Dot6 is a major regulator of cell size and a transcriptional activator of ribosome biogenesis in the opportunistic yeast Candida albicans

In most species, size homeostasis appears to be exerted in late G1 phase as cells commit to division, called Start in yeast and the Restriction Point in metazoans. This size threshold couples cell growth to division and thereby establishes long-term size homeostasis. Our former investigations have shown that hundreds of genes markedly altered cell size under homeostatic growth conditions in the opportunistic yeast Candida albicans, but surprisingly only few of these overlapped with size control genes in the budding yeast Saccharomyces cerevisiae. Here, we investigated one of the divergent potent size regulators in C. albicans, the Myb-like HTH transcription factor Dot6. Our data demonstrated that Dot6 is a negative regulator of Start and also acts as a transcriptional activator of ribosome biogenesis (Ribi) genes. Genetic epistasis uncovered that Dot6 interacted with the master transcriptional regulator of the G1 machinery, SBF complex, but not with the Ribi and cell size regulators Sch9, Sfp1 and p38/Hog1. Dot6 was required for carbon-source modulation of cell size and it is regulated at the level of nuclear localization by TOR pathway. Our findings support a model where Dot6 acts as a hub that integrate directly growth cues via the TOR pathway to control the commitment to mitotic division at G1.

In a eukaryotic organism, cell size homeostasis is maintained through a balanced coordination 76 between cell growth and division. In the last half century, a major focus of cell biology has been 77 the study of cell division, but how eukaryotic cells couple growth to division to maintain a 78 homeostatic size remains poorly understood. In most eukaryotic organisms, reaching a critical 79 cell size appears to be crucial for commitment to cell division in late G1 phase, called Start in 80 yeast Candida albicans is a diploid ascomycete yeast that is an important commensal and 100 opportunistic pathogen in humans. While C. albicans and S. cerevisiae colonize different niches, 101 common biological features are shared between the two yeasts including the morphological trait 102 of budding, and core cell cycle and growth regulatory mechanisms (BERMAN 2006;COTE et al. 103 2009). C. albicans has served as an important evolutionary milestone with which to assess 104 evolutionary conservation of biological mechanism, and recent evidence suggests a surprising 105 extent of rewiring of central signalling, transcriptional and metabolic networks as compared to S.

