Comprehensive Analysis of Vitamin D Receptor Regulators by CRISPR Screening

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.