Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance

Deletion of FKH1 suppresses APC mutant defects

We have extensively used the apc5^CA (chromatin assembly) mutant allele for the bulk of our genetic studies to gain insight into APC function [4-6, 26-29, 33]. We discovered the allele in a screen for chromatin assembly mutants [26]. This allele harbors a 2 bp deletion (₃₇AT₃₈), conferring a temperature sensitive phenotype (ts); growth is impaired at 37°C, versus the optimal growth temperature of 30°C. This allele is predicted to encode a short N-terminal portion of Apc5 (the first 12 Apc5 residues followed by 18 novel residues). However, since APC5 is an essential gene and a short N-terminal portion of Apc5 does not rescue the apc5^CA ts defect [26, 34, 35], we fused the TAP epitope to the C-terminus of the apc5^CA allele and discovered that the ts phenotype is due to an N-terminally truncated protein that likely starts from an internal methionine, and/or undergoes programmed ribosome frameshifting [36] (Fig. S1A). We previously used this allele to show that deletion of both FKH1 and FKH2 was necessary to further impair apc5^CA mutant phenotypes [5]. However, here we show that deletion of only FKH1, and not FKH2, improved APC mutant phenotypes (Fig. 1) implicating Fkh1, but not Fkh2, as a potential APC target. This is consistent with other literature noting that deletion of either FKH1 or FKH2 alone has mild, but opposing and independent phenotypes [37, 38].

We began by examining APC mutant phenotypes, including temperature and oxidative stress sensitivity, and reduced lifespan. Deletion of only FKH1 suppressed ts growth in both apc5^CA and in the more severe apc10Δ mutant (Figs. 1A-C, S1B). The ability to improve apc5^CA ts growth was more apparent when the cells were grown at 40°C. Sensitivity of apc5^CA cells to oxidative stress was also partially alleviated by deletion of FKH1 or FKH2. In contrast, loss of FKH1 in apc10 Δ cells suppressed oxidative stress phenotypes, while deletion of FKH2 was lethal (Fig. 1D). These observations supported our consideration that Fkh1 may serve as an APC target. The remainder of this manuscript will focus on Fkh1-APC interactions in order to understand the molecular mechanisms regulating this interaction.

We had previously observed that overexpression of FKH genes was toxic to cells and induced apoptotic-like cell death [5]. Thus, it is likely that if Fkh1 is accumulating in apc5^CA mutant cells, it may likewise be linked to a shorter lifespan. Our results confirm that deletion of FKH1 completely restored apc5^CA RLS (Fig. 1E) and CLS (Fig. 1F) to wild type (WT) levels. Thus, by removing Fkh1, stress and lifespan defects are reduced. Taken together, these observations suggest that Fkh1 plays a detrimental role in cells experiencing compromised APC function.

Fkh1 accumulates in apc5^CA cells in a ubiquitin- and proteasome-dependent manner

To test the hypothesis that Fkh1 is targeted for ubiquitin-dependent degradation by the APC, we first assessed Fkh1 protein levels in asynchronous WT and apc5^CA cells. We integrated Fkh1-TAP into the WT and apc5^CA strains, expressed under its endogenous promoter. As a control, we followed the protein levels of a known APC targeted protein, Clb2, which is selected for degradation at the end of mitosis and is at minimal abundance in G1 [39]. Like Clb2, Fkh1 was dramatically reduced in G1 arrested cells and accumulated in apc5^CA cells (Figs. 2A, 2B). The accumulation of Fkh1 protein in the apc5^CA strain is most likely a post-translational event, as accumulation of Fkh1 protein did not correlate with increased mRNA transcript (Figs. 2C, 2D).

To determine whether Fkh1 G1 instability was dependent on the proteasome, we expressed endogenous Fkh1-TAP in rpn10Δ cells, which lack the proteasome ubiquitin receptor [40]. In rpn10Δ cells, potentially ubiquitinated proteins accumulate [6, 29], and this was observed for Fkh1-TAP (Fig. 2E). WT Fkh1-TAP expressing cells were next exposed to 75 μM MG132, a proteasome inhibitor, for 3 hours prior to harvesting proteins. In the presence of MG132, both Clb2 and Fkh1-TAP accumulated (Figs. 2F, 2G). This was not cell cycle related, as cells treated with MG312 had the same flow cytometry profile as untreated cells (Fig. 2H). Next, we added cycloheximide (CHX), which halts all protein synthesis, to MG132 treated cells to determine whether MG132 impacts Fkh1 stability or steady state levels. Fkh1-TAP and Clb2 protein levels were both elevated in MG132 treated cells, consistent with stable proteins (Fig. 2I).

