Conversion of a replication origin to a silencer through a pathway shared by a Forkhead transcription factor and an S phase cyclin

Abstract

Silencing of the mating-type locus HMR in Saccharomyces cerevisiae requires DNA elements called silencers. To establish HMR silencing, the origin recognition complex binds the HMR-E silencer and recruits the silent information regulator (Sir)1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2-4. However, silencing is semistable even in sir1Delta cells, indicating that SIR1-independent establishment mechanisms exist. Furthermore, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of FKH1 (FKH1(hc)), a conserved forkhead transcription factor, or by deletion of the S phase cyclin CLB5 (clb5Delta). FKH1(hc) caused only a modest increase in Fkh1 levels but effectively reestablished Sir2-4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1(hc) prolonged the cell cycle in a manner distinct from deletion of its close paralogue FKH2, and it created a cell cycle phenotype more reminiscent to that caused by a clb5Delta. Unexpectedly, and in contrast to SIR1, both FKH1(hc) and clb5Delta established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRDeltaE::ARS). HMRDeltaE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRDeltaE::ARS1 was reduced by clb5Delta or FKH1(hc), whereas ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRDeltaE::ARS1 did not result from formation of Sir2-4 chromatin because sir2Delta did not rescue origin firing in clb5Delta cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2-4 chromatin and late-origin firing through opposing regulation of a common pathway.

Figure 1.

FKH1^hc-silencing required recruitment of Sir3 and the catalytic activity of Sir2. (A) Steady-state levels of a1 and SCR1 RNAs were measured in MATα HMR-SSa sir1Δ cells (CFY762) harboring a 2μ plasmid (lane 1, vector; pCF225), a CEN plasmid with SIR1 (lane 2, SIR1-CEN; pCF99), a 2μ plasmid with FKH1 (lane 3, FKH1-2μ; pCF480), or a CEN plasmid with FKH1 (lane 4, FKH1-CEN; pCF943). The average ratio of a1/SCR1 and SD for three independent experiments is indicated below each lane. FKH1-CEN also reproducibly enhanced silencing slightly as measured by mating assays (Casey, unpublished data). (B) Sir3 association with HMR-SSa and the control locus ADH4 was measured by ChIP with a monoclonal antibody cocktail against Sir3 (α-Sir3). Three isogenic MATα HMR-SSa strains were analyzed that differed only in terms of their SIR1 or SIR3 genotype: SIR1 sir3Δ (CFY1819), SIR1 SIR3 (CFY345), and sir1Δ SIR3 (CFY762). ChIPs were performed on each strain containing either a 2μ plasmid (vector, gray; pCF225) or a 2μ plasmid with FKH1 (FKH1^hc, black; pCF480). The percentage of specific PCR fragment obtained in the immunoprecipitate was determined by videodensitometry analysis using Labworks analysis software (UVP). Data and standard deviations are reported for three independent ChIP experiments. (C) ChIPs were performed as described in B except that multiple regions within the HMR-SSa locus were examined using primer pairs at the positions indicated in the cartoon of HMRa above the figure as described previously (Rusche et al., 2002). Two isogenic MATα HMR-SSa sir1Δ strains were analyzed that differed only in terms of their SIR2 genotype; SIR2 (CFY762) or sir2N345A (CFY1827). Each strain harbored a plasmid containing SIR1 (pCF99) or a 2μ plasmid containing FKH1 (FKH1^hc, pCF480).

Figure 2.