136
Growth conditions and C. albicans Strains 137 The strains used in this study are listed in

228
Dot6 is a negative regulator of START in C. albicans 229 We have previously shown that the transcription factor Dot6 was required for cell size control in 230 C. albicans (SELLAM et al. 2016). A dot6 mutant had a median size that was 21% (41fL) smaller 231 than its congenic parental (52fL) or the complemented strains (51fL) (Figure 1A). Inactivation 232 of DOT6 resulted in a delayed exit from the lag phase (1.5h delay as compared to the WT) 233 ( Figure 1B). However, dot6 had a doubling time comparable to the WT and the complemented 234 strains during the log phase suggesting that the size reduction of dot6 is not a growth rate-235 associated phenotype ( Figure 1B). To ascertain that this effect was mediated at Start, we 236 evaluated two hallmarks of Start, namely bud emergence and the onset of SBF-dependent 237 transcription as a function of cell size in synchronous G1 phase cells obtained by elutriation. As 238 assessed by median size of cultures for which 90% of cells had a visible bud, the dot6 mutant 239 passed Start after growth to 26fL, whereas a parental WT control culture became 90% budded at 240 a much larger size of 61fL ( Figure 1C). Importantly, in the same experiment, the onset G1/S 241 transcription was accelerated in the dot6 strain as judged by the peak in expression of the two 242 representative G1-transcripts, the ribonucleotide reductase large subunit, RNR1 and the cyclin 243 PCL2 (Figure 1D-E) (Figure 2A-C). 255 Furthermore, inactivation of DOT6 in the swi4 mutant resulted in a large size comparable to that 256 of swi4 mutant indicating that Dot6 acts via SBF complex to control Start ( Figure 2D). SWI4 257 deletion is also epistatic to DOT6 regarding the growth rate in liquid YPD medium confirming 258 that both Dot6 and Swi4 act in a common pathway ( Figure 2E). Given the absence of epistatic 259 interaction between Dot6 and the known conserved Ribi and size regulators Sch9, Sfp1 and 260 Hog1, our data uncovered a novel uncharacterized pathway that control the critical cell size 261 threshold in C. albicans (Figure 2F) structural constituents of the ribosome (Figure 3A). This suggest that in contrast to the role of its 274 orthologue in S. cerevisiae, Dot6 in C. albicans is an activator of RiBi. Analysis of promoter 275 region of transcript downregulated in dot6 (transcript with 1.5-fold reduction using 5% FDR-276 Table S3) showed the occurrence of the PAC motif bound by Dot6 in all promoters of genes 277 related to RiBi ( Figure 3B). Furthermore, transcripts downregulated in dot6 exhibited correlation 278 with the set of genes repressed by the TOR complex inhibitor, rapamycin. This suggest that the 279 evolutionary conserved RiBi transcription control by TOR is mediated fully or partially through 280 Dot6. In support of the role of Dot6 in transcriptional control of Ribi genes and thus translation, 281 dot6 mutant exhibited an increased sensitivity to the protein translation inhibitor cycloheximide 282 as compared to the WT and the revertant strains ( Figure 3C). 283 The transcriptional programs characterizing the cell cycle G1/S transition in C. albicans (COTE et 284 al. 2009) were hyperactivated in dot6 mutant, which is a further support of the role of Dot6 as a 285 negative regulator of G1/S transcription and Start ( Figure 3A) Figure S1). This suggest that the Sfp1-Sch9 regulatory circuit had rewired and is 300 unlikely to rely on the nutrient status of the cell to Start control in C. albicans. 301 To assess whether the nutrient-sensitive TOR pathway communicates the nutrient status to Dot6, 302 we first tested whether altering TOR activity by rapamycin could alter the subcellular 303 localization of the Dot6-GFP fusion. In the absence of rapamycin, Dot6-GFP was localized 304 exclusively in the nucleus in agreement with its role as a transcriptional activator under nutrient 305 rich environment (Figure 4A-C). A weak GFP signal was also observed in the nucleolus and the 306 vacuole. When cells were treated with rapamycin, Dot6-GFP was rapidly relocalized to the 307 vacuole and only a small fraction remain in the nucleus (Figure 4D-F). The vacuolar localization 308 of the Dot6-GFP was confirmed by its co localization with the CellTracker Blue-stained 309 vacuoles ( Figure S2). These data suggest that TOR pathway regulates the transcriptional 310 function of Dot6. 311 312 To assess whether the control of Dot6 activity by TOR impacts the cell size of C. albicans, we 313 examined genetic interactions between TOR1 and DOT6 by size epistasis. As TOR1 is an 314 essential gene in C. albicans, we first tried to delete one allele in dot6 homozygous mutant. 315 However, all attempts to generate such mutant were unsuccessful suggesting a haplo-essentiality 316 of TOR1 in dot6 mutant background. Subsequently, we analysed genetic interaction of TOR1 and 317 DOT6 using complex haploinsufficiency (CHI) concept by deleting one allele of each gene and 318 measured size distribution of the obtained mutant. While both DOT6/dot6 and TOR1/Tor1 319 mutants had no disenable size defect, the TOR1/tor1 DOT6/dot6 strain exhibited cell size 320 distribution similar to that of dot6/dot6, suggesting that DOT6 is epistatic to TOR1 (Figure 4G).

321
Similarly, DOT6 was also epistatic to TOR1 with respect to their sensitivity toward rapamycin 322 ( Figure 4H). These data demonstrate that TOR pathway control cell size through Dot6. 323 324 Dot6 is required for carbon-source modulation of cell size 325 The effect of different carbon sources was assessed on the size distribution of the dot6 mutant 326 and the WT. While the cell size of WT and the revertant strains was reduced by 12 % (47.6  0.5 327 fL) when grown under the poor carbon source, glycerol, as compared to glucose (54.2  0.5 fL), 328 dot6 size remain unchanged regardless the carbon source ( Figure 5A-B). Similar finding was 329 obtained when comparing cells growing on the non-fermentable carbon source, ethanol (data not 330 shown). These results demonstrate that the transcription factor Dot6 is required for nutrient 331 modulation of cell size. Furthermore, strain lacking DOT6 was rate-limiting when grown in 332 medium with glycerol as a sole source of carbon as compared to glucose (Figure 5C).

