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Corresponding author: Catherine A. Fox, Department of Biomolecular Chemistry, 587 MSC, 1300 University Ave., University of Wisconsin-Madison, Madison, WI 53706-1532., cfox{at}facstaff.wisc.edu (E-mail)
Communicating editor: F. WINSTON
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The SIR1 gene is one of four specialized genes in Saccharomyces cerevisiae required for repressing transcription at the silent mating-type cassettes, HML
and HMRa, by a mechanism known as silencing. Silencing requires the assembly of a specialized chromatin structure analogous to heterochromatin. FKH1 was isolated as a gene that, when expressed in multiple copies, could substitute for the function of SIR1 in silencing HMRa. FKH1 (Forkhead Homologue One) was named for its homology to the forkhead family of eukaryotic transcription factors classified on the basis of a conserved DNA binding domain. Deletion of FKH1 caused a defect in silencing HMRa, indicating that FKH1 has a positive role in silencing. Significantly, deletion of both FKH1 and its closest homologue in yeast, FKH2, caused a form of yeast pseudohyphal growth, indicating that the two genes have redundant functions in controlling yeast cell morphology. By several criteria, fkh1
fkh2-induced pseudohyphal growth was distinct from the nutritionally induced form of pseudohyphal growth observed in some strains of S. cerevisiae. Although FKH2 is redundant with FKH1 in controlling pseudohyphal growth, the two genes have different functions in silencing HMRa. High-copy expression of CLB2, a G2/M-phase cyclin, prevented fkh1 fkh2-induced pseudohyphal growth and modulated some of the fkh-induced silencing phenotypes. Interestingly, deletions in either FKH1 or FKH2 alone caused subtle but opposite effects on cell-cycle progression and CLB2 mRNA expression, consistent with a role for each of these genes in modulating the cell cycle and having opposing effects on silencing. The differences between Fkh1p and Fkh2p in vivo were not attributable to differences in their DNA binding domains.
DIFFERENTIATION of eukaryotic cells into distinct cell types requires changes in both cellular transcription and cell-cycle progression. The single-celled Saccharomyces cerevisiae has served as a model organism for elucidating many of the fundamental transcription and cell-cycle mechanisms common to all eukaryotes (
Studies of the mechanisms that repress transcription of the silent mating-type cassettes, HML and HMR, have revealed several intriguing connections between this form of transcriptional regulation and cell-cycle progression ( allele confers the -mating phenotype. In normal yeast strains, a silenced copy of the MATa allele resides at HMR and a silenced copy of the MAT allele resides at HML. Mutations that cause defects in silencing lead to the simultaneous expression of both a-mating-type and -mating-type genes, which in turn causes a haploid cell to take on characteristics distinct to the diploid cell type, including the inability to mate. Silencing of HMR and HML requires the combined action of small DNA elements called silencers that flank these loci and several DNA binding proteins that bind to silencers directly (silencer-binding proteins;
The effect of cell-cycle perturbations on the efficiency of transcriptional silencing at HMRa can be observed in strains containing mutations in SIR1 but not in strains containing mutations in any of the other three SIR genes (
We identified FKH1 (Forkhead Homologue One) as a gene that could substitute for the function of SIR1 in silencing when expressed from a high-copy plasmid. FKH1 and its closest homologue in yeast, FKH2, are named for their similarity to an evolutionarily conserved family of transcription factors classified on the basis of their forkhead (winged-helix) DNA binding domains (
The data presented in this article provide evidence for roles for FKH1 and FKH2 in transcriptional silencing and pseudohyphal growth in yeast. Interestingly, although the two genes share a redundant function in preventing pseudohyphal growth, they exhibit different functions in silencing. The roles of FKH1 and FKH2 in pseudohyphal growth and silencing are related to their roles in cell-cycle progression, since both the silencing and pseudohyphal phenotypes caused by loss of FKH function could be modulated by high-copy expression of the G2/M-phase cyclin, CLB2. In addition, mutations in the FKH genes cause measurable changes in cell-cycle progression and levels of CLB2 mRNA consistent with their opposing roles in silencing. The differences between Fkh1p and Fkh2p were not attributable to differences in their DNA binding domains.
