TET-mediated hydroxymethylcytosine at the Pparγ locus is required for initiation of adipogenic differentiation

Background/Objectives:Adipose tissue is one of the main organs regulating energy homeostasis via energy storage as well as endocrine function. The adipocyte cell number is largely determined by adipogenesis. While the molecular mechanism of adipogenesis has been extensively studied, its role in dynamic DNA methylation plasticity remains unclear. Recently, it has been shown that Tet methylcytosine dioxygenase (TET) is catalytically capable of oxidizing DNA 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) toward a complete removal of the methylated cytosine. We investigate whether expression of the Tet genes and production of hydroxymethylcytosine are required for preadipocyte differentiation.Subjects/Methods:Murine 3T3-L1 preadipocytes were used to evaluate the role of Tet1 and Tet2 genes during adipogenesis. Changes in adipogenic ability and in epigenetic status were analyzed, with and without interfering Tet1 and Tet2 expression using small interfering RNA (siRNA). The adipogenesis was evaluated by Oil-Red-O staining and induced expression of adipogenic genes using quantitative polymerase chain reaction (qPCR). Levels of 5-hmC and 5-mC were measured by MassARRAY, immunoprecipitation and GC mass spectrometry at specific loci as well as globally.Results:Both Tet1 and Tet2 genes were upregulated in a time-dependent manner, accompanied by increased expression of hallmark adipogenic genes such as Pparγ and Fabp4 (P<0.05). The TET upregulation led to reduced DNA methylation and elevated hydroxymethylcytosine, both globally and specifically at the Pparγ locus (P<0.05 and P<0.01, respectively). Knockdown of Tet1 and Tet2 blocked adipogenesis (P<0.01) by repression of Pparγ expression (P<0.05). In particular, Tet2 knockdown repressed conversion of 5-mC to 5-hmC at the Pparγ locus (P<0.01). Moreover, vitamin C treatment enhanced adipogenesis (P<0.05), while fumarate treatment inhibited it (P<0.01) by modulating TET activities.Conclusions:TET proteins, particularly TET2, were required for adipogenesis by modulating DNA methylation at the Pparγ locus, subsequently by inducing Pparγ gene expression.