Unraveling the mechanism of thermotolerance by Set302 in Cryptococcus neoformans

ABSTRACT The underlying mechanism of thermotolerance, which is a key virulence factor essential for pathogenic fungi such as Cryptococcus neoformans, is largely unexplored. In this study, our findings suggest that Set302, a homolog of Set3 and a subunit of histone deacetylase complex Set3C, contributes to thermotolerance in C. neoformans. Specifically, the deletion of the predicted Set3C core subunit, Set302, resulted in further reduction in the growth of C. neoformans at 39°C, and survival of transient incubation at 50°C. Transcriptomics analysis revealed that the expression levels of numerous heat stress-responsive genes altered at both 30°C and 39°C due to the lack of Set302. Notably, at 39°C, the absence of Set302 led to the downregulation of gene expression related to the ubiquitin-proteasome system (UPS). Based on the GFP-α-synuclein overexpression model to characterize misfolded proteins, we observed a pronounced accumulation of misfolded GFP-α-synuclein at 39°C, consequently inhibiting C. neoformans thermotolerance. Furthermore, the loss of Set302 exacerbated the accumulation of misfolded GFP-α-synuclein during heat stress. Interestingly, the set302∆ strain exhibited a similar phenotype under proteasome stress as it did at 39°C. Moreover, the absence of Set302 led to reduced production of capsule and melanin. set302∆ strain also displayed significantly reduced pathogenicity and colonization ability compared to the wild-type strain in the murine infection model. Collectively, our findings suggest that Set302 modulates thermotolerance by affecting the degradation of misfolded proteins and multiple virulence factors to mediate the pathogenicity of C. neoformans. IMPORTANCE Cryptococcus neoformans is a pathogenic fungus that poses a potential and significant threat to public health. Thermotolerance plays a crucial role in the wide distribution in natural environments and host colonization of this fungus. Herein, Set302, a critical core subunit for the integrity of histone deacetylase complex Set3C and widely distributed in various fungi and mammals, governs thermotolerance and affects survival at extreme temperatures as well as the formation of capsule and melanin in C. neoformans. Additionally, Set302 participates in regulating the expression of multiple genes associated with the ubiquitin-proteasome system (UPS). By eliminating misfolded proteins under heat stress, Set302 significantly contributes to the thermotolerance of C. neoformans. Moreover, Set302 regulates the pathogenicity and colonization ability of C. neoformans in a murine model. Overall, this study provides new insight into the mechanism of thermotolerance in C. neoformans.

growing evidence suggests that increasing global temperature will inadvertently select for thermotolerant fungi that are more likely to cause opportunistic human disease.A recent evolutionary ecological study of over 1,200 fungal species has shown a strong relationship between opportunistic pathogenicity and thermotolerance (9), and more lineages of opportunistic human pathogens intend to emerge from more thermotolerant fungi (10,11).Notably, the global outbreak of the new heat-resistant pathogenic fungus Candida auris has attracted attention because of its high-temperature resistance, and C. auris is hypothesized to have arisen from high environmental temperatures (12,13).
Cryptococcus neoformans is an environmental basidiomycete yeast causing over 180,000 deaths annually worldwide (14,15).Its distribution in tropical areas and survival in pigeons are closely related to thermotolerance (16).Cryptococcus consists of 37 species, and the most common species associated with human disease are C. neofor mans, Cryptococcus deneoformans, and Cryptococcus gattii (17)(18)(19).Some species, such as Cryptococcus albidus, Cryptococcus laurentii, Cryptococcus liquefaciens, and Cryptococ cus amylolentus, are less virulent due to their temperature sensitivity (20,21).Interest ingly, a recent study showed that the incidence of cryptococcal meningitis is higher in hot environments, suggesting that heat stress may domesticate C. neoformans with a stronger infection ability (22).The importance of thermotolerance has been empha sized in various studies on the molecular mechanisms of C. neoformans, including membrane fluidity and cross-talk between unfolded protein response (UPR) and heat shock response (23)(24)(25)(26)(27).A recent study showed that the C. neoformans CEL1 gene, encoding a lytic polysaccharide monooxygenase, affects thermotolerance and pathoge nicity (28).Our recent research has revealed the role of cryptococcal Hsf3 in the defense of heat-induced reactive oxygen species (ROS) damage by modulating mitochondrial function (29).Thus, multiple pathways are utilized by C. neoformans to protect against heat stress, and the study of fungal heat adaptation mechanisms will help us to understand how heat-resistant fungi such as C. neoformans emerge and prevent more heat-resistant cryptococcal infections in the future.
Herein, we characterize the role of the predicted core subunit Set302 of histone deacetylase complex Set3C in the thermotolerance of C. neoformans.We discovered that Set302 deficiency led to reduced thermotolerance and viability at extreme temperatures, and the expression of ubiquitin-proteasome system (UPS) genes was positively regulated by Set302 and heat.Moreover, Set302 likely reduced the formation of misfolded proteins at high temperatures and performed the same function under proteasome stress.Furthermore, Set302 plays a role in regulating the pathogenicity to host.Overall, our findings emphasize the importance of Set302 in heat resistance and pathogenicity of C. neoformans.

