| BackgroundDNA double-strand breaks(DSBs)are one of the most serious damages to cell survival among all types of DNA damages.The major repair mechanisms for DSBs in eukaryotic cells are homologous recombination(HR)and nonhomologous end-joining(NHEJ).The NHEJ repair process is not limited by the cell cycle,and DSBs can be repaired quickly in a short period of time.Therefore,the NHEJ repair mechanisms occupies the leading and priority position in DSBs repair.Defects in NHEJ core components such as DNA-PKcs,Lig IV,and XLF can cause abnormalities in NHEJ repair mechanisms and increase tumour susceptibility.Studies on Dna-pkcs-/-mice found that the deletion of DNA-PKcs significantly promoted the transformation of normal tissues of mice into precancerous lesions.Although it was found that low expression of DNA-PKcs can promote the transformation of normal tissues into precancerous lesions and increase the susceptibility of tumours,the underlying mechanism regulating DNA-PKcs protein level in normal tissues or cells is not defined.CUL4A(Cullin 4A)protein is a member of the E3 ubiquitin ligase family and forms a CUL4A E3 ubiquitin ligase complex with DDB1.CUL4A regulates a variety of proteins through a ubiquitination mechanism and plays a key role in various biological processes.Studies have reported that CUL4A participates in the regulation of DNA damage repair caused by ultraviolet(UV).CUL4A modifies H2A,H3.and H4 through ubiquitination,regulates the replication of XPC to recruit DNA damage,and thus inhibits UV-induced DNA damage(Nucleotide excision repair)repair mechanisms.Recent studies have found that when DSBs occur in cells,CUL4A was recruited to the DSBs of DNA and participated in the DSB damage repair process through ubiquitinating target proteins.However,the specific function of CUL4A in the NHEJ repair process is unclear.This study found that when DSBs occur in normal cells,CUL4A negatively regulates DNA-PKcs protein level through DTL(Denticleless E3 ubiquitin-protein ligase homolog,also known as DCAF2,L2DTL or CDT2)to inhibit NHEJ repair efficiency,eventually leading to genomic instability and malignant transformation of normal cells.This study can not only clarify the new biological functions of CUL4A in the process of malignant transformation of normal cells,but also provide theoretical basis and potential treatment options for exploring the use of CUL4A as a target for pre-cancerous diagnosis and treatment.Part ?.CUL4A inhibits NHEJ repair by negatively regulating DNA-PKcs[Object]After X-RAY-induced DSBs in cells,mass spectrometry was used to analyse and identify proteins interacting with CUL4A.Functional enrichment analysis was performed on the proteins binding to CUL4A.It is clear that CUL4A participates in the DNA repair process by combining with DNA-PKcs,and further explores the effect of CUL4A on DNA-PKcs regulation and the efficiency of NHEJ repair.[Methods]1.HEK293T was transiently transfected with HA-CUL4A,X-RAY irradiated the cells to induce DSBs,and mass spectrometry was used to identify the proteins that binds to CUL4 A,and functional enrichment analysis of the enriched proteins was perform.2.The CUL4A lentivirus was used to infect breast or pancreatic epithelial cells to construct cell lines that overexpressed CUL4A.3.Ad-Cre adenovirus was used to infect LSL-Cul4aFlox/Flox embryonic fibroblasts(Mouse embryonic fibroblasts,MEFs)to construct Cre;LSL-Cul4aFlox/Frox MEFs overexpressing CUL4A.4.Breast or pancreatic epithelial cells overexpressing CUL4A and Cre;LSL-Cul4aFlox/Flox MEFs were used to induced DSBs by Bleomycin.Neutral comet assay,Western blot and immunofluorescence experiments were used to detect the effect of CUL4A on the accumulation of DSB damage and the level of y-H2AX protein.5.MCF-10A cells containing DNA repair reporter(DRR)was used to transfect I-Scel and CUL4A,and the number of GFP-positive cells was detected by flow cytometry.6.Breast and pancreatic epithelial cells and Cre;LSL-Cul4aFlox/Flox MEFs that stably overexpress or knock out CUL4A were used to induce USBs by Bleomycin.Western blot was used to detect the effects of CUL4A on DNA-PKcs and KU70/80 protein levels.[Results]1.Mass spectrometry identified the proteins that binds to CUL4A and found that CUL4A is involved in the DNA repair process.2.The result of co-immunoprecipitation revealed that CUL4A interacted with DNA-PKcs.3.Successfully constructed breast and pancreatic epithelial cell lines and Cre;LSL-Cul4aFlox/Flox MEFs overexpressing CUL4A.4.Neutral comet assay results showed that overexpression of CUL4A significantly increased the accumulation of DSBs damage in breast and pancreatic epithelial cells and Cre;LSL-Cul4aFlox/Flox MEFs.5.Western blot and immunofluorescence experiments showed that CUL4A significantly increased the expression level of ?