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The genotypes of the yeast strains and the plasmids used in this study are listed in Table 1 and Table 2. Yeast rich medium (YPD), minimal medium (YM), amino acid and base supplements, and standard yeast genetic methods were as described (
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Strain constructions:
All strains were isogenic to W303-1A except as noted. All gene deletions described in this article were constructed as precise substitutions of the relevant gene's entire coding region with the indicated marker gene. DNA fragments for constructing gene deletions were prepared using the fusion polymerase chain reaction (PCR) method (
To construct an isogenic set of MAT HMR-SSIa strains that differed only in their FKH genotype [CFY147 (fkh1::TRP1 fkh2::HIS3), CFY148 (FKH1 FKH2), CFY149 (fkh1::TRP1 FKH2), and CFY150 (FKH1 fkh2::HIS3)], a MATa HMR-SSIa fkh1::TRP1 strain (CFY65) was crossed to a MAT HMR-SSIa fkh2::HIS3 strain (CFY94). The results from this cross were typical of the results from similar crosses in terms of spore viability and the appearance of segregants with a ruffled colony morphology. Specifically, out of 19 tetrads analyzed from this cross, only 8 contained 4 viable spores, indicating a relatively low spore viability for this strain background (W303-1A). The FKH genotype for only 6 out of the 13 dead spores could be accurately deduced from analysis of the remaining live segregants from the tetrad: 2 were fkh1::TRP1, 2 were fkh1::TRP1 fkh2::HIS3, and 2 were wild type. Thus a clear correlation between individual spore viability and FKH genotype was not evident. However, the remaining 62 viable spores indicated an association between the FKH genotype and the ruffled colony morphology. Specifically, 13 of the viable 62 segregants were Trp+ His+ prototrophs and each of these segregants exhibited the ruffled colony morphology. No other segregants exhibited this morphology. In addition this ruffled morphology could be suppressed by transforming these segregants with a plasmid containing either FKH1 or FKH2 (C. A. FOX, unpublished results).
To examine the levels of Fkh1p and Fkh2p expressed from chromosomal copies of FKH1 and FKH2, respectively, three copies of the hemagglutinin epitope (3xHA) were inserted in frame and just upstream of the codon for the C-terminal amino acid for each gene in a MATa strain (CFY145) using the PCR epitope tagging method for S. cerevisiae (
To test whether the FKH1 and FKH2 DNA binding domains were equivalent in vivo, a FKH1 hybrid gene in which the FKH1 DNA binding domain was precisely replaced with the FKH2 DNA binding domain (FKH1FKH2DBD) was introduced at the FKH1 locus in a MATa strain (CFY863). Specifically, an integrating plasmid containing the FKH1FKH2DBD hybrid gene (pCF662) was cleaved at the unique MscI site within the FKH1 gene and the hybrid gene was introduced into a MATa strain (CFY145) by two-step gene replacement. Integrants containing FKH1FKH2DBD were determined by analytical PCR and diagnostic restriction enzyme digests. To construct a strain in which the FKH1FKH2DBD was the only form of FKH, the MATa FKH1FKH2DBD FKH2 strain (CFY863) was crossed to a MAT fkh2::HIS3 strain (CFY95) and the FKH1FKH2DBD genotype of several His+ segregants was determined.
Identification of FKH1 as a high-copy suppressor of a SIR1 defect:
Two identical plasmids that contained FKH1 were identified in the screen discussed in this article. One isolate (pCF337) from the Yep24 library (
Isolation of SIR1, SIR4, and FKH2 genomic clones from the Yep24 library:
In the course of the experiments described in this article, Yep24 genomic clones containing SIR4 (pCF351) and SIR1 were isolated. The SIR4 plasmid was used for experiments described in Fig 2 and behaved identically to previously characterized SIR4 plasmids.
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In the course of investigating fkh1 fkh2-induced yeast pseudohyphal growth, a genomic clone containing FKH2 (pCF561) was isolated from the same Yep24 library used for the silencing screen. This clone behaved identically to a PCR-amplified clone that contained only the FKH2 gene (pCF399), indicating that the FKH2 phenotypes associated with our engineered FKH2 clones were accurate representations of FKH2 function (P. C. HOLLENHORST, unpublished results).
