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CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a cis-Regulatory Element at the TRPV1/TRPV3 Intergenic Region
Authors:Emma Price  Olympia Gianfrancesco  Patrick T. Harrison  Bernhard Frank  Vivien J. Bubb  John P. Quinn
Affiliation:1.Department of Pharmacology and Therapeutics, Institute of Systems, Molecular & Integrative Biology, University of Liverpool, Liverpool L69 3GE, UK; (E.P.); (O.G.); (V.J.B.);2.MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK;3.Department of Physiology, BioSciences Institute, University College Cork, Cork, Ireland;4.Department of Pain Medicine, Walton Centre NHS Foundation Trust, Liverpool L9 7LJ, UK;
Abstract:SINE-VNTR-Alu (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as cis-regulatory elements (CREs); however, the regulatory potential of SVAs has predominantly been demonstrated using bioinformatic approaches and reporter gene assays. The objective of this study was to demonstrate SVA cis-regulatory activity by CRISPR (clustered regularly interspaced short palindromic repeats) deletion and subsequent measurement of direct effects on local gene expression. We identified a region on chromosome 17 that was enriched with human-specific SVAs. Comparative gene expression analysis at this region revealed co-expression of TRPV1 and TRPV3 in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between TRPV1 and TRPV3 coding sequences contained a human specific SVA insertion located upstream of the TRPV3 promoter and downstream of the 3′ end of TRPV1, highlighting this SVA as a candidate to study its potential cis-regulatory activity on both genes. Firstly, we generated SVA reporter gene constructs and demonstrated their transcriptional regulatory activity in HEK293 cells. We then devised a dual-targeting CRISPR strategy to facilitate the deletion of this entire SVA sequence and generated edited HEK293 clonal cell lines containing homozygous and heterozygous SVA deletions. In edited homozygous ∆SVA clones, we observed a significant decrease in both TRPV1 and TRPV3 mRNA expression, compared to unedited HEK293. In addition, we also observed an increase in the variability of mRNA expression levels in heterozygous ∆SVA clones. Overall, in edited HEK293 with SVA deletions, we observed a disruption to the co-expression of TRPV1 and TRPV3. Here we provide an example of a human specific SVA with cis-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.
Keywords:retrotransposon   SVA   cis-regulatory element   TRPV1   TRPV3   CRISPR   gene expression
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