The Role Of CSB Regulating The Expression Of NDN In The Pathogenesis Of Cockayne Syndrome

BackgroundCockayne syndrome(CS)is a severe autosomal recessive genetic disorder characterized by photosensitivity,growth failure,neurological abnormalities,and premature aging.Almost all clinical phenotypes resembling classic CS are associated with mutations in the CSB(ERCC6)gene.The CSB gene encodes a DNA-dependent ATPase protein that is involved in regulating DNA damage repair processes and related gene expression regulation.Therefore,CS was initially thought to be a DNA repair deficiency disease,but in fact,the neurological symptoms manifested in CS are much more severe than DNA repair defects.Increasing evidence suggests that transcriptional dysregulation provides a reasonable explanation for many systemic symptoms of CS and emphasizes the link between transcriptional mechanisms and CS neuropathology.MethodThe importance of the CSB gene as a novel "transcription factors" in the pathogenesis of Cockayne syndrome cannot be ignored.To identify potential transcriptional units regulated by CSB protein and explore their relationship with the pathogenesis of Cockayne syndrome,we identified Necdin(NDN)as a potential important target of CSB protein in multiple cell types through RNA-seq(RNA sequencing).To further obtain more evidence supporting the RNA-seq results,we first validated the effect of CSB on NDN at the transcriptional and translational levels using RT-q PCR and Western blot techniques;demonstrated the DNA 5m C methylation status of the NDN gene using Reduced Representation Bisulfite Sequencing(RRBS-seq);and explored the interaction between CSB protein and NDN using chromatin immunoprecipitation(Ch IP-q PCR)technique.We then studied the effect of abnormal NDN expression on Transcription-Coupled Nucleotide Excision Repair(TC-NER)using cloning formation experiments,Cyclobutane pyrimidine dimer(CPD)detection,new RNA synthesis recovery,and EU experiment(5-ethynyl uridine,EU).We also investigated the role of NDN expression in neuronal differentiation and functional maintenance in the neuroblastoma cell line SH-SY5 Y.Finally,we evaluated the effects of CSB deficiency-induced excessive activation of NDN on mouse phenotypes after injecting adeno-associated virus vector(AAV)interfering with CSB and NDN expression in a mouse model and conducting a series of behavioral experiments.ResultsThe results showed that the expression of NDN increased abnormally when CSB gene was deleted,and decreased again when the expression of CSB was up-regulated.Deletion of methylation of imbloted genes leads to upregulation of NDN expression,and CSB protein is involved in histone modification of NDN to affect its expression.In addition,NDN protein is degraded through proteasome pathway in a CSb-dependent manner.However,abnormal expression of NDN had no effect on TC-NER,but overactivation of NDN caused by deletion of CSB interfered with neurogenesis.Interestingly,cortical inhibition of NDN partially "saved" CSB defects in mice.ConclusionsThrough the above research methods,we found that CSB directly binds to NDN and manipulates active histone marks on NDN gene regulatory elements and affects DNA 5m C methylation processes.Surprisingly,CSB deficiency-induced excessive activation of NDN does not interfere with nucleotide excision repair,but greatly affects neuronal differentiation.In addition,inhibiting NDN partially restores the motor neuron defects in the CSB mouse model.Our data not only reveal the cellular mechanisms of CS and indicate future clinical intervention pathways but also confirm the existence of a communication mechanism between CSB protein and NDN,playing an important role in regulating gene transcription.