If Fkh1 degradation is indeed dependent on the APC E3 activity, then increased APC subunit expression may further decrease Fkh1-TAP levels through enhanced E3 activity. When plasmids expressing copper-inducible promoter (CUP1_prom) driven GST-APC5 (Fig 3A) or GST-APC10 (Fig. 3B) were expressed in Fkh1-TAP cells, we indeed observed that Fkh1-TAP levels decreased as compared to empty vector controls. Interestingly, Fkh1-TAP levels increased in response to copper alone, potentially due to induction of Fkh1 by the heavy metal stress. Taken together, these observations are consistent with the idea that Fkh1-TAP instability is APC, ubiquitin- and proteasome-dependent.

Fkh1 is specifically targeted for degradation at the onset of mitosis by APC^Cdc20

To determine whether Fkh1 stability is correlated with specific positions within the cell cycle, we arrested Fkh1-TAP expressing cells at various points in the cell cycle. CHX was then added to block further protein synthesis with samples removed every 30 minutes to assess protein stability from the arrest point forward (Figs. 4A-D). We observed that Fkh1-TAP was at its lowest level during G1, but remained stable (Figs. 4B, 4E). In contrast, Fkh1 is unstable during mitosis in the WT strain (Figs 4C, 4E). Stable Fkh1-TAP appears to again accumulate during S phase (Figs. 4D, 4E), consistent with cyclical FKH1 mRNA expression throughout the cell cycle, which peaks during S phase [41]. In apc10Δ cells, Fkh1-TAP protein levels in G1 arrested cells, compared to G1 arrested WT cells, was higher when compared to asynchronous cells (Figs. 4F, S4C). Thus, these results indicate that Fkh1-TAP is specifically targeted for degradation at the onset of mitosis by the APC, which is also active at the onset of mitosis.

The APC forms two complexes: a Cdc20-containing complex required for mitotic progression (APC^Cdc20), and a Cdh1-containing complex that is required for G1 maintenance (APC^Cdh1) [25, 42]. Cdc20 and Cdh1 are thought to recruit targets to the APC in order to be ubiquitinated [43]. To confirm that Fkh1 is specifically degraded during mitosis, we integrated Fkh1-TAP into the genomes of cdh1Δ and cdc20-1 cells predicting that a cdc20-1 mitotic mutant would disrupt Fkh1 recruitment and degradation in mitosis. cdh1Δ FKH1-TAP cells were chemically arrested in G1, treated with CHX, then assessed for Fkh1-TAP protein stability. Clb2 can be targeted by either APC^Cdc20 or APC^Cdh1 complex [44, 45], thus serves as a stability control as it should not be degraded at the specific cell cycle points defined by these mutants. We show that in cdh1Δ mutants, Fkh1-TAP levels remain low in G1, whereas Clb2 levels are markedly elevated, as predicted (Figs. 5A-C). This indicates that Fkh1-TAP is not targeted for degradation by the G1 specific APC^Cdh1-since it remains low in cdh1Δ cells. On the other hand, in asynchronous cdc20-1 FKH1-TAP cells, both Clb2 and Fkh1-TAP proteins were clearly stabilized, in contrast to their degradation in WT cells (Figs. 5D, 5E). Lastly, we confirmed that the protein levels of Clb2 and Fkh1-TAP predictably rise and fall throughout the cell cycle following synchronous release of S phase-arrested cells back into the cell cycle. Our analysis confirmed that Fkh1-TAP levels began to drop soon after S phase arrest, prior to Clb2 levels, and began to accumulate before Clb2 (Fig. S2). Taken together, the above observations clearly demonstrate that Fkh1 turnover is cell cycle dependent, depends on the APC^Cdc20 complex, and occurs at the onset of mitosis.

