Kidney Week

Abstract: TH-OR049

In Vivo Deletion of Complex Genomic Enhancers Reveal a Kidney-Specific, Endocrine-Deficient Cyp27b1 Pseudo-Null Mouse and Loss of Reciprocally Regulated Cyp24a1 by FGF23 and PTH

Session Information

• Bone and Mineral Metabolism: Basic Research
  November 07, 2019 | Location: 145, Walter E. Washington Convention Center
  Abstract Time: 06:06 PM - 06:18 PM

Category: Bone and Mineral Metabolism

• 401 Bone and Mineral Metabolism: Basic

Authors

• Meyer, Mark B., University of Wisconsin-Madison, Madison, Wisconsin, United States

Mark B. Meyer, PhD



My research at the University of Wisconsin-Madison focuses on the central question: How does vitamin D regulate the genome? Over the years, we utilized genomic discovery surrounding transcription of vitamin D target genes throughout the body, mainly in skeletal and intestinal cells, where vitamin D acts through its receptor (VDR) to regulate calcium and phosphate intake or resorption. We found that VDR used distal and sometimes numerous genomic enhancers far from the genes they regulate. Often, these coordinated enhancers function in a hierarchical fashion for full regulatory potential. Through genomic manipulation in vivo or in culture, we are able to tease out the mechanism of this complicated hierarchy. More recently, we took a step back from vitamin D target genes and asked the question: What about the in vivo metabolism of vitamin D itself? Two vital genes involved in this process encode the cytochrome P450 enzymes CYP27B1 and CYP24A1, and little was known about their genomic regulation in the kidney by calcium and phosphate sensing hormones. So we set out to discover how these genes were regulated and the intimate relationship they share in this homeostasis. In 2017, and again earlier this year (2019), we published studies related to Cyp27b1 in the Journal of Biological Chemistry and most recently last month we added significant knowledge of the intricate genomic control of Cyp24a1 in vivo. Importantly, we have created unique CRISPR enhancer-deleted animal models with which we can further probe vitamin D metabolism under a variety of situations. We are able to decouple the systemic production of vitamin D through calcium-sensing hormones and now focus in on the effects on the immune system. Our future studies are focused on the regulation of these genes, and others, in non-kidney tissues and cells. We will be able to leverage these unique animals we have created to answer difficult, clinically relevant questions about how vitamin D can help during health, inflammation, and disease.

• Benkusky, Nancy A., University of Wisconsin-Madison, Madison, Wisconsin, United States

Nancy A. Benkusky,

• Lee, Seong Min, University of Wisconsin-Madison, Madison, Wisconsin, United States

Seong Min Lee,

• Jones, Glenville, Queens University, Kingston, Ontario, Canada

Glenville Jones,

• Pike, J. Wesley, University of Wisconsin-Madison, Madison, Madison, Wisconsin, United States

J. Wesley Pike,

Background

Cyp27b1 and Cyp24a1 are reciprocally regulated in the kidney by the key hormones PTH, FGF23, and 1,25(OH)₂D₃. Our recent genomic studies in mice 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(OH)₂D₃. Gross deletion of these segments in mice has severe consequences on skeletal health, but does not affect Cyp27b1 regulation in non-renal target cells (NRTCs).

Methods

Using CRISPR/Cas9-mediated deletions of genomic enhancers (non-coding segments) in mice, we can separate tissue specific responses in both Cyp27b1 and Cyp24a1. We used ChIP-seq from adult human kidney cortex to explore conservation to mouse.

Results

Our current studies reveal a bimodal activity in the M1 intronic enhancer with components responsible for induction of Cyp27b1 by PTH and repression by 1,25(OH)₂D₃. The deletion of both M1 and M21 submodules fully eliminates basal Cyp27b1 expression and regulation in the kidney, leading to a systemic and skeletal phenotype similar to that of the Cyp27b1-KO mouse due to depletion of 1,25(OH)₂D₃ and high PTH. Cyp24a1 levels in the double KO mouse were low due to compensatory regulation by elevated PTH and reduced FGF23. However, expression of Cyp27b1 and retention of its regulation by inflammation (LPS) in the NRTCs remained unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescues this aberrant phenotype and creates an ideal in vivo model with which to study NRTC production of 1,25(OH)₂D₃ and its potential impact on disease. Using a separate set of mouse enhancer deletions, we found that basal as well as PTH and FGF23 regulation of Cyp24a1 in the kidney was controlled by a set of downstream enhancers distinct from those that mediate 1,25(OH)₂D₃. Finally, we confirm the presence of a conserved chromatin landscape for both CYP27B1 and CYP24A1 in human similar to the mouse.

Conclusion

Collectively, these studies define a finely balanced homeostatic control mechanism employed by PTH and FGF23 with catastrophic toxicity protection from 1,25(OH)₂D₃ in the genomic regulation of vitamin D metabolism and its accompanied control of mineral maintenance.

Funding

• NIDDK Support