The Functions And Mechanisms Of Yeast BET Family Proteins In DNA Recombination And Chromosome Segregation

Improper DNA repair or chromosome segregation can lead to genome instability and human diseases.DNA double-stranded breaks(DSBs)are a type of very dangerous DNA lesions.If left unrepaired or repaired incorrectly,DSB can cause adverse consequences including genomic instability,cancer and even cell death.In general,cells use two main mechanisms to repair DSBs,namely homologous recombination(HR)and nonhomologous end joining(NHEJ).Emerging evidence suggests that histone acetylation plays a key role in promoting DSB repair,but the precise mechanisms remain unclear.The BET family proteins specifically recognize acetylated histones via their bromodomains(BRDs).In Saccharomyces cerevisiae,Bdf1 and Bdf2,the only two BET family members,can recognize and bind acetylated histones H3 and H4 via their tandem BRD motifs.In this study,we investigate the role and molecular mechanism of Bdf1 in DNA damage repair and chromosome segregation,and obtained the following results:1)The BRDs of Bdf1 promotes HR and DNA end resection.Drug sensitivity test showed that the function of Bdf1 in DNA damage response largely rely on its BRD motifs.By visualizing the Rad52-YFP foci,we found that disruption of the Bdf1 BRDs by mutating the key residues(bdf1-Y187FY354 F,bdf1-2YF)leads to increased spontaneous DNA damage and impaired DNA repair.Pulsed-field gel electrophoresis(PFGE)showed that Bdf1 BRDs are required for efficient recovery from the DNA damage.Further,we determined the role of the BRDs in HR using an ectopic HR system and found that the HR efficiency is about 80 % in wild type cells while it is reduced to 50 % in the bdf1-2YF mutant.Meanwhile,the repair kinetics of ectopic recombination was slowed down in the mutant,indicating that the BRDs of Bdf1 play an important role in promoting HR.Southern blot analysis revealed that the BRDs of Bdf1 are required to promote processing of the 5’-terminal ends of DSBs.By Ch IP-q PCR,we observed that Bdf1 BRDs promote the chromatin recruitment of the enzymes involved in resection,including Exo1,Dna2 and Sgs1.In addition,we noted that Bdf1 BRDs can facilitate DSB repair by NHEJ.2)Bdf1 interacts with RPA and stimulates RPA loading at DSB ends.Deletion of DOT1 or overexpression of EXO1 in the bdf1-2YF mutant reversed the defect of DNA end resection but not the HR defect,suggesting that Bdf1 BRDs play additional roles in HR.Using mass spectrometry,immunoprecipitation and pull down techniques,we found that Bdf1 interacts with RPA,the single-stranded DNA(ss DNA)binding protein complex,in vitro and in vivo.Bdf1 interacts with RPA through the linker between BRD1 and BRD2 and the C-terminal end,thus facilitating RPA binding at DSBs and HR repair.Disruption of the BRD motifs significantly impaired the recruitment of RPA and the recombinase Rad51 at DSBs.Importantly,overexpression of RPA in the bdf1-2YF mutant largely restored the recruitment of Rad51 and HR repair,but not the resection defect,indicating that the interaction between Bdf1 and RPA is important for promoting RPA recruitment and HR repair.3)The impact of Bdf1 BRDs on different DSB repair pathways and its relationship with Bdf2.In break-induced replication(BIR),disruption of the BRDs in Bdf1 leads to increased BIR and decreased repair by gene conversion.bdf1-2YF mutation also impaired DSB repair by single-strand annealing(SSA).However,the SSA defect can be significantly restored by overexpression of RPA.Moreover,disruption of the BRDs of Bdf1 led to an increased repair by Micro-homology end joining(MMEJ),accompanied by a larger range of DNA deletions at the break site.These results suggest that the BRDs of Bdf1 are important to promote high-fidelity repair and the maintenance of genomic stability.In addition,we noted that Bdf1 and Bdf2 are partially redundant in response to DNA damage,since overexpression of one can partially compensate for the loss of the other in the DNA damage response.However,the role of Bdf1 is more important than Bdf2.Finally,expression of h TAF1,the human homolog of yeast Bdf1,can weakly reverse the defect of DNA damage response in cells lacking Bdf1,suggesting that the function of Bdf1 is conserved in yeast and mammals.4)The BRDs of Bdf1 are important to promote proper chromosome segregation.Disruption of Bdf1 BRDs leads to unequal distribution of duplicated chromosomes into two daughter cells.Notably,the bdf1-2YF mutant showed a synthetic defect with cells lacking the cohesion establishment protein Ctf4 or Ctf18,but is epistatic to cells lacking the establishment of cohesion protein 1(Eco1)in promoting chromosome segregation.We found that Bdf1 can bind to the N-terminal zinc finger domain of Eco1 and interacts with Eco1 in vivo and in vitro.Disruption of their interaction by mutating the residues in Eco1 leads to improper chromosome segregation as seen in the bdf1-2YF mutant.Ch IPq PCR assays revealed that bdf1-2YF mutation impaired the recruitment of cohesin subunits Smc1 and Smc3 in the S phase.Together with previous studies,these results suggest a model that Bdf1 first binds to acetylated histone H4 on chromatin through its BRDs;Meanwhile,Bdf1 physically interacts with Eco1 and facilitates the loading of Eco1 to chromatin.Thus,Bdf1 promotes the assembly of cohesin complex on chromatin to ensure proper chromosome segregation.Our study reveals novel functions of the BET family proteins in DNA repair,chromosome segregation and maintenance of genomic stability,and could provide insight into the mechanism by which histone acetylation regulates DNA recombination and chromosome segregation in human cells.