Inducible transcriptional condensates drive 3D genome reorganization in the heat shock response.

Mammalian developmental and disease-associated genes concentrate large quantities of the transcriptional machinery by forming membrane-less compartments known as transcriptional condensates. However, it is unknown whether these structures are evolutionarily conserved or involved in 3D genome reorganization. Here, we identify inducible transcriptional condensates in the yeast heat shock response (HSR). HSR condensates are biophysically dynamic spatiotemporal clusters of the sequence-specific transcription factor heat shock factor 1 (Hsf1) with Mediator and RNA Pol II. Uniquely, HSR condensates drive the coalescence of multiple Hsf1 target genes, even those located on different chromosomes. Binding of the chaperone Hsp70 to a site on Hsf1 represses clustering, whereas an intrinsically disordered region on Hsf1 promotes condensate formation and intergenic interactions. Mutation of both Hsf1 determinants reprograms HSR condensates to become constitutively active without intergenic coalescence, which comes at a fitness cost. These results suggest that transcriptional condensates are ancient and flexible compartments of eukaryotic gene control.

Shugoshin 2-a new guardian for heat shock transcription.

Takii et al (2019) demonstrate in a recent issue of The EMBO Journal that the pericentromeric protein, SGO2, serves as a novel transcriptional coactivator of HSF1, contributing to PIC assembly and expression of Heat Shock Protein (HSP) genes. This finding highlights repurposing of a protein with a nuclear function to drive transcription of proteotoxic stress machinery genes.

Merging short and stranded long reads improves transcript assembly.

Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet this potential is not fully realized due to inherent limitations of current long-read assembly methods and underdeveloped approaches to integrate short-read data. Here, we critically compare the existing methods and develop a new integrative approach to characterize a particularly challenging pool of low-abundance long noncoding RNA (lncRNA) transcripts from short- and long-read sequencing in two distinct cell lines. Our analysis reveals severe limitations in each of the sequencing platforms. For short-read assemblies, coverage declines at transcript termini resulting in ambiguous ends, and uneven low coverage results in segmentation of a single transcript into multiple transcripts. Conversely, long-read sequencing libraries lack depth and strand-of-origin information in cDNA-based methods, culminating in erroneous assembly and quantitation of transcripts. We also discover a cDNA synthesis artifact in long-read datasets that markedly impacts the identity and quantitation of assembled transcripts. Towards remediating these problems, we develop a computational pipeline to "strand" long-read cDNA libraries that rectifies inaccurate mapping and assembly of long-read transcripts. Leveraging the strengths of each platform and our computational stranding, we also present and benchmark a hybrid assembly approach that drastically increases the sensitivity and accuracy of full-length transcript assembly on the correct strand and improves detection of biological features of the transcriptome. When applied to a challenging set of under-annotated and cell-type variable lncRNA, our method resolves the segmentation problem of short-read sequencing and the depth problem of long-read sequencing, resulting in the assembly of coherent transcripts with precise 5' and 3' ends. Our workflow can be applied to existing datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts.

Chromosome conformation capture that detects novel cis- and trans-interactions in budding yeast.

Chromosome Conformation Capture (3C) has emerged as a powerful approach for revealing the conformation and features of three-dimensional (3D) genomic organization. Yet attainment of higher resolution in organisms with compact genomes presents a challenge. Here, we describe modifications in the 3C technique that substantially enhance its resolution and sensitivity when applied to the 3D genome of budding yeast. Keys to our approach include use of a 4 bp cutter, Taq I, for cleaving the genome and quantitative PCR for measuring the frequency of ligation. Most importantly, we normalize the percent digestion at each restriction site to account for variation in accessibility of local chromatin structure under a given physiological condition. This strategy has led to the detection of physical interactions between regulatory elements and gene coding regions as well as intricate, stimulus-specific interchromosomal interactions between activated genes. We provide an algorithm that incorporates these and other modifications and allows quantitative determination of chromatin interaction frequencies in yeast under any physiological condition.

A critical role for Pol II CTD phosphorylation in heterochromatic gene activation.

