Heat-Shock Factor 1 (HSF1), master regulator of the heat-shock response, facilitates malignant transformation, cancer cell survival, and proliferation in model systems. The common assumption is that these effects are mediated through regulation of heat-shock protein (HSP) expression. However, the transcriptional network that HSF1 coordinates directly in malignancy and its relationship to the heat-shock response have never been defined. By comparing cells with high and low malignant potential alongside their nontransformed counterparts, we identify an HSF1-regulated transcriptional program specific to highly malignant cells and distinct from heat shock. Cancer-specific genes in this program support oncogenic processes: cell-cycle regulation, signaling, metabolism, adhesion and translation. HSP genes are integral to this program, however, many are uniquely regulated in malignancy. This HSF1 cancer program is active in breast, colon and lung tumors isolated directly from human patients and is strongly associated with metastasis and death. Thus, HSF1 rewires the transcriptome in tumorigenesis, with prognostic and therapeutic implications.

HSF1 Drives a Transcriptional Program Distinct from Heat Shock to Support Highly Malignant Human Cancers

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.

Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness

Hsf1 is an ancient transcription factor that responds to protein folding stress by inducing the heat-shock response (HSR) that restore perturbed proteostasis. Hsp70 chaperones negatively regulate the activity of Hsf1 via stress-responsive mechanisms that are poorly understood. Here, we have reconstituted budding yeast Hsf1-Hsp70 activation complexes and find that surplus Hsp70 inhibits Hsf1 DNA-binding activity. Hsp70 binds Hsf1 via its canonical substrate binding domain and Hsp70 regulates Hsf1 DNA-binding activity. During heat shock, Hsp70 is out-titrated by misfolded proteins derived from ongoing translation in the cytosol. Pushing the boundaries of the regulatory system unveils a genetic hyperstress program that is triggered by proteostasis collapse and involves an enlarged Hsf1 regulon. The findings demonstrate how an apparently simple chaperone-titration mechanism produces diversified transcriptional output in response to distinct stress loads.

Cytoplasmic protein misfolding titrates Hsp70 to activate nuclear Hsf1.

HSF1 (heat shock factor 1) is an evolutionarily conserved master transcriptional regulator of the heat shock response (HSR) in eukaryotic cells. In response to high temperatures, HSF1 upregulates genes encoding molecular chaperones, also called heat shock proteins, which assist the refolding or degradation of damaged intracellular proteins. Accumulating evidence reveals however that HSF1 participates in several other physiological and pathological processes such as differentiation, immune response, and multidrug resistance, as well as in ageing, neurodegenerative demise, and cancer. To address how HSF1 controls these processes one should systematically analyze its target genes. Here we present a novel database called HSF1Base (hsf1base.org) that contains a nearly comprehensive list of HSF1 target genes identified so far. The list was obtained by manually curating publications on individual HSF1 targets and analyzing relevant high throughput transcriptomic and chromatin immunoprecipitation data derived from the literature and the Yeastract database. To support the biological relevance of HSF1 targets identified by high throughput methods, we performed an enrichment analysis of (potential) HSF1 targets across different tissues/cell types and organisms. We found that general HSF1 functions (targets are expressed in all tissues/cell types) are mostly related to cellular proteostasis. Furthermore, HSF1 targets that are conserved across various animal taxa operate mostly in cellular stress pathways (e.g., autophagy), chromatin remodeling, ribosome biogenesis, and ageing. Together, these data highlight diverse roles for HSF1, expanding far beyond the HSR.

HSF1Base: A Comprehensive Database of HSF1 (Heat Shock Factor 1) Target Genes.

Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3+/− cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Here, using Drosophila, we show that reduced translation does not cause cell competition. Instead, we identify proteotoxic stress as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3+/− cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, we propose that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection. Baumgartner et al. identify proteotoxic stress as the underlying cause of the loser status in a cell competition model caused by reduced autophagic, proteasomal flux and accumulation of protein aggregates.

Proteotoxic stress is a driver of the loser status and cell competition.

When yeast cells are growing at top speed, they can make 2,000 new ribosomes every minute. These enormous molecular assemblies are the protein-making machines of the cell. Building new ribosomes is one of the most energy-demanding parts of cell growth and, if the process goes wrong, the results can be catastrophic. The proteins that make up the ribosomes themselves are sticky. Left unattended, they start to form toxic clumps inside the compartment that houses most of the cell’s DNA, the nucleus. A protein called Heat shock factor 1, or Hsf1 for short, plays an important role in the cell's quality control systems. It helps to manage sticky proteins by switching on genes that break down protein clumps and prevent new clumps from forming. Hsf1 levels start to rise whenever cells are struggling to keep up with protein production. If it is half-finished ribosomes that are causing the problem, cells can stop making ribosome proteins. The protein in charge of this in yeast is Ifh1. It is a transcription factor that sits at the front of the genes for ribosome proteins, switching them on. When yeast cells get stressed, Ifh1 drops away from the genes within minutes, switching them off again. Yet how this happens, and how it links to Hsf1, is a mystery. To start to provide some answers, Albert et al. disrupted the production of ribosomes in yeast cells and examined the consequences. This revealed a new rescue response, that they named the “ribosome assembly stress response”. Both Hsf1 and Ifh1 are sensitive to the build-up of unfinished ribosomes in the nucleus. As expected, Hsf1 activated when ribosome proteins started to build up, and switched on the genes needed to manage the protein clumps. The effect on Isfh1, however, was unexpected. When the unassembled ribosome proteins started to build up, it was the clumps themselves that pulled the Ifh1 proteins off the genes. The unassembled ribosomes proteins seemed to be stopping their own production. Low levels of clumped ribosome proteins in the nuclei of unstressed cells also helped to keep Hsf1 active and pull Ifh1 off the ribosome genes. It is possible that this provides continual protection against a toxic protein build-up. These findings are not only important for understanding yeast cells; cancer cells also need to produce ribosomes at a very high rate to sustain their rapid growth. They too might be prone to stresses that interrupt their ribosome assembly. As such, understanding more about this process could one day lead to new therapies to target cancer cells.

