Alternative Mechanisms by Which Mediator Subunit MED1/TRAP220 Regulates Peroxisome Proliferator-Activated Receptor γ-Stimulated Adipogenesis and Target Gene Expression

Abstract

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 ␥ (PPAR␥)-stimulated adipogenesis of mouse embryonic fibroblasts (MEFs). However, the molecular mechanisms remain undefined. Here, we show an intracellular PPAR␥-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 PPAR␥ 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 PPAR␥-stimulated adipogenesis. Moreover, in undifferentiated MEFs, MED1/TRAP220 is dispensable both for PPAR␥-mediated target gene activation and for recruitment of Mediator to a PPAR response element on the aP2 target gene promoter. However, PPAR␥ 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 PPAR␥ function. These results indicate that there is a conditional requirement for MED1/TRAP220 and that a direct interaction between PPAR␥ and Mediator through MED1/TRAP220 is not essential either for PPAR␥-stimulated adipogenesis or for PPAR␥ target gene expression in cultured fibroblasts. As Mediator is apparently essential for PPAR␥ 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.Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a key regulator of transcriptional pathways important for adipogenesis (34). PPAR␥ Ϫ/Ϫ mice show a total absence of both brown and white adipose tissue. Furthermore, retrovirus vector-mediated ectopic expression of PPAR␥ alone can stimulate mouse embryonic fibroblasts (MEFs) to undergo adipogenesis. In such cells, the expression of CCAAT/enhancer-binding protein ␣ (C/EBP␣), another key transcriptional regulator of adipogenesis, and adipogenesis markers such as aP2, fatty acid synthase (FAS), and adipsin are induced in a PPAR␥-dependent manner.PPAR␥ and other nuclear hormone receptors comprise a superfamily of DNA binding transcription factors. However, they also require various transcriptional coactivators to activate, in a ligand-dependent manner, transcription of the specific target genes important for cell growth, homeostasis, and differentiation (36). These transcription coactivators often exist as multiprotein complexes. They may act either through chromatin remodeling and histone modification, after recruitment by promoter-bound nuclear receptors, or at steps involving subsequent preinitiation complex formation or function (transcription initiation and elongation). Transcripti...

Key roles for MED1 LxxLL motifs in pubertal mammary gland development and luminal-cell differentiation

“…Furthermore, it recently was found that Mediator can be recruited to ERα target genes by other coactivators such as CCAR1 (37) and to other nuclear receptor-promoter complexes by PGC-1α (38), which also interacts directly with ERα (27). We also have found that the MED1 LxxLL motifs, but not MED1 itself, is dispensable for PPARγ-mediated adipogenesis (15). Therefore, the presence or high level expression of certain other ERα-interacting cofactors in these estrogen-responsive tissues could bypass the requirement for the MED1 LxxLL motifs.…”

“…1A and Fig. S1), which previously was shown to disrupt strong Mediator-nuclear receptor interactions in vitro (14,15). These mutations had no effect on the expression level of the MED1 protein (Fig.…”

“…TRAP220/PBP/DRIP205) subunit (10)(11)(12)(13). Importantly, in relation to the present study, in vitro assays with intact Mediator complexes containing mutated MED1 LxxLL motifs have established direct roles for the LxxLL motifs in strong (ligand-dependent) nuclear receptor-Mediator interactions and in Mediatordependent transcription by nuclear receptors (14,15).…”

The Transcriptional Mediator Subunit MED1/TRAP220 in Stromal Cells Is Involved in Hematopoietic Stem/Progenitor Cell Support through Osteopontin Expression

“…In contrast, mutant II, having NR boxes [MED1(1-703)] showed full recovery of both basal activity and ligand-induced activation. However, mutant III [MED1(592-1587)], incapable of complex formation (8), was unable to recover any activity. Finally, mutant IV, the fulllength MED1 with two inactivated NR boxes (LXXLL to LXXAA), did recover basal transcription but not ligand dependency.…”

Epidermis-Type Lipoxygenase 3 Regulates Adipocyte Differentiation and Peroxisome Proliferator-Activated Receptor γ Activity

“…TRAP220 (Mediator Subunit 1, Med1) can be directly recruited to PPAR␥ in a ligand-dependent manner and is part of the mediator complex shown to be required for adipogenesis (18,19). Furthermore, full PPAR␥ agonists have been shown to recruit TRAP220 more efficiently than partial PPAR␥ agonists (37).…”