Mutation of a highly conserved lysine residue stabilizes Fkh1

If Fkh1 is indeed targeted for degradation by the APC, then mutation of a ubiquitinated lysine (K) should abolish ubiquitination, and stabilize the protein. We searched Fkh1 for a conserved K, and found a highly conserved K at position 373 (Fig. 6A). We mutated the highly conserved K₃₇₃ in the endogenously-TAP tagged FKH1 gene to arginine (R) to generate the Fkh1^K-R strain. Since Fkh2 is believed to compensate for the loss of Fkh1 [37], we deleted FKH2 in these cells to examine the isolated effects of the FHK1 mutation. fkh1^K-R fkh2Δ cells were flocculent, but not as severely as fkh1Δ fkh2Δ cells (Fig. 6B), indicating that the K₃₇₃R mutation impaired Fkh1 function, but did not completely disable it. Next, we tested whether the mutation altered Fkh1 mitotic protein instability. We observed that in cells arrested in mitosis, Fkh1 was unstable, whereas Fkh1^K-R was stable (Figs. 6C, 6D). Lastly, we determined the cycling profile of the Fkh1^K-R protein. The results show that the K₃₇₃R mutation stabilized Fkh1 throughout the cell cycle (Figs. 6E, S3), indicating that K₃₇₃ is required for mitotic Fkh1 instability.

Given the flocculent phenotype of fkh1^K-R fkh2Δ cells (Fig. 6B), consistent with a defective Fkh1 protein, we asked if the K₃₇₃R mutation impaired the core ability of Fkh1: binding promoter DNA. We used chromatin immunoprecipitation (ChIP) in asynchronous cells, with primers designed to amplify the CLB2 and FKH2 promoters, as Fkh1 binds these promoters (Fig. 7) [46]. The high abundance of rDNA, leading to nonspecific interactions with the beads, allows it to be used as a load control. In the bound lanes, Fkh1-TAP was observed at both CLB2 and FKH2 promoters, with Fkh1-TAP abundance clearly increased when FKH2 was deleted (the FKH2 promoter is still present in the FKH2 coding region deletion). Thus, the ability of Fkh1 to compensate for Fkh2 [37] likely involves increased Fkh1 recruitment to promoters in the absence of FKH2. The stable Fkh1^K-R protein was bound to both CLB2 and FKH2 promoters at WT levels, demonstrating that the Fkh1^K-R defect, leading to flocculence in the fkh2Δ background, is not due to impaired promoter binding, at least at CLB2 and FKH2.

Fkh1 interacts physically with Apc5

If the APC targets Fkh1 for ubiquitination and degradation, then it is likely that Apc5 and Fkh1 physically interact. Cells expressing endogenous Fkh1-TAP and plasmid-borne GST-Apc5 were harvested after asynchronous log phase growth. Our results demonstrate that Fkh1 and Apc5 do in fact physically interact in asynchronous cells (Fig. 8A). The control using only GST shows that the TAP antibody does not interact with GST (Fig. 8B). Fkh1 protein levels do fluctuate predictably throughout the cell cycle, demonstrated by G1 arrest followed by synchronous release back into the cell cycle (Fig. S4A). In WT cells, Fkh1-TAP levels were lowest in G1, then steadily increased as the cell cycle progressed. These cell cycle-dependent fluctuations of Fkh1-TAP were blunted and minimized in APC mutants, as Fkh1 protein levels were higher in G1 arrested APC mutants (Figs. S4A, S4C).

We next asked if the Fkh1-Apc5 interaction is cell cycle dependent, perhaps preceding mitosis when Fkh1 is degraded. To determine this, we arrested the Fkh1-TAP cells in S phase, with samples removed for CoIP every 30 minutes after synchronous release back into the cell cycle (Fig. 8C). Apc5 co-immunoprecipitated with Fkh1 throughout the cell cycle, and its levels were proportional to Fkh1. Apc5 likewise continued to be observed in Fkh1^K-R precipitates, with levels also proportional to the amount of Fkh1 IP'd. Our observations therefore suggest that Fkh1 and Apc5 are physically bound throughout the cell cycle, even though Fkh1 degradation only occurs during mitosis, and the stabilizing K₃₇₃R mutation is irrelevant for this interaction.

