Abstract: TH-PO1175
Comprehensive Analysis of Vitamin D Receptor Regulators by CRISPR Screening
Session Information
- CKD: Mechanisms, AKI, and Beyond - 1
November 06, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Hata, Satoshi, Fukushima Kenritsu Ika Daigaku, Fukushima, Fukushima Prefecture, Japan
- Moriya, Reika Flora, Fukushima Kenritsu Ika Daigaku, Fukushima, Fukushima Prefecture, Japan
- Tanaka, Kenichi, Fukushima Kenritsu Ika Daigaku, Fukushima, Fukushima Prefecture, Japan
Background
The vitamin D receptor (VDR) is a nuclear receptor functioning as a transcription factor. It is mainly activated by its ligand, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and various cofactors. VDR regulates gene expression and is involved in physiological processes including bone metabolism, cell proliferation and differentiation, and immune function. Disordered vitamin D metabolism is one of the characteristic features of uremia.
Like other nuclear receptors, VDR exhibits tissue-specific functions, suggesting its activity is regulated not only by ligands but also by a range of upstream regulators, both positive and negative. While many studies have focused on ligands and cofactors, post-translational modifications such as phosphorylation, acetylation, and methylation remain poorly understood. Therefore, it is important to investigate ligand-independent regulatory mechanisms.
We developed a reporter assay to visualize VDR signaling using HaCaT cells derived from keratinocytes and conducted a CRISPR-based screen to comprehensively identify VDR regulators. This study presents an overview of our screening strategy and key findings.
Methods
To visualize both enhancement and suppression of VDR signaling, we cloned a plasmid in which GFP was placed downstream of the CYP24A1 promoter and introduced it into HaCaT cells. To generate a diverse pool of single-gene knockout cells, we transduced these reporter cells with a pooled CRISPR library consisting of lentivirus-packaged guide RNAs (gRNAs). GFP fluorescence was used as a readout to sort cell populations in which presumed positive or negative regulators of VDR had been knocked out.
Results
We identified distinct sets of screen hits corresponding to potential positive and negative regulators of VDR signaling. As expected, the VDR was listed as a top candidate, confirming that the screen was working efficiently. Gene Set Enrichment Analysis (GSEA) was performed to prioritize candidate genes, and a gene set associated with G protein-coupled receptors (GPCRs) was of particular interest. We identified GPCR-X as a putative positive regulator. RT-qPCR analysis showed that GPCR-X synergistically enhanced CYP24A1 expression in the presence of calcitriol.
Conclusion
CRISPR screening yielded multiple candidate genes that may function as upstream regulators of VDR. We are currently validating these candidates and exploring their potential clinical relevance.