Tfs1, transcription elongation factor TFIIS, has an impact on chromosome segregation affected by pka1 deletion in Schizosaccharomyces pombe

The cAMP-dependent protein kinase (PKA) pathway in Schizosaccharomyces pombe plays an important role in microtubule organization and chromosome segregation. Typically, loss of functional Pka1 induces sensitivity to the microtubule-destabilizing drug thiabendazole (TBZ) and chromosome mis-segregation. To determine the mechanism via which Pka1 is involved in these events, we explored the relevance of transcription factors by creating a double-deletion strain of pka1 and 102 individual genes encoding transcription factors. We found that rst2∆, tfs1∆, mca1∆, and moc3∆ suppressed the TBZ-sensitive phenotype of the pka1∆ strain, among which tfs1∆ was the strongest suppressor. All single mutants (rst2∆, tfs1∆, mca1∆, and moc3∆) showed a TBZ-tolerant phenotype. Tfs1 has two transcriptional domains (TFIIS and Zn finger domains), both of which contributed to the suppression of the pka1∆-induced TBZ-sensitive phenotype. pka1∆-induced chromosome mis-segregation was rescued by tfs1∆ in the presence of TBZ. tfs1 overexpression induced the TBZ-sensitive phenotype and a high frequency of chromosome mis-segregation, suggesting that the amount of Tfs1 must be strictly controlled. However, Tfs1-expression levels did not differ between the wild-type and pka1∆ strains, and the Tfs1-GFP protein was localized to the nucleus and cytoplasm in both strains, which excludes the direct regulation of expression and localization of Tfs1 by Pka1. Growth inhibition by TBZ in pka1∆ strains was notably rescued by double deletion of rst2 and tfs1 rather than single deletion of rst2 or tfs1, indicating that Rst2 and Tfs1 contribute independently to counteract TBZ toxicity. Our findings highlight Tfs1 as a key transcription factor for proper chromosome segregation.


