Identification of a tissue-selective heat shock response regulatory network

Abstract

The heat shock response (HSR) is essential to survive acute proteotoxic stress and has been studied extensively in unicellular organisms and tissue culture cells, but to a lesser extent in intact metazoan animals. To identify the regulatory pathways that control the HSR in Caenorhabditis elegans, we performed a genome-wide RNAi screen and identified 59 genes corresponding to 7 positive activators required for the HSR and 52 negative regulators whose knockdown leads to constitutive activation of the HSR. These modifiers function in specific steps of gene expression, protein synthesis, protein folding, trafficking, and protein clearance, and comprise the metazoan heat shock regulatory network (HSN). Whereas the positive regulators function in all tissues of C. elegans, nearly all of the negative regulators exhibited tissue-selective effects. Knockdown of the subunits of the proteasome strongly induces HS reporter expression only in the intestine and spermatheca but not in muscle cells, while knockdown of subunits of the TRiC/CCT chaperonin induces HS reporter expression only in muscle cells. Yet, both the proteasome and TRiC/CCT chaperonin are ubiquitously expressed and are required for clearance and folding in all tissues. We propose that the HSN identifies a key subset of the proteostasis machinery that regulates the HSR according to the unique functional requirements of each tissue.

Figure 1. Genome-wide RNAi screen for HSR regulators.

(A–D) Nomarski and fluorescent images corresponding to the phsp70::gfp reporter strain. (A) Control animals show little reporter expression and only faint autofluorescence of the intestine. (B) Reporter induction in animals exposed to heat shock at 33°C for 1 hour. (C) RNAi knockdown of hsf-1 decreases induction of the reporter by heat shock. (D) RNAi knockdown of hsp-1 causes constitutive induction of the reporter in the absence of heat shock. The scale bar corresponds to 100 µm. (E–F) Quantitation of the effects on endogenous hsp70 and hsp-16.2 genes using qRT-PCR. (E) HSR positive regulators normalized to the heat shocked empty vector control. (F) HSR negative regulators normalized to empty vector control. Averages are from at least three biological replicates and error bars represent SEM.

Figure 2. Tissue-selective induction of the phsp70::gfp reporter by knockdown of negative regulators.

Nomarski and fluorescent images corresponding to whole animals and fluorescent images of the spermatheca, intestine, and muscle tissue are shown. The boundary of the animals, intestine and spermatheca taken from Nomarski images are added as a visual aide to some images. (A–E) RNAi knockdown of daf-21 leads to induction of the reporter in all three tissues. (F–J) RNAi knockdown of cct-1 causes induction only in muscle. (K–O) knockdown of F38A1.8 causes induction in the intestine and spermatheca. Images are taken at different exposures to maximize fluorescence of each image. Scale bars of whole animal images correspond to 100 µm, while scale bars of the images depicting specific tissues correspond to 50 µm. Asterisks denote only autofluorescence.

Figure 3. Validation of tissue-selective effects using small molecules and mutants.

A) Incubation with 100 µM MG132, a small molecule inhibitor of the proteasome, but not DMSO alone, causes tissue-selective induction of the phsp70::gfp reporter in the intestine and spermatheca (arrows), similar to RNAi knockdown of proteasomal subunits. The scale bar corresponds to 200 µm. Asterisks denote only autofluorescence. B) Mutations in T24H7.2, sgt-1, cyn-11, and unc-45 cause induction of the HSR in whole worms measured using qRT-PCR. C) Mutation of T24H7.2, but not unc-45, causes induction in the intestine relative to N2 control animals, measured by qRT-PCR analysis of hsp70 in dissected intestinal tissue. Averages shown are from at least two biological replicates.

Figure 4. Network analysis of HSR regulators.

Shown is a network with HSR negative regulator genes depicted as nodes and interactions as edges. Node shape denotes grouping corresponding to a community detection algorithm based on the structure of the interaction network. Node color corresponds to the tissue-specific phsp70::gfp reporter induction. Cartoons of worms depicting the tissue specificity appear next to nodes containing those colors.

Figure 5. Epistasis analysis of HSR regulators.

The effects of HSR positive regulator knockdown on induction of the reporter by negative HSR regulator knockdown were measured using the phsp70::gfp reporter. (A) Images showing the results from double RNAi with each positive regulator and the negative regulator hsp-1. In each case, knockdown of the positive regulator decreased reporter fluorescence compared to knockdown of hsp-1 alone. (B) Quantitation of the effects of HSR positive regulator knockdown using RNAi on induction of endogenous HSR genes by the HSR negative regulator T24H7.2 mutant reveals that the positive regulators are epistatic to T24H7.2.

Figure 6. HSR regulatory network model.

Each HSR regulator, denoted by common terminology, is indicated as a box and is grouped according to its presence in a multi-subunit complex or functional pathway (i.e., the proteasome or secretory pathway). Positive or negative effects on HSR regulation are indicated by either a green arrow or red line respectively. Positive regulators are further separated from negative regulators by grey shading in the background. At the center of the network, HSF1 integrates signals from the various regulators and establishes a coordinated HSR.