Sheila Teves

Transcription in eukaryotes is primarily performed by three distinct types of RNA polymerases: RNA polymerases I, II, and III (Pol I, Pol II, and Pol III). Decades of research have revealed a comprehensive understanding of the mechanisms of eukaryotic transcription; however, advances in technologies may require a reassessment of previous studies, potentially unveiling previously unattainable insights. Consequently, my thesis focuses on using cutting edge technologies such as super resolution microscopy, next-generation sequencing, and mass spectrometry-based proteomics to delve into the intricate mechanisms of Pol II and Pol III transcription. TATA-box binding protein (TBP) was previously known to be indispensable by all three polymerases for transcription initiation in most eukaryotes. Here, I present evidence for a TBP-family independent mechanism of Pol II transcription in mouse embryonic stem cells, showing that ongoing transcription, gene induction, and TFIID formation are not affected when TBP is depleted, while Pol III transcription is severely impacted. In addition, I demonstrate that heat shock stress leads to a drastic change in the transcription of tRNA genes by Pol III, influenced by the heat shock master regulator HSF1. Lastly, I use super resolution microscopy to explore the dynamics of the general transcription factors through single-particle tracking, demonstrating significant changes in residence time and DNA binding when TBP is depleted. Taken together, my thesis aims to unravel novel perspectives of transcription mechanisms using new technologies, but also to challenge previous paradigms and provide a more comprehensive understanding of gene regulation.

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Transcription by RNA Polymerases (RNA Pol) is initiated by the hierarchal assembly of the Pre-Initiation Complex (PIC) at the promoters of genes. TATA-box binding protein (TBP) isbelieved to be the first factor to bind onto RNA Pol II promoters to recruit the other GeneralTranscription Factors (GTFs) for PIC formation and also plays a role in recruiting the necessaryfactors in the other two major eukaryotic RNA Polymerase (RNA Pol I and RNA Pol III).Decades of in vitro biochemical and in vivo yeast research have shown that TBP is essential fortranscription initiation for the major three eukaryotic RNA Polymerases, but serendipitous TBPresearch in multicellular eukaryotes suggest otherwise. In this thesis, we examined the effects ofTBP depletion in mouse Embryonic Stem Cells (mESCs) and report that acute depletion of TBPhas no global effect on ongoing RNA Pol II and RNA Pol I transcription, but acute depletion ofTBP severely impairs RNA Pol III transcription. We showed that this RNA Pol I and RNA Pol IITBP-independent transcription mechanism is not due to the TBP paralog TRF2 and that for RNAPol II, the TFIID complex can still form and bind onto promoter DNA, despite having alteredbinding dynamics. Additionally, we showed that TBP binds onto RNA Pol I promoters andmitotically bookmark these genes for efficient transcriptional reactivation following mitosis anddepletion of TBP impairs this process. Collectively, these results show how the transcriptionalrole of TBP has diverged throughout evolution and the potential mechanism of TBP-independentRNA Polymerase transcription in mESCs.

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