Abstract: FR-PO569
The TRIM28-PARP1-CTNNB1 Complex Is Associated with the Transcriptional Elongation Machinery for Aqp2 Gene Transcription
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Jang, Hyo-Ju, Kyungpook National University School of Medicine, Daegu, Korea (the Republic of)
- Park, Euijung, National Heart Lung and Blood Institute, Bethesda, Maryland, United States
- Jung, Hyun Jun, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
- Kwon, Tae-Hwan, Kyungpook National University School of Medicine, Daegu, Korea (the Republic of)
Background
Vasopressin enhances water reabsorption in collecting duct cells by increasing the abundance of Aquaporin-2 (AQP2) through transcriptional and post-translational mechanisms. We recently discovered that a complex comprising CTNNB1 and PARP1 acts as a transcriptional regulator of Aqp2 gene transcription in response to vasopressin. In this study, we introduce a Bromodomain protein, Tripartite motif-containing 28 (TRIM28; also known as KAP1), into the transcriptional machinery that controls RNA polymerase II (RNA Pol II) transcription elongation in the AQP2 regulation.
Methods
For in vitro experiments, we used the mouse collecting duct cell line mpkCCDc14, grown on semipermeable filters and treated with dDAVP for 24 h to induce AQP2 expression. We applied siRNA-mediated knockdown of Ctnnb1 or Trim28 using a reverse transfection strategy. RNA sequencing (RNA-Seq) was then conducted on mpkCCD cells with Ctnnb1 knockdown. We employed immunolabeling and immunoblotting to verify TRIM28 protein expression in kidney tissues and immunoprecipitated proteins.
Results
RNA-Seq demonstrated that dDAVP significantly induces Aqp2 expression, which is reduced after Ctnnb1 knockdown. Bioinformatic analysis identified transcriptional regulators related to the promoters of genes differentially expressed after dDAVP treatment or Ctnnb1 knockdown, and highlighted the role of these regulators in RNA Pol II modulation. Bioinformatics and immunoprecipitation assays identified several Bromodomain proteins, including TRIM28, as interacting partners of CTNNB1 and PARP1 associated with RNA Pol II elongation. Immunohistochemistry demonstrated the nuclear presence of TRIM28 in mouse kidney collecting duct cells. Importantly, siRNA-mediated knockdown of Trim28 in mpkCCDc14 cells significantly reduced the dDAVP-induced Aqp2 gene expression, observable at both mRNA and protein levels after 6 and 24 h of treatment. An immunoprecipitation assay further confirmed a direct interaction between TRIM28 and RNA Pol II.
Conclusion
This study reveals TRIM28 as a crucial element of the transcriptional machinery that regulates RNA Pol II activity in the transcription of the Aqp2 gene. These findings provide insights into the transcriptional regulation of Aqp2, highlighting the role of the TRIM28-containing regulatory complex in controlling RNA Pol II function.
Funding
- Government Support – Non-U.S.