FKH1^hc prolonged the cell cycle and attenuated the CLB2 mRNA expression peak. (A) MATa bar1Δ::HIS3 (CFY1265) cells harboring either a 2μ plasmid (vector; pRS426), a 2μ plasmid containing FKH1 (FKH1^hc; pCF480), or a deletion of FKH2 (fkh2Δ; CFY2016) and a 2μ plasmid (vector) were harvested in log phase from medium lacking uracil and arrested in G1 with α-factor. The cells were then released from α-factor arrest into fresh medium, and every 15 min aliquots were harvested for analysis of bud morphology or CLB2 mRNA levels (see B). Unbudded cells were scored in G1 phase, small-budded cells were scored in S phase, and large-budded cells were scored in M phase. A representative graph for each experiment is shown. (B) The ratio of CLB2 mRNA to SCR1 RNA was determined for each of the strains and time points indicated in A. Total RNA (15 μg) as determined by absorbance at 260 λ was analyzed per lane, CLB2 mRNA and SCR1 RNA were probed independently, and the signals quantified by PhosphorImager analysis. Data from a representative experiment are shown. (C) Mating analysis of MATα HMR-SSa sir1Δ cells containing either wild-type FKH2 (CFY762) or a deletion of FKH2 (fkh2Δ; CFY603) and harboring 2μ plasmids with SIR1 (SIR1^hc; pCF345), no insert (vector; pCF225) or FKH1 (FKH1^hc; pCF480). The cells were mixed with an excess of MATa cells (CFY616) and 10-fold serial dilutions were plated on medium to select for the growth of a/α diploids as described in Materials and Methods. The most concentrated samples contained 5 × 10³ cells/μl, and 8 μl was analyzed in each spot. Corresponding growth controls indicated that an equal number of cells were being compared between strains (LC; data not shown). (D) Levels of Fkh1 in MATα HMR-SSa sir1Δ (CFY1649) cells were determined by protein immunoblot with a polyclonal antibody raised against Fkh1 (α-Fkh1). Cells, harboring either an empty 2μ plasmid (pRS426; chromosomal; lanes 1–3) or a 2μ plasmid containing FKH1 (FKH1^hc; pCF480; lanes 4–6), were harvested in log-phase growth in liquid CAS medium. Two-fold serial dilutions of each protein extract were made using protein extract from fkh1Δ cells as diluents (lane 7) to ensure that each lane contained similar total protein amounts that facilitated accurate quantification of Fkh1 levels. The fkh1Δ cells showed no protein band corresponding to Fkh1 in the immunoblot (lane 7). Equal amounts of protein were loaded per lane as determined by Ponceau S staining of the blot before immunoblotting.

Figure 3.

Genetic analyses revealed that FKH1 and CLB5 were functioning in a common pathway. (A) a1/SCR1 RNA ratios were determined by quantitative RNA blot hybridization for an isogenic series of MATα HMR-SSa sir1Δ strains that differed in terms of their CLB genotype (CLB (CFY762); clb1Δ (CFY1614); clb2Δ (CFY1124); clb5Δ (CFY2104); clb6Δ (CFY2279) and in terms of the plasmid they harbored (vector [pRS426], SIR1^hc[pCF34], and FKH1^hc[pCF48]). A normalized ratio of a1 mRNA/SCR1 RNA is shown on the y-axis with a value of 1.0 assigned to the a1/SCR1 ratio calculated for CLB cells transformed with a plasmid harboring SIR1 in one experiment. (B) Mating assays in an isogenic series of MATα HMR-SSa sir1Δ strains that differed in terms of their CLB genotype and in terms of the plasmid they harbored as described in A. In these experiments clb5Δclb6Δ (CFY2268) cells were also analyzed. (C) Because mating assays were more robust on rich media, the same cells analyzed in B were also analyzed in the absence of exogenous plasmids after growth in YPD. (D) An isogenic series of MATα HMR-SSa sir1Δ cells differing in their CLB5 and FKH1 genotypes (CLB5 FKH1 [CFY762]; clb5Δ FKH1 [CFY2104]; and clb5Δ FKH1Δ [CFY2120]) were assessed for silencing by mating assays as described above. Mating was assessed after growth of MATα cells on either synthetic CAS medium to retain a URA3 plasmid (vector) or rich medium in the absence of a plasmid (no plasmid) before selection for diploids. (E) The same cells were assessed for silencing by RNA blot hybridization of a1 mRNA. The a1 mRNA/SCR1 RNA ratio was normalized as in (A). The CLB5 FKH1^hc cells were CFY762 containing a 2μ plasmid with FKH1 (pCF480). The other cells tested in this experiment contained an empty 2μ plasmid (pCF225).

Figure 4.

FKH1^hc did not affect Clb5 levels nor did CLB5 genotype affect Fkh1 levels. (A) Levels of Clb5-3xHA protein were determined by protein immunoblot with anti-HA antibodies in CLB5 (CFY762), clb5Δ (CFY2104), and CLB5–3xHA (CFY2446) cells transformed with either an empty 2μ plasmid (vector) or a 2μ plasmid containing FKH1 (FKH1^hc; pCF480). We analyzed 0.25 OD (optical density at 600 λ) cell equivalents per 4× lane, and 2× and 1× lanes contained twofold and fourfold reductions in that amount of extract, respectively. (B) Levels of Fkh1 protein were determined by protein immunoblot with anti-Fkh1 antibodies in CLB5 (CFY762) and clb5Δ (CFY2104) cells, as indicated (lanes 1–4) and in clb5Δ cells transformed with an empty 2μ plasmid (vector [pCF225]; lanes 5–7) or CLB5 cells transformed with a 2μ plasmid containing FKH1 (FKH1^hc [pCF480]; lanes 8–10). fkh1Δ cells (CFY527) containing vector (pCF225) were analyzed as a negative control (lane 11). We analyzed 0.25 OD cell equivalents per 4× lane and 2× and 1× were twofold and fourfold reductions, respectively, in that amount of extract.