Set3 is widely distributed and conserved in various fungi
Set3C is a histone deacetylase complex, and Set3 is the core subunit for the structural integrity of the complex (31).To probe the distribution of Set3 in fungi, we surveyed 29 Set3 homologous proteins from different fungal phyla.The phylogenetic tree analysis showed that Set3 existed in multiple fungal phyla and that Set3 proteins of the same phylum tended to cluster in an identical group (Fig. 1A).The characterization of Set3 proteins from different fungi showed that, apart from Rhizopus delemar in the Zygomy cota clade, Set3 homologous proteins in Basidiomycota, Pezizomycotina, and Saccharo mycotina contain both the PHD finger and SET domains.Notably, Set3 amino acid sequences of Basidiomycota are much longer than those of other fungal phyla (Fig. 1B).Additionally, we compared the SET domain sequences from representative fungi and mammals and found that all SET domains had a significant E-value of BLASTP hits (E < 1e−6) compared to S. cerevisiae Set3, and there were multiple partially and totally conserved sites (Fig. 1C).Taken together, these results indicate that Set3 is widely distributed in different fungi and its key functional SET domain is well-conserved.

The lack of Set302 results in decreased thermotolerance in C. neoformans
Although Set3 is critical in regulating the stress genes in S. cerevisiae (39), no evidence indicates that it contributes to thermotolerance in pathogenic fungi.To examine the role of the Set3 homologous protein Set302 of C. neoformans in heat resistance, we constructed the set302∆ and set302∆+TEF1p-SET302 strains.The results showed that set302∆ strain exhibited normal growth at 30°C (Fig. 2A; Fig. S1A) and 37°C (Fig. S1B) but displayed a growth defect on YPD agar plate at 39°C (Fig. 2A).The reduced thermotoler ance of set302∆ strain was also observed in YPD liquid medium (Fig. 2B).This growth defect was cured by re-integrating the SET302 gene controlled by the constitutive TEF1 promoter (Fig. 2A and B).Interestingly, this SET302 overexpression strain grew better than wild-type when incubated in liquid YPD medium at 39°C (Fig. 2B).Additionally, when subjected to 50°C for up to 12 minutes, set302∆ strain exhibited reduced viability compared to wild-type, and SET302 overexpression rescued the thermotolerance defect of set302∆ strain (Fig. 2C).These results suggest that Set302 plays a role in thermotoler ance by maintaining survival under heat stress.

