DNA supercoiling impacts alternative transcription start site selection in yeast

Most genes are transcribed from multiple transcription start sites (TSSs), defined as alternative TSSs, which are highly regulated and can lead to various gene regulatory outcomes including changes in translation efficiency and protein isoform expression. Transcription factors and chromatin regulators control alternative TSS selection. DNA supercoiling affects multiple aspects of transcription including transcription initiation; however, its regulatory effect on genes with multiple TSSs is not known. Here, we investigated how DNA supercoiling impacts alternative TSS usage in Saccharomyces cerevisiae. We depleted topoisomerases during early meiosis, where alternative TSS usage is prevalent, and applied an improved TSS sequencing protocol. We show that supercoiling affects alternative TSS usage at almost 600 genes. Increased alternative and aberrant TSS usage was observed near and within open reading frames, likely resulting from transcription-induced supercoiling originating from upstream alternative TSSs. DNA supercoiling had the greatest impact on genes with a dominant alternative TSS, significant spacing between alternative TSSs, and greater overall gene length. Our results establish that DNA supercoiling release during transcription is critical for correct TSS selection.