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THRAP3 Interacts with HELZ2 and Plays a Novel Role in Adipocyte Differentiation
- Publication Year :
- 2013
- Publisher :
- Endocrine Society, 2013.
-
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.
- Subjects :
- Immunoprecipitation
Recombinant Fusion Proteins
Amino Acid Motifs
Biology
Fatty Acid-Binding Proteins
Response Elements
Mass Spectrometry
Mice
Endocrinology
Mediator
3T3-L1 Cells
CEBPB
Adipocytes
Animals
Humans
Molecular Biology
Original Research
Regulation of gene expression
Gene knockdown
Serine-Arginine Splicing Factors
Nuclear Proteins
RNA-Binding Proteins
Cell Differentiation
General Medicine
Molecular biology
Chromatin
Protein Structure, Tertiary
DNA-Binding Proteins
PPAR gamma
Repressor Proteins
Enhancer Elements, Genetic
Gene Expression Regulation
Adipogenesis
Gene Knockdown Techniques
Adiponectin
Chromatin immunoprecipitation
RNA Helicases
Chromatography, Liquid
HeLa Cells
Protein Binding
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....66878291303ed285c36361f676f4276e