Identification of histone mutants with altered cellular response to rapamycin—A collection of 442 yeast strains expressing mutated versions of the core histones were created by replacing each amino acid residue of H2A, H2B, H3, and H4 with alanine. The individual variant carried by a single copy plasmid was introduced into a modified parental strain deleted for both copies of the respective endogenous histone genes . We screened the entire histone mutant collection to identify those that were either sensitive or resistant to rapamycin using spot test analysis. This screen identified a single mutant H2A E65A showing sensitivity to rapamycin, as compared to the WT (Fig. 1A). However, this H2A E65A mutant also showed sensitivity to several other agents such as the DNA damaging agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO)(data not shown), suggesting that H2A E65A might affect multiple cellular functions. In addition, the screen identified eight mutants displaying variable degrees of resistance to rapamycin when compared to the strains expressing WT histones (Fig. 1A). The eight rapamycin-resistant mutants did not show cross-resistance towards other drugs including the DNA damaging agents MMS and 4-NQO, as well as the environmental toxic metalloid arsenite (data not shown), suggesting that these histone residues may be specifically responding to rapamycin.
The histone mutants that were resistant to rapamycin and showed WT responses to other agents were found within histones H2B, H3, and H4, and none was recovered from the set of H2A alleles (Fig. 1). Only one mutant, H2B R95A, was recovered from the histone H2B collection that exhibited prominent resistance to rapamycin amongst all the histone mutants when tested semi-quantitatively by the spot test analysis (Fig. 1A). This H2B R95A mutant was independently checked for growth rate in liquid culture by assessing for cell density at OD 600 throughout 12 hrs in the absence and presence of rapamycin. The H2B WT and H2B R95A mutant cells proliferated at the same rate in the absence of rapamycin (Fig. 1B). However, in the presence of rapamycin growth of the H2B WT was significantly impaired by 2.5 hrs, while the H2B R95A mutant continued to grow (Fig. 1B). This latter analysis is consistent with the H2B R95A mutant being defective in a process that attenuates cell growth upon exposure to rapamycin.
In the case of histone H3, only three rapamycin-resistant mutants H3 G12A, H3 V46A, and H3 E94A were recovered, although these were not as resistant to rapamycin as H2B R95A (Fig. 1A). The H4 mutants (R19A, I21A, L22A, and R36A) were located primarily in the N-terminal part of the protein. The H4 L22A mutant was more resistant to rapamycin as compared to H4 R19A, H4 I21A, and H4 R36A and appeared to be as resistant as the H2B R95A mutant (Fig. 1A). It is noteworthy that the N-terminal tail of H4 makes contact with the H2A-H2B dimer, and plays a major role in many processes including replication-coupled chromatin assembly and gene expression . Collectively, it appears that a narrow set of histone residues can specifically cause resistance to rapamycin when replaced with alanine. Since the histone H2B R95A mutant was strikingly resistant to rapamycin and did not display sensitivity or resistance to other agents , we focused on unraveling its role in response to rapamycin.
Microarray analysis reveals that H2B R95A downregulates several genes in the pheromone pathway—To obtain molecular insights into how H2B R95A brings about rapamycin resistance, we compared the gene expression pattern of the H2B R95A mutant with the H2B WT under normal growth conditions and when the cells were treated with rapamycin (200 ng/ml for 30 mins). A volcano plot of genes differentially expressed under normal conditions revealed that 25 genes, mainly belonging to ribosome biogenesis and RNA processing, were upregulated (P-value of ≤ 0.01) in the H2B R95A mutant as compared to the WT (Fig. 2A, shown in red). More than 50 genes were downregulated in the mutant as compared to the WT (Fig. 2A, shown in green), and of these, 26 belong to the pheromone response pathway . These results suggest that the Arg95 residue of H2B plays a predominant role to ensure proper regulation of the pheromone pathway genes, consistent with the observation of Dai et al that this mutant has a defect in mating .
Rapamycin did not affect the expression profile of these pheromone pathway genes in the H2B WT or H2B R95A mutant using two-dimensional hierarchical clustering (Supplementary Fig. S1). This analysis grouped the non-treated and rapamycin-treated WT and the H2B R95A mutant in distinct clusters, implying that rapamycin did not affect the expression profile of these pheromone pathway genes (Fig. 2B).
We further analyzed the data by comparing genes that were differentially expressed between the WT and the H2B R95A mutant following rapamycin treatment using Venn diagrams (Fig. 2C). The analysis showed that rapamycin treatment caused up-regulation of 70 genes in the H2B R95A mutant and 21 in the H2B WT, with 14 being common to both strains (Fig. 2C). In contrast, a considerable number of genes (354) were significantly down-regulated in the H2B R95A mutant and 86 in the H2B WT with 81 being common to both strains (Fig. 2C). Almost all the differentially expressed genes in the H2B R95A mutant were enriched in the same GO term as the H2B WT and related, e.g., to ribosome biogenesis, RNA processing, RNA maturation, and export from the nucleus (Supplementary Table S2). This analysis suggests that the H2B R95A mutant is still capable of altering the expression of a subset of genes similar to the H2B WT in response to rapamycin.