A ribosome assembly stress response regulates transcription to maintain proteome homeostasis

While expression of ribosomal protein genes (RPGs) in the budding yeast has been extensively studied, a longstanding enigma persists regarding their co-regulation under fluctuating growth conditions. Most RPG promoters display one of two distinct arrangements of a core set of transcription factors (TFs) and are further differentiated by the presence or absence of the HMGB protein Hmo1. However, a third group of promoters appears not to be bound by any of these proteins, raising the question of how the whole suite of genes is co-regulated. We demonstrate here that all RPGs are regulated by two distinct, but complementary mechanisms driven by the TFs Ifh1 and Sfp1, both of which are required for maximal expression in optimal conditions and coordinated downregulation upon stress. At the majority of RPG promoters, Ifh1-dependent regulation predominates, whereas Sfp1 plays the major role at all other genes. We also uncovered an unexpected protein homeostasis-dependent binding property of Hmo1 at RPG promoters. Finally, we show that the Ifh1 paralog Crf1, previously described as a transcriptional repressor, can act as a constitutive RPG activator. Our study provides a more complete picture of RPG regulation and may serve as a paradigm for unravelling RPG regulation in multicellular eukaryotes.

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Mechanisms coordinating ribosomal protein gene transcription in response to stress

Ribosome biogenesis is a complex and energy-demanding process requiring tight coordination of ribosomal RNA (rRNA) and ribosomal protein (RP) production. Alteration of any step in this process may impact growth by leading to proteotoxic stress. Although the transcription factor Hsf1 has emerged as a central regulator of proteostasis, how its activity is coordinated with ribosome biogenesis is unknown. Here we show that arrest of ribosome biogenesis in the budding yeast S. cerevisiae triggers rapid activation of a highly specific stress pathway that coordinately up-regulates Hsf1 target genes and down-regulates RP genes. Activation of Hsf1 target genes requires neo-synthesis of RPs, which accumulate in an insoluble fraction, leading to sequestration of the RP transcriptional activator Ifh1. Our data suggest that levels of newly-synthetized RPs, imported into the nucleus but not yet assembled into ribosomes, work to continuously balance Hsf1 and Ifh1 activity, thus guarding against proteotoxic stress during ribosome assembly.

https://www.biorxiv.org/content/biorxiv/early/2019/01/06/512665.full.pdf

The role of three-dimensional genome organization as a critical regulator of gene expression has become increasingly clear over the last decade. Most of our understanding of this association comes from the study of long range chromatin interaction maps provided by Chromatin Conformation Capture-based techniques, which have greatly improved in recent years. Since these procedures are experimentally laborious and expensive, in silico prediction has emerged as an alternative strategy to generate virtual maps in cell types and conditions for which experimental data of chromatin interactions is not available. Several methods have been based on predictive models trained on one-dimensional (1D) sequencing features, yielding promising results. However, different approaches vary both in the way they model chromatin interactions and in the machine learning-based strategy they rely on, making it challenging to carry out performance comparison of existing methods. In this study, we use publicly available 1D sequencing signals to model chromatin interactions in two human cell lines and evaluate the prediction performance of 5 popular machine learning algorithms: decision trees, random forests, gradient boosting, support vector machines and multi-layer perceptron. Our approach accurately predicts long-range interactions and reveals that gradient boosting significantly outperforms the other four algorithms, yielding accuracies of ∼ 95%. We show that chromatin features in close genomic proximity to the anchors cover most of the predictive information. Moreover, we demonstrate that gradient boosting models trained with different subsets of chromatin features, unlike the other methods tested, are able to produce accurate predictions. In this regard, and besides architectural proteins, transcription factors are shown to be highly informative. Our study provides a framework for the systematic prediction of long-range chromatin interactions, identifies gradient boosting as the best suited algorithm for this task and highlights cell-type specific binding of transcription factors at the anchors as important determinants of chromatin wiring.

Maria Paula Rueda

Papers

A ribosome assembly stress response regulates transcription to maintain proteome homeostasis

Mechanisms coordinating ribosomal protein gene transcription in response to stress

A ribosome assembly stress response regulates transcription to maintain proteome homeostasis

Mechanisms Coordinating Ribosomal Protein Gene Transcription in Response to Stress