Introduction
Cell growth is regulated by the cell cycle, and various environmental conditions affect the individual stages. When cells are exposed to various stressors, the relevant signal transduction pathway is activated to adapt to these conditions and prevent cell death. The activated signal transduction pathway generally regulates the target proteins, including transcription factors, with high probability. The fission yeast, Schizosaccharomyces pombe exhibits the activation of several signal transduction pathways in response to various environmental conditions. In the stress-activated protein kinase (SAPK) pathway, Sty1 is a key kinase activated by osmotic, oxidative, or heat stress (Shiozaki and Russell 1996;Wilkinson et al. 1996;Shieh et al. 1997Shieh et al. , 1998. The target of rapamycin (TOR) pathway consists of two pathways, TORC1 and TORC2 (Morozumi and Shiozaki 2021). In the TORC1 Kouhei Takenaka and Shiho Nishioka authors contributed equally to this work.
In the cAMP/PKA pathway, the proteins Git3 (the G protein-coupled receptor), Gpa2 (the G protein alpha subunit), Git5 (the G protein beta subunit), and Git11 (the G protein gamma subunit), assess environmental glucose conditions. When glucose is abundant, Gpa2 activates adenylate cyclase Cyr1, which synthesizes cAMP from ATP (Kawamukai et al. 1991). Under low-glucose conditions, two cAMP-dependent protein kinase regulatory subunits (Cgs1) bind with two protein kinase A catalytic subunits (Pka1) to form a heterotetramer complex, and Pka1 is held in the inactivated form (Gupta et al. 2011a). When cAMP binds with Cgs1, Pka1 is released from the hetero-tetramer complex and is activated (DeVoti et al. 1991;Maeda et al. 1994;Gupta et al. 2011b;Matsuo and Kawamukai 2017). Activated Pka1 regulates target proteins, including transcription factor Rst2. Rst2 is negatively regulated via phosphorylation by Pka1 (Higuchi et al. 2002;Inamura et al. 2021). The cAMP/PKA pathway also plays a role in the transition to meiosis (Maeda et al. 1994;Wu and McLeod 1995;Matsuo et al. 2008), chronological aging (Roux et al. 2006), chromosome segregation (Tanabe et al. 2019), and maintenance of the microtubule structure (Tanabe et al. 2020). Loss of functional Pka1 leads to the formation of the thiabendazole (TBZ)-sensitive phenotype and chromosome mis-segregation (Tanabe et al. 2019). These phenotypes are rescued by the overexpression of Mal3, which is an end-binding 1 (EB1) homologous protein (Tanabe et al. 2019). Mal3 has three domains: the calponin homology (CH) domain at the N-terminus, a coiledcoil domain in the middle region, and an EB1 domain at the C-terminus (Hayashi and Ikura 2003). Binding of the CH domain of Mal3 with microtubules is responsible for the suppression of pka1∆ upon exposure to the toxic compound TBZ (Tanabe et al. 2019). The loss of Pka1 function causes growth defects at a high concentration of TBZ; however, the mechanism remains to be elucidated.
Tfs1 is homologous to the transcription elongation factor IIS (TFIIS), which regulates a component of RNA polymerase II (RNAPII) preinitiation complexes (Kim et al. 2007;Lock et al. 2018). TFIIS forms a complex with RNAPII and induces RNA cleavage. When RNAPII is positioned and backtracked onto the template DNA, TFIIS facilitates the restart of transcription elongation (Izban and Luse 1992;Kettenberger et al. 2003). In S. pombe, Tfs1-dependent transcription is involved in the suppression of gross chromosomal rearrangements by heterochromatin .
In this study, we identified rst2∆, tfs1∆, mca1∆, and moc3∆ as suppressors of TBZ-sensitive pka1∆ strains by screening the double-deletion strain of pka1 and 102 individual genes encoding transcription-related factors in the S. pombe. We also showed that tfs1∆ rescues chromosome mis-segregation mediated by pka1∆. Tfs1 overexpression enhanced the TBZ-sensitive phenotype and chromosome mis-segregation. Finally, we investigated the genetic relationship between Rst2 and Tfs1. Taken together, our findings suggest that Pka1 negatively regulates Tfs1 to adapt to the TBZ toxicity in S. pombe.

Yeast strains, media, and genetic methods
The S. pombe strains used in this study are listed in Table 1. Standard yeast culture media and genetic methods were used (Petersen and Russell 2016;Murray et al. 2016). S. pombe cultures were grown on either YES medium (0.5% yeast extract, 3% glucose, 225 mg/L adenine, 225 mg/L uracil, 225 mg/L leucine, 225 mg/L histidine, and 225 mg/L lysine), YE medium (0.5% yeast extract, 3% glucose), or synthetic minimal medium (EMM) with appropriate auxotrophic supplements (Petersen and Russell 2016).

Plasmid construction and induction of expression under the nmt1 promoter
To construct pREP3X-Tfs1, the oligonucleotide primers TFS1-SF and TFS1-BR were used to amplify an 882 bp fragment containing the complete tfs1 protein coding sequence from S. pombe cDNA. The amplified tfs1 gene fragment was digested with SalI and BamHI and integrated into the corresponding sites of pREP3X (Forsburg 1993). To construct pREP81-Tfs1, pREP3X-Tfs1 was digested with SalI and BamHI and integrated into the corresponding sites of pREP81 (Basi et al. 1993).
Wild-type cells were transformed with the pREP3Xderived plasmids (LEU2 marker) and transformants were selected on EMMU (EMM containing uracil but lacking leucine) plates containing 15 µM thiamine to repress gene expression from the nmt1 promoter. To induce the overexpression of tfs1 under the nmt1 promoter, cells were grown on EMMU containing 15 µM thiamine for 2 days at 30 °C, and were then transferred onto EMMU without thiamine, and incubated for 1 day at 30 °C. The cells were then spotted

