Friday, May 9, 2025
5/9/2025
Genome Architecture and Gene Control in Response to Stress - Genome Architecture and Gene Control in Response to Stress The 3D topology of the genome plays a critical role in transcriptional regulation in health, disease and development. The genome adopts discrete structural and regulatory domains that are maintained across cell types and even organisms. However, while it is becoming clear that alterations in this standard topology play important roles in both development and disease, very little is known about the mechanisms that dynamically control the restructuring process. Moreover, it is unknown whether (or how) the topology of the genome contributes to the coordination of expression of genes critical to the cell’s response to stress. We have established a system in which we can induce dramatic architectural rearrangements concerted within and between genes and synchronized across a population of cells. The system, the heat shock response in the budding yeast S. cerevisiae, allows us to leverage the powerful genetic tractability of this organism to define the factors and uncover the mechanisms that drive genome architecture and nuclear reorganization. Moreover, Heat Shock Protein (HSP) genes and the transcriptional regulator that controls their expression, Heat Shock Factor 1 (Hsf1), are evolutionarily conserved and critical for health and disease. Using a highly sensitive and quantitative version of chromosome conformation capture (3C) that our laboratory developed, termed Taq I - 3C, we have obtained evidence that in response to acute thermal stress, HSP genes undergo intense intragenic interactions that include looping between UAS and promoter elements, promoter and terminator regions and regulatory and coding regions. Even more striking, they engage in frequent intra- and interchromosomal interactions, coalescing into discrete intranuclear foci. Genes that are heat shock-activated by an alternative transcription factor (TF), Msn2, likewise loop yet do not appear to coalesce, either with themselves or with Hsf1-target genes. Likewise, robustly transcribed, constitutively expressed genes undergo intragenic looping yet these genes too do not appear to coalesce. In addition to their distinctive coalescence, the intragenic and intergenic restructuring/reorganization of Hsf1-target genes is remarkably dynamic: detectable within 60 sec, peaking within 2.5 min and attenuating within 30 min. These observations raise important questions. To address these, we propose three aims: Aim 1: Elucidate the 3D topology of the yeast genome during heat shock and other stresses, and the role played by Hsf1 and Pol II in orchestrating these changes. We will utilize cutting-edge deep sequencing-based approaches, principally Taq I – Hi-C, to reveal heat shock- and factor-dependent chromosomal topology dynamics across the genome. We will define the role of Hsf1 and the Pol II, and other factors identified in Aims 2 and 3, in driving 3D genome architecture in cells exposed to thermal stress. We will confirm key findings using Taq I - 3C. Aim 2: Elucidate determinants of Pol II and Hsf1 in driving HSP gene coalescence and test notion that HSP condensates assemble through liquid-liquid phase separation. We will identify the functional domains within Hsf1 and Pol II that are responsible for driving HSP genes into coalesced foci in cells exposed to acute HS, and explore the biophysical nature of the dynamic HSP condensates. Aim 3: Unveil the roles of transcriptional coactivators, chromatin remodelers and architectural proteins in driving the specific and dynamic interactions within and between Hsf1-target genes. We will test that hypothesis that HSP gene coalescence represents the concerted action of multiple cofactors – recruited by Hsf1 and acting in concert with Pol II – and investigate the contribution made by select factors, focusing on those that are preferentially recruited to HSP genes in coalescence-competent cells. We will exploit the Taq I-3C assay in combination with an array of powerful yeast genetic techniques – conditional nuclear depletion, conditional protein degradation, genome-editing – to interrogate the role of these factors. Together, the experiments proposed will reveal both mechanistic insight and a broad, genome-wide perspective on the dynamic, Hsf1-dependent 3D genome remodeling that occurs during the yeast heat shock response. They will set up a future exploration of the biological significance of HSP gene coalescence, informed by results of experiments proposed here.
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