Plasmid constructions:
To measure the level of expression of Fkh1 and Fkh2 proteins, two clones were constructed that contained FKH1-3xHA (pCF547) and FKH2-3xHA (pCF665), respectively, in pRS426. To construct the high-copy plasmid containing FKH1-3xHA (pCF547), a fragment containing the 3' region of FKH1-3xHA was amplified by high-fidelity PCR from total genomic DNA prepared from a yeast strain harboring a chromosomal copy of FKH1-3xHA (CFY480) and cloned into the BclI/NheI sites of the FKH1 plasmid (pCF480), creating FKH1-3xHA in pRS426 (pCF547). To construct the high-copy plasmid containing FKH2-3xHA (pCF665), a fragment containing the entire FKH2-3xHA locus was amplified by high-fidelity PCR from total genomic DNA prepared from a yeast strain harboring a chromosomal copy of FKH2-3xHA (CFY-854). The amplified fragment was cloned into the SmaI site of pRS426 to create FKH2-3xHA in pRS426 (pCF665). The FKH1-3xHA and FKH2-3xHA plasmids each provided wild-type FKH function (P. C. HOLLENHORST, unpublished results).
A high-copy plasmid containing CLB2 was constructed by synthesizing the CLB2 gene by high-fidelity PCR amplification of total yeast genomic DNA prepared from W303-1A and cloning it into pRS426 (pCF633).
To construct the plasmids used to examine the cellular localization of Fkh1p (pCF587) and the role of the Fkh1p DNA binding domain in FKH1 function (pCF569, pCF574, pCF589, and pCF662), three parent plasmids were constructed (pCF543, pCF555, and pCF557). High-fidelity PCR was used to generate FKH1 fragments that were combined using standard recombinant techniques to generate the following two parent FKH1 clones: (1) An FKH1 clone in pRS426 identical to pCF480 except for a SmaI site engineered at the 5' end of the FKH1 DNA binding domain (pCF543) and (2) an FKH1 clone in pRS426 identical to pCF480 except for a SmaI site engineered at the 3' end of the FKH1 DNA binding domain (pCF555). Fragments from pCF543 and pCF555 were combined to generate a third parent FKH1 clone in pRS426 (pCF557) identical to pCF480 except that it contained two SmaI sites flanking the coding region for the FKH1 DNA binding domain. Each SmaI site introduced a codon for a single glycine residue into the recombinant FKH1 such that this engineered Fkh1p contained one glycine inserted after the proline at position 291 and one after the proline at position 420. This engineered FKH1 functioned identically to wild-type FKH1 (M. MIELKE, unpublished results). To construct the FKH1-Green Fluorescent Protein (FKH1-GFP) fusion gene, the entire coding region for GFP was amplified by high-fidelity PCR from pSP65T (; pCF569). To determine whether the fkh1DBD encoded a stable mutant protein, the coding region for the 3xHA C-terminal epitope was introduced into the FKH1DBD clone (pCF569) to generate FKH1DBD-3xHA in pRS426 (pCF589). To determine whether a Fkh1p containing the Fkh2p DNA binding domain in place of its own possessed FKH1 function, a FKH1FKH2DBD hybrid gene in pRS426 was generated (pCF574). Specifically, the coding region for the FKH2 DNA binding domain was amplified by high-fidelity PCR and cloned into the SmaI site of FKH1DBD (pCF569) to create a FKH1FKH2DBD hybrid gene in pRS426 (pCF574). To construct an integrating version of this hybrid gene, a fragment containing the FKH1FKH2DBD hybrid gene from pCF574 was cloned into pRS406 (pCF662).
