Related Experiment Videos
Inhibition of nuclear receptor signalling by poly(ADP-ribose) polymerase
T Miyamoto1, T Kakizawa, K Hashizume
1Department of Geriatrics, Endocrinology and Metabolism, Shinshu University School of Medicine, Matsumoto 390-8621, Japan. miyamoto@hsp.md.shinshu-u.ac.jp
Abstract:
Mammalian poly(ADP-ribose) polymerase (PARP) is a nuclear chromatin-associated protein with a molecular mass of 114 kDa that catalyzes the transfer of ADP-ribose units from NAD+ to nuclear proteins that are located within chromatin. We report here the identification of a novel property of PARP as a modulator of nuclear receptor signalling. PARP bound directly to retinoid X receptors (RXR) and repressed ligand-dependent transcriptional activities mediated by heterodimers of RXR and thyroid hormone receptor (TR). The interacting surface is located in the DNA binding domain of RXRalpha. Gel shift assays demonstrated that PARP bound to TR-RXR heterodimers on the response element. Overexpression of wild-type PARP selectively blocked nuclear receptor function in transient transfection experiments, while enzyme-defective mutant PARP did not show significant inhibition, suggesting that the essential role of poly(ADP-ribosyl) enzymatic activity is in gene regulation by nuclear receptors. Furthermore, PARP fused to the Gal4 DNA binding domain suppressed the transcriptional activity of the promoter harboring the Gal4 binding site. Thus, PARP has transcriptional repressor activity when recruited to the promoter. These results indicates that poly(ADP-ribosyl)ation is a negative cofactor in gene transcription, regulating a member of the nuclear receptor superfamily.
Insights
Poly(ADP-ribose) polymerase (PARP) acts as a novel repressor of nuclear receptor signaling. Its enzymatic activity is crucial for inhibiting gene transcription mediated by nuclear receptors like retinoid X receptors (RXR).
Area of Science:
- Molecular Biology
- Gene Regulation
- Cellular Signaling
Background:
- Mammalian poly(ADP-ribose) polymerase (PARP) is a chromatin-associated protein involved in DNA repair and other nuclear processes.
- Nuclear receptors, such as retinoid X receptors (RXR) and thyroid hormone receptors (TR), regulate gene expression in response to ligands.
- The precise role of PARP in modulating nuclear receptor signaling pathways remained largely unexplored.
Purpose of the Study:
- To investigate the potential role of PARP as a modulator of nuclear receptor signaling.
- To determine if PARP directly interacts with nuclear receptors and affects their transcriptional activity.
- To elucidate the mechanism by which PARP influences gene transcription regulated by nuclear receptors.
Main Methods:
- Co-immunoprecipitation and gel shift assays to assess PARP binding to nuclear receptors (RXR and TR) and their DNA response elements.
- Transient transfection experiments using wild-type and enzyme-defective PARP mutants to evaluate effects on nuclear receptor-mediated transcription.
- Reporter gene assays with Gal4-fused PARP to determine its intrinsic transcriptional repressor activity.
Main Results:
- PARP directly binds to retinoid X receptors (RXR), specifically within the DNA binding domain of RXRalpha.
- PARP represses ligand-dependent transcriptional activity of TR-RXR heterodimers by binding to their response elements.
- Enzymatically active PARP, but not an enzyme-defective mutant, significantly inhibited nuclear receptor function, indicating the importance of poly(ADP-ribosyl)ation.
- PARP functions as a transcriptional repressor when recruited to a promoter, independent of its interaction with nuclear receptors.
Conclusions:
- PARP acts as a novel negative cofactor in gene transcription, specifically modulating nuclear receptor superfamily signaling.
- The poly(ADP-ribosyl)ation enzymatic activity of PARP is essential for its role in repressing nuclear receptor-mediated gene expression.
- These findings reveal a new layer of regulation in nuclear receptor signaling, involving PARP-mediated transcriptional repression.