Transcriptome profiles governed by Set302 and heat stress
UPR and heat shock proteins (HSPs) are key for thermotolerance in fungi and mammals (25,27,46).The assay of phenotypes regulated by the UPR pathway, including growing ability with DTT treatment as endoplasmic reticulum stress and the integrity of cell membrane as detected by SDS, revealed no differences in growing ability between wild-type and set302∆ strains (Fig. S2A).We also examined the phenotype regulated by the calcineurin pathway, which is important for the growth of C. neoformans under heat stress (25).However, we found no growth defect in the set302∆ strain under calcium chloride (Fig. S2A) or cyclosporin A treatments (Fig. S2B).Additionally, the lack of SET302 did not affect the HSP gene expression levels (Fig. S2C).Cryptococcal heat shock factor 3 (CnHsf3) was discovered as a regulator of thermotolerance by inhibiting mitochondrial ROS overload under heat stress (29).However, Set302 did not regulate CnHSF3 gene expression (Fig. S2D).These results suggest that Set302 affects the thermotolerance of C. neoformans independently of these known pathways.
To elucidate how Set302 contributes to thermotolerance, we performed an RNA-seq analysis of the wild-type and set302∆ strains at 30°C and 39°C (Fig. 3A).We found that the correlation of gene expression between normal and high temperatures in the wild-type strain was low, indicating a significant switch in the gene expression pattern during temperature shift.Interestingly, we observed that the correlation of gene expression between wild-type and set302∆ strains at high temperatures was lower than that under normal conditions, suggesting a more crucial role for Set302 under heat stress (Fig. 3B).Next, we performed a detailed analysis of differentially expressed genes to identify the genes regulated by temperature and Set302.The RNA-seq data revealed that the expression of many genes changed from 30°C to 39°C, including 1,265 up-regulated genes and 772 down-regulated genes.A total of 154 genes showed altered expression at 30°C in the absence of SET302, including 102 down-regulated genes and 52 up-regulated genes.Intriguingly, the expression of 2,073 genes changed at 39°C due to the lack of SET302, consisting of 1,245 down-regulated genes and 828 up-regulated genes (Fig. 3C; Table S1).As the set302∆ strain only showed defective growth at 39°C, we aimed to identify the genes responsive to heat stress and dependent on Set302 only at 39°C, but not at 30°C.The results showed that only 8 and 65 identical genes were up-and downregulated by the SET302 deficiency at 30°C and 39°C, respectively, indicating that Set302 has different functions at 30°C and 39°C.Furthermore, 627 genes responsive to heat stress were up-regulated and 1,059 genes responsive to heat stress were down-regulated in set302∆ strain at 39°C, but not at 30°C (Fig. 3D).We speculated that the altered expression of these genes (627 and 1,059) may contribute to the defective thermotoler ance of set302∆ strain.We found no effect of Set302 on the gene expression of the known thermotolerance pathway in C. neoformans after comparing thermotolerancerelated genes from CryptoNet with the differentially expressed genes between wild-type and set302∆ strains (Fig. S3).
To clarify how Set302 affects thermotolerance in C. neoformans, we examined the 627 up-regulated and 1,059 down-regulated genes in set302∆ strain at 39°C, but not at 30°C.A Gene Ontology (GO) analysis revealed that Set302 has a broad effect on various primary biological functions and cellular components, including translation, RNA binding, plasma membrane, and ribosome at 39°C (Fig. 4A; Table S2).However, no difference in membrane integrity was detected between wild-type and set302∆ strains (Fig. S2A).Notably, a subset of genes positively regulated by Set302 were enriched in the ubiquitin-dependent protein catabolic process, which is related to UPS (Fig. 4A; Table S2).UPS has been reported to play an important role in fungal survival under various conditions (47)(48)(49).Further investigation showed that the expression of UPS-related genes was positively regulated by Set302 at 39°C.Moreover, these genes were asso ciated with terms, such as ubiquitin-mediated proteolysis and ubiquitin-conjugating enzyme activity, highlighting their functional connectivity (Fig. 4B).Surprisingly, we observed a similar change in their expression levels under heat stress to that observed in set302∆ strain at 39°C. (Fig. 4B).This observation was further confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), which demonstrated that the expression of these genes increased in response to heat stress (Fig. 4C) and decreased in set302∆ strain at 39°C (Fig. 4D).These findings suggest a potential relationship between the function of UPS, Set302, and thermotolerance.

The role of Set302 in the degradation of misfolded proteins under heat stress
UPS has been implicated in regulating the degradation of misfolded proteins.For example, α-synuclein is a protein associated with Parkinson's disease that undergoes misfolding and is primarily eliminated by UPS (50).Additionally, the yeast model overexpressing α-synuclein has been used to investigate the accumulation of misfolded proteins and the resulting cellular toxicity (51).To investigate whether the degradation of misfolded proteins regulated by Set302-mediated UPS function contributes to thermo tolerance in C. neoformans, we created a GFP-labeled α-synuclein-overexpressed model in wild-type and set302∆ strains.In this C. neoformans model, the fluorescence foci of the aggregates were observed if GFP-labeled α-synuclein was misfolded.Almost no fluorescent aggregates were caused by misfolded α-synuclein at 30°C in both strains, suggesting that misfolded α-synuclein rarely forms under normal conditions.However, at 39°C, a small fraction of wild-type strain cells contained fluorescent aggregates, suggest ing that this elevated temperature leads to misfolding of α-synuclein (Fig. 5A).Wild-type strain exhibited approximately 10% of cells with foci, and of those with foci, the average number of foci per cell was about 4. In contrast, the percentage of cells with foci for set302∆ strain was about 70%, and the average number of foci per cell in those cells with foci was about 6, which were both higher than those of wild-type, suggesting that the lack of SET302 contributed to the increased aggregate formation (Fig. 5B).To assess the effect of misfolded protein formation on the ability of the fungus to grow, we examined the phenotypes of wild-type and set302∆ strain overexpressing α-synuclein at 30°C and 39°C.As a result, no difference in growing ability was observed between wild-type and set302∆ strain at 30°C, which was consistent with the observation of fluorescent aggre gates.However, the misfolded protein aggregation strikingly reduced the growing ability of C. neoformans at 39°C (Fig. 5C).Notably, inhibited growth was more pronounced in the set302∆ strain than in the wild-type strain under heat stress (Fig. 5C and D).Collectively, these results indicate that the absence of SET302 results in a higher accumulation of misfolded proteins, such as α-synuclein, under heat stress.