Immunoblot analysis of chromosomal and overexpressed versions of Fkh1p-3xHA and Fkh2p-3xHA:
The level of Fkh1p-3xHA or Fkh2p-3xHA in crude yeast extracts was determined as described previously (
RNA blot analysis:
Total yeast RNA was prepared and RNA blot hybridization was performed with probes for a1, SIR4, CLB2, or SCR1, as indicated and as described previously (
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To identify new genes that could provide insights into SIR1 function and the relationship between silencing and cell-cycle progression, we performed a genetic screen to isolate genes that, when expressed at a high copy number, enhanced silencing in a sir1-101 strain. We exploited this recessive hypomorphic allele of SIR1 ( allele, and thus contributed to a sensitized genetic background. The synthetic HMR-E silencer (HMR-SSa), a simplified version of the HMR-E silencer that provides full silencing to HMRa in combination with the HMR-I silencer, requires SIR1 for efficient silencing. Thus a MAT HMR-SSa sir1-101 strain is unable to mate because the simultaneous expression of both a and genes causes the nonmating phenotype of a diploid (
Multicopy expression of FKH1 enhanced silencing in strains containing defects in SIR1:
To test whether multicopy expression of FKH1 enhanced silencing in a strain containing a complete deletion of SIR1, a MAT HMR-SSa sir1::LEU2 strain was transformed with a high-copy plasmid containing FKH1. Multiple copies of either SIR1 or FKH1 conferred the -mating phenotype to this strain, consistent with restored silencing at HMRa (Fig 1A, compare SIR1 and FKH1 to vector). Further analysis indicated that FKH1 was responsible for the enhanced mating efficiency in this mutant yeast strain; a plasmid containing only FKH1 enhanced the mating efficiency of this mutant strain to the same degree as the plasmid isolated from the genomic library. Thus FKH1 restored the ability to mate to this sir1 mutant strain.
As a second measure of the ability of FKH1 to restore silencing to HMRa, the levels of a1 mRNA were analyzed directly by RNA blot hybridization in a MAT HMR-SSa strain harboring either a sir1-101 or a sir1 allele (Fig 1B). In the absence of silencing at HMRa, a1 mRNA is expressed ( strains containing either sir1-101 or sir1, indicating that by this criterion sir1-101 behaved similarly to a sir1 allele (Fig 1B, lanes 1 and 4). Multicopy expression of wild-type SIR1 in either sir1 mutant strain restored full silencing to HMRa as indicated by the disappearance of a1 mRNA (Fig 1B, lanes 3 and 6). Multicopy expression of FKH1 restored some silencing to HMRa in both sir1 mutant strains as indicated by a reduction in the level of a1 mRNA (Fig 1B, lanes 2 and 5). However, FKH1 reduced the levels of a1 mRNA more efficiently in the strain harboring sir1-101 than in the strain harboring sir1::LEU2 (Fig 1B, lanes 2 and 5). Thus, multicopy expression of FKH1 could substitute only partially for SIR1 function in silencing. These data also provide evidence that the sir1-101 allele provided some residual SIR1 function, consistent with the previously published characterization of this allele (
The data presented above indicate that multicopy expression of FKH1 reduced the levels of a1 mRNA expressed from HMRa in strains containing defects in SIR1, consistent with a role for FKH1 in silencing. Two additional experiments provided evidence that FKH1 was mediating its effects on a1 mRNA levels through a bona fide silencing mechanism. First, multicopy expression of FKH1 failed to enhance silencing by HMR-SSa in sir2, sir3, or sir4 strains (M. MIELKE, unpublished results). The SIR2, SIR3, and SIR4 genes encode structural components of silenced chromatin and a requirement for these genes is a hallmark of silencing. Second, we determined whether multicopy expression of FKH1 could silence a gene other than a1 at HMR, since another hallmark of silencing is that it is not gene specific (-mating-type genes at an HMR locus controlled by the synthetic silencer (matap HMR-SS sir1-106; Fig 1C). The -mating-type genes, controlled by a different promoter than the a1 genes (p HMR-SS sir1-106 strain had an a-mating-type and mated with the -mating-type lawn when it expressed multicopy SIR1 because SIR1 silenced HMR, and a-mating is the default mating pathway (-mating-type because the genes at HMR were not silenced (Fig 1C, vector). In contrast, when this strain contained multicopy FKH1, silencing was restored; the strain expressing FKH1 mated with the -mating-type lawn, indicating that the genes present at HMR were silenced in a significant fraction of the cell population (Fig 1C, FKH1). The bimating phenotype indicated that the plasmid containing FKH1 did not silence the genes at HMR as efficiently as the plasmid containing SIR1, consistent with the data obtained from RNA blot hybridization of HMRa (Fig 1B). Taken together, these data indicate that multicopy expression of the FKH1 partially substituted for the function of SIR1 in silencing HMR.