-H2AX protein and the proportion of y-H2AX positive cells.6.The MCF-10A cell line stably expressing DRR was successfully constructed,and the results of flow cytometry showed that overexpression of CUL4A significantly inhibited the repair efficiency of NHEJ.7.Western blot results showed that overexpression of CUL4A negatively regulated the level of DNA-PKcs protein.[Conclusions]1.CUL4A binds to DNA-PKcs and participates in the DNA repair process.2.CUL4A negatively regulates DNA-PKcs protein level.inhibits the repair efficiency of NHEJ,and increases the damage accumulation of DSBs in normal cells.Part ?.DTL mediates CUL4A to degrade DNA-PKcs and increase DSB[Object]The results of our Part I study showed that CUL4A can negatively regulate the level of DNA-PKcs protein,inhibit the repair efficiency of NHEJ,and increase the accumulation of cell DSBs damage.Therefore,this section will explore the mechanism through which CUL4A negatively regulates the level of DNA-PKcs protein,inhibits the repair efficiency of NHEJ and increases the accumulation of DSBs damage.[Methods]1.HEK293T cells were transiently transfected with HA-DTL,and mass spectrometry was used to identify the proteins that interact with DTL.2.HEK293T cells were transiently transfected with Myc-CUL4A,and co-immunoprecipitation and Western blot were used to detect whether DTL interacted with the CUL4A/DNA-PKcs complex.3.HEK293T cells were transiently transfected with Myc-CUL4A/DTL shRNA or Flag-DTL/CUL4A shRNA,and immunoprecipitation and Western blot were used to detect CUL4A through DTL to recognize DNA-PKcs.4.HEK293T cells were transiently transfected with DTL point mutants(DTL R171H and DTL R246H),and co-immunoprecipitation was used to detect the effect of the interaction between DTL and CUL4A or DNA-PKcs.5.HEK293T cells were transiently transfected with Myc-CUL4A and Flag-DTL,and Western blot and qRT-PCR were used to detect changes in the protein and mRNA levels of DNA-PKcs by CUL4A or DTL.6.Cycloheximide and proteasome inhibitors were used to treat CUL4A or DTL overexpressing cells,and Western blot was used to detect the effect of CUL4A/DTL on the stability of DNA-PKcs protein.7.HEK293T cells were transiently transfected with Myc-CUL4A and Flag-DTL,and immunoprecipitation was used to detect the effect of CUL4A or DTL on the ubiquitination of DNA-PKcs.8.DTL-overexpressing breast and pancreatic epithelial cells,Cre;DtlFlox/Flox MEFs and DTL knocking out breast epithelial cells were used to induce DSBs by Bleomycin and Western blot to detect the effect of DTL on DNA-PKcs and KU70/80 protein levels.9.Breast epithelial cells that stably overexpressing DTL and DTL point mutants were used to induce DSBs by X-RAY and Western blot was used to detect the effect of DTL point mutation on DNA-PKcs and KU70/80 protein levels.10.MCF-10A cells containing DRR were used to detect the effect of DTL on the repair efficiency of NHEJ by flow cytometry.11.DTL-overexpressing breast or pancreatic epithelial cells and MEFs were used to induce DSBs by Bleomycin,Western blot,immunofluorescence and neutral comet assays were used to detect the effect of DTL on the level of ?-H2AX protein and the accumulation of DSB damage.12.After induction of DSBs in breast epithelial cells,nuclear/plasma protein separation experiment and Western blot experiments were used to detect the protein expression,intracellular distribution of CUL4A and DTL,and the co-localization of CUL4A or DTL and ?-H2AX/DNA-PKcs.13.Chromatin separation experiment was used to detect the accumulation of CUL4A,DTL,DNA-PKcs and KU70/80 proteins on chromatin after DSBs occurred in epithelial cells.[Results]1.Mass spectrometry analysis and immunoprecipitation showed that DTL also combined with DNA-PKcs.2.The result of co-immunoprecipitation found that DTL was present in the complex of CUL4A and DNA-PKcs.3.Co-immunoprecipitation results showed that CUL4A bound to DNA-PKcs through DTL.4.DTL R246 regulated the interaction of DTL with CUL4A-DDB1 complex.5.Co-immunoprecipitation results showed that CUL4A degraded DNA-PKcs through DTL ubiquitin-proteasome pathway.6.DTL R246 regulated the ubiquitination level of DNA-PKcs protein by CUL4ADTL.7.CUL4ADTL ubiquitinated DNA-PKcs through K48 ubiquitin connection.8.Western blot results showed that DTL negatively regulated the level of DNA-PKcs protein.9.Western blot results showed that DTL R246 was involved in the negative regulation of DNA-PKcs protein level.10.The results of flow cytometry showed that DTL significantly inhibited the repair efficiency of NHEJ,and the DTL R246H mutation could reverse the inhibitory effect of DTL on the repair efficiency of NHEJ.11.The results of the neutral comet experiment found that DTL significantly increased the damage accumulation of DSBs in breast and pancreatic epithelial cells and Cre;DtIFlox/Flox MFEs.12.Western blot and immunofluorescence experiments showed that DTL significantly increased the level of y-H2AX protein and the proportion of ?