How gene activation works in heterochromatin, and how the mechanism might differ from the one used in euchromatin, has been largely unexplored. Previous work has shown that in SIR-regulated heterochromatin of Saccharomyces cerevisiae, gene activation occurs in the absence of covalent histone modifications and other alterations of chromatin commonly associated with transcription.Here we demonstrate that such activation occurs in a substantial fraction of cells, consistent with frequent transcriptional bursting, and this raises the possibility that an alternative activation pathway might be used. We address one such possibility, Pol II CTD phosphorylation, and explore this idea using a natural telomere-linked gene, YFR057w, as a model. Unlike covalent histone modifications, we find that Ser2, Ser5 and Ser7 CTD phosphorylated Pol II is prevalent at the drug-induced heterochromatic gene. Particularly enriched relative to the euchromatic state is Ser2 phosphorylation. Consistent with a functional role for Ser2P, YFR057w is negligibly activated in cells deficient in the Ser2 CTD kinases Ctk1 and Bur1 even though the gene is strongly stimulated when it is placed in a euchromatic context. Collectively, our results are consistent with a critical role for Ser2 CTD phosphorylation in driving Pol II recruitment and transcription of a natural heterochromatic gene - an activity that may supplant the need for histone epigenetic modifications.

Primordial super-enhancers: heat shock-induced chromatin organization in yeast.

Specialized mechanisms ensure proper expression of critically important genes such as those specifying cell identity or conferring protection from environmental stress. Investigations of the heat shock response have been critical in elucidating basic concepts of transcriptional control. Recent studies demonstrate that in response to thermal stress, heat shock-responsive genes associate with high levels of transcriptional activators and coactivators and those in yeast intensely interact across and between chromosomes, coalescing into condensates. In mammalian cells, cell identity genes that are regulated by super-enhancers (SEs) are also densely occupied by transcriptional machinery that form phase-separated condensates. We suggest that the stress-remodeled yeast nucleome bears functional and structural resemblance to mammalian SEs, and will reveal fundamental mechanisms of gene control by transcriptional condensates.

Heat Shock Factor 1 Drives Intergenic Association of Its Target Gene Loci upon Heat Shock.

Transcriptional induction of heat shock protein (HSP) genes is accompanied by dynamic changes in their 3D structure and spatial organization, yet the molecular basis for these phenomena remains unknown. Using chromosome conformation capture and single-cell imaging, we show that genes transcriptionally activated by Hsf1 specifically interact across chromosomes and coalesce into diffraction-limited intranuclear foci. Genes activated by the alternative stress regulators Msn2/Msn4, in contrast, do not interact among themselves nor with Hsf1 targets. Likewise, constitutively expressed genes, even those interposed between HSP genes, show no detectable interaction. Hsf1 forms discrete subnuclear puncta when stress activated, and these puncta dissolve in concert with transcriptional attenuation, paralleling the kinetics of HSP gene coalescence and dissolution. Nuclear Hsf1 and RNA Pol II are both necessary for intergenic HSP gene interactions, while DNA-bound Hsf1 is necessary and sufficient to drive heterologous gene coalescence. Our findings demonstrate that Hsf1 can dynamically restructure the yeast genome.

Heat Shock Protein Genes Undergo Dynamic Alteration in Their Three-Dimensional Structure and Genome Organization in Response to Thermal Stress.

Three-dimensional (3D) chromatin organization is important for proper gene regulation, yet how the genome is remodeled in response to stress is largely unknown. Here, we use a highly sensitive version of chromosome conformation capture in combination with fluorescence microscopy to investigate Heat Shock Protein (HSP) gene conformation and 3D nuclear organization in budding yeast. In response to acute thermal stress, HSP genes undergo intense intragenic folding interactions that go well beyond 5'-3' gene looping previously described for RNA polymerase II genes. These interactions include looping between upstream activation sequence (UAS) and promoter elements, promoter and terminator regions, and regulatory and coding regions (gene "crumpling"). They are also dynamic, being prominent within 60 s, peaking within 2.5 min, and attenuating within 30 min, and correlate with HSP gene transcriptional activity. With similarly striking kinetics, activated HSP genes, both chromosomally linked and unlinked, coalesce into discrete intranuclear foci. Constitutively transcribed genes also loop and crumple yet fail to coalesce. Notably, a missense mutation in transcription factor TFIIB suppresses gene looping, yet neither crumpling nor HSP gene coalescence is affected. An inactivating promoter mutation, in contrast, obviates all three. Our results provide evidence for widespread, transcription-associated gene crumpling and demonstrate the de novo assembly and disassembly of HSP gene foci.

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor.

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results 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 in AA strains.

Chromatin.

Chromatin is a complex of proteins, RNA and DNA that constitutes the physiological state of the genome. Its basic structure is essentially the same in nearly all eukaryotes, from single-celled yeasts to the most complex multicellular organisms (exceptions include the chromatin of dinoflagellates and vertebrate sperm). Its fundamental role is to package the genome in a sufficiently compact form that allows comparatively very large molecules of DNA to fit inside the cell's nucleus. In human cells, the contour length of the DNA molecules comprising the largest chromosomes is nearly 10,000 times the diameter of the nucleus (typically on the order of 5-10 microns). How is this compaction accomplished? Through multiple layers of folding.