Multicopy expression of FKH1 did not increase levels of SIR4 mRNA:
Previous studies indicate that increasing the dosage of SIR4 enhances silencing at HMR in a strain that lacks SIR1 ( HMR-SSa sir1-101 strain. RNA blot hybridization indicated that multicopy SIR4 expression silenced HMRa in a MAT HMR-SSa sir1 strain (Fig 2A, compare lanes 1–3). However, low-copy expression of SIR4 failed to enhance silencing in this strain (Fig 2A, compare lanes 1 and 4). In contrast to SIR4, multicopy expression of SIR2 or SIR3 failed to silence HMR-SSa in this strain (M. MIELKE, unpublished results).
One possible role for FKH1 in silencing was that it functioned in transcription of SIR4, consistent with the proposed role of the Fkh1p as a transcription factor. Therefore we measured SIR4 mRNA levels in a strain transformed with either a multicopy plasmid encoding SIR4 or FKH1 or a low-copy plasmid encoding SIR4. The level of SIR4 mRNA in the strain expressing high-copy FKH1 was below the level of SIR4 mRNA required for silencing HMRa in this sir1 mutant strain (Fig 2B), indicating that multicopy expression of FKH1 did not enhance silencing by increasing the level of SIR4 mRNA.
FKH1 and FKH2 have different functions in silencing:
The data presented above indicate that multicopy expression of FKH1 could enhance silencing. If these data reflect a natural role for FKH1 in silencing, then one prediction is that a deletion of FKH1 would cause a defect in silencing. Therefore one copy of FKH1 was deleted from a diploid strain in which one HMRa locus was controlled by the synthetic version of the HMR-E silencer and lacked the HMR-I element (MATa/MAT fkh1::HIS3/FKH1 HMR-SSIa/HMRa), and the segregants that resulted from sporulation and dissection of this strain were analyzed. From over 20 tetrads analyzed, every spore was viable, and the growth and morphology of individual segregants were indistinguishable, indicating that the FKH1 gene was not essential. Qualitative analysis of the mating properties of MAT HMR-SSIa fkh1::HIS3 segregants indicated that silencing was not affected dramatically (C. A. FOX, unpublished results). However, the sensitivity of HMR-SSIa permits the detection of small changes in silencing at the level of a1 mRNA expression (Ia locus as demonstrated by the small increase in levels of a1 mRNA compared to an isogenic wild-type strain (Fig 3A, compare lanes 5 and 6). These data are consistent with a positive role for FKH1 in silencing.
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One explanation for the small role of FKH1 in silencing at HMRa and its nonessential role in haploid yeast growth was that FKH1 has overlapping functions with another gene(s). In fact, a query of the yeast genome database revealed a second gene, FKH2, with a high degree of similarity to FKH1. The two genes are 44% identical over the length of FKH1 and 75% identical within their conserved DNA binding domains. This sequence similarity raised the possibility that the two genes might share overlapping functions that could complicate analysis of the role of FKH1. Therefore, to analyze FKH2 and its possible overlapping function with FKH1, one copy of FKH2 was deleted from the diploid strain described above. Analysis of the segregants from over 20 tetrads obtained from sporulation and dissection of this strain (MATa/MAT fkh2::HIS3/FKH2 HMR-SSIa/HMRa) indicated that FKH2 was not required for haploid yeast growth. Unexpectedly, based on the strong sequence similarity between FKH1 and FKH2, a deletion of FKH2 reduced the levels of a1 mRNA expressed by HMR-SSa, consistent with a negative role for FKH2 in silencing HMRa (Fig 3A, compare lanes 4 and 6; a very faint band corresponding to a1 mRNA could be detected in the original autoradiogram; also see Fig 7, below). Moreover, deletion of both FKH1 and FKH2 caused an even further reduction in a1 mRNA levels (Fig 3A, lane 3, and see Fig 7, below). Thus, the silencing phenotypes associated with loss of FKH1 and FKH2 were not predicted from their sequence similarities. In particular, rather than having overlapping functions in silencing, these data indicated that FKH1 and FKH2 had opposing functions in silencing.
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One simple prediction based on the data described above was that multicopy expression of FKH2 would fail to enhance silencing at HMRa. Significantly, in contrast to multicopy expression of either SIR1 or FKH1, multicopy expression of FKH2 failed to enhance silencing in a MAT HMR-SSa sir1-102 strain (Fig 3B). Thus, multicopy expression of FKH2 failed to substitute for SIR1 function in silencing HMRa, consistent with the view that FKH2 behaved differently from FKH1.
