Genomic mechanisms controlling renal vitamin D metabolism

Abstract

Vitamin D metabolism centers on regulation in the kidney of CYP27B1 induction by PTH, suppression by FGF23 and 1,25(OH)₂D₃, and reciprocal CYP24A1 suppression by PTH, and induction by FGF23 and 1,25(OH)₂D₃. This coordinated genomic regulation through enhancer modules results in the production and dynamic maintenance of circulating endocrine 1,25(OH)₂D₃ which, together with PTH and FGF23, controls mineral homeostasis. We discovered enhancers near Cyp27b1 in the mouse kidney located within intronic regions of Mettl1 and Mettl21b genes. These kidney-specific enhancers ("M1", "M21") control Cyp27b1. Through CRISPR/Cas deletion, we found that PTH activation of Cyp27b1 is lost with deletion of M1, whereas FGF23 suppression is lost with deletion of M21. The combination of both deletions (M1/M21-DIKO) eliminated the suppression by 1,25(OH)₂D₃. Cyp24a1 activation by 1,25(OH)₂D₃ is controlled by a promoter proximal pair of VDREs as well as a distal region - 35 to - 37 kb (DS2). We also found that FGF23 activation and PTH suppression of Cyp24a1 was located in a region - 21 to - 37 kb downstream (DS1). More recently, using in vivo ChIP-seq in mouse kidney, we demonstrate that PTH activation rapidly induces increased recruitment of pCREB and its coactivators, CBP and CRTC2, to the M1 and M21 enhancers near the Cyp27b1 gene. At distal enhancers of the Cyp24a1 gene, PTH suppression promotes dismisses CBP with only minor changes in pCREB and CRTC2 occupancy, all of which correlate with a suppression of basal histone acetylation across this locus and reduced transcripts. Surprisingly, we find that 1,25(OH)₂D₃ suppression increases the occupancy of CRTC2 in the M1 enhancer, a novel observation for CRTC2 and/or 1,25(OH)₂D₃ action. The suppressive actions of 1,25(OH)₂D₃ and FGF23 at the Cyp27b1 gene are associated with a reduction in CBP recruitment at these enhancers. Although FGF23-regulated transcription factors remain unknown, we hypothesize that VDR occupancy induced at the M1 and M21 enhancers by 1,25(OH)₂D₃ likely disrupts or competes with the active conformation of these CREB modules thereby preventing full induction by PTH. Our findings show coactivators such as CRTC2 and CBP contribute to Cyp27b1 and Cyp24a1 transcription and provide molecular insight into the coordinated mechanistic actions of PTH, FGF23, and 1,25(OH)₂D₃ in the kidney that regulate mineral homeostasis.

Keywords: 1,25(OH)(2)D(3); CRISPR/Cas9; ChIP-seq; Cyp24a1; Cyp27b1-KO; Cytochrome P450; Fibroblast growth factor 23 (FGF23); Gene regulation; Parathyroid hormone (PTH); Vitamin D.