Cyclin C regulates adipogenesis by stimulating transcriptional activity of CCAAT/enhancer-binding protein α

Abstract

Brown adipose tissue is important for maintaining energy homeostasis and adaptive thermogenesis in rodents and humans. As disorders arising from dysregulated energy metabolism, such as obesity and metabolic diseases, have increased, so has interest in the molecular mechanisms of adipocyte biology. Using a functional screen, we identified cyclin C (CycC), a conserved subunit of the Mediator complex, as a novel regulator for brown adipocyte formation. siRNA-mediated CycC knockdown (KD) in brown preadipocytes impaired the early transcriptional program of differentiation, and genetic KO of CycC completely blocked the differentiation process. RNA sequencing analyses of CycC-KD revealed a critical role of CycC in activating genes co-regulated by peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Overexpression of PPARγ2 or addition of the PPARγ ligand rosiglitazone rescued the defects in CycC-KO brown preadipocytes and efficiently activated the PPARγ-responsive promoters in both WT and CycC-KO cells, suggesting that CycC is not essential for PPARγ transcriptional activity. In contrast, CycC-KO significantly reduced C/EBPα-dependent gene expression. Unlike for PPARγ, overexpression of C/EBPα could not induce C/EBPα target gene expression in CycC-KO cells or rescue the CycC-KO defects in brown adipogenesis, suggesting that CycC is essential for C/EBPα-mediated gene activation. CycC physically interacted with C/EBPα, and this interaction was required for C/EBPα transactivation domain activity. Consistent with the role of C/EBPα in white adipogenesis, CycC-KD also inhibited differentiation of 3T3-L1 cells into white adipocytes. Together, these data indicate that CycC activates adipogenesis in part by stimulating the transcriptional activity of C/EBPα.

Keywords: C/EBPα; Mediator complex; PPARγ; adipocyte; adipogenesis; brown adipocyte; cell differentiation; cyclin C; gene expression; transcription coregulator.

Figure 1.

Screen for novel regulators of brown adipogenesis. A–C, establishment of mouse brown preadipocyte lines (A) and qRT-PCR analyses of Fabp4 (B) and Ucp1 (C) mRNA levels during differentiation. D, the shRNA screen scheme. E, biological process analyses of positive hits. F, enriched transcriptional cofactors.

Figure 2.

CycC in brown fat. A and B, immunoblotting of the indicated proteins during brown preadipocyte differentiation (A) and in BAT from 3- or 24-month-old (M) old male C57Bl/6J mice (B). C, densitometry data of B (*, p < 0.05; #, p < 0.01 versus 3 M; n = 3). D and E, real-time RT-PCR (D) and immunoblotting (E) analyses of the indicated genes at 22 °C and 4 °C (6-h exposure; &, p < 0.001 versus 22 °C; n = 5). F, CycC immunostaining of brown preadipocytes before (Day 0) and after (Day 7) differentiation. Scale bar = 50 μm.

Figure 3.

CycC-KD impairs brown adipogenesis. A, qRT-PCR analyses of CycC-KD. B and C, immunoblotting of the indicated proteins in siRNA-treated cells before (B) or after 2 days of differentiation (C). D, oil red O staining of siRNA-treated cells after day 5 of differentiation. Scale bar = 100 μm). E—G, qRT-PCR analyses of CycC-KD effects at day 2 and/or day 5 of brown preadipocyte differentiation. *, p < 0.05; #, p < 0.01; &, p < 0.001 versus NS-siRNA (n = 3).

Figure 4.

CycC-KO blocks brown adipogenesis. A, qRT-PCR analyses of Ccnc in brown preadipocytes. B—E, oil red O staining (B), qRT-PCR (C and D) and immunoblotting (E) analyses of CycC-KO on brown preadipocyte differentiation at day 8. F and G, CycC overexpression in CycC-KO brown preadipocytes analyzed by qRT-PCR (F) and immunoblotting (G). H and I, effects of CycC overexpression on differentiation in CycC-KO brown preadipocytes at day 10 as analyzed by oil red O staining (H) and qRT-PCR (I). J—M, effects of CycC overexpression in WT brown preadipocytes (J) on adipogenesis at day 7 as analyzed by qRT-PCR (K), oil red O staining (L), and semiquantitative measurement of lipids (M). N, immunoblotting analyses of CycC overexpression in WT brown preadipocytes. Scale bars = 100 μm. *, p < 0.05; #, p < 0.01; &, p < 0.001 versus control (n = 3).

Figure 5.

CycC is required to activate the PPAR pathway in brown adipogenesis. A, number of genes affected by CycC-KD at day 2 of differentiation. B, biological process analyses of CycC-KD down-regulated genes (black bars, numbers of genes; red bars, p values). C, KEGG pathway analyses of CycC-KD down-regulated genes. D, heat map of down-regulated genes in the PPAR pathway. E, qRT-PCR analyses of indicated genes. #, p < 0.01; &, p < 0.001 versus NS-siRNA (n = 3).

Figure 6.

CycC is not required for PPARγ to activate targets in brown adipocytes. A—E, effects of PPARγ2 overexpression (A) or treatment with rosiglitazone (Rosi, 0.5 μm) in CycC-KO brown preadipocytes on differentiation (day 8) as analyzed by oil red O staining (B and D) (scale bars = 100 μm) and qRT-PCR (C and E). F and G, effects of PPARγ2 overexpression on the indicated genes (F) and the activity of a synthetic PPARγ-responsive promoter (PPRE) (G) in WT and KO cells. *, p < 0.05; #, p < 0.01, and &, p < 0.001 versus WT (n = 4).

Figure 7.

CycC is required for C/EBPα transcriptional activity. A, number of defined PPARγ and/or C/EBPα target genes affected by CycC-KD. B—F, effects of C/EBPα overexpression (B) in CycC-KO brown preadipocytes on gene expression (C), differentiation (day 8) as analyzed by oil red O staining (D) (scale bars = 100 μm) and qRT-PCR (E), and the activity of a synthetic C/EBP-responsive promoter (3×CBE) (F). G, co-IP analyses of HA-tagged CycC binding to FLAG-tagged C/EBPα in HEK293T cells. H, effects of CycC knockout on the activities of indicated TADs by luciferase reporter assays. *, p < 0.05; #, p < 0.01; &, p < 0.001 versus WT (n = 4).

Figure 8.

CycC-KD impairs white adipogenesis. A—C, effects of C/EBPβ overexpression (A; asterisk, nonspecific signal) in CycC-KO brown preadipocytes on differentiation (day 8) as assayed by oil red O staining (B) and qRT-PCR (C). D—H, effects of CycC-KD (D and E) on 3T3-L1 cell differentiation (day 8) as analyzed by oil red O staining (F), semiquantitative measurement of lipids (G), and qRT-PCR (H). Scale bars = 100 μm. *, p < 0.05; #, p < 0.01; &, p < 0.001 versus control (n = 3).