Degradation of Fkh1 in mitosis is required for normal cell cycle progression through G1

The demonstration that the APC is required to target Fkh1 for degradation leads to the next question: what is the biological significance of this interaction? We hypothesized that Fkh1 degradation in mitosis is necessary to facilitate ordered cell cycle progression into G1. Furthermore, we propose that this progression involves Fkh1-dependent transcriptional control of Clb2, based on a combination of several observations: asynchronous apc5^CA mutants exhibit high Clb2 and Fkh1 protein levels (Figs. 2A, 2B) [6, 28]; G1 arrested apc5^CA mutants exhibit very low Clb2 levels (Figs. 9A, S4A); and Fkh proteins normally repress CLB2 expression during G1 [19, 23]. Together, this raises the possibility that the higher levels of Fkh1 protein (in G1 arrested APC mutants) could be keeping Clb2 protein levels low through transcriptional repression. To test this, WT, apc5^CA and apc5^CA fkh1Δ fkh2Δ cells were arrested in G1, then released back into the cell cycle with samples removed to determine Clb2 protein levels (Fig. 9A). Our hypothesis predicts that removal of Fkh1/2 function in the mutant APC background will return Clb2 protein levels to near normal. In apc5^CA mutants, Clb2 was not apparent until after 90 minutes, and it took 120 minutes for cells to begin to move into G2/M. However, in apc5^CA fkh1Δ fkh2Δ cells, Clb2 expression was clear after 15 minutes, with a synchronous movement of cells into S phase as early as 30 minutes (Figs. 9A, 9C). In WT cells, Clb2 protein levels begin to accumulate again after 60 minutes, which coincided with movement of cells from G1 to G2/M (Figs. 9A, 9B). These experiments, therefore, provide the biological relevance of Fkh1 degradation in mitosis as being essential for normal cell cycle progression.

Stabilization of Fkh1 alters chronological lifespan and genomic stability

Additional important functions of the yeast Forkhead proteins includes lifespan and genomic stability, and here we assess whether Fkh1 protein stability is biologically important in these processes. Fkh1-TAP and Fkh1-TAP fkh2Δ cells exhibited near superimposable lifespans, when measured for chronological lifespan (CLS) (Fig. 10A). In contrast, fkh1^K-R fkh2Δ cells have a remarkably shorter lifespan, with 99% of the cell population dead by four days, compared to 99% dead by day nine in the FKH1 WT strains. The presence of WT FKH2 in fkh1^K-R cells partially abrogated the shortening of the K-R lifespan, supporting its compensatory effect for Fkh1 function.

Color sectoring of yeast colonies is a marker of genomic instability, and we serendipitously observed that individual cells plated during the CLS experiment were sectoring, depending on genetic background. Colonies derived from the WT and fkh2Δ strains did not sector (Fig. 10B). Samples of sectored colonies are shown in the inset for Fig. 10B. However, the red fkh1^K-R FKH2 mutant colonies generated significant numbers of sectored red:white, and white colonies. This occurs when the red phenotype of ade2 mutants revert to white, due to spontaneous genomic mutation [47]. Mutations leading to red:white sectoring occur after plating, whereas the white colonies are due to mutations occurring prior to plating. The longer the cells sat in stationary phase, the more red:white colony sectoring occurred (Fig. 10B). Strikingly, it was most prevalent in the short-lived fkh1^K-R fkh2Δ cells. Pink speckled colonies began to appear suggesting that mutations causing color change were ongoing and persistent despite being in stationary phase. Thus, the lifespan defect in Fkh1^K-R cells appears to be linked with severe genomic instability.

Lastly, we questioned if stress response was impaired in the Fkh1^K-R mutant, along with its shortened lifespan and elevated genomic instability. We directly assessed this by spot diluting cells onto rich media (YPD) or complete media (CM) in the presence of temperature and carbon (2% vs 0.1% glucose) stress. Growth on 0.1% glucose requires metabolic adaptation to utilize alternative carbon sources; complete media (CM) is a much more nutritionally restricted growth medium than YPD; and higher temperatures contribute further to adverse growth conditions (Fig. 10C). In general we observed that fkh1^K-R FKH2 cells were ts regardless of plate conditions. The shorter-lived fkh1^K-R fkh2Δ were more resilient, and were only ts when carbon stress was introduced (0.1% glucose). This investigation therefore confirms that Fkh1 degradation also plays an important biological role in carbon and temperature stress responses, which may be important for controlling genomic stability, as well as in the previous lifespan requirement. In conclusion, we have shown that Fkh1 is targeted for degradation by the APC^Cdc20 complex in mitosis, and have identified a highly conserved lysine that when mutated results in reduced CLS, genomic instability and ts growth.

