Research Mentor(s): Yadav Wagley
Authors: David Ntsiful, Yadav Wagley, Ph.D., Kurt Hankenson, DVM, Ph.D.
EPDR1 is a novel human osteoblast effector gene identified by intersecting BMD GWAS data with functional genomics in differentiating human mesenchymal stem cells (MSC). The open chromatin region harboring the GWAS-BMD variants rs1524068, rs6975644, and rs940347 functions as an osteoblast-specific EPDR1 enhancer. Additionally, loss of EPDR1 function biases hMSC towards adipogenic differentiation. Jagged1 (JAG1), a canonical Notch ligand, is a critical regulator of normal skeletal homeostasis as loss-of-function mutations in JAG1 are associated with Alagille syndrome, which manifests multiple skeletal defects, low bone mass, and bone fragility in affected individuals. Herein, we aim to evaluate the role of EPDR1 during JAG1-mediated human osteoblast differentiation and examine whether EPDR1 GWAS-enhancer responds to canonical Notch signaling. hMSC were plated onto JAG1-coated plates using osteopermissive cell culture medium. Gene expression analyses showed an increase in the expression of Notch target genes (HEY1, HEY2, and HES1), suggesting canonical Notch signal transduction in hMSC. Cells stimulated with JAG1 showed enhanced ALP staining and deposited calcium in the extracellular matrix indicating osteoblast differentiation. A temporal evaluation of EPDR1 expression in JAG1 stimulated hMSC showed enhanced expression in a dose and a time-dependent fashion, suggesting a tight correlation with Notch pathway activation. To dissect the role of the EPDR1 GWAS-enhancer, CRISPR-Cas9 genome editing was performed using a one-step transfection of Cas9 protein and synthetic guides targeting all three proxy SNPs. We have validated deletion of the genomic region encompassing EPDR1enhancer using a nested PCR approach and are currently analyzing the effect of JAG1 on these genome-edited cells. A decrease in EPDR1 expression is expected in genome-edited cells. To further explore the role of GWAS-BMD variants in the regulation of EPDR1, we will perform in-silico analysis of Notch signal transducer, RBPJ, within the SNP region as well as the promoter regions. Follow-up chromatin immunoprecipitation experiments will be conducted to demonstrate RBPJ binding in those EPDR1 regulatory regions. To facilitate the development of preclinical animal models to study the in vivo function of the EPDR1 gene, Epdr1 expression was evaluated during osteoblastogenesis and adipogenesis of murine MSC. Our results showed a transient upregulation of Epdr1 during osteoblastogenesis that followed comparable temporal expression kinetics with Alpl, Runx2, and Sp7 expression, whereas Epdr1 expression gradually declined as murine MSC differentiated into mature adipocytes. Collectively, these results supportEPDR1’s role in terminal MSC fate specification in both human and mouse models. With a more robust characterization of Epdr1 in human and murine cells, further studies can be undertaken to elucidate the understanding of EPDR1 function in vivo for bone mineral density regulation during development and pathological conditions.