-H2AX positive cells.13.Western blot results showed that the expression levels of CUL4A and DTL proteins increased significantly after DSBs occurred in the cells,and they tended to accumulate in the nucleus.14.Immunofluorescence staining revealed that after DSBs occurred in the cells,both CUL4A and DTL could be recruited to DSBs and co-localized with y-H2AX.15.Immunofluorescence staining revealed that the binding site of CUL4ADTL and DNA-PKcs was located in the nucleus,and the binding of CUL4ADTL and DNA-PKcs increased after DSBs occurred in the cells.16.The results of co-immunoprecipitation found that the ubiquitination of DNA-PKcs by CUL4ADTL also occurred in the nucleus,and the level of DNA-PKcs ubiquitination by CUL4A and DTL increased significantly after DSBs occurred in the cell.17.Chromatin separation experiments found that after DSBs occurred in breast epithelial cells,overexpression of CUL4A or DTL significantly reduced the accumulation of DNA-PKcs protein on chromatin[Conclusions]1.CUL4A specifically recognizes and ubiquitinates DNA-PKcs protein through DTL.2.DTL negatively regulates DNA-PKcs to inhibit NHEJ repair efficiency.3.CUL4ADTL binds and ubiquitinates DNA-PKcs in the nucleus.4.CUL4ADTL are involved in regulating DNA-PKcs shedding on chromatin.Part ?.CUL4ADTL increases genomic instability and promotes malignant transformation[Object]The results of the first and second parts show that CUL4A degrades DNA-PKcs through DTL ubiquitination in normal cells,inhibits NHEJ repair efficiency and increases the accumulation of DSBs damage.This section will continue to explore the impact of CUL4A on genome stability and malignant transformation of normal cells,and further clarify the relationship between CUL4A,DNA-PKcs and ?-H2AX in human precancerous tissues.[Methods]1.Hoechst staining of Cre;LSL-Cul4aFlox/Flox and Cre;DtlFlox/Flox MEFs was used to detect the number of primary nuclei.2.Giemsa staining was used to observe the chromosome morphology in breast epithelial cells overexpressing CUL4A or DTL.3.Cells stably overexpressing CUL4A or DTL were irradiated with low dose of X-RAY,MTT and clone formation experiments were used to detect the effect of CUL4A or DTL on cell proliferative potential.4.Western blot and immunohistochemical staining were used to detect changes in CUL4A,DNA-PKcs and ?-H2AX protein levels in normal pancreatic tissues,pancreatic precancerous tissues and gastric mucosal intestinal metaplasia tissues.The correlation between these proteins were analysed.[Results]1.Hoechst staining results showed that CUL4A/DTL significantly increased the proportion of multinucleated cells in primary cells.2.The results of Giemsa staining show that CUL4A/DTL affected the morphological changes of chromosomes in normal cells.3.The results of MTT and clone formation experiments showed that CUL4A or DTL significantly increased cell proliferative potential.4.Western blot results showed that compared with normal pancreatic tissue,CUL4A protein level in pancreatic precancerous lesions was significantly increased,DNA-PKcs protein levels was significantly reduced,and ?-H2AX protein level was significantly increased,and found that CUL4A protein level is negatively correlated with DNA-PKcs protein level,and positively correlated with ?-H2AX protein level.5.Immunohistochemical staining revealed that the level of CUL4A protein in gastric precancerous lesions was negatively correlated with the level of DNA-PKcs protein,and positively correlated with the level of ?-H2AX protein.[Conclusions]1.CUL4A/DTL significantly increase the number of multinuclear cells.2.CUL4A/DTL significantly increase chromosome instability.3.CUL4A and DTL significantly increase the malignant transformation of breast epithelial cells.4.The expression of CUL4A protein in the precancerous lesions of the pancreas and stomach is negatively correlated with the level of DNA-PKcs protein and positively correlated with the level of ?-H2AX protein. |
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