title: THRAP3 interacts with HELZ2 and plays a novel role in adipocyte differentiation.
abstract: Using yeast two-hybrid screen, we previously isolated HELZ2 (helicase with zinc finger 2, transcriptional coactivator) that functions as a coregulator of peroxisome proliferator-activated receptorγ (PPARγ). To further delineate its molecular function, we here identified thyroid hormone receptor-associated protein3 (THRAP3), a putative component of the Mediator complex, as a protein stably associating with HELZ2 using immunoprecipitation coupled with mass spectrometry analyses. In immunoprecipitation assays, Thrap3 could associate with endogenous Helz2 as well as Pparg in differentiated 3T3-L1 cells. HELZ2 interacts with the serine/arginine-rich domain and Bcl2 associated transcription factor1-homologous region in THRAP3, whereas THRAP3 directly binds 2 helicase motifs in HELZ2. HELZ2 and THRAP3 synergistically augment transcriptional activation mediated by PPARγ, whereas knockdown of endogenous THRAP3 abolished the enhancement by HELZ2 in reporter assays. Thrap3, similar to Helz2, is evenly expressed in the process of adipogenic differentiation in 3T3-L1 cells. Knockdown of Thrap3 in 3T3-L1 preadipocytes using short-interfering RNA did not influence the expression of Krox20, Klf5, Cebpb, or Cebpd during early stages of adipocyte differentiation, but significantly attenuated the expression of Pparg, Cebpa, and Fabp4/aP2 and accumulation of lipid droplets. Pharmacologic activation of Pparg by troglitazone could not fully restore the differentiation of Thrap3-knockdown adipocytes. In chromatin immunoprecipitation assays, endogenous Helz2 and Thrap3 could be co-recruited, in a ligand-dependent manner, to the PPARγ-response elements in Fabp4/aP2 and Adipoq gene enhancers in differentiated 3T3-L1 cells. These findings collectively suggest that Thrap3 could play indispensable roles in terminal differentiation of adipocytes by enhancing PPARγ-mediated gene activation cooperatively with Helz2.
authors.affiliation: Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Katano-Toki
authors.forename: Tetsurou
authors.initials: T
authors.lastname: Satoh
authors.forename: Takuya
authors.initials: T
authors.lastname: Tomaru
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yoshino
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishizuka
authors.forename: Sumiyasu
authors.initials: S
authors.lastname: Ishii
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ozawa
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Shibusawa
authors.forename: Takafumi
authors.initials: T
authors.lastname: Tsuchiya
authors.forename: Tsugumichi
authors.initials: T
authors.lastname: Saito
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shimizu
authors.forename: Koshi
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Shuichi
authors.initials: S
authors.lastname: Okada
authors.forename: Masanobu
authors.initials: M
authors.lastname: Yamada
authors.forename: Masatomo
authors.initials: M
authors.lastname: Mori
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism*; D052242:Adiponectin / Q000235:genetics; D020816:Amino Acid Motifs; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002853:Chromatography, Liquid; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D050556:Fatty Acid-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D047495:PPAR gamma / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry; D020218:Response Elements / Q000235:genetics; D000068103:Serine-Arginine Splicing Factors
chemical_list: D052242:Adiponectin; C539097:Bclaf1 protein, mouse; D004268:DNA-Binding Proteins; D050556:Fatty Acid-Binding Proteins; D009687:Nuclear Proteins; D047495:PPAR gamma; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D000068103:Serine-Arginine Splicing Factors; D020365:RNA Helicases