The deletion of Set302 leads to decreased growth of C. neoformans under proteasome stress
Heat stress influences UPS function by affecting its activity and efficiency (52,53).Our data suggest that Set302 is important to the UPS pathway by affecting the degradation of misfolded proteins under heat stress in C. neoformans; thus, we speculated that it also functions under other UPS stress.To explore this possibility, we assessed growth in the presence of the proteasome inhibitor MG132, which is commonly used to disturb ubiquitin-mediated protein degradation (54).The results showed that the set302∆ strain had slightly lower growth than the wild-type strain when treated with MG132 (Fig. 6A).Additionally, MG132 effectively inhibited the growth of the wild-type strain under heat stress (Fig. 6B), indicating that UPS plays a role in the thermotolerance of C. neoformans.
The fluorescence observations of GFP-labeled α-synuclein revealed that MG132 exacer bated the formation of aggregates similar to heat stress, indicating that the elimination of misfolded proteins, such as α-synuclein, is also dependent on UPS function in C. neoformans (Fig. 6C).The absence of SET302 caused a higher percentage of cells with fluorescent aggregates, exhibiting a similar effect as heat stress (Fig. 6D).MG132 also inhibited the growth of wild-type strain overexpressing α-synuclein significantly more than set302∆ strain overexpressing α-synuclein at 39°C (Fig. 6E).We hypothesized that this was attributed to the finding that the functional integrity of UPS in wild-type strain is stronger than that of set302∆ strain, which was damaged under heat stress, and there fore, set302∆ strain was less inhibited than wild-type when treated with MG132.Wildtype strain overexpressing GFP-labeled α-synuclein also displayed stronger growth than the set302∆ strain without MG132 treatment at 39°C (Fig. 6E).This observation agrees the wild-type strain at 39°C, and the right chart represents the increased fold change of gene expression in the wild-type strain in response to heat stress.
(C) Quantitative RT-PCR was performed using total RNA from wild-type strains at 30°C or 39°C, respectively, and the gene expression of representative UPS genes was measured.(D) Quantitative RT-PCR was performed using total RNA from wild-type and set302∆ strains at 39°C and the gene expression of representative UPS genes was measured.
with the findings from the spot-assay of these two strains at 39°C.Taken together, these results suggest that Set302 contributes to the degradation of misfolded proteins when cells are exposed to various UPS stress conditions.

The lack of Set302 reduces virulence and tissue colonization of C. neoformans
In addition to thermotolerance, capsule, and melanin are also crucial to the pathogenic ity of C. neoformans (55).The results of the microscopic images and quantitative analysis of capsule size showed that the set302∆ strain displayed a smaller surface capsule than the wild-type, suggesting that Set302 contributes to capsule size (Fig. 7A and B).Defective melanin production was also observed in the set302∆ strain compared to the wild-type and the SET302 complement rescued this melanin defect in the set302∆ strain (Fig. 7C).By analyzing the differentially expressed genes in RNA-seq and comparing them with the capsule-and melanin-related genes gained from Cryptonet database, we found that the lack of SET302 changed the expression of six capsule formation-related genes and six melanin production-related genes only at high temperature, but not at normal condition.Among these genes, five capsule formation-related genes and five melanin production-related genes are responsive to heat stress (Fig. S4).This result suggests that Set302 may affect these virulence factors by influencing the expression level of related genes under heat stress such as in the host environment, thereby contributing to the pathogenicity of C. neoformans.Due to the defective phenotype of these critical virulence factors of the set302∆ strain, we aimed to investigate the effect of Set302 on C. neoformans pathogenicity.Using a murine inhalation model of cryptococcosis, we found that female C57BL/6 mice with an intranasal infection by set302∆ strain showed significantly prolonged survival than those by wild-type, and rescuing the SET302 gene overcame the reduced virulence caused by the lack of SET302 (Fig. 7D).Furthermore, set302∆ strain exhibited markedly reduced colony forming units in the lung and brain of infected mice compared to wild-type strain, and the rescue of SET302 increased the colonization capacity of set302∆ strain (Fig. 7E).The histopathological assessment with Periodic Acid-Schiff staining showed fewer fungal cells in the lung of mice with the infection by set302∆ strain (Fig. 7F).Overall, these results reveal that Set302 contributes to the colonization in the infected tissues and the pathogenicity of C. neoformans to the host.

