Epigenetics of adipocyte commitment. Regulation of Pparγ and Zfp423 expression

Obesity is the most frequent metabolic disease worldwide and the major risk factor for metabolic disorders such as insulin resistance and type 2 diabetes. In response to overfeeding, excess lipids are stored in the adipocytes, leading to inappropriate adipose cell expansion (hypertrophic obesity), which is associated with local inflammation and dysregulated and insulin resistant adipose tissue. Hypertrophic obesity is also characterized by an inability to recruit and differentiate precursor cells into mature adipocytes. This is not due to lack of these precursor cells but instead is a consequence of an impaired ability to induce commitment and promote differentiation of available precursor cells, by inactivating inhibitory pathways, and/or activating pathways needed to commit and/or differentiate. Very recently, the zinc finger protein Zfp423 has been identified as a transcriptional regulator of preadipocyte determination. Zfp423 protein, indeed, directly binds the peroxisome proliferator-activated receptor gamma (Pparγ) promoter, thus promoting the gene transcription of the master regulator of adipocyte differentiation. Furthermore, Zfp423 protein is the point of convergence between the anti-adipogenic Wnt1-inducible-signaling pathway protein 2 (Wisp2) pathway and the pro-adipogenic bone morphogenetic protein 4 (Bmp4) pathway. In light of this, it is now clear that alterations impacting on Zfp423 and Pparγ protein and/or gene expressions are at least in part responsible for the restricted adipogenesis observed in hypertrophic obesity. Increasing evidence sustains that even adipogenesis might be regulated by an epigenetically induced gene transcription reprogramming. Thus, in light of all these observations, I aimed to establish which are the molecular mechanisms regulating the gene expression of Zfp423 and of its downstream target Pparγ looking at epigenetic changes involvement. Experiments were performed in two cell models: 3T3-L1 and NIH-3T3 cells, which are committed to and uncommitted to adipocyte lineage, respectively. The mRNA expression of both Zfp423 and Pparγ genes are increased in 3T3-L1 compared with NIH-3T3 cells. Furthermore, the expression differences among the two cellular models are not dependent on differences in the promoter DNA sequence of both genes, but rather to epigenetic mechanisms and chromatin remodeling. Indeed, performing methylation studies by bisulfite sequencing and nucleosomes positioning and occupancy analysis by micrococcal nuclease (MNase) digestion, I revealed that the DNA methylation status and the nucleosomes occupancy of Zfp423 and Pparγ promoter regions are increased in NIH-3T3 compared with 3T3-L1 cells. Finally, chemically-induced demethylation of the Zfp423 and Pparγ promoters by DNA-methyltransferase (Dnmt) inhibitor 5-Azacyditine (AZA) treatment promotes adipocyte terminal differentiation in the uncommitted NIH-3T3 cell line. 9 In conclusion, I demonstrated that Zfp423 and Pparγ genes, which are involved in adipocyte commitment and differentiation, are transcriptionally regulated by DNA methylation and dynamic chromatin remodeling, and that the modulation of the methylation status of the promoter region of these two genes is relevant to the regulation of adipocyte commitment and terminal differentiation in vitro.