Fkh1 is an APC substrate

Genetic interactions between FKH and APC mutants have been previously established, as deletion of both FKH1 and FKH2 exacerbates APC mutant cell cycle, lifespan and stress response phenotypes [5]. Nonetheless, while deletion of both FKH1 and FKH2 impair apc5^CA defects, we show that mutation of only FKH1 suppressed these defects (Fig. 1). Mutations that suppress APC defects have been proposed to define potential APC substrates, as removal of these proteins blocks their ability to accumulate and impair cell cycle function [32]. In addition, many proteins that activate the APC, later become APC substrates [24, 25]. Fkh1, therefore, constituted a likely APC substrate. Our previous observation that the Fkhs act upstream of the APC, as activators [5], provided further evidence to suggest that Fkh1 had the potential to be an APC substrate. Our results presented here provide compelling evidence to support this hypothesis, as Fkh1 1) accumu-lates and is stabilized in APC and proteasome mutants, and with proteasome inhibitors (Fig. 2), 2) is reduced when APC subunits are overexpressed (Fig. 3), 3) is unstable during mitosis (Fig. 4), 4) is specifically stable in cdc20-1, but not cdh1Δ mutants (Fig. 5), and 5) is stabilized when a highly conserved lysine is mutated to arginine (Fig. 6). The stabilized Fkh1^K-R protein resulted in reduced yeast CLS, increased genomic instability and increased sensitivity to stress (Fig. 10). Previous work from the Aparicio laboratory showed that Fkh1 and Fkh2 are required for early replication origin clustering, acting by facilitating long-range origin interactions [55]. More recently this lab showed that Fkh protein levels limit early origin firing, and that Fkh overexpression resulted in an increase in origin firing in early S phase. This increase was established in late G1 and slowed the progression of the replication fork [56]. Considering these observations, the inappropriate stabilization of Fkh1 during mitosis and G1 could lead to early firing of replication origins, thus further destabilizing cell cycle progression. Taken together, our results clearly show that the inability to target Fkh1 for degradation following mitotic onset has dire consequences to the cell.

Although Fox proteins are known in higher eukaryotic systems to be targeted for degradation, it was not at all clear in yeast how Fox proteins are regulated. Our detection of a physical interaction between Fkh1 with Apc5 lasting throughout the cell cycle supports the idea that Fkh1 is an APC substrate (Fig. 8). It is interesting that Apc5 and Fkh1 remain physically associated throughout the cell cycle, but that Fkh1 is only targeted for degradation during mitosis. One possible explanation for this association may be to allow the flexibility to rapidly respond to stress regardless of the cell cycle position. Both the APC and Fkh1 respond to stress [5, 57], thus it is possible a tight association of the two throughout the cell cycle allows for a rapid response to stress through the coordinated action of these two stress response pathways.

Accumulation of Fkh1 during mitosis delays cell cycle re-entry

We have observed that Clb2 accumulates in asynchronous apc5^CA cells, yet does not accumulate in G1 arrested apc5^CA or apc10Δ cells (Figs. 2A, 4F, 9A, S4A). Knowing that Fkh1 can repress CLB2 transcription in G1, we considered whether the low levels of Clb2 protein during G1 in APC mutants is a consequence of simultaneously elevated Fkh1 levels during G1. Our findings presented in Fig. 9 demonstrate that Clb2 protein synthesis and cell cycle progression through G1 was indeed delayed in apc5^CA cells, supporting this concept. Interestingly, deletion of FKH1 and FKH2 in these cells not only reversed this effect, but accelerated Clb2 protein synthesis and cell cycle re-entry. Therefore, cell cycle progression through mitosis and G1 could rely on the downregulation of Fkh1.

In summary, our work presented here illuminates how Fkh1 function is regulated in a cell cycle-dependent manner. We identified a highly conserved Fkh1 lysine residue (K373) that regulates cell cycle-dependent protein stability, cellular lifespan, and genomic stability. This provides valued insight into how this family of conserved stress responsive transcription factors is regulated and what occurs when this regulation fails. Much remains to be learned regarding how the Fkhs interact in yeast with each other and with their binding partners. Our results therefore pave the way for future studies in higher eukaryotic systems.

