title: [DNA hypermethylation at C/EBPα gene promoter relieves a PPARγ-associated repression of HDAC1].
abstract: OBJECTIVE
  To explore the molecular mechanisms of C/EBPα for regulating the osteogenesis of C3H10T1/2 cells.
  
  
  METHODS
  We constructed an in vitro osteogenic differentiation model and investigated the mRNA and protein expression profile of PPARγ2 during C3H10T1/2 osteogenesis by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Next we constructed an adenovirus vector loading siRNA against PPARγ2 and employed a specific antagonist of PPARγ, G3335, to examine the effect of PPARγ on activating the expression of C/EBPα. Then we performed ChIP to test PPARγ2 binding on -1 286 bp/-1 065 bp region of C/EBPα promoter and explore the role of PPARγ in the dependence of histone hypoacetylation on DNA methylation in -1 286 bp/-1 065 bp region of C/EBPα promoter.
  
  
  RESULTS
  During the osteogenic differentiation, PPARγ mRNA and protein expression initially increased and then decreased in double-phase reactions. siRNA and G3335 induced down-regulation of PPARγ activity inhibited C/EBPα in C3H10T1/2 cells up-regulation of osteogenic differentiation. ChIP results show that DNA methylation could weaken the binding section PPARγ2 to -1 286 bp/-1 065 bp capabilities.
  
  
  CONCLUSIONS
  This study provides a deeper insight into the molecular mechanisms underlying the osteogenesis of MSCs regulated by C/EBPα in synergy with PPARγ. PPARγ contributes to C/EBPα expression through binding on -1 286 bp/-1 065 bp region of its promoter during the osteogenesis of C3H10T1/2 cells. And DNA hypermethylation in-1 286 bp/-1 065 bp region during the terminal stage of osteogenesis prevents PPARγ from binding on it so that PPARγ-associated repression of HDAC1 is relieved to down-regulate the acetylation status of -1 286 bp/-1 065 bp region. Meanwhile DNA methylation and histone acetylation are linked by PPARγ for regulating the differentiation of MSCs.
authors.affiliation: Department of Orthopedics, First Municipal People's Hospital, Shanghai 200080, China. Email: sawboneszhao@163.com.
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Zhao
authors.forename: Zhaoliang
authors.initials: Z
authors.lastname: Zhang
authors.forename: Jipeng
authors.initials: J
authors.lastname: Li
authors.forename: Aihua
authors.initials: A
authors.lastname: Zhang
mesh_terms: D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D019175:DNA Methylation; D056284:Histone Deacetylase 1 / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D010012:Osteogenesis; D047495:PPAR gamma / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D047495:PPAR gamma; D012333:RNA, Messenger; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1