Research ArticleMetabolism

Transcriptional activation of lipogenesis by insulin requires phosphorylation of MED17 by CK2

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Science Signaling  21 Feb 2017:
Vol. 10, Issue 467, eaai8596
DOI: 10.1126/scisignal.aai8596
  • Fig. 1 USF1 directly interacts with MED17.

    (A) Immunoblot (IB) of proteins from coimmunoprecipitation (Co-IP) of 293FT cells overexpressing FLAG-tagged MED17 (75 kDa) and hemagglutinin (HA)–tagged USF1 (37 kDa). n = 6 independent experiments. (B) IB of proteins from Co-IP of liver nuclear extracts using either MED17 or USF1 antibodies. n = 6 independent experiments. (C) Left: Diagram of USF1 deletion constructs. Right: GST-USF1 proteins in bacterial cell lysates were detected by Coomassie staining (top). GST pull-down assays were performed with GST-USF1 proteins and 35S-labeled MED17 (bottom). n = 5 independent experiments. (D) Left: Diagram of MED17 deletion constructs. Right: 6xHis-MED17 proteins in bacterial cell lysates were detected by immunoblotting (top). His-tag pull-down assays were performed with 6xHis-MED17 proteins and 35S-labeled USF1 (bottom). n = 5 independent experiments. (E and F) FASN promoter activity in 293FT cells overexpressing the indicated proteins (means ± SEM). *P < 0.05, different from FASN; #P < 0.05, different from FASN + USF1; +P < 0.05, different from FASN + USF1 + MED17. n = 6 wells of cells per group. luc, luciferase.

  • Fig. 2 MED17 is recruited to the FASN promoter in a feeding- and insulin-dependent manner.

    (A to D) ChIP using the indicated antibodies of 293FT cells transfected with indicated vectors. n = 5 wells of cells per group. (A) Cells were transfected with −444-FASN-luc, FLAG-MED17, and either HA-USF1 or empty vector. (B) Cells were transfected with −444-FASN-luc, FLAG-MED17, and either USF1 siRNA or scrambled siRNA. (C) Cells were transfected with FLAG-MED17, HA-USF1, and either −444-FASN-luc or −65m-FASN-luc. (D) Cells were transfected with −444-FASN-luc, HA-USF1, and either FLAG-MED17 [wild type (WT)] or FLAG-MED17 (1 to 621). (E) Left: Map of the HepG2 FASN promoter region showing the relative enrichment of USF1, MED17, and POL II. Agarose gel image of Re-ChIP of FASN promoter in HepG2 cells (middle) and quantification by quantitative polymerase chain reaction (qPCR) (right) using primers targeting the −65 E-box region. n = 5 dishes of cells per group. LXRE, liver X receptor response element; TSS, transcription start site; Ab1, antibody 1. (F) ChIP-qPCR of fatty acid synthetic and oxidative promoters in HepG2 cells using the indicated antibodies. n = 5 dishes of cells per group. (G) ChIP-qPCR analysis of fatty acid synthetic and oxidative promoters in livers of fasted and refed mice. n = 5 mice per group. (A to G) Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 Differential phosphorylation of MED17 is required for FASN promoter activation.

    (A and B) FASN promoter activity and representative blots of 293FT cells transfected with the indicated vectors. *P < 0.05, different from FASN; #P < 0.05, different from FASN + USF1; +P < 0.05, different from FASN + USF1 + MED17. n = 8 wells of cells per group for (A) and n = 5 wells of cells per group for (B). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) IB of immunoprecipitated MED17 from nuclear extracts of livers from fasted or refed mice (n = 5 mice per group) or serum-starved or insulin-treated HepG2 cells (n = 4 independent experiments) using phosphorylated Ser53 (P-Ser53) and total MED17 antibodies.

  • Fig. 4 CK2-mediated phosphorylation of MED17 at Ser53 is required for its recruitment and activation of FASN promoter in response to insulin.

    (A) In vitro phosphorylation assays with WT, S53A, or S55A forms of MED17 and recombinant CK2. Reactions were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and either immunoblotted with the indicated antibodies or stained with phosphospecific or total protein dye. n = 4 independent experiments. (B) IB of 293FT cells overexpressing MED17. Cells were transfected with either empty vector (EV) or CK2α and CK2β. Cells were immunoblotted using antibodies against P-Ser53, MED17 (75 kDa), and CK2α (45 kDa). n = 5 independent experiments. (C) IB of 293FT cells transfected with CK2α and CK2β and WT, S53A, or T570D forms of MED17. n = 4 independent experiments. (D) IB of HepG2 cells that were serum-starved overnight, pretreated with CX-4945 or vehicle for 30 min, and then treated with insulin for 30 min. n = 5 independent experiments. (E) FASN promoter activity in 293FT cells overexpressing the indicated proteins (means ± SEM). *P < 0.05, different from FASN; #P < 0.05, different from FASN + USF1; +P < 0.05, different from FASN + USF1 + MED17. n = 8 wells of cells per group. (F) qPCR of HepG2 cells that were serum-starved, pretreated with CX-4945, and then treated with insulin [means ± SEM; different from (−) insulin + vehicle at *P < 0.05, **P < 0.01 and (+) insulin + vehicle at P < 0.05]. n = 7 wells of cells per group. (G) ChIP-qPCR of the FASN promoter from HepG2 cells infected with MED17 (WT) or MED17 (S53A) adenovirus (Ad). n = 5 dishes of cells per group. (H) ChIP-qPCR of HepG2 cells serum-starved overnight and then pretreated with CX-4945 for 30 min before 8-hour treatment with insulin. n = 5 dishes of cells per group. (I) ChIP-qPCR of chromatin from serum-starved/insulin-treated HepG2 cells at the FASN proximal promoter region and 8500 bp downstream of the transcription start site. n = 5 dishes of cells per group. (G to I) Means ± SEM. *P < 0.05, ***P < 0.001.

