CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a <i>cis</i>-Regulatory Element at the <i>TRPV1/TRPV3</i> Intergenic Region



Price, Emma, Gianfrancesco, Olympia, Harrison, Patrick T, Frank, Bernhard, Bubb, Vivien J ORCID: 0000-0003-2763-7004 and Quinn, John P ORCID: 0000-0003-3551-7803
(2021) CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a <i>cis</i>-Regulatory Element at the <i>TRPV1/TRPV3</i> Intergenic Region. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 22 (4). 1911-.

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Abstract

SINE-VNTR-<i>Alu</i> (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as <i>cis</i>-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 <i>cis</i>-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 <i>TRPV1</i> and <i>TRPV3</i> in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between <i>TRPV1</i> and <i>TRPV3</i> coding sequences contained a human specific SVA insertion located upstream of the <i>TRPV3</i> promoter and downstream of the 3' end of <i>TRPV1</i>, highlighting this SVA as a candidate to study its potential <i>cis</i>-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 <i>TRPV1</i> and <i>TRPV3</i> 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 <i>TRPV1</i> and <i>TRPV3</i>. Here we provide an example of a human specific SVA with <i>cis</i>-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.

Item Type: Article
Uncontrolled Keywords: retrotransposon, SVA, cis-regulatory element, TRPV1, TRPV3, CRISPR, gene expression
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 17 Mar 2021 11:41
Last Modified: 09 Feb 2024 00:32
DOI: 10.3390/ijms22041911
Open Access URL: https://doi.org/10.3390/ijms22041911
Related URLs:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3117545