Figure 5.

FKH1^hc or clb5Δ could use nonsilencer replication origins in place of the HMR-E silencer. (A) Structures and sequences of the E-silencer variations used in these experiments. HMRΔE:ARS contains either ARS1 or ARSH4 in the orientation shown as a complete substitute for HMR-SS. (B) Mating assays were performed with an isogenic series of MATα sir1Δ strains that differed in terms of their HMR-E (E) and/or HMR-I (I) silencers at HMRa, as indicated to the left of the figure and described in the text. The cells contained the following silencers: HMR-SSa (CFY1649) in place of an 800-base pair deletion that includes HMR-E (McNally and Rine, 1991; Palacios DeBeer and Fox, 1999); HMR-SSa as described above, and a 335-base pair deletion of that includes HMR-I (CFY110) (Fox et al., 1995); HMR-SSa with a mutation in the Rap1 binding site (CFY2221) (McNally and Rine, 1991); a deletion of an 800-base pairs region, including HMR-E (CFY2133); deletion of an 800-base pairs region including HMR-E replaced with ARSH4 (CFY2071) or ARS1 (CFY2237) as shown in A. Each strain was transformed with a 2μ plasmid containing SIR1 (SIR1^hc [pCF345]), an empty 2μ plasmid (vector [pCF225]), or a 2μ plasmid containing FKH1 (FKH1^hc [pCF480]). Mating was assessed by drop tests in which 10-fold dilutions of MATα cells being tested were mixed with an excess of MATa cells (CFY616) and grown on medium that selected for diploids retaining the plasmids. (C) Sir3-directed ChIPs were performed on MATα sir1Δ HMRΔE::ARS1 + HMR-I cells (CFY2071) or on MATα sir1Δ HMR-SSa + HMRΔI (CFY35) cells transformed with either SIR1 or FKH1^hc as indicated. (D) Sir3-directed ChIPs were used to determine Sir3 association with a number of ARSs compared with HMR-SSa in sir1Δ cells transformed with vector, SIR1, or FKH1^hc. (E) Mating assays were performed with an isogenic series of MATα cells that differed in terms of their silencers at HMRa (as indicated on left of figure and described in A) and their SIR1 or CLB5 genotypes (as indicated at the top of the figure). The various strains used in these experiments were as follows: SIR1 CLB5 with HMR-SSa (CFY345); HMR-SSaΔI (CFY35); HMR containing an 800-base pair deletion that includes HMR-E replaced with either ARSH4 or ARS1 (CFY325 and CFY321, respectively). sir1Δ CLB5 versions with the silencers as listed for SIR1 CLB5 (CFY1649, CFY110, CFY2071 and CFY2237); and sir1Δ clb5Δ with versions with the same silencers as listed (CFY2104, CFY2230, CFY2193, CFY2236).

Figure 6.

clb5Δ or FKH1^hc suppressed HMRΔE::ARS1 origin activity. (A) 2-D origin mapping experiments were performed in MATα HMRΔE::ARS1 sir1Δ cells that contained either CLB5 (CFY2237) or clb5Δ (CFY2236). Origin activity at HMRa was assessed with an HMR-specific probe, whereas origin activity at the native ARS1 locus was assessed with an ARS1-specific probe. Two different exposures of HMRΔE::ARS1 blots are shown (dark and light) to facilitate comparison of this origin activity in CLB5 versus clb5Δ cells. (B) 2-D origin mapping experiments with the cells used in A were repeated side by side with clb5Δ sir2Δ cells (CFY2481). (C) The DNA content in actively dividing populations of isogenic yeast cells that varied in terms of their SIR2 or CLB5 genotypes as indicated was determined by flow cytometry. (D) 2-D origin mapping experiments were performed in MATα HMRΔE::ARS1 sir1Δ CLB5 cells containing either an empty 2μ plasmid (vector; pCF225) or a 2μ plasmid with FKH1 (FKH1^hc; pCF480).

Figure 7.

Two models for how FKH1 and CLB5 modulated origin activity and silencing at HMRa (A) FKH1 positively regulates (arrow) and CLB5 negatively regulates (flat head) a common pathway or target referred to as T. T in turn negatively regulates origin firing by normally late-firing replication origins (or origins placed within chromosomal domains that cause late firing). Robust origin firing is incompatible with the assembly of Sir2–4 chromatin. (B) FKH1 and CLB5 regulate target T as described in A, but origin firing is not causally linked to Sir2–4 chromatin assembly. Rather firing by late replication origins is negatively regulated, whereas Sir2–4 is positively regulated by target T.