The glucocorticoid receptor ligand-binding domain confers drug-gated protein regulation in C. elegans

Controlling protein activity and localization is a key tool in modern biology. Mammalian steroid receptor ligand-binding domains (LBDs) fusions have been used in a range of organisms and cell-types to inactivate proteins of interest until the cognate steroid ligand is applied. Here, we demonstrate that the glucocorticoid receptor LBD confers ligand-gated control of a heterologous gene expression system (Q system) and the DAF-16 transcription factor in C. elegans. These experiments demonstrate provide a powerful tool for temporal control of protein activity, and will bolster existing tools used to modulate gene expression and protein activity in this animal.


Introduction 39
The ability to temporally and spatially control gene expression and protein 40 activity/localization are essential tools in modern genetics. Heterologous systems, such 41 as Gal4/UAS have been widely used in Drosophila melanogaster, allowing researchers 42 exquisite control over the time and tissue in which the transgene is expressed (Brand 43 and Perrimon 1993). The Q system, which adapts the Neurospora crassa transcriptional 44 activator, QF, and its repressor, QS, to gene activation via de-repression of QS by 45 quinic acid, offers an additional system for transgene expression analysis, lineage 46 tracing, and mosaic analysis in mammalian cells and in fly models (Potter et al. 2010; 47 Riabinina et al. 2015). Other methods to control gene expression include tissue-specific 48 expression of the Cre recombinase fused to the estrogen receptor ligand-binding 49 domain (Feil et al. 1996), CRISPR interference and CRISPR activation (Qi et al. 2013; In C. elegans, the most common method to induce gene expression is to fuse heat 57 shock promoters upstream of a gene of interest; following acute heat shock, gene 58 expression is robustly induced (Stringham et al. 1992). Modifications to this approach 59 include the FLP-OUT system (Voutev and Hubbard 2008), and the HSF-1 system, 60 which allows for some tissue-specificity of gene induction (Bacaj and Shaham 2007). 61 169 The remaining microscopy was performed on a Zeiss Axioplan 2 fluorescent 170 microscope attached to Xenon excitation lamps and green/red fluorescence filter sets. imaging. In populations expressing the hsp-16.48 promoter, mixed-staged animals were 179 gently washed off of NGM plates, and resuspended in M9 with vehicle or 100μM dex. A 180 30min, 33°C heat shock was performed in a thermocycler, and animals were incubated 181 at room temperature for an additional 3.5 hours on a bench top rotator before imaging. 182 To assess vulva morphology, populations were synchronized by extracting eggs from 183 gravid adults by alkaline lysis followed by hatching M9+0.05% gelatin for 24-48 hours at 184 25°C. Next, L1 hatchlings were released from the starvation-induced L1-diapause by 185 feeding, and subsequently grown to the mid-L4 larval stage. L4 larvae were then 186 washed off of plates, and treated with vehicle or 100μM dex for three hours rotating on a 187 bench top before imaging. Vulval morphology was assessed in mCherry positive 188 animals in late L4 larvae or young adults, at 16X-25X magnification.

Statistical Analysis 204
Relative gene expression was calculated by determining the fold-change variation over 205 control (vehicle) samples using the Comparative C T method (Schmittgen and Livak 206 2008). Regression analysis was utilized to determine C T values, and the mean C T value 207 from reactions preformed in triplicate was used to determine the average fold-change 208 from the ama-1 internal control. Error bars were calculated using the error propagation 209 of standard deviations to the logarithmic scale. The comparisons between vehicle and 210 dex-treated animals (and thus limited to two conditions) were performed using an 211 unpaired, two-tailed Student's T-test, or a Chi-square test. All p-values were calculated 212 using the GraphPad Prism 6.0 software. 213 214

Data Availability 215
Strains generated in this study will be available via the Caenorhabditis Genetics Center 216 (CGC) at the University of Minnesota-Twin Cities and/or through direct request to 217 J.D.W. The destination vectors described in this study (pGM32DEST, pGM34DEST, 218 and pGM48DEST) will be available via Addgene. 219

