SDHD promoter mutations ablate GABP transcription factor binding in melanoma

SDHD encodes subunit D of the succinate dehydrogenase complex, an integral membrane protein. Across cancer types, recurrent SDHD promoter mutations were reported to occur exclusively in melanomas, at a frequency of 4-5%. These mutations are predicted to disrupt consensus ETS-transcription factor binding sites and are correlated with both reduced SDHD gene expression and poor prognosis. However, the consequence of these mutations on SDHD expression in melanoma is still unclear. Here, we found that expression of SDHD in melanoma correlated with the expression of multiple ETS-transcription factors, particularly in SDHD promoter wild-type samples. Consistent with the predicted loss of ETS-transcription factor binding, we observed that recurrent hotspot mutations resulted in decreased luciferase activity in reporter assays. Furthermore, we demonstrated specific GABPA and GABPB1 binding to probes containing the wild-type promoter sequences, with binding disrupted by the SDHD hotspot promoter mutations in both quantitative mass spectrometry and band-shift experiments. Finally, using siRNA-mediated knockdown across multiple melanoma cell lines, we determined that loss of GABPA resulted in reduced SDHD expression at both RNA and protein levels. These data are consistent with a key role for GABPA/B1 as the critical ETS-transcription factors deregulating SDHD expression in the context of highly recurrent promoter mutations in melanoma, and warrant a detailed search for other recurrent promoter mutations that create or disrupt GABPA consensus sequences.

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