Transcriptional study of mutations in Mediator complex subunits or his partner NIPBL causing genetic diseases

Le Médiateur (MED) est un complexe multi-protéique dont le principal rôle est de transmettre à la machinerie transcriptionnelle de base les différents signaux fournis par les facteurs fixés sur des séquences d’ADN spécifique , permettant ainsi une régulation fine de l’expression des gènes. Des mutations dans le MED ou ses partenaires, comme NIPBL, sont à l’origine de diverses maladies telles que des malformations congénitales, des troubles neuro développementaux ou des cancers.A partir de cellules provenant de patients portant différentes mutations dans les sous-unités MED12 ou MED17 du MED ou dans NIPBL, nous avons observé une altération du niveau d’expression de certains gènes qui dépend de la localisation de la mutation et de la nature de leur activation. Ces variations de l’expression des gènes sont la conséquence d’un défaut dans la formation du complexe de transcription et du remodelage de la chromatine (modifications post-traductionnelles des histones). Outre une meilleure appréhension du rôle des sous-unités MED12 et MED17 du MED ainsi que NIPBL, sur la transcription des gènes, ma thèse a permis de mieux comprendre l’étiologie des maladies associées à une mutation dans ces protéines.Mediator (MED) is a multi-protein complex whose main role is to convey to basal transcriptional machinery the different signals from factors bound at specific DNA sequences , allowing thus a fine regulation of gene expression. Mutations in MED or its partners, like NIPBL, cause various diseases, such as congenital malformations, neurodevelopmental disorders or cancers. Using cells from patients carrying different mutations in the MED subunits, MED12 or MED17, or in NIPBL, we observed an alteration of the expression of studied genes which depend on the position of the mutation and on the nature of the activation. These variations of gene expression are the consequence of a defect in transcription complex formation, as well as in chromatin remodeling(post-translational histones modifications). In addition to better comprehend the role of the MED subunits MED12 and MED17, and of NIPBL on gene transcription, my thesis helped to better understand the ethiology of the disorders associated with mutations in these proteins

Étude transcriptionnelle des mutations dans le Médiateur ou dans son partenaire NIPBL à l'origine des maladies génétiques

Mediator (MED) is a multi-protein complex whose main role is to convey to basal transcriptional machinery the different signals from factors bound at specific DNA sequences , allowing thus a fine regulation of gene expression. Mutations in MED or its partners, like NIPBL, cause various diseases, such as congenital malformations, neurodevelopmental disorders or cancers. Using cells from patients carrying different mutations in the MED subunits, MED12 or MED17, or in NIPBL, we observed an alteration of the expression of studied genes which depend on the position of the mutation and on the nature of the activation. These variations of gene expression are the consequence of a defect in transcription complex formation, as well as in chromatin remodeling(post-translational histones modifications). In addition to better comprehend the role of the MED subunits MED12 and MED17, and of NIPBL on gene transcription, my thesis helped to better understand the ethiology of the disorders associated with mutations in these proteins.Le Médiateur (MED) est un complexe multi-protéique dont le principal rôle est de transmettre à la machinerie transcriptionnelle de base les différents signaux fournis par les facteurs fixés sur des séquences d’ADN spécifique , permettant ainsi une régulation fine de l’expression des gènes. Des mutations dans le MED ou ses partenaires, comme NIPBL, sont à l’origine de diverses maladies telles que des malformations congénitales, des troubles neuro développementaux ou des cancers.A partir de cellules provenant de patients portant différentes mutations dans les sous-unités MED12 ou MED17 du MED ou dans NIPBL, nous avons observé une altération du niveau d’expression de certains gènes qui dépend de la localisation de la mutation et de la nature de leur activation. Ces variations de l’expression des gènes sont la conséquence d’un défaut dans la formation du complexe de transcription et du remodelage de la chromatine (modifications post-traductionnelles des histones). Outre une meilleure appréhension du rôle des sous-unités MED12 et MED17 du MED ainsi que NIPBL, sur la transcription des gènes, ma thèse a permis de mieux comprendre l’étiologie des maladies associées à une mutation dans ces protéines

Transcriptional study of mutations in Mediator complex subunits or his partner NIPBL causing genetic diseases

