MED14 tethers mediator to the N-terminal domain of peroxisome proliferator-activated receptor gamma and is required for full transcriptional activity and adipogenesis.
The Mediator subunit MED1/TRAP220/DRIP205/PBP interacts directly with many nuclear receptors and was long thought to be responsible for tethering Mediator to peroxisome proliferator-activated receptor (PPAR)-responsive promoters. However, it was demonstrated recently that PPARgamma can recruit Mediator by MED1-independent mechanisms. Here, we show that target gene activation by ectopically expressed PPARgamma and PPARalpha is independent of MED1. Consistent with this finding, recruitment of PPARgamma, MED6, MED8, TATA box-binding protein (TBP), and RNA polymerase II (RNAPII) to the enhancer and proximal promoter of the PPARgamma target gene Fabp4 is also independent of MED1. Using a small interfering RNA (siRNA)-based approach, we identify MED14 as a novel critical Mediator component for PPARgamma-dependent transactivation, and we demonstrate that MED14 interacts directly with the N terminus of PPARgamma in a ligand-independent manner. Interestingly, MED14 knockdown does not affect the recruitment of PPARgamma, MED6, and MED8 to the Fabp4 enhancer but does reduce their occupancy of the Fabp4 proximal promoter. In agreement with the necessity of MED14 for PPARgamma transcriptional activity, we show that knockdown of MED14 impairs adipogenesis of 3T3-L1 cells. Thus, MED14 constitutes a novel anchoring point between Mediator and the N-terminal domain of PPARgamma that is necessary for functional PPARgamma-mediated recruitment of Mediator and transactivation of PPARgamma subtype-specific target genes.
Grøntved
Lars
L
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Madsen
Maria S
MS
Boergesen
Michael
M
Roeder
Robert G
RG
Mandrup
Susanne
S
D041721:3T3-L1 Cells / Q000378:metabolism; D050156:Adipogenesis / Q000235:genetics*; D000818:Animals; D004742:Enhancer Elements, Genetic / Q000235:genetics; D050556:Fatty Acid-Binding Proteins / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D056913:Mediator Complex Subunit 1 / Q000378:metabolism*; D051379:Mice; D047495:PPAR gamma / Q000737:chemistry* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
C493411:Fabp4 protein, mouse; D050556:Fatty Acid-Binding Proteins; C538856:Med1 protein, mouse; C122171:Med14 protein, mouse; D056913:Mediator Complex Subunit 1; D047495:PPAR gamma; D021122:Protein Subunits; D034741:RNA, Small Interfering; D014157:Transcription Factors; D008156:Luciferases