CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a <i>cis</i>-Regulatory Element at the <i>TRPV1/TRPV3</i> Intergenic Region
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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-02-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/22/4/1911 |
| Summary: | 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. |
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| ISSN: | 1661-6596 1422-0067 |