H2B R95A mutant drastically reduces the expression of the STE5 gene and its encoded protein—In the pheromone response pathway, the scaffold protein Ste5 and its associated kinases Ste11, Ste7, Fus3, and Kss1 are recruited to the α-factor receptor Ste2 located at the plasma membrane when cells are exposed to α-factor. This brings the MAPKKK Ste11 in proximity with another membrane-bound complex containing the Ste20 kinase [30, 32, 33]. Ste20 phosphorylates Ste11 which then activates the MAPKK Ste7, which in turn activates the MAPKs Kss1 and Fus3 [30, 32, 33]. The activated Fus3 kinase translocates to the nucleus leading to the downregulation of the G1 cyclins including Cln1 and Cln2 causing cell cycle arrest [34, 35]. Since Ste5 is a crucial component that allows the assembly and communication between the kinases, and mutants deleted for the STE5 gene cannot transmit a signal along the pheromone pathway to activate the MAPKs , we checked whether STE5 gene expression is indeed downregulated by H2B R95A mutant as revealed by the microarray data (Fig. 2). Total RNA was isolated from H2B WT and the H2B R95A mutant and the cDNAs derived from reverse transcriptase were used to quantify STE5 gene expression by quantitative PCR and normalized against the ACT1 gene. The analysis revealed that the STE5 gene was dramatically downregulated in the H2B R95A mutant as compared to the H2B WT, consistent with the microarray data (Fig. 3A and Fig. 2A, respectively).
We next checked whether the Ste5 protein would also be similarly downregulated in the H2B R95A mutant. To do this, we introduced a single-copy plasmid pSTE5-GFP expressing Ste5 from its promoter, as a GPF fusion protein, into the H2B WT and H2B R95A strains and monitored Ste5-GFP expression level by immunoblot analysis with anti-GFP antibodies, as no commercial Ste5 antibody is available . The Ste5-GFP was highly expressed in the H2B WT strain, but only weakly expressed in the H2B R95A mutant (Fig. 3B, lane 2 vs. 5). Under the extraction conditions, some of the native Ste5-GFP protein was apparently proteolytically processed to a lower molecular weight isoform of ~ 80 kDa (Fig. 3B, shown by an asterisk). A control protein GFP-Apn1 driven from the galactose-inducible promoter GAL1 under the non-induced condition was expressed at the same level in both the H2B WT and H2B R95A strains (Fig. 3B lane 3 and 6, respectively). These findings support the notion that the pheromone response pathway is disrupted in the H2B R95A mutant.
ste5 null mutant displays resistance to rapamycin—To determine whether Ste5 is involved in controlling the cellular response to rapamycin, we used a different and most common parental background strain BY4741 and its isogenic ste5Δ mutant and test for rapamycin resistance using spot test analysis. As shown in Fig. 4, the ste5Δ mutant displayed resistance to rapamycin as compared to the parent BY4741, suggesting that Ste5 is required to channel the rapamycin signal to trigger growth arrest. Similar results were obtained when the STE5 gene was deleted from the W303 parental background (Supplementary Table S1). In contrast, the ste2Δ mutant lacking the transmembrane α-factor receptor protein Ste2 did not show resistance to rapamycin when compared to the WT (Fig. 4). Based on this observation, it would appear that Ste2 is not required for the recruitment of Ste5 and its associated proteins to the plasma membrane to signal the response by rapamycin.
Rapamycin induces G1 arrest in the H2B WT, but not in the H2B R95A mutant—When WT cells are challenged with α-factor, the signal is transmitted along the pheromone pathway leading to cell cycle arrest in the G1 phase. If rapamycin uses this same pathway to signal G1 arrest , then it is anticipated that this arrest would be disrupted in the H2B R95A mutant. Exponentially growing asynchronous H2B WT cells were treated with rapamycin (200 ng/ml for 60 mins), then washed to remove the drug, followed by post-treatment recovery in fresh media, and the samples taken at the indicated time were processed by Fluorescence-Activated Cell Sorting (FACS) analysis (Fig. 5). The H2B WT cells were rapidly arrested in the G1 phase following the rapamycin treatment (Fig. 5A, second panel) and remained in the G1 phase during the duration of the sampling (90 mins) in the fresh media without rapamycin (Fig. 5A, third, fourth, and fifth panels). In contrast, the asynchronous population of the H2B R95A mutant cells failed to arrest in the G1 phase following rapamycin treatment (Fig. 5B vs. 5A). This observation is consistent with the H2B R95A mutant ability to grow in the presence of rapamycin (Fig. 1). We interpret these findings to indicate that rapamycin may transmit a signal through the pheromone response pathway to arrest the cell cycle in the G1 phase.
