DNA Methylation Biphasically Regulates 3T3-L1 Preadipocyte Differentiation

Abstract

Better understanding the mechanisms underlying adipogenesis may provide novel therapeutic targets in the treatment of obesity. Most studies investigating the mechanisms underlying adipogenesis focus on highly regulated transcriptional pathways; little is known about the epigenetic mechanisms in this process. Here, we determined the role of DNA methylation in regulating 3T3-L1 adipogenesis in early and late stage of differentiation. We found that inhibiting DNA methylation pharmacologically by 5-aza-2'-deoxycytidine (5-aza-dC) at early stage of 3T3-L1 differentiation markedly suppressed adipogenesis. This inhibition of adipogenesis by 5-aza-dC was associated with up-regulation of Wnt10a, an antiadipogenic factor, and down-regulation of Wnt10a promoter methylation. In contrast, inhibiting DNA methylation by 5-aza-dC at late stage of differentiation enhanced the lipogenic program. The differential effects of 5-aza-dC on adipogenesis were confirmed by gain or loss of function of DNA methyltransferase 1 using genetic approaches. We further explored the molecular mechanism underlying the enhanced lipogenesis by inhibition of DNA methylation at late stage of differentiation. The Srebp1c promoter is enriched with CpG sites. Chromatin immunoprecipitation assays showed that DNA methyltransferase 1 bound to the methylation region at the Srebp1c promoter. Pyrosequencing analysis revealed that the DNA methylation at the key cis-elements of the Srebp1c promoter was down-regulated in adipogenesis. Further, luciferase reporter assays showed that the Srebp1c promoter activity was dramatically up-regulated by the unmethylated promoter compared with the fully methylated promoter. Thus DNA methylation appears to exert a biphasic regulatory role in adipogenesis, promoting differentiation at early stage while inhibiting lipogenesis at late stage of 3T3-L1 preadipocyte differentiation.

Epigenetic programming of Dnmt3a mediated by AP2α is required for granting preadipocyte the ability to differentiate

“…15, 29, 30, 31, 32, 33, 34, 35 Our previous study has reasoned that the stage of CI might provide a special window for 3T3-L1 cells to establish an adipogenic competency program through epigenetic modifications. Dnmt3a has been implicated in epigenetic regulation in adipogenesis in vitro and in vivo .…”

Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis

“…Previously, we demonstrated (14) that preadipocytes and MSCs must down-regulate Thy1 expression to differentiate into adipocytes. To determine whether DNA methylation regulates Thy1 expression during adipogenesis, we first pharmacologically inhibited DNA methyltransferase in bone marrow-derived mouse MSCs (mMSCs) before differentiation into adipocytes (28). mMSCs were treated daily with either DMSO (vehicle) or the DNA methyltransferase inhibitor, 5-aza-dC, for 12 d and were concurrently treated with either medium alone or the ACT medium, starting at d 3 (Fig.…”

“…In the most widely used and wellaccepted model of in vitro adipogenesis, the murine 3T3-L1 preadipocyte line, global DNA methylation has been shown to increase during adipogenesis (26,27). Interestingly, addition of the DNA methylation inhibitor 5aza-29-deoxycytidine (5-aza-dC) at the onset of adipogenic differentiation can completely block adipocyte formation, suggesting that DNA methylation changes are essential for adipogenesis (27,28). Specific changes in regional DNA methylation are also critical for adipogenesis, and blocking those patterns can alter the differentiation pathway of precursor cells toward osteoblastogenesis, rather than adipogenesis (24,29,30).…”

Epigenetic regulation of Neuregulin-1 tunes white adipose stem cell differentiation

“…Much previous work utilized 3T3-L1 cells, which are of an unknown lineage from mouse, not human, and are a transformed cell line, not primary cultures [47] . Nonetheless, epigenetic regulation of adipogenic differentiation has been documented in several 3T3-L1 studies [48,49] .…”