DISCUSSION
In this study, we aimed to characterize the function of the histone deacetylase complex Set3C in response to heat stress in C. neoformans.In S. cerevisiae, the core subunit Set3 is vital to the assembly and integrity of the Set3C complex (31).The Set3 protein contains a SET domain, which potentially mediates protein-protein interaction, and a PHD domain, which binds the methylated tails of histone 3 (32,56).Analysis of the Set3 protein homolog Set302 in C. neoformans revealed the presence of both the PHD and SET domains.Interestingly, the PHD and SET domains are commonly present, except in R. delemar, where the PHD domain is absent, suggesting that R. delemar Set3 may have a distinct role from other fungi owing to the lack of a PHD domain.One study showed that the phylogenies based on SET domains in Ascomycetes do not appear to consistently support any specific evolutionary relationship among fungi, animals, and plants.An evolutionary tree analysis of the Set3 proteins from Saccharomycotina fungi indicated a high degree of homology between Set3 and Set4, which could be due to the whole-genome duplication event in the history of Saccharomyces (57).Our study analyzed the phylogenetic tree of the Set3 proteins from different fungal phyla, including Saccharomycotina, and showed that the Set3 proteins tended to cluster based on evolutionary branches, suggesting a higher degree of evolutionary conservation among the Set3 proteins.It is noteworthy that the Set3 proteins from Basidiomycota, including C. neoformans, Ustilago maydis, and Coprinopsis cinerea, have longer protein sequences than those in other fungi, suggesting subtle differences in the Set3 proteins between Basidiomycota and other phyla.
Previous findings demonstrated that in S. cerevisiae, the deletion of Set3 does not affect growth or stress response (31).However, this leads to faster meiosis I, meiosis II, and ascus formation progression.In Magnaporthe oryzae, the absence of SET3 strongly reduces virulence and impairs conidiation (41).In Fusarium graminearum, Set3C affects virulence through the interaction with the cAMP-PKA pathway (58).In the human pathogenic fungus C. albicans, the Set3C complex plays a crucial role in regulating switching modulation and yeast-filament transition caused by a hyperactive cAMP/PKA pathway, thereby affecting the virulence of C. albicans (43,44).However, based on the transcriptomics data, we found that the deletion of SET302 in C. neoformans had little effect on the cAMP/PKA pathway.Some minor changes in gene expression, such as GPR4 and Gpa1 occurred, but most cAMP/PKA pathway-related genes, particularly the core regulator of cAMP production, Cac1, and the downstream execution factor, Pka, remained unchanged (Table S1).
In this study, we found that the set302∆ strain was equally viable with the wild-type, exhibiting a similar phenotype to those in S. cerevisiae, M. oryzae, and C. albicans.However, the set302∆ strain displayed defective thermotolerance in solid and liquid media conditions and decreased viability under the extreme temperature pretreatment, indicating that Set3C-mediated thermotolerance is controlled by the ability to survive, not the growth rate.We also determined that the deletion of SET302 reduced but did not abolish the thermotolerance of C. neoformans, suggesting that Set3C complex is not indispensable for thermotolerance, but plays a partial role and is redundant with other factors.Intensive efforts have been made to elucidate the key regulators govern ing thermotolerance in C. neoformans (24,25,27,29).However, Set302 plays a role in thermotolerance independent of these pathways mentioned above.
UPS is critical for selectively digesting misfolded or useless proteins marked by ubiquitin in all eukaryotes to maintain a properly functioning proteome.UPS involves the attachment of ubiquitin to target proteins, which are then recognized and degraded by the 26S proteasome (59)(60)(61)(62).In pathogenic fungi, UPS regulates the expression and activity of many pathogenic factors that are crucial for virulence, stress response, and fungal morphology (48,62,63).In this study, we found that multiple UPS-related pathways responded to heat stress and were regulated by Set302 at high temperatures.Heat stress can lead to abnormal protein folding, resulting in an imbalance of protein homeostasis (46) and UPS-mediated degradation is the predominant mechanism for the clearance of misfolded proteins, such as α-synuclein (50,64,65).In this study, we found that the lack of SET302 led to increased formation of α-synuclein aggregates under heat stress.Research in the model organism S. cerevisiae has demonstrated that the formation of misfolded proteins impairs growing ability (66).Similarly, protein aggregation caused by misfolded proteins significantly inhibits the thermotolerance of C. neoformans, and the set302∆ strain displayed a greater sensitivity to protein aggregation-induced damage than the wild-type strain, suggesting a role of Set302 in protecting C. neoformans from damage caused by misfolded proteins under heat stress.We speculated that Set302 is a potential gene regulator that maintains UPS function under heat stress by affecting the expression of UPS-related genes, including E3 ubiquitin-protein ligases and proteasome subunits.
Heat stress induces the ubiquitination of many cellular proteins followed by proteasomal degradation (67).Previous studies have reported that the α2 subunit of the 26S proteasome and various E3 ubiquitin ligases are associated with enhanced thermotolerance (68)(69)(70).Additionally, heat stress can function as a UPS stressor by inactivating the proteasome and inhibiting the assembly of the proteasome complex (71).We demonstrated here that the inhibition of UPS with MG132 decreased C. neoformans thermotolerance.Notably, the set302∆ strain exhibited increased protein aggregation formed by α-synuclein compared to the wild-type when treated with MG132, and this protein aggregation exerted a stronger inhibitory effect on thermo tolerance of the set302∆ strain, similar to the phenotype observed under heat stress conditions, suggesting that Set302 is a universal regulator for UPS function in the elimination of misfolded proteins under different UPS stressors.However, explaining how Set302 affects UPS function will require further studies.
Many studies have shown a close relationship between the major virulence factors, including thermotolerance, capsule, and melanin, and the pathogenicity of C. neofor mans (17,18,20,21,29,55).In this study, the set302∆ strain also exhibited a defective capsule and melanin formation.Oliver W. Liu et al. showed that Set302 has a slight inhibitory effect on capsule formation (72), whereas Yunfang Meng et al. reported that set302∆ strain has no apparent capsular defect (45).We speculated that this difference is due to differences in the capsule-inducing conditions.The capsule-inducing condi tions used in our study that mimic the host environment led to differences in capsule formation.Furthermore, in a murine inhalation model, the set302∆ strain with deficiencies in these virulence factors displayed significantly reduced virulence and a lower fungal burden in lung and brain tissues compared to the wild-type strain.These findings are consistent with the observed effects on the pathogenicity of M. oryzae and C. albicans, further illustrating the significance of Set3C complex in the pathogenicity of pathogenic fungi.Although the deletion of SET302 led to defective thermotolerance, Set302 was not particularly critical for thermotolerance, suggesting that Set302 may play a partial role in thermotolerance and that the reduction in pathogenicity may result from a combination of these major virulence factors, including thermotolerance, capsule, and melanin.

