title: Inhibition of SIRT1 increases EZH2 protein level and enhances the repression of EZH2 on target gene expression.
abstract: OBJECTIVE
  To study the regulatory rolesof SIRT1 on EZH2 expression and the further effects on EZH 2’ s repression of target gene expression.
  
  
  METHODS
  The stable SIRT1 RNAi and Control RNAi HeLa cells were established by infection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation(ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells.
  
  
  RESULTS
  Western blot results showed that EZH2 protein level increased upon SIRT1 depletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter regionof EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases.
  
  
  CONCLUSIONS
  Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expression.
authors.affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College , Beijing 100005, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Lu
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Xiang
authors.initials: X
authors.lastname: Lü
authors.forename: Xue-song
authors.initials: XS
authors.lastname: Wu
authors.forename: De-pei
authors.initials: DP
authors.lastname: Liu
authors.forename: Chih-chuan
authors.initials: CC
authors.lastname: Liang
mesh_terms: D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D000071221:Enhancer of Zeste Homolog 2 Protein; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D063151:Polycomb Repressive Complex 2; D012097:Repressor Proteins / Q000502:physiology*; D056564:Sirtuin 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; C447939:SIRT1 protein, human; D056564:Sirtuin 1