334
To check whether Dot6 localization is modulated by carbon sources, the subcellular localization 335 of the Dot6-GFP fusion was tested in cells that grew in poor (glycerol) or in the absence of 336 carbon sources. Neither the absence or the quality of carbon sources altered the nuclear 337 localization of Dot6 (data not shown). This suggest that Dot6 govern the carbon-source 338 modulation of cell size through a mechanism that is independent from its cellular relocalization 339 340 The CTG-clade specific acidic domain of Dot6 is not required for size control 341 Our analysis unexpectedly reveals that Dot6 switched between activator and repressor 342 transcriptional regulator of Ribi between C. albicans and S. cerevisiae, respectively. Sequence 343 examination of C. albicans Dot6 protein revealed a C-terminal aspartate-rich domain that is 344 similar to acidic activation domains of transcriptional activators. This Dot6 D-rich domain was 345 found specifically in C. albicans and other related species of the CTG clade, and it was absent in 346 Dot6 orthologs in S. cerevisiae and other ascomycetes (Figure 6A). tissues and organs ( Figure S3). This reinforce the fact that control of cell size homeostasis is an 372 important attribute for this C. albicans to persist inside its host. 373 374 We found that Dot6 is a major regulator of cell size in C. albicans as compared to S. cerevisiae critical SBF complex, that control the the G1/S transition, and also with the TOR growth and 384 Ribi machineries, which might explain the influential role of Dot6 in size control. 385 386 Our findings support a model where Dot6 acts as a hub that might integrate directly growth cues 387 via the TOR pathway to control the commitment to mitotic division at G1. This regulatory 388 behavior is similar to the p38/HOG1 pathway that controls the Ribi regulon through the master 389 transcriptional regulator, Sfp1, and acts upstream the SBF transcription factor complex to control 390 division (SELLAM et al. 2016). Meanwhile, our genetic interaction analysis showed that the dot6 391 hog1 double mutant had an additive small size phenotype suggesting that both Dot6 and Hog1 392 act in parallel. This finding emphasizes that, in C. albicans, multiple signals are integrated at the 393 level of G1 machinery to optimize adaptation to different conditions. Contrary to the p38/HOG 394 pathway, Dot6 were required for both growth and size adjustment in response to glycerol 395 suggesting that this transcription factor provides a nexus for integrating carbon nutrient status to 396 the ribosome synthesis and Start machineries (Figure 7).  DOT6 is epistatic to TOR1 with respect to their sensitivity toward rapamycin. Strains were 573 grown on in SC medium at 30°C for 24 hours. Growth was calculated as percentage of OD 600 of 574 rapamycin-treated cells relatively to the non-treated controls. Results are the mean of three 575 replicates. Size distribution of log-phase cultures of the indicated WT (CAI4) and sch9 strains grown in 605 synthetic glucose (black curve) and glycerol (red) medium. 606 607 Figure S2. Localization of Dot6-GFP in the vacuole when TOR pathway is compromised. 608 Dot6-GFP fluorescence was visualized using confocal microscopy in cells treated with the TOR 609 pathway inhibitor, rapamycin. Vacuoles were stained using CellTracker Blue CMAC dye. Red 610 and blue arrows indicate Dot6-GFP florescence in the vacuole and the nucleus, respectively. 611 612 Figure S3. Dot6 is required for size homeostasis of hyphal cells. 613 Length of at least 20 hyphal cells of both WT (SFY87) and dot6 mutant grown on YPD 614 supplemented with 10% fetal bovine serum (FBS) at 37°C. Bars represent the means  standard 615 errors of the means. *, P < 0.0003 using a two-tailed t-test. 616 617 Table S1. Strains used in the current study and their genotypes. 618 619 Table S2. Primer sequences used in the current study. 620 Table S3. Gene Set Enrichment Analysis (GSEA) of dot6 mutant transcriptome 621 622 Table S4. Transcripts differentially expressed in dot6 mutant using a 1.5-fold change cut-off and 623 a 5% false discovery rate.