PPARgamma is a master regulator of adipogenesis and fat metabolism. It belongs to the nuclear receptor super-family of ligand activated transcription factors. Highly selective PPARgamma ligands are being used in clinic to treat millions of patients with type 2 diabetes. However, the molecular mechanism by which these ligands act as anti-diabetes agents has largely remained unclear. The tripartite nature of the nuclear receptor biology suggests that the biological effect of a ligand is determined by the combinatorial collaboration among these three parts: ligand, nuclear receptor, and cofactors (coactivators or corepressors) recruited by ligand-bound nuclear receptor on the target gene promoters. ? ? To understand the molecular mechanism by which ligand-activated PPARgamma transcriptionally regulates fat metabolism, we started using proteomic approaches to isolate and characterize transcription cofactors for PPARgamma2. As expected, GST-PPARgammaLBD (ligand binding domain) pulled down subunits of the TRAP/Mediator transcription coactivator complex and RNA polymerase II from HeLaS nuclear extracts. In addition, PTIP (Pax transactivation domain-interacting protein), a 120KDa nuclear protein with multiple BRCT domains, and Mybbp1a (p160 Myb-Binding Protein), a PGC1a-interacting protein, were also identified among others. In reporter gene assay, PTIP showed robust transcriptional coactivator activity for PPARgamma2, an activity comparable with that of PGC1a. ? ? PTIP has been implicated in DNA damage response. However, its normal cellular functions remain unclear. To understand the cellular function of PTIP, we affinity purified PTIP associated proteins from cells. We show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4/ALR, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Further, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5 and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1 and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a role of PTIP in the regulation of gene expression.? ? We are currently investigating the molecular mechanism by which PTIP functions as a transcriptional coactivator of PPARgamma. Specifically, we are investigating whether the PTIP-associated MLL3- and MLL4-containing HMT complexes are involved in the regulation of PPARgamma activity.
