Alternative mechanisms by which mediator subunit MED1/TRAP220 regulates peroxisome proliferator-activated receptor gamma-stimulated adipogenesis and target gene expression.
Mediator is a general coactivator complex connecting transcription activators and RNA polymerase II. Recent work has shown that the nuclear receptor-interacting MED1/TRAP220 subunit of Mediator is required for peroxisome proliferator-activated receptor gamma (PPARgamma)-stimulated adipogenesis of mouse embryonic fibroblasts (MEFs). However, the molecular mechanisms remain undefined. Here, we show an intracellular PPARgamma-Mediator interaction that requires the two LXXLL nuclear receptor recognition motifs on MED1/TRAP220 and, furthermore, we show that the intact LXXLL motifs are essential for optimal PPARgamma function in a reconstituted cell-free transcription system. Surprisingly, a conserved N-terminal region of MED1/TRAP220 that lacks the LXXLL motifs but gets incorporated into Mediator fully supports PPARgamma-stimulated adipogenesis. Moreover, in undifferentiated MEFs, MED1/TRAP220 is dispensable both for PPARgamma-mediated target gene activation and for recruitment of Mediator to a PPAR response element on the aP2 target gene promoter. However, PPARgamma shows significantly reduced transcriptional activity in cells deficient for a subunit (MED24/TRAP100) important for the integrity of the Mediator complex, indicating a general Mediator requirement for PPARgamma function. These results indicate that there is a conditional requirement for MED1/TRAP220 and that a direct interaction between PPARgamma and Mediator through MED1/TRAP220 is not essential either for PPARgamma-stimulated adipogenesis or for PPARgamma target gene expression in cultured fibroblasts. As Mediator is apparently essential for PPARgamma transcriptional activity, our data indicate the presence of alternative mechanisms for Mediator recruitment, possibly through intermediate cofactors or other cofactors that are functionally redundant with MED1/TRAP220.
Nuclear Receptor Biology Section, Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. kaig@niddk.nih.gov
Kai
K
Ge
Young-Wook
YW
Cho
Hong
H
Guo
Teresa B
TB
Hong
Mohamed
M
Guermah
Mitsuhiro
M
Ito
Hong
H
Yu
Markus
M
Kalkum
Robert G
RG
Roeder
D050156:Adipogenesis; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004706:Endodeoxyribonucleases / Q000378:metabolism / Q000502:physiology*; D050556:Fatty Acid-Binding Proteins / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology; D005786:Gene Expression Regulation; D056913:Mediator Complex Subunit 1; D051379:Mice; D018345:Mice, Knockout; D047495:PPAR gamma / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation
C493411:Fabp4 protein, mouse; D050556:Fatty Acid-Binding Proteins; C538855:MED1 protein, human; C538856:Med1 protein, mouse; D056913:Mediator Complex Subunit 1; D047495:PPAR gamma; D014157:Transcription Factors; D004706:Endodeoxyribonucleases; C118712:MBD4 protein, human