One explanation for the inability for multicopy expression of FKH2 to enhance silencing was that some mechanism prevented the overexpression of the Fkh2 protein. Therefore, we compared the levels of Fkh1p and Fkh2p in a population of yeast cells expressing FKH1 or FKH2 fused to the coding region of three tandem copies of the hemagglutinin epitope (3xHA; Fig 3C). Both FKH1-3xHA and FKH2-3xHA provided for wild-type FKH function (P. C. HOLLENHORST, unpublished results; see MATERIALS AND METHODS). The levels of both Fkh1p-3xHA and Fkh2p-3xHA were elevated relative to their normal wild-type levels when either fusion gene was expressed from a high-copy-number plasmid (Fig 3C, compare "chromosomal" to "2 micron"). The levels of Fkh2p-3xHA appeared to be lower than the levels of Fkh1p-3xHA in these experiments, but the larger size of Fkh2p compared to Fkh1p could have contributed to a reduced transfer efficiency of Fkh2p. Regardless, the Fkh2p-3xHA levels could be substantially increased over wild-type levels when FKH2-3xHA was expressed from a high-copy plasmid, suggesting that the inability for FKH2 to enhance silencing was not due to an inability to generate a higher level of Fkh2p. Taken together, these data indicate that FKH1 and FKH2 behaved differently in silencing HMRa.
FKH1 and FKH2 had redundant functions in preventing pseudohyphal growth:
The silencing data indicate that, despite their strong sequence similarity, FKH1 and FKH2 had opposite effects on silencing HMRa. Significantly, a cross between a strain containing a deletion of FKH1 (MATa fkh1::TRP1 HMR-SSIa) and a strain containing a deletion of FKH2 (MAT fkh2::HIS3 HMR-SSIa) indicated that the two FKH genes did indeed share overlapping functions in controlling another form of yeast cell differentiation. Specifically, segregants containing deletions in both FKH1 and FKH2 (fkh1::TRP1 fkh2::HIS3) gave rise to colonies with ruffled edges and a chalky appearance and texture. Furthermore, diploids that were homozygous for deletions in both FKH1 and FKH2 also exhibited this colony phenotype (C. A. FOX, unpublished results). Therefore, FKH1 and FKH2 have redundant functions in controlling yeast colony morphology.
The ruffled colony phenotype observed in yeast strains containing deletions in both FKH1 and FKH2 suggested that the individual cell morphology in these strains might be different from wild-type strains. In liquid culture, yeast strains harboring deletions in both FKH1 and FKH2 exhibited a clumpy, flocculent phenotype characteristic of yeast strains that grow pseudohyphally (C. A. FOX, unpublished results; fkh2 strain grew similarly to pseudohyphal yeast cells, cells were viewed under light microscopy (Fig 4A). Cells containing a deletion of both FKH1 and FKH2 had an elongated morphology relative to wild-type cells or cells containing a deletion of either FKH1 or FKH2 alone. Furthermore, the elongated cells grew in chains in a manner similar to characterized pseudohyphal growth in some strains of S. cerevisiae (
One documented characteristic of pseudohyphal yeast cells is that many of the cells within a colony penetrate or invade solid agar media. This agar penetration causes a "scar" of imbedded cells to be left on the medium after the surface cells are washed off (
1279B, which in its haploid form exhibits pseudohyphal growth under glucose starvation ( fkh2 strain (Fig 4B). Significantly, the strain containing deletions in both FKH1 and FKH2 (W303-1A, fkh1 fkh2) caused agar scarring to a degree similar to that caused by strain 1279B, whereas the wild-type strain used in these studies caused no agar scarring (W303-1A, wild type). A strain containing a deletion of either FKH1 or FKH2 alone exhibited no agar scarring in an analogous experiment (C. A. FOX, unpublished results). Analysis of the plates after washing indicated that the scarring was due to a large number of cells that had penetrated beneath the agar surface. Thus by the second criterion of agar penetration, FKH1 and FKH2 have redundant functions in preventing yeast pseudohyphal growth.
