Defining Novel GRE Binding Motifs in Genes Related to the Wnt Signaling Pathway



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The glucocorticoid receptor (GR) is a nuclear hormone receptor known to directly and indirectly modulate a wide array of downstream signaling pathways in response to ligand binding. Given its near ubiquitous presence and high conservation across vertebrate species, GR presents a vital research focus as pertaining to transcriptional control of the inflammatory response. In the endothelium, GR negatively regulates vascular inflammation; accordingly, endothelial GR knockout in the Apoe mouse model has been shown to increase the extent and severity of atherosclerotic lesions compared to those of the control group. In further exploration of GR regulation mechanisms, ChIP-seq in mouse lung endothelial cells revealed that multiple genes in the Wnt signaling pathway are characterized by high-fidelity GR binding. The limited understanding of endothelial GR regulation of Wnt led us to search for novel glucocorticoid response elements (GRE) in Wnt-related genes identified from our genomic data. A putative motif (5’-CCTCCCAAGTGCTGGGATT-3’) was detected and cloned into basic and promoter versions of the pGL3 luciferase reporter vector. Using the Wnt-related gene frzb, additional clones containing various combinations of the motif, the intron in which it is found, and protein coding regions were obtained from bacmid/cDNA library PCR and Gibson assembly. A luciferase assay will be performed on transfected and dexamethasone-treated HEK293 cells to assess the glucocorticoid receptor binding affinity in each of the constructs. Ultimately, we anticipate that this study will help elucidate molecular mechanisms of the Wnt signaling pathway and how they contribute to endothelial cell functions and vascular disease development. This project was completed with contributions from Julie Goodwin and Kariona Grabińska from the Yale School of Medicine, Section of Nephrology.