Genomic Mechanisms Governing Mineral Homeostasis and the Regulation and Maintenance of Vitamin D Metabolism

Abstract

Our recent genomic studies identified a complex kidney-specific enhancer module located within the introns of adjacent Mettl1 (M1) and Mettl21b (M21) genes that mediate basal and PTH induction of Cyp27b1, as well as suppression by FGF23 and 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. The tissue specificity for this regulatory module appears to be localized exclusively to renal proximal tubules. Gross deletion of these segments in mice has severe consequences on skeletal health, and directly affects Cyp27b1 expression in the kidney. Deletion of both the M1 and M21 submodules together almost completely eliminates basal Cyp27b1 expression in the kidney, creating a renal specific pseudo-null mouse, resulting in a systemic and skeletal phenotype similar to that of the Cyp27b1-KO mouse caused by high levels of both 25-hydroxyvitamin D₃ [25(OH)D₃] and PTH and depletion of 1,25(OH)₂D₃. Cyp24a1 levels in the double KO mouse also decrease because of compensatory downregulation of the gene by elevated PTH and reduced FGF23 that is mediated by an intergenic module located downstream of the Cyp24a1 gene. Outside of the kidney in nonrenal target cells (NRTCs), expression of Cyp27b1 in these mutant mice was unaffected. Dietary normalization of calcium, phosphate, PTH, and FGF23 rescues the aberrant phenotype of this mouse and normalizes the skeleton. In addition, both the high levels of 25(OH)D₃ were reduced and the low levels of 1,25(OH)₂D₃ were fully eliminated in these mutant mice as a result of the rescue-induced normalization of renal Cyp24a1. Thus, these hormone-regulated enhancers for both Cyp27b1 and Cyp24a1 in the kidney are responsible for the circulating levels of 1,25(OH)₂D₃ in the blood. The retention of Cyp27b1 and Cyp24a1 expression in NRTCs of these endocrine 1,25(OH)₂D₃-deficient mice suggests that this Cyp27b1 pseudo-null mouse will provide a model for the future exploration of the role of NRTC-produced 1,25(OH)₂D₃ in the hormone's diverse noncalcemic actions in both health and disease. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: 1,25(OH)2D3; CRISPR/Cas9; CYTOCHROME P450; CYTOKINES; ChIP‐seq; Cyp24a1; Cyp27b1‐KO; FIBROBLAST GROWTH FACTOR 23; GENE REGULATION; GENETIC ANIMAL MODELS; PARATHYROID HORMONE; PTH/Vit D/FGF23; TRANSCRIPTIONAL REGULATION; VITAMIN D.

Fig 1

The interregulatory nature of the mineralotropic hormones PTH, FGF23, and 1,25(OH)₂D₃ (1,25D₃) and their genes (via expression of Pth, Fgf23, Cyp27b1, and Cyp24a1). 1,25(OH)₂D₃ feedback regulates their own expression. Arrows indicate the direction and nature (+/−) of regulation. Kidney, intestine, and bone represent the direct mineral regulating targets of the individual hormones.

Fig 2

Extended bacterial artificial chromosome (BAC) clone transgenes that contain mouse Cyp27b1 and Cyp24a1 genetic loci recapitulate the hormonal regulation by exogenous PTH, FGF23, and 1,25(OH)₂D₃ regulation seen for endogenous genes in mice. (A) Schematic depiction of mouse transgene structures. (B) Hormonal regulation. Transgenic mice were prepared and selected as recently reported.^{( 22 )} Animals were injected with PTH (230 ng/g body weight [BW]; blue), FGF23 (50 ng/g BW; green), or 1,25(OH)₂D₃ (10 ng/g BW; black) and tissues harvested at 1, 3, and 6 hours, respectively. Transgene‐derived Cyp27b1 and Cyp24a1 transcripts were quantitated using probes that required the presence of the internal ribosome entry‐site module incorporated into the BAC clones. Data are derived from four to six mice per group, and presented as the means ± SEM, *p < 0.05.

Fig 3

Schematic representation of the genomic enhancers for Cyp27b1 and Cyp24a1. (A) The locations of enhancers for Cyp27b1 are shown in purple and designated M1 and M21 with the gene dense region of the Cyp27b1 locus, and mediated by PTH (M1; blue), FGF23 (M21; green), and 1,25(OH)₂D₃ (M1 and M21; yellow). (B) The locations of enhancers for Cyp24a1 are shown in purple and designated PP1, DS1, and DS2 within the Cyp24a1 gene locus, and mediated by PTH and FGF23 (DS1; blue and green) and 1,25(OH)₂D₃ (PP1 and DS2; yellow). Figure modified from Meyer and Pike.^{( 26 )}

Fig 4

Single cell genome analyses of chromatin accessibility in renal and nonrenal cell types in the mouse. (A) ChIP‐seq analysis of VDR binding at the kidney cortex and intestine documenting the locations of the regulatory submodules (gray‐shaded dropdown bars) that mediate regulation by PTH (M1; purple) and FGF23 (M21, purple) as well as 1,25(OH)₂D₃ (M1 and M21). Data from Meyer et al.^{( 8} , ¹⁹ , ^{20 )} The upper panel indicates the chromosomal location of Cyp27b1 and Cyp24a1, genome scale, and the identity of adjacent genes. Arrows indicate the direction of gene transcription. As seen, the overall module is restricted to the kidney, where it is dispersed within single introns of the Mettl1 and Mettl21b genes, and absent in intestine (and all other tissues; see 8, 19, 20 ⁾). (B,C) Single‐cell ATAC‐seq (scATAC‐seq) analysis by Cusanovich and colleagues.^{( 32 )} Raw data were reanalyzed and displayed for only the Cyp27b1 and Cyp24a1 gene loci for (B) ATAC‐seq analysis of individual renal cell types and (C) analysis of representative nonrenal cell types (emphasis on immune cell types). As seen, peaks representative of an open chromatin state align with each of the individual components that comprise the kidney‐specific module (M1 and M21) and are restricted to renal proximal tubules (two separate analyses). No other renal or nonrenal cells types retain this chromatin regulatory pattern.

Fig 5

Vitamin D metabolism in the kidney. Schematic diagram depicting the regulation of vitamin D metabolism and serum calcium and phosphate homeostasis in the kidney. Our genetic models (black) and previously existing models (gray) are overlaid on or near the pathways they disrupt. Figure modified from Meyer and Pike.^{( 26 )}