Functional interrogation of the elovl5 gene regulation in Atlantic salmon

Abstract

Long-chain polyunsaturated fatty acids (LC-PUFA) and cholesterol are important for different physiological pathways including immunology. As these cannot be synthesized in the teleosts including Salmon itself, food supplement is considered the prime source in this case. Two duplicated genes in salmon elovl5a and elovl5b, derived from the WGD event at about 80 Mya, are important in the synthesis of LC-PUFAs. Differential gene regulation has been shown for these genes in previous studies, including highly divergent tissue regulation. This tissue-specific expression might be the result of different binding patterns of major lipid-metabolism transcriptional regulators. According to Carmona-Antoñanzas et al., 2016, elovl5a equally responded to the two sterol regulatory element-binding proteins (srebp-1 and Srebp-2) while elovl5b responded stronger to only Srebp-2. In this thesis, we investigated the roles of different transcription factors in the gene expression for the two salmon elovl5 gene copies. We first conducted a reporter-promoter assay to measure the effect of the Lxr-Srebp regulatory pathway on elovl5a and elovl5b regulation. Next, we conducted a CRISPR-Cas9 knockout experiment to assess the importance of predicted binding sites of Lxr, Srebp-1, Srebp-2, and NY-F for the transcriptional regulation of elovl5b. Though our findings were not completely conclusive, induction of the Lxr-Srebp pathway increased the expression of both elovl5 promoters in the SHK-1 cells. Elovl5a was showing stronger gene expression compared to elovl5b, moreover synthetic (elovl5a ATAC and elovl5b ATAC) were stronger in upregulation than the native promoters. In the CRISPR-cas knockout experiments, we found that mutating Srebp-1, Srebp-2, and NF-Y binding motifs in the elovl5 promoter decreased elovl5b expression. In addition, our experiments indicate that Srebp binding sites were potentially important for regulating elovl5b gene through the Lxr-pathway as knock out of these binding sites led to ablation of the Lxr-agonist effect. In conclusion, our experiments shed light on the gene regulatory mechanisms of elovl5 genes, supporting previous findings regarding the Srebp pathway-mediated regulation in non-salmonid cell lines. Moreover, the novel approach tested for targeted CRISPR-based knock-down experiments will pave the way for more effective interrogation of gene regulatory mechanisms in the years to come.