Nuclear Localization Of Beclin 1 Promotes DNA Damage Repair Independent Of Autophagy

Objective:Beclin 1 is a well-established core mammalian autophagy protein that is embryonically indispensable and has been presumed to suppress oncogenesis via an autophagy-mediated mechanism.Here,we show that Beclin 1 is a prenatal primary cytoplasmic protein but rapidly relocated into the nucleus during postnatal development in mice.Nevertheless,it remains an open question whether the tumor suppressive phenotypes associated with beclin1 deletions are a direct consequence of defective autophagy or whether they indicate the involvement of alternative,previously unidentified functions of Beclin 1.Methods:To construct a beclin1 knockout HeLa cell line using a CRISPR/ Cas9 system to study the role of nuclear Beclin 1.Cas9 plasmids containing a guide RNA(gRNA)that targeted exon 5 and exon 6 of beclin1.We performed the HR and NHEJ repair reporter assays to determine which of these pathways might be regulated by beclin1.This observation was further confirmed in a Western blot assay to measure the levels of critical proteins in these DSB damage repair pathway.We performed co-immunoprecipitation and immunoblotting analysis to determine whether Beclin 1 regulates the formation of the complexes(MNR complex,DNA-PK complex).To understand how Beclin 1 is localized to the nucleus,we performed domain mapping of Beclin 1 by constructing a series of beclin1 mutants in which beclin1 was fused with a HA-tag to determine which region is potentially responsible for its nuclear localization.Western blot analyses of respective cytoplasmic and nuclear proteins showed that the beclin1 mutants that lacked both of the short domains including residues 0-50 and 254-278 lost their nuclear localization capability,but presence of either of the domains was sufficient for nuclear localizationTo determine whether Beclin 1 regulates DNA DSB repair in an autophagy-dependent manner,we examined whether beclin1 deletion affects the autophagy response using image flow cytometry to visually and statistically analyze LC3 b spot counts,a marker used for measuring the formation of autophagasomes.To further evaluate the autophagy response after exposure to IR,we randomly chose two beclin1 knockout clones(KO15 and KO40)and exposed the cells to 6 Gy of IR.Besides,cells were serum-starved or exposed to bafilomycin A1.We next examined the structure of autophagosomes using electron microscopy.And investigated the autophagy flux of cells with LC3/ p62-mCherry-GFP plasmid.To elucidate the mechanism by which Beclin 1 exerts its effect on DNA DSB repair,we pulled down nuclear proteins associated with Beclin 1 in NIH3T3 cells which ectopically expressed Flag-tagged Beclin 1.The identities of the Beclin 1-interacting proteins were determined using mass spectrometry analysisResults:To explore the localization of Beclin 1 in cells under physiological conditions,we examined the subcellular distribution of Beclin 1 in hepatic cells at different developmental stages.The results from our confocal microscopic analysis showed that during embryonic development and in neonatal mice,Beclin 1 is primarily located in the cytoplasm and plasma membrane,with a very small portion located in the nucleus.Thereafter,Beclin 1 progressively redistributed into the nucleus.When the mice were 15 days old,roughly half of the total Beclin 1 in hepatocytes was located in the nucleus.At postnatal day 20,the majority of Beclin 1 relocated into the nucleus,and much less Beclin1 remained in the cytoplasm.This finding was confirmed by immunoblot,which demonstrates that while total cellular levels of Beclin 1 were relatively stable during the course of mouse development.This pattern,in which the majority of Beclin 1 was localized in the nucleus,was similarly sustained in adult mice not only in hepatocytes but also in other tissues such as the heart and kidney.We construct a beclin1 knockout HeLa cell line using a CRISPR/ Cas9 system to study the role of nuclear Beclin 1.Cas9 plasmids containing a guide RNA(gRNA)that targeted exon 5 and exon 6 of beclin1 were transfected into Hela cells,and single cell clones were analyzed.A total of 18 randomly selected clones that harbored a gRNA targeting beclin1 were analyzed using polymerase chain reaction to show mutant bands in gel electrophoresis.Beclin 1 expression was undetectable in 5 clones that were examined using Western blot analysis Sequencing analysis of these 5 clones demonstrated that the loss of Beclin 1 expression resulted from multiple indels that occurred at the targeted locus in beclin1,which led to frame shifts and a loss of gene function.Exposure to ionizing radiation(IR)caused a transient increase in nuclear localization of Beclin 1,suggesting that Beclin 1 may be involved in regulation of DNA damage response to radiation.In consistent with this hypothesis,we found that the beclin1 knockout cells showed a marked increase in the cells with the Ser139-phosphorylated histone H2AX(?-H2AX)foci that are considered as a biomarker of DNA DSBs at 1 h post IR,compared with wild-type cells.Knocking out of beclin1 led to a significant reduction in the repair of DNA DSBs by both of these pathways.Specifically,the beclin1 knockout cells showed approximately 60-fold and 30-fold reductions in DNA DSB repair by the NHEJ and HR pathways,respectively,compared with wild-type cells.The results showed that the beclin1 knockout cells expressed a lower level of the NHEJ repair protein DNA Ligase 1 than wild-type cells.Similarly,the beclin1 knockout cells exhibited lower levels of the HR repair proteins Rad 51 and Rad 52,and phosphorylated p95/NBS1 in response to IR,than wild-type cells.Furthermore,while the downstream proteins in the DNA DSB repair pathways,including p53,Chk1 and Chk2,were phosphorylated in wild-type cells in response to IR,these phosphorylation events were significantly reduced in the beclin1 knockout cells.The results showed that wild-type cells are able to form both the MNR complex and DNA-PK complex,but knockout of beclin1 significantly attenuated the formation of the DNA-PK complex,with slight effect on the MNR complex.The identities of the Beclin 1-interacting proteins were determined using mass spectrometry analysis,which identified DNA topoisomerase II? as one of the major Beclin 1-interacting proteins in the nucleus.This finding was confirmed by immunoprecipitation,which demonstrated that epitope-tagged Beclin 1 was able to specifically bind to DNA topoisomerase II?,and this interaction was intensified after exposure to IR.Furthermore,after exposure to IR,DNA topoisomerase II? and Beclin 1 were found to be colocalized with the DSB repair protein p53 binding protein 1(53bP1)or ?-H2 AX.Strikingly,depletion of DNA topoisomerase II? by gene silencing completely prohibited Beclin 1 from localization to the DNA break sites,suggesting that DNA topoisomerase II? may recruit Beclin 1 to the DNA damage site through its direct interaction to Beclin 1 to promote DNA damage repair.Conclusion:1)Beclin 1 is progressively relocalized to the nucleus during development.2)Domains including residues 1-50 and 254-278 are involved in Beclin 1 nuclear localization.3)Loss of Beclin 1 resulted in reduced DNA damage repair.4)Beclin 1 regulates DNA DSB repair independent of autophagy.5)Beclin 1 promotes DNA DSB repair via a direct interaction with DNA topoisomerase II?.Our work in this study establishes that Beclin 1 plays an autophagy-independent role in maintaining genomic integrity by promoting a cell's capacity for DNA damage repair.