RNF20/40 Interacts With P53 To Regulate The Molecular Mechanism Of Gene Transcription

Tumor suppressor gene mutation is a critical event in the process of cancer.As one of the most important tumor suppressor genes in vivo,p53 mutation frequency in human cancer is as high as 50%.p53 mutation is also the main cause of human hereditary disease li-fraumeni syndrome.The most common mutation form of p53 is missense mutation.The resulting mutant p53 not only lost the anti-cancer function of wild-type p53,but also acquired a series of functions similar to those of oncogenes,which promoted the progress of tumors.Histone ubiquitination,as an important epigenetic modification form,is widely involved in the regulation of chromatin structure,gene transcription,DNA damage response and other physiological processes.Histone ubiquitination leads to errors in chromatin folding and assembly,which affects gene transcription and DNA damage repair,and ultimately leads to genomic instability and tumorigenesis.About 1-5%of ribosomal histone H2B is ubiquitinated,which is the most abundant protein in cells.Lysine monoubiquitination at position 120 of histone H2B（H2BK120ub1）is the most important form of histone ubiquitination in the modification of H2B ubiquitination.H2BK120ub1 is very conserved in evolution,from low eukaryotic yeast to higher mammals.Although H2BK120ub1 has been discovered for more than 30years,its physiological role in the body is still poorly understood.Different from other protein ubiquitination,H2BK120ub1 does not participate in the enzymatic hydrolysis process dependent on proteasome,but mainly plays a role in changing the topological structure of chromatin and mediating other histone modifications so as to coordinate and participate in various physiological processes.In 2003,genetic screening revealed that a Ring domain protein BRE1 in yeast is the E3 ubiquitin ligase that ubiquitinates H2B.Through homologous gene comparison,it was further found that RNF20 and RNF40 are homologous genes of BRE1 in mammals.Subsequent studies have suggested that RNF20 and RNF40 form a protein complex that synergizes with E2ubiquitin transferase RAD6 to catalyze ubiquitination of H2B in vivo.However,relevant studies have also found that RNF20 and RNF40 only have limited ubiquitin ligase activity in vitro,suggesting that there may be a key cofactor that binds to RNF20 and RNF40 to promote its ubiquitin ligase activity.In order to explore the molecular mechanism of regulating H2BK120ub1,a novel functional protein WAC regulating H2BK120ub1 was discovered by tandem affinity purification ofRNF20/RNF40 interacting proteins.form a functional protein complex（RNF20/RNF40/WAC）and promotes the E3 ubiquitin ligase activity of RNF20 and RNF40,thereby regulating H2BK120ub1.There is increasing evidence that H2BK120ub1 dependent on RNF20/RNF40/WAC is closely related to genomic stability and tumorigenesis.First,we constructed a series of point mutations in the p53 DNA-binding domain.These mutations occur frequently in cancer and are closely related to the development of cancer.We identified the interaction between a series of p53 mutants and RNF20/40 by immunoprecipitation and other methods.After further analysis,it was found that the key site of mutation between RNF20/40 and p53 was R282W.Then,we identified the interaction between p53 mutation and MDM2,and found that the key site of the interaction between p53 and MDM2 was R248W.Subsequently,immunofluorescence staining assay was used to identify the effect of p53 and RNF20/40 on p53 nuclear localization due to mutations at the key sites of the interaction between p53 and RNF20/40.The results showed that these mutations of p53 had no effect on its localization.Doxorubicin hydrochloride is known to cause double-stranded DNA breakage,These two key mutated plasmids were transferred into cells of HCT116p53^+/+and HCT116p53^-/-,and the transcription levels of p53 target genes p21,MDM2 and PUMA were analyzed by QPCR after DOX treatment.Then,we induced DNA damage in HCT116,HCT116RNF20^-/-and HCT116RNF40^-/-cells after DOX and VP16 treatment,and analyzed the effect of RNF20/40 on the stability of p53 by Western blot.