The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level.While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay.Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Background Temperature influences biology at all levels, from altering rates of biochemical reactions to determining sustainability of entire ecosystems. Although extended exposure to elevated temperatures influences organismal phenotypes important for human health, agriculture, and ecology, the molecular mechanisms that drive these responses remain largely unexplored. Prolonged, mild temperature stress (48 h at 28 °C) has been shown to inhibit reproduction in Caenorhabditis elegans without significantly impacting motility or viability. Results Analysis of molecular responses to chronic stress using RNA-seq uncovers dramatic effects on the transcriptome that are fundamentally distinct from the well-characterized, acute heat shock response (HSR). While a large portion of the genome is differentially expressed ≥ 4-fold after 48 h at 28 °C, the only major class of oogenesis-associated genes affected is the vitellogenin gene family that encodes for yolk proteins (YPs). Whereas YP mRNAs decrease, the proteins accumulate and mislocalize in the pseudocoelomic space as early as 6 h, well before reproduction declines. A trafficking defect in a second, unrelated fluorescent reporter and a decrease in pre-synaptic neuronal signaling indicate that the YP mislocalization is caused by a generalized defect in endocytosis. Molecular chaperones are involved in both endocytosis and refolding damaged proteins. Decreasing levels of the major HSP70 chaperone, HSP-1, causes similar YP trafficking defects in the absence of stress. Conversely, increasing chaperone levels through overexpression of the transcription factor HSF-1 rescues YP trafficking and restores neuronal signaling. Conclusions These data implicate chaperone titration during chronic stress as a molecular mechanism contributing to endocytic defects that influence multiple aspects of organismal physiology. Notably, HSF-1 overexpression improves recovery of viable offspring after exposure to stress. These findings provide important molecular insights into understanding organismal responses to temperature stress as well as phenotypes associated with chronic protein misfolding.

The heat shock response (HSR) is a cellular stress response that senses protein misfolding and restores protein folding homeostasis, or proteostasis. We previously identified an HSR regulatory network in Caenorhabditis elegans consisting of highly conserved genes that have important cellular roles in maintaining proteostasis. Unexpectedly, the effects of these genes on the HSR are distinctly tissue-specific. Here, we explore this apparent discrepancy and find that muscle-specific regulation of the HSR by the TRiC/CCT chaperonin is not driven by an enrichment of TRiC/CCT in muscle, but rather by the levels of one of its most abundant substrates, actin. Knockdown of actin subunits reduces induction of the HSR in muscle upon TRiC/CCT knockdown; conversely, overexpression of an actin subunit sensitizes the intestine so that it induces the HSR upon TRiC/CCT knockdown. Similarly, intestine-specific HSR regulation by the signal recognition particle (SRP), a component of the secretory pathway, is driven by the vitellogenins, some of the most abundant secretory proteins. Together, these data indicate that the specific protein folding requirements from the unique cellular proteomes sensitizes each tissue to disruption of distinct subsets of the proteostasis network. These findings are relevant for tissue-specific, HSR-associated human diseases such as cancer and neurodegenerative diseases. Additionally, we characterize organismal phenotypes of actin overexpression including a shortened lifespan, supporting a recent hypothesis that maintenance of the actin cytoskeleton is an important factor for longevity.

Approximately 75% of breast cancers are driven by the estrogen receptor alpha (ER), and despite the advent of endocrine therapy to block ER signaling pathways, a significant portion of women develop resistance to these drugs. The pioneer factor FOXA1 has been shown to facilitate nearly all DNA-binding events of ER in response to estrogen in ER+ breast cancer (ER+BC). Notably, up-regulation of FOXA1 is a hallmark of endocrine-resistant phenotypes and has been shown to reprogram enhancer elements, leading to an altered transcriptome. However, FOXA1 is a critical pioneer factor for multiple nuclear hormone receptors, aside from ER, and is implicated in regulation of important factors such as HER2 and the androgen receptor (AR). With the diverse array of breast cancer molecular subtypes displaying complex interplay between ER, HER2, AR, PR, and other hormone receptors, describing the complete ensemble of FOXA1 binding partners in various contexts, such as endocrine-resistant tumors, is of increasing importance. To define a comprehensive catalog of FOXA1 binding partners under basal conditions, we generated MCF-7 cell lines stably expressing constructs of FOXA1 fused at its N- or C-terminus to the biotin ligase miniTurbo. Using proximity labeling coupled with mass-spectrometry, we have comprehensively cataloged binding partners of FOXA1, including many expected proteins such as ER, AR, MLL3, YAP1, and GATA-3. Moreover, we have discovered more than 150 previously unidentified binding partners of FOXA1, which may exert profound effects on FOXA1 function. Importantly, high hazard ratios and significant dependencies are associated with several of these new binding partners, such as subunits of a previously described histone deacetylase (HDAC) complex containing genetic suppressor element 1 (GSE1) and lysine-specific histone demethylase 1A (KDM1A). Genomic approaches are currently underway to characterize where in the genome FOXA1 is interacting with these novel proteins and to guide future exploration into the physiological significance of these interactions. Integrating biochemical, molecular, and genomic approaches, we have potentially highlighted new mechanisms of FOXA1, which could have significant clinical impact in the future. Citation Format: Rosemary N. Plagens, Shen Li, Christine A. Mills, Laura Herring, Hector L. Franco. Identifying FOXA1 Binding Partners using Proximity Labeling [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-08.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level.While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay.Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.