[DNA hypermethylation at C/EBPα gene promoter relieves a PPARγ-associated repression of HDAC1]

To explore the molecular mechanisms of C/EBPα for regulating the osteogenesis of C3H10T1/2 cells.

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.

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.

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.