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.