Yeast strains and plasmids

The S. cerevisiae strains used in this study are shown in Table 1. All strains were isogenic derivatives of S288c strains except where noted. BY4741 (YTH1029) and the rpn10Δ::kanMX6 and cdh1Δ::kanMX6 derivatives, were obtained from the ResGen Library (provided by W. Xiao, U of Saskatchewan). The cdc20-1 strain and its associated WT (W303) were a kind gift from A. Amon (David Koch Institute for Integrative Cancer Research at MIT). PCR based methods were used to integrate the TAP::HIS3 cassette into the various cells used. Cells harboring deletions marked by the same phenotypic marker were generated by crossing. The plasmid encoding GAL-driven FKH1-HA (hemaglutinin epitope tag) was obtained from Open Biosytems and kindly provided by W. Xiao. The copper-driven (50 μM CuSO₄ for 2 hours) GST-APC5, GST-APC10, and the empty vector control (pYEX4T-1) were obtained from the Exclone Library.

Media and methods

Yeast growth media used in this study, yeast/peptone/dextrose (YPD) and drop out (DO) supplemented with required amino acids, was described previously [5, 28]. Yeast Minimal Media (MM) used in chronological lifespan (CLS) experiments contained 0.17% yeast nitrogen base/0.5% ammonium sulfate/0.003% essential amino acids, and 2% glucose unless otherwise indicated, with the pH adjusted using sodium hydroxide. Yeast Complete Media (CM) was also used in CLS experiments (0.17% yeast nitrogen base/0.5% ammonium sulfate/2% glucose/0.13% dropout powder including all amino acids, and 1 sodium hydroxide tablet). Methods including spot dilutions, Westerns, reverse transcriptase PCR, E. coli and yeast transformations, flow cytometry, DNA mini-preps, oxidative stress plate assays, and replicative and chronological lifespan assays were all performed according to our previous publications [5, 28, 29, 58]. For MG132 experiments, cultures were started in YPD to ensure log phase growth, then transferred to synthetic media (0.17% yeast nitrogen base without ammonium sulfate) supplemented with 0.1% proline, essential amino acids, and 2% glucose, with addition of 0.003% SDS the next day for an additional 3 hours at 30°C. At this point 75 μM MG132 (Enzo Life Sciences) or the drug vehicle (dimethyl sulfoxide; DMSO) were added, and cells were incubated at 30°C for 3 hours before harvesting and storage at −80°C. Genomic instability was determined by scoring the number of red, white, red/white sectored and pink speckled colonies following stationary phase growth. Four separate plates were counted for each strain on each day. Antibodies against Clb2 (Santa Cruz Biotechnology), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Sigma), α-tubulin (Sigma), and Tandom Affinity Purification (TAP; Pierce) where purchased from the indicated suppliers. The GST J90 GST antibody was described previously [33].

TAP Co-immunoprecipitation (Co-IP)

Lysates were generated using a standard beadbeat protocol [26] in RIPA buffer lacking SDS. TAP tagged proteins were IPed using IgG Sepharose 6 Fast Flow beads (GE Healthcare), essentially as described [33], and eluted lysates were analyzed by western blot to identify co-purified proteins. To generate lysates used in Co-IP, frozen cell pellets were resuspended in 2X cell volume of ice-cold RIPA (Radio Immuno Precipitation Assay Buffer) lysis buffer containing non-ionic detergents, a 1:500 dilution of protease inhibitor cocktail (BioVision), 10 μM n-ethylmaleimide, 1.2 mM dithiothreitol (DTT), and 5 mM EDTA. Iced 0.5 mm glass beads were added to the suspension and the frozen cells were mechanically disrupted using a Mini-Beadbeater (Biospec products). Lysates were clarified and total protein concentration was determined using the Bradford method (BioRad). Protein lysates were added to washed IgG Sepharose 6 Fast Flow beads (GE Healthcare) and rotated end-over-end at 4°C for 90 minutes. Following incubation, beads were washed 2X with lysis buffer, and proteins were eluted off beads by boiling in 2X Laemmli sample buffer and then SDS-PAGE was used to separate proteins for western analyses.