A drug-inducible expression tool for C. elegans 221
To engineer a heterologous, drug-inducible gene expression system for C. elegans, we 222 modified the QF transcriptional activator by fusing the ligand-binding domain (LBD) from 223 the human glucocorticoid receptor (GRα; NCBI Gene ID: 2908) at the C-terminus (QF-224 GR; Figure 1A). We chose the glucocorticoid receptor as C. elegans does not have any 225 clear orthologs (Antebi 2015), and ligand binding by the GR LBD is very specific, 226 whereas estrogen receptor ligand-binding domain exhibits promiscuity (Eick et al. 227 2012). QF-GR should be inactive until the synthetic, non-metabolized GR ligand 228 dexamethasone (dex) is added (Scherrer et al. 1993). QF-GR, was cloned into a 229 plasmid with a contiguous splice leader (SL) and an mCherry reporter, which marks 230 cells in which QF-GR is expressed. Though designed for Gateway cloning ( Figure S1A We first tested if the GR ligand, dex, could be effectively absorbed into live animals. We 238 used a fluorescein-dex (F-dexa) conjugate previously used to monitor the uptake of dex 239 in other systems (Maier et al. 2005). As uptake of molecules in C. elegans can occur via fold decrease in brood sizes observed between the rescued transgenics and wild-type 287 animals (*p<0.05, ANOVA test). Finally, we did not observe any abnormalities in 288 morphology, foraging behavior, or developmental timing of the animals cultured in dex 289 (data not shown). 290 291 Next, we performed a time course to assess how quickly GFP was detectable by 292 fluorescence microscopy following dex exposure. Mixed-stage animals were cultivated 293 on plates containing either vehicle or 100μM dex, and scored hourly for GFP and 294 mCherry expression. After two hours of drug-treatment, 20% (n=25) of animals 295 exhibited GFP fluorescence, as compared to no vehicle-treated animals (n=21; *p<0.03, 296 T-Test) ( Figure 1C). Using qPCR to measure GFP and mCherry transcript levels, we 297 observed a 3.5-fold increase in GFP levels in dex-treated animals, as compared to 298 vehicle-control animals ( Figure 1D); mCherry mRNA levels did not change significantly 299 within the same dex and vehicle-treated populations. In contrast, after 30 minutes of 300 ligand exposure, we did not observe any significant changes in GFP transcript levels 301 ( Figure S1C). GFP was detected initially in the nervous system, and most 302 predominately in unidentified tail neurons and the ventral cord. Notably, both the 303 percentage of GFP-positive animals, and the tissue-distribution of GFP expression 304 increased over time, with 80% of animals (n=38) expressing GFP in multiple tissues 305 after eight hours of cultivation on dex-treated plates, as compared to 0% (n=27) of 306 vehicle-treated populations (*p<0.001, T-Test). We similarly expressed QF-GR from an 307 alternative ubiquitous driver, eef-1A.1, and observed dex-specific GFP induction in 308 transgenic animals ( Figure S1C). QF-GR expression from the eef-1A.1 promoter drove 309 significant reporter expression predominately in hypodermal, intestinal, and vulval 310 tissues after two hours of dex-exposure in 27% (n=26) of animals, as opposed to 0% 311 (n=22) of vehicle-treated animals (*p<0.04, T-Test) ( Figure S1D). In contrast to 312 transgenics expressing QF-GR from the pro-1 promoter, we did not observe GFP in the

QF-GR allows tissue-specific transgene expression 321
Having established that the GR LBD inactivated QF and that dex exposure allowed for 322 QF activation, we next asked if QF-GR could drive tissue-specific transgene expression. 323 We expressed QF-GR in vulval and hypodermal tissues, using the tissue-specific egl-17 324  Figure 2C). We scored transgenic animals exposed for two hours to either vehicle or 348 100μM dex, and then assessed the behavioral quiescence of adult animals as 349 described in Methods. We observed an approximate 30-fold difference in behavioral 350 quiescence of dex-treated adults, as compared to vehicle-exposed adults (*p<0.005, T-351 Test). Specifically, 17% (n=181) of adults exhibited behaviors consistent with lin-3c-352 induced quiescence after a two hour treatment of dex, as compared to 0.4% (n=180) of 353 vehicle-treated animals ( Figure 2C). These observations indicate that the forced 354 expression of lin-3/EGF can be achieved with dex-inducible QF-GR. 355 356 A powerful application of Gal4-UAS in Drosophila is for the conditional cell ablation by 357 expressing death genes, such as Reaper (White et al. 1994). In C. elegans, the peel-1 358 gene encodes a sperm-specific toxin, which destroys both germ and somatic cells in a 359 cell autonomous manner. Accordingly, ubiquitous overexpression of the peel-1 gene 360 results animal lethality (Seidel et al. 2011). To test if our system could be used to 361 conditionally induce animal lethality, we attempted to induce multi-tissue expression of 362 peel-1 using pro-1p::QF-GR; however, we were unable to propagate the transgenic 363 lines due to toxicity of the arrays, even after multiple trials with various gene dosages 364 (data not shown). Therefore, we then asked if, alternatively, our system could be utilized

