Objective:The ataxia telangiectasia mutated(ATM)gene is a master regulator in cellular DNA damage response.The dysregulation of ATM expression is frequent in breast cancer,and is known to be involved in the carcinogen...Objective:The ataxia telangiectasia mutated(ATM)gene is a master regulator in cellular DNA damage response.The dysregulation of ATM expression is frequent in breast cancer,and is known to be involved in the carcinogenesis and prognosis of cancer.However,the underlying mechanism remains unclear.The bioinformatic analysis predicted a potential antisense transcript ATM-antisense(AS)from the opposite strand of the ATM gene.The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.Methods:Single strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus.qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples.Luciferase reporter gene assays,biological mass spectrometry,ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression.Immunofluorescence and host-cell reactivation(HCR)assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair.Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.Results:The ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter.The reduced ATM-AS level led to the abnormal downregulation of ATM expression,and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro.The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples,and the patient prognosis.Conclusion:The present study demonstrated that ATM-AS,an antisense transcript located within the ATM gene body,is an essential positive regulator of ATM expression,and functions by mediating the binding of KAT5 to the ATM promoter.These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in breast cancer,and enrich our understanding of how an antisense transcript regulates its host gene.展开更多
Natural antisense transcripts （NAT） and alternative polyadenylation （APA） of messenger RNA （mRNA） are important contributors of transcriptome complexity, each playing a critical role in multiple biological proce...Natural antisense transcripts （NAT） and alternative polyadenylation （APA） of messenger RNA （mRNA） are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense （S-AS） gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA （pA） site in sense gene RNASEH2C, which generated longer 3＇ untranslated region （3＇UTR） and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene＇s protein level in RNASEH2C.KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.展开更多
基金supported by the National Natural Science Foundation of China(No.81802670 and No.82072580).
文摘Objective:The ataxia telangiectasia mutated(ATM)gene is a master regulator in cellular DNA damage response.The dysregulation of ATM expression is frequent in breast cancer,and is known to be involved in the carcinogenesis and prognosis of cancer.However,the underlying mechanism remains unclear.The bioinformatic analysis predicted a potential antisense transcript ATM-antisense(AS)from the opposite strand of the ATM gene.The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.Methods:Single strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus.qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples.Luciferase reporter gene assays,biological mass spectrometry,ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression.Immunofluorescence and host-cell reactivation(HCR)assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair.Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.Results:The ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter.The reduced ATM-AS level led to the abnormal downregulation of ATM expression,and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro.The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples,and the patient prognosis.Conclusion:The present study demonstrated that ATM-AS,an antisense transcript located within the ATM gene body,is an essential positive regulator of ATM expression,and functions by mediating the binding of KAT5 to the ATM promoter.These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in breast cancer,and enrich our understanding of how an antisense transcript regulates its host gene.
基金We thank Drs. Li Jin, Feng Qian, Jun Zhu and Hongjie Yao for constructive suggestions of this manuscript. The vectors of CRISPR/ Cas9 are the generous gifts from Drs. Yangming Wang and Yong- ming Wang. This work was supported by the National Basic Research Program （973 Program） （Nos. 2013CB530700 and 2015CB943000 to T. N.） and National Natural Science Foundation of China （Grant Nos. 31471192 and 31521003 to T. N.）.
文摘Natural antisense transcripts （NAT） and alternative polyadenylation （APA） of messenger RNA （mRNA） are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense （S-AS） gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA （pA） site in sense gene RNASEH2C, which generated longer 3＇ untranslated region （3＇UTR） and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene＇s protein level in RNASEH2C.KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.
