Related Experiment Videos
Promyelocytic leukemia protein PML inhibits Nur77-mediated transcription through specific functional interactions
Wen-Shu Wu1, Zhi-Xiang Xu, Ruixiang Ran
1Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Abstract:
The promyelocytic leukemia protein PML is a tumor and growth suppressor and plays an important role in a multiple pathways of apoptosis and regulation of cell cycle progression. Our previous studies and others also documented a role of PML in transcriptional regulation through its association with transcription coactivator CBP and transcription corepressor HDAC. Here, we showed that PML is a potent transcriptional repressor of Nur77, an orphan receptor and a member of the steroid receptor superfamily of proteins. We found that PML represses Nur77-mediated transactivation through a physical and functional interaction between the two proteins. PML interacts with Nur-77 in vitro in a GST-pull down assay and in vivo by coimmunoprecipitation assay. PML/Nur77 colocalized in vivo in a double immunofluorescent staining and confocal microscopic analysis. Our study further showed that the coiled-coil domain of PML interacts with the DNA-binding domain of Nur77 (amino acids 267-332). Electrophoretic mobility shift assay demonstrated that PML interferes with Nur77 DNA binding in a dose-dependent manner. This study indicates that PML interacts with the DNA-binding domain of Nur77 and represses transcription by preventing it from binding to the target promoter. This study supports a role of PML/Nur77 interaction in regulating cell growth and apoptosis.
Insights
The promyelocytic leukemia protein (PML) acts as a transcriptional repressor of Nur77. PML binds to Nur77, inhibiting its DNA binding and repressing transcription, which impacts cell growth and apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The promyelocytic leukemia protein (PML) is a known tumor suppressor involved in apoptosis and cell cycle regulation.
- PML has established roles in transcriptional regulation via interactions with coactivators like CBP and corepressors like HDAC.
- Nur77 is an orphan receptor and a member of the steroid receptor superfamily, implicated in various cellular processes.
Purpose of the Study:
- To investigate the role of PML as a transcriptional regulator of Nur77.
- To elucidate the molecular mechanisms underlying the interaction between PML and Nur77.
- To determine the functional consequences of PML-Nur77 interaction on gene transcription.
Main Methods:
- GST-pull down assays to assess in vitro interaction.
- Coimmunoprecipitation assays to confirm in vivo interaction.
- Double immunofluorescence staining and confocal microscopy for colocalization studies.
- Electrophoretic mobility shift assays (EMSA) to evaluate DNA binding interference.
Main Results:
- PML physically interacts with Nur77 both in vitro and in vivo.
- PML and Nur77 colocalize within the cell.
- The coiled-coil domain of PML interacts with the DNA-binding domain of Nur77.
- PML inhibits Nur77's ability to bind to its target promoter in a dose-dependent manner, thereby repressing Nur77-mediated transactivation.
Conclusions:
- PML functions as a potent transcriptional repressor of Nur77.
- The interaction between PML and Nur77's DNA-binding domain is crucial for transcriptional repression.
- This PML-Nur77 interaction plays a significant role in the regulation of cell growth and apoptosis.