Current Research Projects
- Chromosome Conformation and 3D Nuclear Architecture of Heat Shock Protein (HSP) Genes
- Genome-wide Organization of HSP Genes and Identification of Molecular Determinants Underlying the Formation of Transcription Factories
- Role of Mediator in the Transcriptional Regulation and 3D Genomic Organization of Hsf1 Target Genes
- Genetic and Epigenetic Determinants of Heat Shock Factor 1 Occupancy
Chromosome Conformation and 3D Nuclear Architecture of Heat Shock Protein (HSP) Genes
Participants: Surabhi Chowdhary and Amoldeep S. Kainth
Three-dimensional chromatin organization is important for proper gene regulation, yet how the genome is remodeled in response to stress is largely unknown. In this project, we are using chromosome conformation capture (3C) in combination with fluorescence microscopy to study the chromosome conformation and 3D nuclear architecture of Heat Shock Factor 1 (Hsf1)-regulated HSP genes in the budding yeast Saccharomyces cerevisiae. We have discovered the existence of dramatic intragenic folding interactions that occur between UAS and promoter, promoter and terminator (gene ‘looping’), and regulatory and coding regions (gene ‘crumpling’) of these genes following exposure to heat. Such interactions are prominent within 60 sec of thermal upshift, peak within 2.5 min and attenuate by 30 min. They tightly correlate with HSP gene transcriptional activity. With similar kinetics and dynamics, HSP genes engage in intra- and interchromosomal interactions involving their UAS, promoter, coding and terminator regions, coalescing into discrete intranuclear foci. Genes regulated by an alternative stress-inducible activator, Msn2/Msn4, loop and crumple in response to heat shock yet interestingly do not coalesce. This work provides evidence for novel transcription-dependent gene ‘crumpling’ and demonstrate the de novo assembly and disassembly of transcriptionally active foci.
Chowdhary, S., Kainth, A.S. and Gross, D.S. 2017. Heat Shock Protein genes undergo dynamic alteration in their three-dimensional structure and genome organization in response to thermal stress. Mol. Cell. Biol. 37: e00292-17.
Chowdhary, S., Kainth, A.S. and Gross, D.S. 2019. Chromosome conformation capture that detects novel cis- and trans-interactions in budding yeast. Methods, Epub ahead of print.
Left: Circos plot summarizing intragenic interaction frequencies detected within HSP genes in 10 min heat shocked (HS) cells. Similar looping and folding interactions have been observed in other (non-HSP) transcriptionally active genes. Right: Circos plot summarizing cis- and trans- intergenic interactions tested within and between different categories of genes. Depicted is the interactome of 10 min HS cells. Strikingly, intergenic interactions have been detected only between Hsf1-regulated genes, as genes regulated by alternative thermal stress responsive factors Msn2 and Msn4 show no detectable interactions in 10 min HS cells.
Genome-wide Organization of HSP Genes and Identification of Molecular Determinants Underlying the Formation of Transcription Factories
Participants: Amol Kainth, Surabhi Chowdhary, Linda S. Rubio and Vickky Pandit
Collaborators: David Pincus (The University of Chicago), Dan Capurso and Yijun Ruan (The Jackson Laboratory for Genomic Medicine)
In mammalian cells, it has been observed that certain types of genes – e.g., those involved in erythroid differentiation, immunoglobulin production, or response to the cytokine TNF-α – cluster together when they are transcriptionally activated into so-called transcription factories. Exactly how and why they do this is unknown. Recent work from our laboratory has revealed that heat-shock responsive genes in budding yeast regulated by the conserved activator Hsf1 likewise coalesce into prominent intranuclear foci upon their transcriptional activation. Such coalescence is rapid, robust yet evanescent, and involves physical interactions between both chromosomally linked and unlinked HSP genes. It is also absolutely dependent on Hsf1 and active transcription (RNA Polymerase II). To extend our understanding of this phenomenon, we are mapping HSP gene interactions genome-wide and identifying the DNA sequences and protein factors that drive Hsf1-regulated genes into coalesced intranuclear foci in response to thermal and other proteotoxic stresses (e.g., ethanol). To do so, we are employing a combination of biochemistry, yeast genetics, 3C, Hi-C, ChIA-PET and fluorescence microscopy.
Chowdhary, S., Kainth, A.S., Pincus, D. and Gross, D.S. 2019. Heat Shock Factor 1 Drives Intergenic Association of Its Target Gene Loci Upon Heat Shock. Cell Reports 26: 18-28.