Cell cycle arrest

Cell cycle arrest was performed according to our standard procedures [6, 21]. Hydroxyurea (Sigma) was used at 0.3 M for approximately 3 hours for S phase arrest. For mitotic arrest, Nocodazole (Calbiochem) was used for approximately 3 hours, with 5 μg/ml added initially and then an additional 2.5 μg/ml added after 90 minutes. For G1 arrest of BAR1 strains, cells were first washed 2X with fresh YPD, and then resuspended in YPD at pH 3.5 containing 2 μg/ml of α-factor (Zymo Research) and cultured for approximately 3 hours, with an additional 2 μg/ml added after 90 min. Cultures were considered arrested once >90% of the cells were in the desired phase of the cell cycle according to bud morphology. To release cells from arrest, cultures were washed twice with fresh media. To stop protein synthesis Cycloheximide (CHX; Calbiochem) was used at 10 μg/ml. Arrests were confirmed by flow cytometry (Saskatoon Cancer Center/Pharmacy & Nutrition), as performed previously [5, 26].

Quantitative PCR (Q-PCR)

Cells were cultured in YPD at 30°C, and samples were collected in mid log phase (ie.OD₆₀₀=0.46-0.90). Equivalent cell amounts were harvested for each sample, for a total of 6×10⁸ total cells per sample. Total RNA was extracted using the hot phenol method with DNA digestion completed using DNAse I (Fermentas) in the presence of RNAse inhibitors (Fermentas). The quality of RNA was assessed spectrophotometrically by measuring the OD_260/280 ratio. Only samples that measured a ratio between 1.8-2.0 were used. The RNA integrity was assessed by visual inspection of the 28S/18S subunits on a formaldehyde agarose gel. First-strand synthesis was performed on 1 μg of total RNA using RevertAid Reverse Transcriptase (Fermentas) following manufacturers’ recommendations. Gene-specific primers targeted against yeast ACT1 and FKH1 (Table 2) were designed based on full-length sequences available from the Saccharomyces Genome Database (SGD). The expression of FKH1 was normalized with reference to the ACT1 endogenous control gene. Standard PCR reactions were completed in the mid-linear range for each message using our previously published protocol [29].

Chromatin Immunoprecipitation (ChIP)

CHIP was performed with modifications to those previously described [59]. Following cross-linking of the cell lysate (1% formaldehyde at room temperature for 15 min), and quenching of the cross-linker (5 min incubation in 125 mM glycine) cells were washed 2× with iced TBS (20 mM Tris HCL pH 7.6, 150 mM NaCl) and frozen at −80°C. Frozen cells were resuspended in lysis buffer (50 mM Hepes-KOH pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 1:500 dilution of protease inhibitor cocktail (BioVision)) and lysed by mechanical disruption using iced 0.5 mm glass beads with two 30-sec pulses in a Mini-BeadBeater (Biospec Products). Lysates were sonicated 5 × 10 sec at output 2 (Branson Sonifier 250). Sonicated lysates were clarified and total protein was quantified using the Bradford method (BioRad). Equivalent amounts of total protein were incubated with washed IgG Sepharose 6 Fast Flow beads (GE Healthcare) and rotated end-over-end at 4°C for 90 minutes. The IPs were washed 2× with lysis buffer, 1x with lysis buffer with 500 mM NaCl, 1x with CHIP wash buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% Na deoxycholate, and 1 mM EDTA), and 1x with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted from the beads by incubating for 15 min at 65°C with 50 mM Tris-HCl pH 8.0, 10 mM EDTA, and 1% SDS. The beads were then washed with TE/0.67% SDS and the wash was pooled with the eluted sample. Cross-links were reversed by overnight incubation at 65°C, followed by protein digestion with Proteinase K (Sigma), and RNA digestion with RNAse A (Sigma). DNA was phenol-extracted, ethanol-precipitated, and resuspended in H₂O. Crude input samples were made up to 250 μL with TE/1% SDS and the cross-links were reversed and the DNA purified as indicated above.

Equivalent amounts of input DNA for each sample were used in PCR. Two sets of primers were used in each reaction. Primer sets were designed to amplify the promoter region of CLB2 or FKH2, or part of the RDN1 coding sequence, which served as a loading control. PCR involved an initial denaturation of 30 sec at 98°C, then 36-40 cycles at: 98°C for 15 sec, 55°C for 20 sec, and 72°C for 20 sec, with a final extension at 72°C for 5 min. Control PCR reactions were carried out with a range of cycles to ensure that we were in the linear portion of the assay.

Statistics

The Standard Error of the Mean was calculated in Microsoft Excel and was used for error bars. Significance was calculated using the Student T-test for comparing 2 Independent Means (http://www.socscistatistics.com).