fkh1 fkh2-induced pseudohyphal growth is distinct from nutritionally induced pseudohyphal growth:
Pseudohyphal growth exhibited under nutritional starvation in yeast strain 1279b requires several genes, including FLO11. In particular, Flo11p, a cell-surface flocculin, is a critical terminal gene product required for the pseudohyphal cell morphology and agar scarring exhibited by strain 1279b ( fkh2-induced pseudohyphal growth observed in W303-1A also required FLO11. Therefore, we constructed a strain that harbored complete deletions of FLO11, FKH1, and FKH2 in W303-1A and determined whether this strain formed pseudohyphae and penetrated solid agar media (Fig 5). Significantly, a strain lacking FKH1, FKH2, and FLO11 (fkh1 fkh2 flo11) formed pseudohyphae and penetrated solid agar as efficiently as a strain lacking FKH1 and FKH2 but containing wild-type FLO11 (fkh1 fkh2 FLO11), indicating that FLO11 was not required for the pseudohyphal growth associated with deletion of the FKH genes. In a separate set of experiments, we also demonstrated that STE12, another gene required for pseudohyphal growth in strain 1279b ( fkh2-induced pseudohyphal growth or agar penetration in strain W303-1A (C. A. FOX, unpublished results). Thus, although the pseudohyphal growth caused by deletion of both FKH1 and FKH2 was morphologically similar to the pseudohyphal growth described for strain 1279B, it was distinct by at least two genetic criteria.
Multicopy expression of CLB2 prevented fkh1 fkh2-induced pseudohyphal growth:
Pseudohyphal differentiation in yeast is characterized by growth during the G2/M phase of the cell cycle ( fkh2-induced pseudohyphal growth could be abrogated by multicopy expression of CLB2. Significantly, multicopy expression of CLB2 completely suppressed the formation of elongated cells and pseudohyphae associated with the loss of FKH function (Fig 6A, compare vector to CLB2). In addition, fkh1 fkh2-induced agar penetration was also abolished (Fig 6B). Thus, multicopy expression CLB2 abolished fkh1 fkh2-induced pseudohyphal growth, providing evidence that the pseudohyphal phenotype associated with loss of the FKH genes was related to yeast cell-cycle progression.
Multicopy expression of CLB2 prevented fkh1 fkh2-enhanced silencing:
Previous studies indicated that mutations in CLB2 enhance silencing at HMRa ( fkh2-induced pseudohyphal growth, we postulated that the enhanced level of silencing observed in a fkh1 fkh2 strain might be abrogated by multicopy expression of CLB2. Therefore we measured the a1 mRNA levels in an isogenic set of MAT HMR-SSIa strains (HMR-SSIa) that differed only by their FKH1 or FKH2 genotypes and the plasmid that they contained (Fig 7). Specifically, the same set of strains was transformed with either a 2-µm plasmid (vector) or a 2-µm plasmid containing the CLB2 gene (CLB2). As a control in these experiments, the a1 mRNA levels from two isogenic MAT strains containing wild-type HMRa (HMRa) and differing only in their SIR2 genotype were also measured.
As discussed above, deletion of FKH1 (fkh1) reduced silencing, whereas deletion of FKH2 (fkh2) enhanced silencing as measured by a reduction in a1 mRNA levels (Fig 7, compare lanes 2, 3, and 4). Deletion of both FKH1 and FKH2 (fkh1 fkh2) enhanced silencing further than deletion of FKH2 alone (fkh2); a1 mRNA was undetectable even after a long exposure of the RNA blot in a fkh1 fkh2 strain (Fig 7, compare lanes 1 and 2). Thus the selective growth conditions used to retain the plasmid in these experiments yielded results similar to those observed under rich growth conditions.
If multicopy expression of CLB2 could abrogate the silencing phenotypes caused by deletion of FKH2 (fkh2) or deletion of both FKH1 and FKH2 (fkh1 fkh2), then the fkh2 and fkh1 fkh2 strains harboring a CLB2 plasmid should express more a1 mRNA than these same strains harboring vector alone. Significantly, the level of a1 mRNA expressed from these strains was markedly increased in the presence of multicopy CLB2 (Fig 7, compare lanes 7, 8, and 10 to lanes 1, 2, and 4). Thus, multicopy CLB2 expression abrogated the pseudohyphal growth and silencing phenotypes caused by the simultaneous deletions of FKH1 and FKH2 and the silencing phenotype caused by deletion of FKH2 alone. Multicopy expression of CLB2 did not significantly affect silencing in either the wild-type or fkh1 strains (Fig 7, compare lanes 2 and 3 to lanes 8 and 9), supporting the observation that FKH1 and FKH2 functioned differently in silencing. Moreover, these data raise the possibility that the silencing and pseudohyphal growth phenotypes caused by simultaneous deletion of both FKH1 and FKH2 were associated with similar changes in the cell cycle.
