Topoisomerases regulate alternative transcription start site selection in yeast

Most genes are transcribed from multiple transcription start sites (TSSs), also known 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 depletion of topoisomerases, which resolve DNA supercoiling events, impacts alternative TSS usage in Saccharomyces cerevisiae. We depleted topoisomerases (Top1 and Top2) during early meiosis, where alternative TSS usage is prevalent, and applied an improved TSS sequencing protocol. We show that supercoiling affects alternative TSS usage of almost 600 genes. Increased alternative and aberrant TSS usage were observed near and within open reading frames, likely resulting from transcription-induced supercoiling originating from upstream alternative TSSs. Top1/Top2 co-depletion strongly affected genes with highly used, widely spaced alternative TSSs. Our correlative analyses support a model where DNA supercoiling release during transcription is critical for correct TSS selection.