The Function Of Bdfl Protein In DNA Recombination And Gene Expression

DNA double-strand break(DSB)is one of the most cytotoxic lesions that can result in mutagenesis or cell death if left unrepaired or repaired inappropriately.Cells use two major pathways for DSB repair:nonhomologous end joining(NHEJ)and homologous recombination(HR).The repair of DSB involves both posttranslational modification of histones and recruitment of DNA repair proteins at the site of damage.Histones acetylation plays an important role in DSB repair by relaxing the chromatin structures and facilitating the binding of DNA repair proteins.The bromodomain-containing protein is a class of proteins that specifically recognizes and binds to acetylated lysines on histones.In recent years,more and more studies show that such proteins are involved in DNA repair and are associated with multiple types of cancers.In Saccharomyces cerevisiae,as a member of the BET family,Bdf1 contains two tandem bromodomains and a C-terminal domain.Its bromodomains bind to acetylated lysine of the histone H3 and H4 tails and the C-terminal domain interacts with TFIID to elicit PIC assembly and to promote transcription.Moreover,Bdf1 corresponds to the C-terminal half of its human homolog protein hTAF1 and is involved in regulating multiple cellular processes including DNA damage response.However,the molecular details by which Bdf1 contributes to DNA damage response remain unknown.1)In this study,we created a colletion of mutant bdf1 strains in which each bromodomain was deleted or mutated.The expression of each mutant protein was detected by western blot.The results showed that deletion of bromodomain or C-terminal clearly decreased the expression of mutant proteins,but point mutations in bromodomains did not.This suggests that bromodomains and C-terminal domain play an important role in maintaining protein stability.2)By using spotting assay,we observed that deletion of either of the two bromodomains caused a slightly decrease in resistance to DNA-damaging agents,particularly for the bromodomain ?.However,deletion of both bromodomains simultaneously caused a much greater defect in resistance to DNA damaging agents,suggesting that the two bromodomains share redundant functions in response to DNA damages.Consistently,the strain with Y187F or Y354F point mutation,which disrupted the structure of bromodomain ? and ?,respectively,exhibited a minor defect in resistance to DNA damages,while the mutant carrying both point mutations displayed a moderate to severe defects,as seen in the mutant with both bromodomains deleted.Therefore,it appears that tyrosine 187 and 354 are highly conserved and play a redundant function in response to DNA damages.It suggests that the function of Bdfl in DNA damage response may rely on its interaction with acetylated histones.3)In order to identify the Bdfl domains that are important for HR,we assessed the efficiency of HR in these mutant strains by gene targeting.The results showed that deletion or disruption of bromodomain ? caused a severe defect in HR while deletion of bromodomain ? did not,suggesting that bromodomain I plays a key role in HR.Moreover,the role of Bdfl in HR appeared to be independent of its association with TFIID.4)RNA-seq experiments provided a global view of the transcriptional alterations caused by deletion of BDF1.GO term and KEGG analysis revealed that the genes differentially expressed between two strains are mainly involved in carbohydrate and amino acids metabolic,biosynthesis of various metabolites,plasma membrane transport,autophagy,protein processing in endoplasmic reticulum,protein synthesis and folding.In the absence of damage,Bdfl regulates the transcription levels of a number of genes involved in NHEJ,HR,mismatch repair,base/nucleotide excision repair,post-replication repair and photoreactivation repair.5)After DSB induction,the pattern of differentially expressed genes between WT and bdfl cells was drastically changed.Interestingly,these differentially expressed genes are mostly not involved in DNA repair but rather they act in other processes such as cell-cycle progression,environmental stress responses,carbohydrate metabolism and energy metabolism.In addition,Bdfl promoted the transcription of RNR3,HUG1,HO and YOR338W after DSB,suggesting that Bdf1 may participate in response to DSB partially by promoting the transcription of those genes.However,a detailed analysis showed that the expression levels for the genes involved in NHEJ and HR repair pathways were not significantly changed,suggesting that Bdfl may control HR at additional levels.This study characterized the Bdfl domains important for HR,and analyzed the alteration in transcriptional patterns between WT and bdfl cells.This study will provide clues for further study of the function and mechanisms of Bdfl in DNA repair.