Mini-chromosomal loss assay
This assay was performed as previously described (Niwa et al. 1986;Tanabe et al. 2019). Two independent Ade + isolates of the wild type, pka1∆, tfs1∆, pka1∆ tfs1∆, pka1∆ rst2∆, and pka1∆ rst2∆ tfs1∆ strains, were analyzed for the stability of the mini-chromosome16 (Ch16) containing the ade6 gene, on the YE or adenine-limited EMMGU plates, in the presence or absence of 7.5 µg/mL TBZ. The host ade6-M210 cells become Ade + by allelic complementation with ade6. Cells were plated on YE or adenine-limited EMMGU plates in the presence or absence of 7.5 µg/mL TBZ and incubated at 30°C for 3 to 5 days and then at 4°C for 1 to 2 overnight periods to allow deepening the red color of the Ade − colonies. The frequency of chromosome loss was determined by counting the total colonies and the red colonies.

Monitoring the mis-segregation of chromosome II
This analysis was performed as previously described (Yamagishi et al. 2012). Centrosome-tagged GFP (cen2-GFP) was used to analyze the mis-segregation of chromosome 2 (Yamamoto and Hiraoka 2003). To investigate the frequency of cen2-GFP mis-segregation at anaphase, cells were treated by the addition of hydroxyurea (HU) for 4 h and arrested at the G1/S phase. Cells were washed three times by water and released at a low temperature (18°C) without HU. Cells were incubated for 8 h and analyzed for the localization of cen2-GFP by fluorescence microscope.

Fluorescence microscopy of GFP fusion protein
S. pombe cells were grown in YES liquid medium to the mid-log phase at 30 °C. GFP-tagged Tfs1 and cen2 were visualized in living cells, and images were taken by a BX51 microscope (Olympus) equipped with a DP74 digital camera (Olympus).

Preparation of cell lysates and detection of 13Myc fusion protein using immunoblotting
S. pombe cell lysates were prepared as previously described (Matsuo et al. 2004). Lysate proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which western blot analysis was performed using a Western Lightning enhanced chemiluminescence (ECL) Pro detection system (PerkinElmer) according to the supplier's instructions. Mouse monoclonal anti-Myc (9E10; diluted 1:1000) and mouse monoclonal anti-Cdc2 (Y100.4; diluted 1:1000) antibodies were purchased from Santa Cruz Biotechnology. Horseradish-peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) was used as a secondary antibody.

Isolation of suppressors of TBZ sensitivity in the pka1∆ strain
The S. pombe pka1∆ strain shows a TBZ-sensitive phenotype and chromosome mis-segregation (Tanabe et al. 2019).
To determine the mechanism by which the loss of Pka1 induces the TBZ-sensitive phenotype, we focused on the transcription-related proteins that may be regulated by Pka1. We selected the 107 transcription-related gene knockouts from version 4 of the Bioneer genome-wide deletion mutant library, which consists of 3,400 S. pombe non-essential gene deletion mutants. We first analyzed the TBZ-sensitive phenotype of 107 transcription-related gene knockouts (Kim et al. 2010) and found that five gene knockouts (gsf1∆, iws1∆, cbf11∆, spt6∆, and tup12∆) induced the TBZ-sensitive phenotype (Table S2). Next, we performed doublegene knockouts in 102 TBZ-none-sensitive strains with the pka1∆ strain (Table S3). These 102 double-gene knockouts were derived by crossing individual gene knockouts with the pka1∆ strain. We then tested the suppressive effects of the TBZ-sensitive phenotype of the pka1∆ strain using transcription-related gene knockouts. Sixteen gene knockouts rescued the TBZ-sensitive phenotype of the pka1∆ strain (Table S2). Since it is known that the Bioneer genome-wide deletion mutant library contains unexpected gene deletions or mutations in other loci in certain proportions, we re-generated the candidate gene knockout strains, re-checked the suppression of pka1∆, and isolated rst2∆, tfs1∆, mca1∆, and moc3∆ strains.