The QS repressor and quinic acid together restrain the activity of QF-GR 379
Neurospora QS protein inhibition of QF activity is relieved by addition of quinic acid (Wei 380 et al. 2012). To test whether QS could inhibit the activity of QF-GR, we co-expressed 381 QF-GR and QS, and monitored QUAS::GFP reporter activity. As expected, we failed to 382 detect GFP fluorescence in animals cultured in either vehicle only or vehicle plus quinic 383 acid (n≥57) ( Figure 3B). Following dex-treatment in the absence of quinic acid, we 384 observed GFP expression in only 0.9% and 1.0% (n≥85) of animals. However, when 385 transgenic animals were co-treated with both dex and quinic acid, we observed that 386 40% and 54% (n≥80) of animals expressed GFP ( Figure 3B). Using qRT-PCR to assess 387 induction of transgene transcripts, after a two hour vehicle-treatment, the QF-GR 388 activator increased GFP levels approximately 28-fold above basal expression; addition 389 of 100μM dex increased GFP expression to 98-fold above basal expression (Figure 3). 390 As before, QS suppressed the expression of GFP mRNAs, as transcript levels were 391 only 1.7-fold higher in both vehicle and dex-treated populations relative to basal 392 expression ( Figure 3C). After pre-treatment with quinic acid for approximately 24 hours, 393 we measured a small, but significant 2.3-fold increase in GFP transcript expression after 394 an acute, two hour dex-treatment. This dex-specific amplification was further intensified 395 after a 49 hour treatment with both dex and quinic acid. We identified an 11-fold 396 increase in GFP mRNAs in dex-treated animals, and only a 1.2-fold increase in GFP 397 transcripts in vehicle-treated animals, as compared to the basal activity from the 398 quas::GFP reporter. These data suggest that QS can repress transcriptional activity 399 even in the presence of dex, and that QS de-repression and dex activation provide dual 400 regulation, analogous to numerous bacterial operons that are under both negative and 401 positive control. 402 403

The GR-LBD adduct modulates DAF-16 and GFP activity in vivo. 404
Finally, we wished to determine whether the GR LBD could be more broadly used to 405 control the activity and localization of other C. elegans proteins. We fused the GR LBD 406 adduct to eGFP under the control of a heat-shock-inducible promoter (hsp-16.48), which 407 drives expression predominately in the muscle and hypodermis (Stringham et al. 1992). 408 After a four hour treatment with 100μM dex, GFP expression was detected only in the 409 hypodermal nuclei of 100% (n=78) of animals; in contrast, GFP was visible in both the 410 cytoplasm and nucleus in 55% (n=94) of vehicle-treated animals ( Figure 4A). This result 411 suggests that the GR LBD adduct may function to restrict localization of the linked 412 protein to the cytoplasm in the absence of ligand, and upon dex-binding, translocation to 413 the nucleus occurs more readily (Picard and Yamamoto 1987). daf-2(-) mutants or wildtype animals (*p<0.03, T-Test). Together, these results suggest 432 that addition of the GR LBD adduct allows ligand-gated control of DAF-16 activity. 433

Conclusions 434
We found that the QF-GR system was sufficient to drive dex-inducible, tissue-specific 435 expression of a GFP reporter, the toxin gene peel-1, and ubiquitous expression of lin-436 3c. We further demonstrate that addition of a GR LBD onto DAF-16 was sufficient to 437 confer ligand-inducible activity of this protein, a potentially generalizable method of 438 regulating the activity and/or localization of nuclear and cytoplasmic proteins in C.  Figure  782