Cobb, JohnSkuplik, Isabella2022-01-252022-01-252022-01Skuplik, I. (2022). Cis-regulatory control of the human short stature homeobox gene in the developing limb (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/114328Disruption of the human short stature homeobox (SHOX) gene causes shortening of the middle limb segment (zeugopod), affecting up to 1:1000 individuals. SHOX deficiencies are caused by coding lesions, or by the deletion of non-coding sequences surrounding the gene. Several deletion intervals downstream of SHOX are predicted to disrupt enhancers that activate transcription during limb development. However, the precise locations and activities of these enhancers remain to be identified. In this work, we investigated the cis-regulatory landscape of the human SHOX gene. We systematically screened a recurrent 47.5 kb short-stature deletion interval downstream of SHOX for the presence of limb enhancers. Human genomic sequences were placed upstream of a lacZ reporter gene and tested for their ability to activate expression in transgenic mice. This revealed the presence of a zeugopodal enhancer downstream of SHOX (the ZED). Using primary cell luciferase assays, we further delineated the minimal active sequence and identified putative HOX9/11 binding sites required for its activity. Next, we developed the domestic cat as an emerging model to characterize enhancer and gene interactions at the endogenous locus. Rodent genomes lack the SHOX gene, while other commonly used laboratory models lack conservation of the ZED and other non-coding sequences at the SHOX locus. We demonstrated that cats are an effective model to identify enhancers. Cat genomes display a synteny of genes and conserved non-coding elements in the pseudoautosomal region 1 where SHOX is located. Using whole-mount in situ hybridization, we validated the expression of SHOX in the zeugopod of cat embryos. Next, we employed the circular chromosome conformation capture (4C) technique to identify SHOX cis-interacting sequences in embryonic limb tissue. Human orthologous sequences were identified through sequence conservation; and human short-stature deletions and enhancer chromatin signatures were used to delineate enhancer candidates. Finally, we confirmed the limb enhancer activity of three human and cat sequences in transgenic mice. Our findings provide a potential explanation for the pathogenicity of certain non-coding deletions downstream of SHOX. This work also uncovers similarities in the regulation of Shox genes (Shox and Shox2) and the development of limbs in cats, humans, and mice.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.short stature homeoboxmouse modellimb development4C-SeqSHOX genesenhancersgene regulationcat modelGeneticsBiology--MolecularHuman Developmentdoctoral thesis10.11575/PRISM/39540