The rst2∆, tfs1∆, mca1∆, and moc3∆ strains induced TBZ tolerance
We next tested whether the isolated double knockouts had a suppressive effect on the TBZ-sensitive phenotype induced by pka1∆ in the presence of TBZ (18 µg/mL) and also at a higher concentration of TBZ (20 µg/mL) to compare TBZ tolerance between wild-type strain and mutants. The pka1∆ rst2∆, pka1∆ tfs1∆, pka1∆ mca1∆, and pka1∆ moc3∆ double mutants exhibited growth comparable to that of the wildtype strain upon exposure to 20 µg/mL TBZ, whereas the pka1∆ single mutants showed a TBZ-sensitive phenotype in the same conditions (Fig. 1a). Rst2 is a zinc finger C2H2type transcription factor that is negatively regulated by the cAMP/PKA pathway (Higuchi et al. 2002;Takenaka et al. 2018;Inamura et al. 2021). Our results suggest that Rst2 also plays a role in response to TBZ toxicity. Tfs1 is a transcription elongation factor TFIIS which facilitates the restart of RNAPII after backtracking RNAPII ).

Loss of functional Tfs1 rescued chromosome mis-segregation induced by pka1∆
The loss of functional Pka1 causes chromosome mis-segregation (Tanabe et al. 2019). Since deletion of the tfs1 gene rescued the TBZ-sensitive phenotype induced by pka1∆, we next analyzed whether tfs1∆ suppresses chromosome mis-segregation induced by pka1∆ by performing a minichromosome loss assay. Mini-chromosome Ch16 contains the ade6 gene on a non-essential chromosome, and the strain harbors the ade6-M210 mutation. The colonies are white in color when normal segregation of the mini-chromosome occurs, whereas they appear red after mis-segregation of mini-chromosomes on the medium containing a low concentration of adenine (Niwa et al. 1986;Tanabe et al. 2019).
The pka1∆ strain showed a high frequency of chromosome mis-segregation in the presence of TBZ, and the tfs1∆ strain considerably rescued this phenotype (Fig. 3a). We additionally analyzed chromosome segregation using the cen2-GFP strain, in which the centromere locus of chromosome II is marked with GFP. The pka1∆ strain showed approximately 5% chromosome mis-segregation, and tfs1∆ clearly rescued the chromosome mis-segregation induced by pka1∆ (Fig. 3b). Thus, tfs1∆ rescues chromosome mis-segregation induced by pka1∆, in addition to the suppressive effect of TBZ sensitivity.

Localization of Tfs1 was slightly altered in the presence of TBZ
Next, we analyzed the localization of Tfs1 in the presence of TBZ. We generated a tfs1-GFP strain by tagging GFP at the C-terminus of Tfs1 to visualize its localization. Tfs1-GFP localized to the nucleus and translocated into the cytoplasm in the wild-type and pka1∆ strains under normal growth conditions, indicating that Tfs1-GFP localization was not affected by pka1∆ (Fig. 4a). The nuclear localization of Tfs1-GFP was confirmed via DAPI staining (Fig. 4b).
In the presence of TBZ, Tfs1-GFP was diffusely localized to the cytoplasm and highly accumulated around and in the nucleus, however, its localization to the nucleoplasm was suppressed ( Fig. 4a and b). Next, we determined whether pka1∆ affects Tfs1 expression in the presence of TBZ. Under normal growth conditions, Tfs1-13Myc was detected as a single band in wild-type and pka1∆ strains (Fig. 4c). The expression level of Tfs1-13Myc was not significantly different between the wild-type and pka1∆ cells in the presence or absence of TBZ (Fig. 4c and d). Thus, Tfs1 expression was not affected by TBZ or pka1 deletion.

