The Role And Mechanism Of DCAF8L2 In The Regulation Of BARD1 Protein Stability

BARD1 tumor suppressor forms stable heterodimer with BRCA1 through their RING domain.The BARD1-BRCA1 heterodimer has been implicated in pleiotropic biological processes including DNA damage repair,replication fork protection,cell cycle and checkpoint control,and chromatin remodeling,disruption of each of these functions could lead to genome instability,a hallmark of cancer cell and malignancy.Previous studies have shown that BARD1 mutation is an important risk factor associated with familial breast cancer.Since BARD1 is one of the most important regulator of BRCA1,the haploinsufficiency of which was reported to be a pathogenic cause for sporadic breast cancer,the loss or reduction of BARD1 would inevitably lead to a decrease in BRCA1 protein level and hence affect the tumor suppressor function of BARD1-BRCA1 heterodimer.Thus,negative regulators of BARD1 could be tumor promoting risk factors that may play important role in the pathogenesis of breast cancer.Identification of these yet-unknown BARD1 regulators is important for our understanding the mechanism underlying breast carcinogenesis.In this study,we identified DCAF8L2 is an E3 ligase of BARD1 and could target BARD1 for ubiquitination and subsequent degradation through UPS.DCAF8L2(DDB1 and CUL4 Associated Factor 8 Like 2)is encoded by DCAF8L2 gene located on chromosome Xp21.3,and is a paralogue of DCAF8(DDB1 and CUL4 Associated Factor 8).By promoting the degradation of BARD1,DCAF8L2 can also indirectly downregulate the BRCA1 protein level.To further understand the relationship between DCAF8L2 and BARD1-BRCA1 protein homeostasis,and as well as breast cancer pathogenesis,we performed comprehensive experiments and the results are presented as following.First,by performing mass spectrometry assays,we discovered that DCAF8L2 interacts with BARD1,but not BRCA1.We further verified this interaction in the T47 D and MCF7 cells by using immunoprecipitation assay.GST-Pull down assay showed that DCAF8L2 could interact with BARD1 RING domain.Then,we used si RNA/sh RNA to deplete DCAF8L2 in T47 D and MCF7 cells to examine the effect of DCAF8L2 on BARD1 protein level.The results showed that BARD1 protein level was increased upon DCAF8L2 depletion,with concomitant upregulation of BRCA1.In contrast,forced expression of DCAF8L2 in MCF10 A and He La cells caused a reduction in both BARD1 and BRCA1 protein level.These results demonstrated that DCAF8L2 could negatively regulate BARD1 and BRCA1 as well.Next,we found that DCAF8L2 could ubiquitinate BARD1 by performing in vivo and in vitro ubiquitination assays.DCAF8L2 ubiquitinates BARD1 and ultimately causes the degradation of both BARD1 and BRCA1 by UPS.This study also investigated whether DCAF8L2 could affect the biological function of BARD1-BRCA1 by regulating their protein homeostasis.By performing HR reporter assay,we demonstrated that DCAF8L2 impair homologous recombination efficiency by negatively regulating BARD1-BRCA1 protein level.Immunofluorescent staining showed that forced overexpression of DCAF8L2 impair the recruitment of BARD1-BRCA1 as well as their downstream DNA damage repair factors RPA32 and RAD51 at DNA damage sites,which in turn decreased DNA damage repair efficiency.However,the recruitment of DNA damage repair factors such as RNF8 and RAP80 that are upstream of BARD1-BRCA1 were not affected by DCAF8L2 overexpression.Then,sensitivity assays were preformed,the results showed that DCAF8L2-mediated downregulation of BARD1-BRCA1 sensitizes cells to DNA damage events.Finally,we investigated whether DCAF8L2 is a potential risk factor involved in the pathogenesis of breast cancer.The results showed that DCAF8L2 is highly expressed in some breast cancer cell lines,such as MCF7 and T47 D,and DCAF8L2 protein level was negatively correlated with the expression of BARD1 and BRCA1.Immunohistochemical experiments on human breast tissue sections demonstrate that DCAF8L2 is highly expressed in myoepithelial cells and some breast cancer cells,whereas an inverse pattern in the expression of BARD1 was observed in these myoepithelial cells.In contrast,DCAF8 and DCAF8L1,the other two members of the DCAF8 s,were observed to be expressed mainly in luminal epithelial cells,or both luminal epithelial and myoepithelial cells,respectively.Collectively,we demonstrated that DCAF8L2 acts as an BARD1 E3 ligase to downregulate BARD1 and BRCA1 protein level through UPS.DCAF8L2 participates in BRCA/BARD1-mediated biological functions,including DNA damage repair by HR.Our study revealed that DCAF8L2 impairs cells homologous recombination repair efficiency,thus forced overexpression of DCAF8L2 renders tumor cells more sensitive to DNA damage events.Meanwhile,our study also demonstrated that the expression of DCAF8L2 is closely associated with breast cancer through negatively regulating tumor suppressor BARD1-BRCA1 protein level.Taken together,our study illustrated the inner relationship among DCAF8L2,BARD1-BRCA1 protein homeostasis,and breast cancer pathogenesis.Our study will have important implications for the prevention and treatment of breast cancer.