  • Fig. 5 Phosphorylation of Ser53 in MED17 is required for lipogenesis by insulin in vivo.

    (A and B) MED17 mRNA abundance (left) and representative blots (middle) in HepG2 cells (A) (n = 5 dishes of cells per group) or livers of mice (B) (n = 5 mice per group) after infection with either control Ad or Ad-MED17-S53A. mRNA abundance of the indicated genes (right). Dashed lines indicate image splicing to remove unnecessary lanes. (C) Percent of newly synthesized palmitate (left) and hepatic triglyceride content (right) in livers of control Ad or Ad-MED17-S53A–infected mice (n = 5 mice per group). DNL, de novo lipogenesis. (D) Left: Representative blot of MED17 after infection of HepG2 cells with either control Ad or Ad-shMED17 for MED17 knockdown. Right: mRNA abundance of the indicated fatty acid synthetic or oxidative genes (n = 5 dishes of cells per group). GFP, green fluorescent protein. (E) MED17 mRNA abundance (left) and representative blot (middle) from livers of mice injected with either control or Ad-shMED17 Ad. Right: mRNA abundance of the indicated fatty acid synthetic or oxidative genes (n = 5 mice per group). (F) Nascent RNA abundance of fatty acid synthetic or oxidative genes (n = 5 mice per group). (G) Percent of newly synthesized palmitate (left), hepatic triglyceride content (middle), and representative images Oil Red O staining with 50-μm scale bars (right) (n = 5 mice per group) of livers from mice administered control or shMED17 Ad. (A to G) Means ± SEM. *P < 0.05, **P < 0.01.

  • Fig. 6 CK2 phosphorylates MED17 to promote lipogenesis in response to insulin in vivo.

    (A) Left: CK2α1 mRNA abundance in livers from mice injected with control Ad or Ad-shCK2α1. Middle: Representative blot of MED17 and CK2α1. Right: mRNA abundance of fatty acid synthetic or oxidative genes (n = 5 mice per group). (B) Hepatic triglyceride content of mice infected with either control Ad or Ad-shCK2α1 (n = 5 mice per group). (C) mRNA abundance for the indicated genes from livers of mice treated with either vehicle or the CK2 inhibitor CX-4945(n = 5 mice per group). (D) Hepatic triglyceride content in mice treated with either vehicle or CX-4945 (n = 5 mice per group). (A to D) Means ± SEM. *P < 0.05, **P < 0.01. (E) Insulin signaling pathways for USF1-mediated transcriptional activation of lipogenesis through recruitment of coregulators, Mediator complex, and POL II and general transcription machinery.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/467/eaai8596/DC1

    Fig. S1. Coimmunoprecipitation experiments with USF1 and Mediator subunits.

    Fig. S2. Additional experiments for ChIP assays in transfected 293FT cells.

    Fig. S3. MED6 and MED7 ChIP experiments using HepG2 cells.

    Fig. S4. Relative abundance of FASN, USF1, and MED17 mRNA and protein in mice and cells.

    Fig. S5. Immunoblot of phosphoserine and MED17 from liver nuclear extracts.

    Fig. S6. In vitro phosphorylation assay for MED17 using p38 MAPK.

    Fig. S7. Triglyceride accumulation in HepG2 cells after knockdown of MED17 or CK2.

    Fig. S8. Adenovirally mediated overexpression of MED17 and rescue experiments.

    Fig. S9. FASN protein abundance in the livers of shMED17-, shCK2-, or S53A-adenovirus–infected mice.

    Fig. S10. MED17 phosphorylation and FASN mRNA abundance in the liver of ob/ob mice.

    Table S1. Primers used for ChIP.

  • Supplementary Materials for:

    Transcriptional activation of lipogenesis by insulin requires phosphorylation of MED17 by CK2

    Jose A. Viscarra, Yuhui Wang, Il-Hwa Hong, Hei Sook Sul*

    *Corresponding author. Email: hsul{at}berkeley.edu

    This PDF file includes:

    • Fig. S1. Coimmunoprecipitation experiments with USF1 and Mediator subunits.
    • Fig. S2. Additional experiments for ChIP assays in transfected 293FT cells.
    • Fig. S3. MED6 and MED7 ChIP experiments using HepG2 cells.
    • Fig. S4. Relative abundance of FASN, USF1, and MED17 mRNA and protein in mice and cells.
    • Fig. S5. Immunoblot of phosphoserine and MED17 from liver nuclear extracts.
    • Fig. S6. In vitro phosphorylation assay for MED17 using p38 MAPK.
    • Fig. S7. Triglyceride accumulation in HepG2 cells after knockdown of MED17 or CK2.
    • Fig. S8. Adenovirally mediated overexpression of MED17 and rescue experiments.
    • Fig. S9. FASN protein abundance in the livers of shMED17-, shCK2-, or S53A-adenovirus–infected mice.
    • Fig. S10. MED17 phosphorylation and FASN mRNA abundance in the liver of ob/ob mice.
    • Table S1. Primers used for ChIP.

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    Citation: J. A. Viscarra, Y. Wang, I.-H. Hong, H. S. Sul, Transcriptional activation of lipogenesis by insulin requires phosphorylation of MED17 by CK2. Sci. Signal. 10, eaai8596 (2017).

    © 2017 American Association for the Advancement of Science

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