Project description:SUMOylation is a posttranslational protein modification which is characterized by the covalent attachment of a small 11kDa protein, called Small Ubiquitin-like MOdifier (SUMO). SUMOylation plays a pivotal role in a multitude of cellular pathways including cellular responses upon DNA damage. Here, we identified multiple proteins which are SUMOylated in U2OS cells in response to ultraviolet light (UV) irradiation and ionizing radiation (IR). We show that the SUMOylation response upon UV irradiation was more pronounced compared to the response upon IR. The major SUMOylation target upon UV-irradiation was the transcription-coupled nucleotide excision repair (TC-NER) protein, Cockayne Syndrome B (CSB). This protein plays an important role in the repair of UV-induced lesions in actively transcribed genes. In a second proteomic approach we identified SUMOylation-dependent and independent protein interactors of the N-terminus of CSB. Here, we uncovered that the affinity of multiple RNA polymerase-associated proteins towards CSB is influenced by SUMOylation. Finally, we set out to identify ubiquitination events upon UV-irradiation which are influenced by the CSA-ubiquitin ligase complex, which is also involved in TC-NER and is closely connected to CSB, because mutations in either CSA or CSB result in the same phenotype, Cockayne syndrome. We found that RPB1, the major subunit of RNA polymerase II, was ubiquitinated in a CSA-dependent manner upon UV which finally led to its degradation.
Project description:In our study we aimed to characterise the effects of p53-induced liver senescence on murine kidneys. We induced liver senescence in 8-12 weeks old male mice by compromising hepatocellular Mdm2 functionality through systemic administration of the AAV8-TBG-Cre viral vector (ΔMdm2Hep mice). We observed transmision of the senescent phenotype from the senescent liver to the genetically unmanipulated kidney. To further elucidate the mechanisms that mediate this transmission phenotype and to characterise its effects on the kidney, we performed single-cell RNA sequencing (scRNA-seq) on the kidneys of 3 ΔMdm2Hep and 3 control mice. We observed the upregulation of several signalling pathways in specific renal cell compartments (e.g. TGFβ in the tubular compartment and JAK-STAT in the mesenchymal compartment). Our results show that liver senescence has a different effect on the renal transcriptome depending on the cellular compartment.
Project description:Unbiased single-cell RNA-sequencing in freshly-dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidneys epithelial cells undergoing both mesenchymal transition and senescence.
Project description:MUC1 kidney disease mouse model generated by knock-in of the human mutated MUC1-fs gene. The mice were treated for 7 days, daily with either vehicle control or BRD4780, a compound showed to be beneficial to disease cells in-vitro. The kidneys were then removed and the bulk RNA was sequenced. The goal of this study was to undersand the effect of the compound on the diseased kidneys and to gain insights to its mechanism of action.
Project description:Endogenous aldehydes induce inter-strand crosslinks (ICL) and DNA-protein crosslinks (DPC). While DNA-repair and aldehyde-clearance systems cope with cellular toxicity, deficiencies in these mechanisms cause genome-instability disorders. The FA-pathway, defective in Fanconi anemia (FA), specifically removes ICL. In contrast, SPRTN, compromised in Ruijs-Aalfs syndrome, eliminates DPC during replication. However, AMeDS patients lacking aldehyde-detoxification display combined features of FA and Cockayne syndrome, associated with transcription-coupled repair (TCR) deficiency, suggesting a novel repair mechanism for aldehyde-induced DNA lesions in active genes. In this report, we demonstrate efficient resolution of aldehyde-induced transcription-blocking lesions by TCR. Mass-spectrometry and DPC-seq identify the TCR complex and additional factors involved in DPC removal and formaldehyde-induced damage tolerance. Notably, TFIIS-dependent cleavage of stalled-RNAPII transcripts exclusively protects against formaldehyde-induced damage. A mouse-model lacking both aldehyde-clearance and TCR pathways confirms endogenous DPC accumulation in transcribed regions. These findings highlight the importance of DPC removal in preventing transcription-roadblocks and contribute to understanding disorders related to aldehyde clearance and TCR deficiencies.
Project description:Bulk RNAseq of kidneys from 5 months-old mice invalidated for Nphp1 which is the main gene responsible for Nephronophtisis, a genetic kidney disease