In the control experiment, exponentially growing cultures were treated with α-factor for 3 hrs, washed free of the α-factor, and the cell cycle release was monitored over time post-treatment in fresh media using FACS. The asynchronous cell populations for both the H2B WT and the H2B R95A mutant showed a similar proportion of cells in the G1 and G2 phases (Supplementary Fig. S3A). As expected, following α-factor treatment, the H2B WT cells were arrested in the G1 phase and re-entered the cell cycle upon removal of the α-factor (Supplementary Fig. S3A). In contrast, the H2B R95A mutant failed to arrest in the G1 phase, even after prolonged exposure to the α-factor, and continued to progress normally in the cell cycle (Supplementary Fig. S3B). This finding confirms that the H2B R95A mutant is indeed defective in the pheromone response pathway.
Fus3, a downstream component of the pheromone pathway, is expressed in the H2B WT, but not detected in the H2B R95A mutant—The MAP kinases Kss1 (43 kDa) and Fus3 (41 kDa) are downstream components of the pheromone response pathway that can be phosphorylated. Under conditions of starvation, Kss1 (43 kDa) can act on the Ste12 transcriptional activator to turn on filamentation genes. Likewise, Fus3 when activated, for example, by α-factor can also regulate the function of the transcription factor Ste12 that controls expression of the mating genes leading to G1 arrest. The phosphorylated form of Kss1 and Fus3 is readily detected by immunoblot analysis when probed with anti-ERK1/2 antibodies . This antibody recognizes the active phosphorylated form of Kss1 and Fus3, as well as other kinases such as Slt2 (52 kDa), which is required is to maintain the integrity of the cell wall . We examined the phosphorylation status of Kss1 and Fus3 and whether these two MAPKs would be altered in the H2B R95A mutant in comparison to the H2B WT in response to rapamycin. As expected, the anti-ERK1/2 antibody detected the phosphorylated Kss1 and Fus3 in the H2B WT strain using 4 to 20 % gradient SDS polyacrylamide gel, although better resolution can be seen on standard 12 % gel (Fig. 6, lane 4 vs. lane 1). Neither of the MAPKs was visibly detected in the H2B R95A mutant as compared to the H2B WT (Fig. 6, lane 1 vs. lane 4). This latter finding is consistent with the microarray data revealing that multiple genes of the pheromone pathway, including FUS3, are downregulated in the H2B R95A mutant (Fig. 2A). Treatment of the H2B WT cells with rapamycin did not show any noticeable increase in the Kss1 or Fus3 phosphorylation status (Fig. 6 lanes 5 and 6 vs. 4), excluding the possibility that rapamycin mode of action is similar to that of α-factor triggering MAPKs activation. It is noteworthy that the anti-ERK1/2 antibody can also detect the Slt2 kinase that responds to various stress conditions including heat shock and oxidative stress . The Slt2 kinase was present at nearly the same level in the H2B WT and the H2B R95A mutant, and unaffected by rapamycin treatment (Fig. 6 lanes 4 to 6 vs. lanes 1 to 3), suggesting that the H2B R95A mutant is not likely to harbor global downregulation of signaling pathways. Taken together, the above results suggest that the inability of the H2B R95A mutant to arrest in the G1 phase in response to rapamycin is consistent with the downregulation of signaling components of the pheromone response pathway.
Rapamycin treatment accumulates the cyclin Cln2 at a higher level in the ste5Δ mutant, as compared to the WT—If rapamycin-induced G1 arrest involves the downregulation of the G1 cyclins (Cln1-3)[16, 17], we anticipate that such regulation would be compromised in the ste5Δ mutant allowing the cells to confer resistance to rapamycin. We examined the level of the G1 cyclin Cln2 in the parent BY4741 strain and the isogenic ste5Δ mutant, both expressing CLN2-TAP at the endogenous locus to monitor the level of Cln2-TAP with the TAP antibody, which detects the Protein A portion of the TAP tag . Briefly, the parental and ste5Δ mutant cells were grown in liquid YPD and treated in the log phase without and with an acute dose of rapamycin (200 ng/ml), samples taken at zero, 30, 60, and 120 mins, and processed by TCA extraction for immunoblot analysis. Upon rapamycin treatment, Cln2-TAP was unexpectedly induced at least 3-fold in the ste5Δ mutant within 30 mins and further accumulated by 120 mins (Fig. 7A, lanes 5 to 8 and quantified as in Fig. 7B, and supplementary Fig. S3 for another independent experiment). In contrast, there was a modest induction of Cln2-TAP in the parental BY4741 strain after 30 min of rapamycin treatment, which continued to accumulate by 120 mins (Fig. 7A, lanes 1 to 4 and quantified as in Fig. 7B, and supplementary Fig. S3), but not to the same extent as the ste5Δ mutant. While this finding suggests that there could be a strain-specific response towards rapamycin, due to the lack of downregulation of Cln2-TAP, it strongly indicates that the early upregulation of Cln2 might be associated with the resistance of the ste5Δ mutant towards rapamycin.