Elgood Hunt, Eleanor; (2025) Role of alternative transcription start sites in regulating gene expression during cell fate transitions. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Over 50% of mammalian genes are transcribed from multiple transcription start sites (TSS). TSS selection is often regulated during development and in a tissue-dependent manner. Misregulation of TSS usage is instead associated with diseases including neurodevelopmental disorders and cancer. Use of alternative TSSs can change the 5’ untranslated region of transcripts with resulting effects on transcript stability and translation efficiency. In certain cases, protein isoforms with differing functions can also be produced. Finally, transcription from one TSS can directly regulate in cis, expression from another TSS of the same gene, in a process known as transcriptional interference. Controlling where transcription is initiated therefore represents an important and widespread mechanism of gene regulation. However, despite the prevalence of alternative TSSs in mammals, the underlying regulatory mechanisms, effects on gene expression and physiological roles are not well understood. To this end, I developed a technique which detects the TSSs of nascent RNA, allowing TSS usage to be determined without interfering RNA stability effects. Applying this technique to a neuronal differentiation system, enabled the genome-wide identification of genes, including Ash2l, which showed switches in TSS usage. Ash2l is a core component of the COMPASS family histone methyltransferase complexes. Ash2l is predominantly expressed from two alternative TSSs, which are regulated in a cell-type and development-specific manner. I showed that, in mouse embryonic stem cells, transcription from the distal TSS inhibits proximal TSS expression, requiring the chromatin regulators SETD2 and FACT. Inhibiting the distal TSS affected the ability of mouse embryonic stem cells to differentiate into motor neurons, while inhibiting the proximal TSS slowed the growth rate of fibroblasts. Together, these approaches have highlighted the different roles that alternative TSSs can play in the cell, and have identified different mechanisms by which alternative TSS usage may be regulated.

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