Tfs1 overexpression induced the TBZ-sensitive phenotype and chromosome mis-segregation
Since the tfs1∆ strain showed the TBZ-tolerant phenotype, we next analyzed whether Tfs1 overexpression induces the TBZ-sensitive phenotype. Wild-type cells harboring pREP3X-Tfs1 exhibited a minor TBZ-sensitive phenotype, whereas control cells harboring the vector showed optimal growth on the medium containing 18 µg/mL TBZ (Fig. 5a).
Next, we analyzed the effect of tfs1 overexpression on chromosome segregation in the mini-chromosome Ch16containing the wild-type strain (TTP101). Under normal growth conditions, tfs1 overexpression induced chromosome mis-segregation at a low frequency (approximately 0.6%), whereas tfs1-overexpressing cells showed a high frequency (approximately 2.5%) of chromosome missegregation in the presence of TBZ (Fig. 5b). Thus, high expression of tfs1 enhances chromosome mis-segregation induced by the microtubule-destabilizing drug TBZ.  Overexpression of Tfs1 causes the TBZ-sensitive phenotype and chromosome mis-segregation. a Wild-type (PR109) cells harboring pREP3X (vector) or pREP3X-tfs1 were cultured for 1 day in EMMU at 30°C to induce expression from the nmt1 promoter. Culture dilutions were made as described in Fig. 1a, and each dilution was spotted onto EMMU in the presence or absence of 18 µg/ mL TBZ. All plates were incubated for 5 days at 30°C b Wild-type (PR109) cells harboring pREP3X (vector) or pREP3X-tfs1 were cultured for 1 day in EMMU at 30°C to induce expression from the nmt1 promoter. Cultured cells were platted on the EMMGU plate containing 5 mg/mL adenine and 7.5 µg/mL TBZ. Plates were incubated for 4 days at 30°C. Red colonies and sector colonies were counted. Experiments were performed three times: averages with S.D. are shown. Double asterisks (**) indicate P-value < 0.01 for comparing with the wild-type strain harboring vector without TBZ

Tfs1 and Rst2 synergistically regulated chromosome segregation
Since the transcription factor Rst2 is regulated by the cAMP/ PKA pathway (Higuchi et al. 2002;Takenaka et al. 2018;Inamura et al. 2021), we next analyzed whether Rst2 is involved in the function of Tfs1. To this end, we compared the effects of pka1 deletion in rst2∆, tfs1∆, and rst2∆ tfs1∆ in the presence of TBZ. While the pka1∆ strain exhibited the TBZ-sensitive phenotype in the presence of 18 µg/mL TBZ, the pka1∆ rst2∆, pka1∆ tfs1∆, and pka1∆ rst2∆ tfs1∆ strains showed optimal growth on the medium containing 18 µg/mL TBZ and even on the medium containing 20 µg/ mL TBZ (Fig. 6a). Growth inhibition of pka1∆ strain by TBZ was considerably rescued by rst2∆ tfs1∆ compared with that obtained after the deletion of rst2∆ or tfs1∆ (Fig. 6a).
The pka1∆ rst2∆ tfs1∆ strain showed a TBZ-tolerant phenotype compared with that of wild-type strain cultured on the medium containing 18 µg/mL and 20 µg/mL TBZ. These results indicate that Rst2 and Tfs1 play a synergistic role in the response to the microtubule-destabilizing drugs in conjunction with the cAMP/PKA pathway. Finally, we analyzed whether rst2 and/or tfs1 deletion synergistically affected chromosome segregation. The pka1∆ strain showed chromosome mis-segregation in the presence of TBZ. Under the same growth conditions, the pka1∆ rst2∆, pka1∆ tfs1∆, and pka1∆ rst2∆ tfs1∆ mutants exhibited proper chromosome segregation, similar to that in the wild-type strain (Fig. 6b).