Fungal strains and culture conditions
C. neoformans var.grubii wild-type strain H99 was used for analysis in this study.Strains were cultured in YPD (1% yeast extract, 2% peptone, 2% dextrose) medium.For the selection of transformants, G418 and hygromycin B were added to the YPD agar medium with a final concentration of 200 µg/mL and 200 units/mL, respectively.The strains were grown at 30°C for normal culture unless it was stated at a specific temperature.The stress medium was supplemented with 0.4 M calcium chloride, 0.02% SDS, 20 mM DTT, or 100 µg/mL cyclosporin A. 100 μM MG132 was added to the YPD medium for proteasome activity detection.L-DOPA agar and Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% of fetal bovine serum (FBS) were used for melanin and capsule production.For the growth curve experiment, the strains were cultured in a YPD medium with the initial OD 600 value of 0.05 and incubated at indicated conditions.The OD 600 value was measured using a MultiskanGO microplate reader (Thermo).

Construction of fungal strains
The strains are listed in Table S3.The manipulation of C. neoformans and protocols were described previously (73).SET302 gene (CNAG_06591) was knocked out by the homol ogous replacement of its open reading frame with a drug-resistance cassette.Primer pairs CDp2419/CDp2420 and CDp2421/CDp2422 amplified SET302's flanking regions, while M13F/M13R primers amplified the NEO marker from the pHA-NEO vector.Then set302∆::NEO construct was obtained by CDp2419/CDp2422 primer pair and transformed into H99 strain.CDp2477/CDp2478 primer pair was used for the diagnostic PCR.
For SET302 complementation, SET302 upstream and TEF1p sequences were amplified using GXD85/86 and GXD87/88 primer pairs and cloned into the pHA-HYG vector.The open reading frame of SET302 and SET302 downstream amplified using GXD91/92 primer pair and GXD89/90 primer pair were cloned into the above-constructed vector.The cassette was amplified using the GXD85/90 primer pair and transformed into the set302∆ strain.Diagnostic PCR was performed using the GXD87/CDp2478 primer pair.
For the GFP overexpressing strain, the GFP coding sequence was amplified using the CDp2665/CDp2671 primer pair and cloned at the PacI site of the pHYG plasmid.For GFP-α-synuclein overexpressing strain, GFP and -α-synuclein coding sequences were amplified using CDp2665/CDp2666 and CDp2674/CDp2673 primer pair and then cloned at the PacI site of the pHYG plasmid.The reconstructed plasmids were transformed into H99 or set302∆ strains.All primers were listed in Table S4.

In vitro assay for thermotolerance at extreme temperature
The strains were cultured overnight at 30°C.Then the strains were inoculated to OD 600 = 1 in the preheated tubes at a 50°C hot block.Aliquots were removed at the indicated time and placed immediately on ice.A hundred dilutions were plotted onto YPD plates and incubated at 30°C for 2 days to determine the number of colony-forming units.

