Functional analysis of recurrent non-coding variants in human melanoma

Abstract

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused in on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in a human A375 melanoma cell line and observed decreased expression of CDC20, changes in migration capabilities, and an altered transcriptional state previously associated with neural crest transcriptional programs and melanoma initiation. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes.

The promoter mutation paucity as part of the dark matter of the cancer genome

“…1c). Moreover, our analysis also detected the previously-reported 13,14 CDC20 G529A pSNV in a single patient with elevated CDC20 expression and a recurrence in 11 other samples, 2 of which had RNA-Seq data available.…”

“…By luciferase reporter assay, a significant upregulation was reported in the kidney cell line HEK293 while a consistent but insignificant effect was observed in the melanoma cell line M14. However, in another study, a statistically-significant reduction in luciferase activity within HEK293 was reported 14 . Within our study, we also functionally validated the recurrent CDC20 G529A pSNV within the HEK293 cell line and observed a significant upregulation, agreeing with He et al 13 .…”

“…Rather than creating a TFBS, the CDC20 G529A mutation was found to disrupt the binding of the well-conserved repressor ELK4 motif, which results in an upregulation of the downstream gene 13 . Subsequent in vitro validation showed that this pSNV led to a 50% increase in luciferase activity although this result was debated in another study 14 . Beyond these examples, little evidence for recurrent oncogenic promoter mutations is available.…”

Cross-species analysis reveals unique and shared roles of Sox9 and Sox10 (SOXE family) transcription factors in melanoma

“…We then applied our recently published method to look for recurrently mutated non-coding regions in human melanoma and identified a mutation in the intron of SOX9-AS1 ( chr17:70114939.70114939.G.A) that is detected in 6 out of 140 whole-genome sequenced cutaneous melanomas (4%, Figure 2A) 25 . We cloned a 300 bp region containing either the WT sequence or the recurrently mutated sequence upstream of a minimal promoter driving luciferase and observed a statistically significant decrease in reporter activity across all 3 melanoma cell lines assayed (Figure 2B).…”

“…Recurrent mutations upstream of SOX9 were identified using methods previously described 25 . For luciferase assays, we synthesized 300 bp sequences corresponding to WT and mutant hotspots with the variant centered at position 150 and ligated into a luciferase vector with a minimal promoter (pGL3-Promoter, E1761).…”