Genes regulated by Hsf1 intricately fold upon their activation and preferentially interact, coalescing into transcriptionally active foci. In acutely heat-shocked cells, physical interactions take place between Hsf1-regulated HSP genes located throughout the genome. Here are depicted HSP genes (colored pretzels with yellow spheres) residing on the left and right arms of Chr. XII (green ribbon). Such interactions are highly specific, as they exclude neighboring genes regulated by heterologous activators (brown pretzel with red sphere), and are remarkably robust, as they can circumvent the physical barrier imposed by the nucleolus (condensed chromatin domain, upper right).
Role of Mediator in the Transcriptional Regulation and 3D Genomic Organization of Hsf1 Target Genes
Participants: Amol Kainth, Anandhakumar Jayamani, Yara Moustafa, Surabhi Chowdhary and Sunyoung Kim
Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. We have recently evaluated the role of this coactivator in Hsf1-driven transcription and found that it is rapidly and robustly recruited to the UAS/promoter regions of HSP genes in response to heat shock. Mediator occupancy peaks within 2.5 min of exposure to stress, and begins to dissipate shortly thereafter. Interestingly, Hsf1 uses both its N- and C-terminal activation domains to recruit Mediator, engaging in physical and functional interactions with the Med15 Tail module subunit. Using the ‘Anchor Away’ technique to conditionally deplete select subunits within Mediator and its reversibly associating Cdk8-Kinase module (CKM), we have additionally found that Mediator’s Tail module is highly dynamic and that a subcomplex consisting of Med2, Med3 and Med15 can be independently recruited to the UAS regions of Hsf1-activated genes. In addition, and contrary to current models, we have observed that Hsf1 can recruit the CKM independently of core Mediator, and that core Mediator has a role in regulating post-initiation events. Our results therefore suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 Tail Triad, and the four-subunit CKM can be independently recruited by activated Hsf1 to its target genes. We are currently exploring the role that Mediator, and in particular its Tail Triad, might play in the intergenic coalescence of HSP genes that takes place upon heat shock.
Anandhakumar J., Moustafa Y.W., Chowdhary S., Kainth A.S. and Gross D.S. 2016. Evidence for multiple Mediator complexes in yeast independently recruited by activated Heat Shock Factor. Mol. Cell. Biol. 36: 1943-1960.
Left: Existence of multiple Mediator complexes in vivo. Those marked with an asterisk (*) have been detected at upstream regulatory regions of heat shock-induced HSP genes.
Genetic and Epigenetic Determinants of Heat Shock Factor 1 Occupancy
Participants: Jayamani Anandhakumar and Alexander Erkine
Collaborators: David Pincus, Pratt Thiru (Whitehead Institute) and Michael Guertin (University of Virginia)
The heat shock transcriptional response is a universal response of organisms to thermal, chemical and oxidative stress. In eukaryotes from yeast to human, proteotoxic conditions activate Heat Shock Factor 1 (Hsf1), which is responsible for the transcriptional induction of genes that encode molecular chaperones and other cytoprotective heat shock proteins (HSPs). Such induction is usually transient as Hsf1 is subject to negative feedback regulation by molecular chaperones, particularly Hsp70 and Hsp90. Our earlier work has suggested that the ability of Hsf1 to bind and remodel chromatin is critical to its biological function in the budding yeast Saccharomyces cerevisiae. In this project our goal is to gain deeper insight into the biology of yeast Hsf1 through mapping its genome-wide occupancy under basal, acutely inducing and chronically inducing conditions, as well as through a determination of the role played by the pre-set chromatin landscape in fostering Hsf1 binding and subsequent, heat shock-dependent chromatin remodeling.
Pincus, D ., Anandhakumar, J., Thiru, P., Guertin, M.J., Erkine, A.M. and Gross, D.S. 2018. Genetic and epigenetic determinants establish a continuum of Hsf1 occupancy and activity across the yeast genome. Mol. Biol. Cell 29: 3168-3182.
Left: Hsf1 ChIP-seq reveals differential basal and heat-shock-inducible binding across the Hsf1 regulon. Top, Normalized ChIP signal at 43 core loci occupied under all conditions tested: non-heat shock (NHS), 5 min HS and 120 min HS. Bottom, Normalized ChIP signal at 31 loci occupied only following heat shock. A key distinction between the core and inducibly occupied loci is the presence of accessible, nucleosome-depleted chromatin at the core loci conducive to basal Hsf1 occupancy.