The fluorescence signal detection
GFP or GFP-α-synuclein overexpressing strains were cultured in a YPD medium at indicated conditions.Fungal cultures were washed twice, resuspended by sterile PBS, and imaged using a Nikon DS-Ri1 microscopy.The percentage of fungal cells with fluorescence foci was quantified from 30 cells with GFP fluorescence signal, and the number of fluorescence foci per cell was counted from 15 cells with foci, both from three independent experiments.

Total RNA preparation and quantitative RT-PCR
The strains were grown to the mid-log phase in YPD medium at 30°C or 39°C.Cells were centrifuged at 1,000 × g for 5 minutes at 4°C and washed twice with ice-cold ddH2O.Total RNA was extracted using the Total RNA Kit I (Omega).cDNA was synthesized using the Reverse Transcript All-in-one Mix (Mona), followed by genomic DNA removal using TURBO DNA-free (Invitrogen).The primers are listed in Table S4.The CFX96 real-time system (Bio-Rad) was used for data acquisition.ACT1 was used as a normalization control.The relative gene expression levels were calculated using the ΔΔCt method.

Phylogenetic and protein domain analysis
The domain analysis was performed using Interpro (http://www.ebi.ac.uk/interpro/).The phylogenetic tree was constructed by MEGA5 using the neighbor-joining method and displayed using Figtree software.Multiple sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).The conservation sites were calculated using Jalview software.The Blastp hits were calculated by NCBI using the protein sequence of the SET domain of S.cerevisiae Set3 (YKR029C) as the template.

RNA-seq and data analysis
Three independent samples of wild-type and set302∆ strains were inoculated in 10 mL YPD broth at 30°C overnight.The fungal strains were subcultured in 100 mL YPD medium with an OD 600 of 0.2 until the OD 600 reached 1 at 30°C or 39°C, respectively.The fungal strains were washed with PBS three times and frozen in liquid nitrogen.Total RNA was extracted using TRIzol regent (Invitrogen Life Technologies).The process protocol for RNA-seq and data analysis was described previously (29).

Animal experiments
Animal survival assay, fungal burden analysis, and histopathology analysis were conducted as described previously (29).

Bioinformatic methods
GO analysis was conducted using KOBAS 3.0.Heatmap was generated using the pheatmap package and the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values was transformed using the Log 2 (FPKM + 1) formula.The correlation analysis was calculated using the corrplot package.Packages were operated in R.3.4.3.The Upset plot was conducted using Evenn.

Statistical analysis
Statistical analysis was conducted with GraphPad Prism 8.0.1.Two-group comparisons and mouse survival data were analyzed by two-tailed Student's t-test and Log-rank test, respectively.Growth curve differences were assessed using two-way ANOVA.A P-value <0.05 indicated significance (*P < 0.05; **P < 0.01; ***P < 0.001).In transcriptome analysis, genes with fold changes >1.5 or <0.67 and P < 0.05 were considered differentially expressed.

FIG 1
FIG 1 Set3 is broadly distributed and conserved across diverse fungi phyla.(A) Phylogenetic tree of Set3 amino acid sequences of representative fungi in distinct phyla.The tree was constructed using a neighbor-joining algorithm by MEGA5 and displayed using Figtree software.(B) The characterization of PHD and SET domain of Set3 protein from representative fungi.The domains were predicted using InterPro (http://www.ebi.ac.uk/interpro/).The blue and green (Continued on next page)

FIG 1 (
FIG1 (Continued)    boxes indicate the PHD and SET domains, respectively.The numbers represent the position of amino acids.(C) Multiple sequence alignment of SET domains in Set3 proteins.The SET domain sequences of Set3 proteins from the representative fungi from different phyla and mammals were used for alignment by Clustal Omega.The E-value was calculated by NCBI Blastp using the SET domain sequence of S.cerevisiae Set3 (YKR029C) as the template.Jalview software was employed for the calculation of conservation sites.

FIG 2
FIG 2 Set302 deficiency results in reduced thermotolerance of C. neoformans.(A) Spot dilution assays with wild-type (H99), set302∆, and set302∆+TEF1p-SET302 strains were performed on YPD agar and incubated at the indicated temperature for 2 days.(B) Growth curve of fungal cells in liquid cultures.Wild-type, set302∆, and set302∆+TEF1p-SET302 strains were grown in YPD liquid media at 39°C.Cell growths (absorbance at 600 nm) were measured at indicated time points.(C) The viability assay of wild-type, set302∆, and set302∆+TEF1p-SET302 strains.The strains were plated onto YPD agar after the pretreatment at an extreme temperature of 50°C for the indicated time and incubated for 2 days at 30°C.CFUs were counted and normalized by the CFU of wild-type without heat treatment.

