clamped state). Opening of the SMC3/RAD21 interface (open-engaged state) leads to a stiffening of the SMC3 proximal coiled coil that constricts, together with that of SMC1A, the DNA binding chamber of the ATPase module. Our results reveal that the plasticity of the ATP-dependent interface between the SMC1A and SMC3 ATPase domains enables the structural rearrangements occurring between the engaged, clamped and open-engaged states, while keeping the ATP gate shut.

biochemistry↗

bioRxiv2021

The histone methyltransferase NSD3 contributes to cohesin loading during mitotic exit

Sister chromatid cohesion guarantees the correct transmission of chromosomes to daughter cells, and this multi-step process occurs throughout the cell cycle. Loading of the core cohesin complex onto chromatin takes place during mitotic exit, cohesion establishment happens during DNA replication, and the timely removal of cohesin occurs during mitosis. While cohesion establishment and mitotic cohesion dissolution have already been explored, the regulation of cohesin loading is not as well understood. Here, we report that the histone-lysine N-methyltransferase NSD3 is an essential factor in sister chromatid cohesion and mitotic progression, and that this occurs before and not after entry into mitosis. We establish that NSD3 interacts with the cohesin loader complex kollerin (NIPBL/MAU2), and that at mitotic exit it ensures proper levels of both MAU2 and cohesin itself on chromatin. In accordance with this newly described function in cohesin loading, we also show that NSD3 associates with chromatin in early anaphase, prior to the loading recruitment of MAU2 and RAD21, and that it then dissociates from chromatin when prophase begins. Going further, we also demonstrate that of the two NSD3 variants existing in somatic cells, it is the long isoform that is responsible for regulating kollerin and cohesin chromatin-loading, and that this isoforms methyltransferase activity is required for efficient sister chromatid cohesion. Based on these observations, we propose that NSD3-dependent methylation contributes to sister chromatid cohesion by ensuring the proper recruitment of kollerin and thus loading of cohesin.

molecular biology↗

bioRxiv2020

Aurora-A phosphorylates splicing factors and regulates alternative splicing

Aurora-A kinase is well known to regulate progression through mitosis. However, the kinase also performs additional functions that could explain the failure of its inhibitors to be effective in cancer treatments. To identify these functions, we applied a proteomics approach to search for interactors of Aurora-A. We found a large number of proteins involved in pre-mRNA splicing, strongly suggesting an important role for Aurora-A in this biological process. Consistently, we first report the subcellular localization of Aurora-A in nuclear speckles, the storehouse of splicing proteins. We also demonstrate direct interaction of Aurora-A with RRM domain-containing splicing factors such as hnRNP and SR proteins and their phosphorylation in vitro. Further, RNA-sequencing analysis following pharmacological inhibition of Aurora-A resulted in alternative splicing changes corresponding to 505 genes, including genes with functions regulated by Aurora-A kinase. Finally, we report enrichment of RNA motifs within the alternatively spliced regions affected by Aurora-A kinase inhibition which are bound by Aurora-A interacting splicing factors, suggesting that Aurora-A regulates alternative splicing by modulating the activity of these interacting splicing factors. Overall our work identified Aurora-A as a novel splicing kinase and for the first time, describes a broad role of Aurora-A in regulating alternative splicing.

molecular biology↗

bioRxiv2020

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers critically implicated in human pathologies, with CHD6 being one of its least studied members. Here, we discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. We show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS-mutation affects CHD6 protein folding and impairs its ability to recruit co-factors in response to DNA damage or autophagy stimulation. This leads to an accumulation of DNA damage burden and to senescence-like phenotypes. By combining genomics and functional assays, we describe for the first time a molecular mechanism for the chromatin control of autophagic flux and genotoxic stress surveillance that applies broadly to human cell types and explains HSS onset.

cell biology↗

bioRxiv2018

MAU2 and NIPBL variants in Cornelia de Lange syndrome reveal MAU2-independent loading of cohesin and uncover a protective mechanism against early truncating mutations in NIPBL

Cornelia de Lange syndrome (CdLS) is a rare developmental disorder caused by mutations in genes related to the cohesin complex. For its association with chromatin, cohesin depends on a heterodimer formed by NIPBL and MAU2, which interact via their respective N-termini. Variants in NIPBL are the main cause of CdLS and result in NIPBL haploinsufficiency.\n\nUsing CRISPR, we generated cells homozygous for an out-of-frame duplication in NIPBL. Remarkably, alternative translation initiation rescued NIPBL expression in these cells and produced an N-terminally truncated NIPBL that lacks MAU2-interaction domain, causing a dramatic reduction of MAU2 protein levels. Strikingly, this protective mechanism allows nearly normal amounts of cohesin to be loaded onto chromatin in a manner that is independent of functional NIPBL/MAU2 complexes and therefore in contrast to previous findings.\n\nWe also report the first pathogenic variant in MAU2, a deletion of seven amino acids important for wrapping the N-terminus of NIPBL within MAU2. The mutation causes dramatic reduction of MAU2 heterodimerization with NIPBL, hence undermining the stability of both proteins.\n\nOur data confirm NIPBL haploinsufficiency as the major pathogenic mechanism of CdLS and give new insights into the molecular mechanisms responsible for this neurodevelopmental disorder. Our work also unveils an alternative translation-based mechanism that protects cells from out-of-frame variants of NIPBL and that may be of relevance in other genetic conditions.

molecular biology↗

bioRxiv2018

Integrated Clinical and Omics Approach to Rare Diseases: Novel Genes and Oligogenic Inheritance in Holoprosencephaly

PurposeHoloprosencephaly (HPE) is a pathology of forebrain development characterized by high phenotypic and locus heterogeneity. Seventeen genes are known so far in HPE but the understanding of its genetic architecture remains to be refined. Here, we investigated the oligogenic nature of HPE resulting from accumulation of variants in different relevant genes.\n\nMethodsExome data from 29 patients diagnosed with HPE and 51 relatives from 26 unrelated families were analyzed. Standard variant classification approach was improved with a gene prioritization strategy based on clinical ontologies and gene co-expression networks. Clinical phenotyping and exploration of cross-species similarities were further performed on a family-by-family basis.\n\nResultsWe identified 232 rare deleterious variants in HPE patients representing 180 genes significantly associated with key pathways of forebrain development including Sonic Hedgehog (SHH) and Primary Cilia. Oligogenic events were observed in 10 families and involved novel HPE genes including recurrently mutated genes (FAT1, NDST1, COL2A1 and SCUBE2) and genes implicated in cilia function.\n\nConclusionsThis study reports novel HPE-relevant genes and reveals the existence of oligogenic cases resulting from several mutations in SHH-related genes. It also underlines that integrating clinical phenotyping in genetic studies will improve the identification of causal variants in rare disorders.

genetics↗