Discussion
The microtubule-destabilizing drugs TBZ and methyl benzimidazole-2-yl carbamate (MBC) cause chromosome missegregation or abnormal microtubule organization. Upon the loss of functional Pka1 in S. pombe, cells show chromosome mis-segregation and abnormal microtubule organization (Tanabe et al. 2019). We hypothesized that Pka1 has a common target of the transcription factor responsible for the TBZ sensitivity and chromosome mis-segregation and we explored this possibility in this study. We created double-gene deletion strains of pka1 and 102 individual genes encoding transcription-related proteins and tested TBZ sensitivity. We identified the knockouts of four transcriptionrelated genes (rst2, tfs1, mca1, and moc3) that suppressed the growth defects induced by pka1∆ upon the addition of TBZ. We observed that these single mutants (rst2∆, tfs1∆, mca1∆, and moc3∆ mutants) exhibited the TBZ-tolerant phenotype (Fig. 1b), which enabled the strains to reverse TBZ-sensitive phenotype in the pka1∆ strain (Fig. 1a). The TBZ-tolerant phenotype of the moc3∆ strain was consistent with previous results (Goldar et al. 2005). Although the tfs1∆ and rst2∆ strains show normal growth after exposure to 10 µg/mL and 18 µg/mL TBZ, respectively (Takenaka et al. 2018;Okita et al. 2019), the mca1∆ strain has not been analyzed in this respect. Tfs1 contains the TFIIS domain at its N-terminus and the Zn finger domain at its C-terminus. The presence of one of the Tfs1 domains resulted in partial function, and the lack of two domains resulted in a complete loss-of-function in the reversibility of the TBZ-tolerant phenotype in the pka1∆ tfs1∆ strain (Fig. 2) Fig. 6 Loss of Rst2 and Tfs1 cooperatively enhance the suppression of pka1∆ on TBZ. a Wild type (PR109), pka1∆ (YMP36), rst2∆ (YMP130), tfs1∆ (KTP32), pka1∆ rst2∆ (YMP170), pka1∆ tfs1∆ (KTP40), and pka1∆ rst2∆ tfs1∆ (KTP50) cells were cultured for 1 day on YES at 30°C. Culture dilutions were made as described in Fig. 1a, and each dilution was spotted onto YES in the presence or absence of 18 µg/mL or 20 µg/mL TBZ. All plates were incubated for 3 days at 30°C. b Wild-type (TTP101), pka1∆ (TTP104), pka1∆ tfs1∆ (KTP105), pka1∆ rst2∆ (YMP975), and pka1∆ rst2∆ tfs1∆ (YMP977) cells were cultured for 1 day in the YES liquid medium. Cultured cells were platted on the YE plate containing 7.5 µg/mL TBZ. Plates were incubated for 4 days at 30°C. Red colonies and sector colonies were counted. Experiments were performed three times: averages with S.D. are shown. Double asterisks (**) indicate P-value < 0.01 for comparing with the pka1∆ strain on YE + 7.5 µg/ mL TBZ is required in the formation of the complex with RNAPII (Cermakova et al. 2023) and the Zn finger domain binds to DNA and RNA (Klug 1999). Since each domain is responsible for a part of the TFIIS function, both strains harboring pREP81-Tfs1 (6-80∆: a TFIIS domain) or pREP81-Tfs1 (1-245: a Zn finger domain) showed partial restoration of the TBZ-tolerant phenotype of the pka1∆ tfs1∆ strain. We found that tfs1∆ rescued not only the TBZ-sensitive phenotype ( Fig. 1a) but also chromosome mis-segregation (Fig. 3) induced by pka1∆. tfs1 overexpression generated a TBZsensitive phenotype and enhanced chromosome mis-segregation (Fig. 5). These findings suggest that Tfs1 is activated in pka1∆; however, we did not observe a clear difference in the Tfs1 expression and localization in the pka1∆ strain compared with that in the wild-type strain (Fig. 4). Although there is no direct evidence that Tfs1 is activated in the pka1∆ strain, we cannot exclude this possibility because the phosphorylation of Tfs1 has been demonstrated via proteomics analysis of S. pombe whole proteins (Kettenbach et al. 2015). Tfs1-dependent transcriptional regulation is a key target of heterochromatin for maintaining centromeres integrity . Heterochromatin ensures sister chromatid cohesion at centromeres (Bernard et al. 2001) and prevents chromosome mis-segregation ). The heterochromatin-defective