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. 2019 Dec;16(12):1785-1793.
doi: 10.1080/15476286.2019.1658508. Epub 2019 Aug 27.

ZFP217 regulates adipogenesis by controlling mitotic clonal expansion in a METTL3-m6A dependent manner

Qing Liu  1 , Yuanling Zhao  1 , Ruifan Wu  1 , Qin Jiang  1 , Min Cai  1 , Zhen Bi  1 , Youhua Liu  1 , Yongxi Yao  1 , Jie Feng  1 , Yizhen Wang  1 , Xinxia Wang  1
Affiliations

ZFP217 regulates adipogenesis by controlling mitotic clonal expansion in a METTL3-m6A dependent manner

Qing Liu et al. RNA Biol. 2019 Dec.

Abstract

Obesity is becoming a global problem. Research into the detailed mechanism of adipocyte development is crucial for the treatment of excess fat. Zinc finger protein 217 plays roles in adipogenesis. However, the underlying mechanism remains unclear. Here, we demonstrated that ZFP217 knockdown prevented the mitotic clonal expansion process and caused adipogenesis inhibition. Depletion of ZFP217 increased the expression of the m6A methyltransferase METTL3, which upregulated the m6A level of cyclin D1 mRNA. METTL3 knockdown rescued the siZFP217-inhibited MCE and promoted CCND1 expression. YTH domain family 2 recognized and degraded the methylated CCND1 mRNA, leading to the downregulation of CCND1. Consequently, cell-cycle progression was blocked, and adipogenesis was inhibited. YTHDF2 knockdown relieved siZFP217-inhibited adipocyte differentiation. These findings reveal that ZFP217 knockdown-induced adipogenesis inhibition was caused by CCND1, which was mediated by METTL3 and YTHDF2 in an m6A-dependent manner. We have provided novel insight into the underlying molecular mechanisms by which m6A methylation is involved in the ZFP217 regulation of adipogenesis.

Keywords: METTL3; ZFP217; adipogenesis; mRNA m6A; mitotic clonal expansion.

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Figures

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Graphical abstract
Figure 1.
Figure 1.
ZFP217 knockdown inhibit 3T3-L1 adipogenesis. (a) ZFP217 knockdown efficiency in 3T3-L1 cell lines. (b) Oil red O staining of control and ZFP-depleted cells after two-day post-confluence 3T3-L1 preadipocytes incubated with the hormonal induces (IBMX, dexamethasoneand insulin). Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (c) The adipogenesis marker genes mRNA expression was analysed using qPCR. (d) Cells were collected by RIPA lysate after 24 h of induced and subjected to Western blot analyses using the corresponding antibodies. The right panel shows the relative protein levels quantified by densitometry and normalized to GAPDH. The data are presented as the mean ± SD of triplicate tests. *P< 0.05, **P< 0.01, ***P< 0.001 compared to the control group.
Figure 2.
Figure 2.
ZFP217 knockdown induced cell-cycle arrest in G0G1 phase. (a) Flow cytometry analysis of cell-cycle progression of control and ZFP217-depleted cells. Cells were harvested at 24 h after MDI initiated and stained. The percentages of cells in the G1, S and G2 phase were calculated using ModFit LT software. (b) Cell lysates were obtained after 24 h of induction and subjected to Western blot analyses using the corresponding antibodies. The right panel shows the relative protein levels quantified by densitometry and normalized to GAPDH. (c) The MCE-related genes mRNA expression was analysed using qPCR. The data are presented as the mean ± SD of triplicate tests. *P< 0.05, **P< 0.01, ***P< 0.001 compared to the control group.
Figure 3.
Figure 3.
ZFP217 regulate m6A methyltransferase METTL3 expression. (a) The MCE and adipogenesis related protein expression. Cell lysates were obtained after 24 h of induction and subjected to Western blot to analysis MCE-related proteins. (b) Flow cytometry analysis of cell-cycle progression of several groups. The percentages of cells in the G1, S and G2 phase were calculated using ModFit LT software. (c) Oil Red O staining of control, ZFP217 knockdown, METTL3 knockdown and ZFP217+ METTL3 knockdown cells after MDI-induced for 8days. (d) ZFP217 knockdown increased METTL3 expression in 3T3-L1 preadipocytes during MCE. Total cell lysates were prepared and analysed by Western blot. Western blot analysis of each total protein was done in parallel. Immunoblots are a representative image of three independent experiments. The data are presented as the mean ± SD of triplicate tests. *P< 0.05, **P< 0.01, ***P< 0.001 compared to the control group.
Figure 4.
Figure 4.
METTL3mediates gene and protein expression of CCND1 in an m6A-dependent manner. (a) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m6A levels of CCND1 mRNA in each group after MDI-induced for 24 h. (b) Western blot analysis of METTL3 and CCND1 in cells after transfected with control, wild-type (WT) and mutant (MUT) METTL3 plasmid and MDI-induced for 24 h. The data are presented as the mean ± SD of triplicate tests. *P< 0.05, **P< 0.01, ***P< 0.001 compared to the control group.
Figure 5.
Figure 5.
CCND1 is a target of YTHDF2 which could influence mRNA stability. (a) RIP analysis of the interaction of CCND1 with FLAG in 3T3-L1 cells transfected with control or YTHDF2-FLAG. Enrichment of CCND1 with FLAG was measured by qPCR and normalized to input. (b) Western blot analysis of FLAG and YTHDF2 in cells transfected with control and YTHDF2-FLAG plasmid. The right panel shows the relative protein levels quantified by densitometry and normalized to GAPDH. (c) Lifespans of CCND1 expression in cells transfected with the YTHDF2 overexpression plasmid compared with those of cells transfected with empty vector. Relative mRNA levels quantified by qPCR. (d) Western blot analysis of CCND1 in control, ZFP217 knockdown or YTHDF2 knockdown cells after MDI-induced for 24 h. The right panel shows the relative protein levels quantified by densitometry and normalized to GAPDH. (e) ORO staining of control, ZFP217 knockdown and ZFP217+ YTHDF2 knockdown cells after MDI-induced for 8 days. The data are presented as the mean ± SD of triplicate tests. *P< 0.05, **P< 0.01, ***P< 0.001 compared to the control group.
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This work is supported by the National Natural Science Foundation of China [Grant No. 31572413], the National Key Research and Development Program [2018YFD0500405], the Natural Science Foundation of Zhejiang Province [No. LZ17C1700001].

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