FIG 3 FIG 4
FIG 3 Transcriptome profiles governed by Set302 and heat stress.(A) Transcriptomics schematics for wild-type and set302∆ strains.Wild-type and set302∆ strains were grown in YPD medium overnight at 30°C and subcultured in fresh YPD medium with an OD 600 of 0.2 until the OD 600 reached 1 at 30°C or 39°C, respectively, for RNA-seq.(B) The correlation analysis of gene expression patterns between wild-type and set302∆ strains from RNA-seq.The corrplot package was used to conduct the correlation analysis.(C) Volcano plot of differentially expressed genes between different strains at 30°C or 39°C.(D) Venn diagrams depict the comparison between the number of differentially expressed genes among the different groups in Fig. 3C.

FIG 5
FIG 5 The lack of Set302 results in the declined degradation process of misfolded protein under heat stress.(A) The representative fluorescence photographs of GFP or GFP-labeled α-synuclein overexpressed in wild-type and set302∆ strains at 30°C and 39°C.Fungal cells were harvested at 30°C or 39°C and observed by fluorescence microscopy.Scale bar = 10 microns.(B) The statistics of fluorescence foci number per cell and the percentage of cells with fluorescence foci from wild-type and set302∆ strains overexpressing GFP or GFP-labeled α-synuclein at 39°C, respectively.(C) Spot dilution assays with wild-type and set302∆ strains overexpressing the control vector, GFP or GFP-labeled α-synuclein were performed onto YPD agar and incubated at 30°C or 39°C for 2 days, respectively.(D) The relative quantitative analysis of wild-type and set302∆ strains with overexpressed GFP or GFP-labeled α-synuclein colony spot at 39°C.The signal intensity of colony spot was calculated using ImageJ and the intensity ratio of wild-type or set302∆ strains with GFP-labeled α-synuclein relative to those with GFP was calculated.

FIG 6
FIG 6 The deletion of Set302 leads to decreased growth for C. neoformans under proteasome stress.(A) Growth curve of fungal cells in liquid cultures.Wild-type and set302∆ strains were grown in YPD liquid media with or without 100 µM MG132 at 30°C.Cell growths (absorbance at 600 nm) were quantified at indicated time points.(B) Growth curve of fungal cells in liquid cultures.Wild-type and set302∆ strains were grown in YPD liquid media with or without 100 µM MG132 at 39°C.Cell growths (absorbance at 600 nm) were measured at indicated time points.(C) The representative fluorescence photographs of GFP or GFP-labeled α-synuclein overexpressed in wild-type and set302∆ strains with or without 100 µM MG132.Fungal cells were harvested and observed by fluorescence microscopy.Scale bar = 5 microns.(D) The statistics of the percentage of cells with fluorescence foci from wild-type and set302∆ strains overexpressing GFP or GFP-labeled α-synuclein with 100 µM MG132 treatment, respectively.(E) Quantification of fungal growth in liquid cultures.Wild-type and set302∆ strains overexpressing GFP-labeled α-synuclein were grown in YPD liquid media with or without 100 µM MG132 at 39°C.Cell growths (absorbance at 600 nm) were measured at indicated time points.

FIG 7
FIG 7 The absence of Set302 reduced the formation of virulence factors and the pathogenicity of C. neoformans.(A) Representative microscopy images of capsule assay.Strains were grown in Dulbecco's Modified Eagle Medium (DMEM)+10% fetal bovine serum (FBS) medium at 37°C for 2 days followed by India ink staining and photographing.Scale bars = 5 microns.(B) The capsule sizes of wild-type and set302∆ strains (n = 20) were measured and compared.(C) The melanin production assay of wild-type, set302∆, and set302∆+TEF1p-SET302 strains.Strains were spotted on the YPD agar plate with L-DOPA and incubated at 37°C for 2 days.(D) Animal survival analysis and the Kaplan-Meier survival plot of wild-type, set302∆, and set302∆+TEF1p-SET302 strains.Cells were grown overnight in YPD liquid medium, harvested, washed with PBS, and inoculated into mice via intranasal instillation.Animals were sacrificed at defined endpoints of mortality and survival was plotted.Significance was determined using the Log-rank (Mantel-Cox) test.(E) The colony forms units of surviving fungal cells per gram from infected organs.The infected lung and brain with wild-type, set302∆, and set302∆+TEF1p-SET302 strains were extracted, homogenized, and plated onto YPD agar.The colony-forming units were counted and normalized to organ weight.(F) Histopathological staining of lung tissues infected by wild-type, set302∆, and set302∆+TEF1p-SET302 strains with Periodic Acid-Schiff.The lung tissues were captured under a ×10 lens (scale bars = 100 µm).Fungal cells were indicated with arrows.