strains (clr4∆ and ago1∆) exhibit chromosome mis-segregation and the TBZ-sensitive phenotype (Sadeghi et al. 2015;Okita et al. 2019), and concomitant deletion of tfs1 restores its phenotype (Nakagawa and Okita 2019). Tfs1/TFIIS induces transcriptional elongation, promotes the dissociation of proteins from the chromosome, and forms DNA-RNA hybrids. Consequently, Tfs1 causes chromosome mis-segregation using repetitive DNA sequences Nakagawa and Okita 2019). The tfs1∆ strain showed suppressed transcription elongation, resulting in proper chromosome segregation. Although we did not evaluate the role of Pka1 in heterochromatin structure, the role of Tfs1 is considered to be related to our finding that tfs1∆ rescued the TBZ-sensitive phenotype and chromosome mis-segregation induced by pka1∆. Together, these observations support the hypothesis that Tfs1 is hyperactivated in the pka1∆ strain and that hyperactivated Tfs1 causes chromosome mis-segregation. However, further analysis is required to determine how Pka1 affects the Tfs1 function.
Multiple copies of Tfs1 and Pka1 suppress growth defects caused by overexpression of the prz1 gene, which encodes a transcription factor that responds to calcium signaling (Koike et al. 2012). Prz1 is highly expressed in pka1∆ cells, which is likely why pka1∆ cells are sensitive to growth in the presence of calcium (Matsuo and Kawamukai 2017). The tfs1∆ strain was similarly sensitive to growth in the presence of calcium (our unpublished data).
Notably, both Tfs1 and Pka1 function as counteracting factors toward inhibition by prz1 overexpression. However, the role of TBZ sensitivity is in the opposite direction: Tfs1 expression induces sensitivity to TBZ, whereas the activation of Pka1 induces tolerance to TBZ. Pka1 has been found to have many targets based on the observation that many genes are upregulated in pka1∆ cells (Nishida et al. 2019). Combined with the assumption that Tfs1 affects gene transcription, it is not surprising that Pka1 and Tfs1 function in the same pathway, but not always.
The cAMP/PKA pathway negatively regulates the transcription factor Rst2 (Higuchi et al. 2002;Inamura et al. 2021). The rst2∆ tfs1∆ mutant strongly restored the TBZsensitive phenotype induced by pka1∆ compared with that obtained using single knockouts of rst2∆ or tfs1∆ (Fig. 6a). rst2 overexpression causes growth inhibition (Koike et al. 2012;Takenaka et al. 2018), but it is not rescued by tfs1 overexpression (Koike et al. 2012), suggesting that the target genes of these two transcription factors are different. These observations support our hypothesis that Tfs1 is involved in TBZ sensitivity (or tolerance) separately from the cAMP/PKA pathway, leading to the regulation of Rst2.
The cAMP/PKA pathway in S. pombe is important for facilitating proper chromosome segregation; the TBZ-sensitive phenotype of pka1∆ was restored via the deletion of at least four transcription factors. Among them, the role of Tfs1 is crucial because it enhances the TBZ-sensitive phenotype and chromosome mis-segregation. Our study demonstrated that Tfs1 is a key transcription factor for mediating proper chromosome segregation.
Author contribution KT designed the experiments, performed the experiments, made the yeast strains, and analyzed the data; SN performed the experiments, made the yeast strains, and analyzed the data; YN performed the experiments; MK analyzed the data and provided advice; YM planned the study, designed the experiments, made the plasmids and strains, performed the experiments, and analyzed the data. YM drafted the original manuscript. MK and YM reviewed and edited the original manuscript.
Funding The authors thank the faculty of Life and Environmental Sciences in Shimane University for help in financial support for publication. This work was supported by a JSPS KAKENHI Grant Number JP18K05438 (to YM) and JP19K222831 (to MK).
Data availability Additional information is provided in the Supplementary Material.