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Abstract

The heat shock response (HSR) is a cellular stress pathway primarily regulated by the transcription factor Hsf1. Traditionally, Hsf1 activation has been viewed as a simple on/off switch, controlled by the chaperone protein Hsp70. However, in this study, I demonstrate that Hsf1 activation is more nuanced: it can function as a graded, tuneable response, modulated by both multivalent interactions with Hsp70 and phosphorylation. During thermal stress, I observed that Hsf1 forms biophysically dynamic clusters with Mediator and RNA Polymerase II (Pol II). These clusters, or condensates, serve as membrane-less hubs that concentrate transcriptional machinery, promoting the efficient expression of HSR target genes. Notably, these condensates facilitate the spatial reorganization of HSR target genes, effectively bringing them together during stress. In mammalian systems, transcriptional condensates are known to enable high-level gene expression, particularly in genes involved in development and cell identity, however, it was unclear whether these condensates are evolutionarily conserved or play a role in 3D genome organization. In this thesis, I identify transcriptional condensates within the yeast HSR, suggesting that they may represent an ancient, adaptable strategy in eukaryotic gene regulation. I also describe the stepwise process of condensate assembly: it begins with Hsf1 partially dissociating from Hsp70, allowing Mediator to bind. Further Hsp70 dissociation and Hsf1 hyperphosphorylation then expose multivalent binding sites that enhance this assembly. However, Hsf1-Mediator condensates alone do not recruit Pol II; Pol II condensation only occurs in response to a separate environmental signal, providing an additional layer of control over Hsf1-regulated genes. These findings highlight the role of HSR transcriptional condensates as flexible regulatory hubs, with full assembly and Pol II recruitment enabling active, long-range interactions across the genome in response to thermal stress. This dynamic, modular process underscores the sophisticated regulation possible within eukaryotic stress responses.

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