Le Médiateur (MED) est un complexe multi-protéique dont le principal rôle est de transmettre à la machinerie transcriptionnelle de base les différents signaux fournis par les facteurs fixés sur des séquences d’ADN spécifique , permettant ainsi une régulation fine de l’expression des gènes. Des mutations dans le MED ou ses partenaires, comme NIPBL, sont à l’origine de diverses maladies telles que des malformations congénitales, des troubles neuro développementaux ou des cancers.A partir de cellules provenant de patients portant différentes mutations dans les sous-unités MED12 ou MED17 du MED ou dans NIPBL, nous avons observé une altération du niveau d’expression de certains gènes qui dépend de la localisation de la mutation et de la nature de leur activation. Ces variations de l’expression des gènes sont la conséquence d’un défaut dans la formation du complexe de transcription et du remodelage de la chromatine (modifications post-traductionnelles des histones). Outre une meilleure appréhension du rôle des sous-unités MED12 et MED17 du MED ainsi que NIPBL, sur la transcription des gènes, ma thèse a permis de mieux comprendre l’étiologie des maladies associées à une mutation dans ces protéines.Mediator (MED) is a multi-protein complex whose main role is to convey to basal transcriptional machinery the different signals from factors bound at specific DNA sequences , allowing thus a fine regulation of gene expression. Mutations in MED or its partners, like NIPBL, cause various diseases, such as congenital malformations, neurodevelopmental disorders or cancers. Using cells from patients carrying different mutations in the MED subunits, MED12 or MED17, or in NIPBL, we observed an alteration of the expression of studied genes which depend on the position of the mutation and on the nature of the activation. These variations of gene expression are the consequence of a defect in transcription complex formation, as well as in chromatin remodeling(post-translational histones modifications). In addition to better comprehend the role of the MED subunits MED12 and MED17, and of NIPBL on gene transcription, my thesis helped to better understand the ethiology of the disorders associated with mutations in these proteins

CSB-Dependent Cyclin-Dependent Kinase 9 Degradation and RNA Polymerase II Phosphorylation during Transcription-Coupled Repair

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A stable XPG protein is required for proper ribosome biogenesis: Insights on the phenotype of combinate Xeroderma Pigmentosum/Cockayne Syndrome patients

International audienceNucleotide Excision Repair is one of the five DNA repair systems. More than 30 proteins are involved in this process, including the seven XP proteins. When mutated, the genes coding for these proteins are provoking the rare disease Xeroderma Pigmentosum , which causes cutaneous defects and a high prevalence of skin cancers in patients. The CSA and CSB proteins are also involved in Nucleotide Excision Repair, and their mutation leads to Cockayne Syndrome, another rare disease, causing dwarfism, neurodegeneration, and ultimately early death, but without high skin cancer incidence. Some mutations of ERCC5 , the gene coding for XPG, may give rise to a combined Xeroderma Pigmentosum and Cockayne Syndrome. A defect in Nucleotide Excision Repair alone cannot explain all these phenotypes. XPG has been located in the nucleolus, where ribosome biogenesis happens. This energy-consuming process starts with the transcription of the ribosomal DNA in a long ribosomal RNA, the pre-rRNA 47S, by RNA Polymerase 1. 47S pre-rRNA undergoes several cleavages and modifications to form three mature products: the ribosomal RNAs 18S, 5.8S and 28S. In the cytoplasm, these three products will enter the ribosomes’ composition, the producers of protein in our cells. Our work aimed to observe ribosome biogenesis in presence of an unstable XPG protein. By working on Xeroderma Pigmentosum/ Cockayne Syndrome cell lines, meaning in the absence of XPG, we uncovered that the binding of UBF, as well as the number of unresolved R-loops, is increased along the ribosomal DNA gene body and flanking regions. Furthermore, ribosomal RNA maturation is impaired, with increased use of alternative pathways of maturation as well as an increase of immature precursors. These defective processes may explain the neurodegeneration observed when the XPG protein is heavily truncated, as ribosomal homeostasis and R-loops resolution are critical for proper neuronal development

XAB2 dynamics during DNA damage-dependent transcription inhibition

International audienceXeroderma Pigmentosum group A-binding protein 2 (XAB2) is a multifunctional protein playing a critical role in distinct cellular processes including transcription, splicing, DNA repair, and messenger RNA export. In this study, we demonstrate that XAB2 is involved specifically and exclusively in Transcription-Coupled Nucleotide Excision Repair (TC-NER) reactions and solely for RNA polymerase 2 (RNAP2)-transcribed genes. Surprisingly, contrary to all the other NER proteins studied so far, XAB2 does not accumulate on the local UV-C damage; on the contrary, it becomes more mobile after damage induction. XAB2 mobility is restored when DNA repair reactions are completed. By scrutinizing from which cellular complex/partner/structure XAB2 is released, we have identified that XAB2 is detached after DNA damage induction from DNA:RNA hybrids, commonly known as R-loops, and from the CSA and XPG proteins. This release contributes to the DNA damage recognition step during TC-NER, as in the absence of XAB2, RNAP2 is blocked longer on UV lesions. Moreover, we also demonstrate that XAB2 has a role in retaining RNAP2 on its substrate without any DNA damage

β-Actin and Nuclear Myosin I are responsible for nucleolar reorganization during DNA Repair

Abstract During DNA Repair, ribosomal DNA and RNA polymerase I (rDNA/RNAP1) are reorganized within the nucleolus. Until now, the proteins and the molecular mechanism governing this reorganisation remained unknown. Here we show that Nuclear Myosin I (NMI) and Nuclear Beta Actin (ACTβ) are essential for the proper reorganisation of the nucleolus, after completion of the DNA Repair reaction. In NMI and ACTβ depleted cells, the rDNA/RNAP1 complex can be displaced at the periphery of the nucleolus after DNA damage but cannot re-enter within the nucleolus after completion of the DNA Repair. Both proteins act concertedly in this process. NMI binds the damaged rDNA at the periphery of the nucleolus, while ACTβ brings the rDNA back within the nucleolus after DNA repair completion. Our results reveal a previously unidentified function for NMI and ACTβ and disclose how these two proteins work in coordination to re-establish the proper rDNA position after DNA repair

Cell-type specific concentration regulation of the basal transcription factor TFIIH in XPBy/y mice model

Background: The basal transcription/repair factor TFIIH is a ten sub-unit complex essential for RNA polymerase II (RNAP2) transcription initiation and DNA repair. In both these processes TFIIH acts as a DNA helix opener, required for promoter escape of RNAP2 in transcription initiation, and to set the stage for strand incision within the nucleotide excision repair (NER) pathway. Methods: We used a knock-in mouse model that we generated and that endogenously expresses a fluorescent version of XPB (XPB-YFP). Using different microscopy, cellular biology and biochemistry approaches we quantified the steady state levels of this protein in different cells, and cells imbedded in tissues. Results: Here we demonstrate, via confocal imaging of ex vivo tissues and cells derived from this mouse model, that TFIIH steady state levels are tightly regulated at the single cell level, thus keeping nuclear TFIIH concentrations remarkably constant in a cell type dependent manner. Moreover, we show that individual cellular TFIIH levels are proportional to the speed of mRNA production, hence to a cell's transcriptional activity, which we can correlate to proliferation status. Importantly, cancer tissue presents a higher TFIIH than normal healthy tissues. Conclusion: This study shows that TFIIH cellular concentration can be used as a bona-fide quantitative marker of transcriptional activity and cellular proliferation

Nucleolar Reorganization After Cellular Stress is Orchestrated by SMN Shuttling Between Nuclear Compartments

ABSTRACT SMA is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein SMN. Within the nucleus, SMN localizes to Cajal bodies (CBs), which have been shown to be associated with nucleoli, nuclear organelles dedicated to the first steps of ribosome biogenesis. The highly organized structure of the nucleolus can be dynamically altered by genotoxic agents. After a genotoxic stress, RNAP1, Fibrillarin (FBL) and nucleolar DNA are exported to the periphery of the nucleolus and once DNA repair is fully completed the organization of the nucleolus is restored. We found that SMN is required for the restoration of nucleolar structure after genotoxic stress. Unexpectedly, during DNA repair, SMN shuttles from the CBs to the nucleolus. This shuttling is important for the nucleolar homeostasis and relies on the presence of Coilin, FBL and the activity of PRMT1