Deletion of the FKH genes affected cell-cycle progression and CLB2 mRNA expression:
The data discussed above indicate that multicopy expression of CLB2 suppressed the pseudohyphal growth and some of the silencing phenotypes caused by deletion of the FKH genes, raising the possibility that deletion of the FKH genes caused defects in cell-cycle progression and CLB2 expression. To test these possibilities, cell-cycle progression and CLB2 mRNA levels were measured in an isogenic set of MATa strains that differed only in their FKH genotype (Fig 8). A growing liquid culture was synchronized in G1 phase by -factor arrest, released from arrest into fresh medium, and at 15-min intervals cell-cycle progression was monitored by counting the number of cells in the G1 (no buds), S (small buds), and G2/M (large buds) phases of the cell cycle (Fig 8A). CLB2 mRNA levels were also measured at each interval by RNA blot hybridization (Fig 8B).
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Deletion of either FKH1 (fkh1 FKH2) or FKH2 (FKH1 fkh2) caused subtle but measurable changes in cell-cycle progression compared to an isogenic wild-type strain (FKH1 FKH2). Specifically, deletion of FKH1 caused a slight increase in progression through the S and G2/M phases of the cell cycle such that the peak of cells in the second G1 phase occurred slightly earlier than the corresponding peak in the wild-type strain (Fig 8A). In addition, the fkh1 FKH2 strain progressed more rapidly and synchronously through S phase and into G2/M phase than did the wild-type strain. In contrast, deletion of FKH2 (FKH1 fkh2) reduced the rate of progress through the cell cycle relative to the wild-type and fkh1 FKH2 strains (Fig 8A, FKH1 fkh2). The filamentous morphology of the isogenic fkh1 fkh2 strain prevented a similar analysis of this strain. However, vigorous sonication of an asynchronously growing fkh1 fkh2 strain indicated that the majority of cells released from filaments had a large two-budded morphology. In contrast, after exposure to -factor, a large number of cells released from filaments after sonication had a single-budded morphology, suggesting that these cells had responded to -factor and arrested in the G1 phase (C. A. FOX, unpublished results). These observations are consistent with the majority of individual cells in an asynchronously growing fkh1 fkh2 culture exisiting in the G2/M phase of the cell cycle. Taken together, these data indicated that reductions in FKH gene function altered cell-cycle progression. Moreover, deletion of either FKH1 or FKH2 alone caused detectable and opposite effects on cell-cycle progression.
Analysis of CLB2 mRNA levels during cell-cycle progression revealed that deletion of the FKH genes also altered CLB2 expression (Fig 8B). In the G1 phase, all four strains expressed very low levels of CLB2 mRNA, as expected (-factor, each strain exhibited a different expression pattern for CLB2 mRNA. Deletion of FKH1 (fkh1 FKH2) elevated the levels of CLB2 mRNA at each time interval relative to wild type, although cycling of CLB2 mRNA was similar. Significantly, the CLB2 mRNA levels in the fkh1 strain did not return to their low G1-phase levels as they did in the wild-type strain during the course of this experiment, although the fkh1 cells continued to cycle similarly to the wild-type strain (Fig 8A). In contrast, deletion of FKH2 (FKH1 fkh2) reduced the levels of CLB2 mRNA at most time intervals. Interestingly, CLB2 mRNA was detected early after release from -factor, but this level remained constant until CLB2 mRNA levels peaked sharply and much later at 90 min. Deletion of both FKH1 and FKH2 (fkh1 fkh2) dramatically reduced the levels of CLB2 mRNA. A shallow cycling of CLB2 mRNA was still observable in this strain, although compared to the other strains in this experiment, cycling of CLB2 mRNA was less evident. Thus reductions in FKH gene function altered CLB2 mRNA expression. Moreover, deletion of either FKH1 or FKH2 alone caused opposite effects on the levels of CLB2 mRNA expressed at most intervals during cell-cycle progression.
The Fkh1p was nuclear and required its DNA binding domain for function:
To test whether Fkh1p functioned through its DNA binding domain, we determined whether Fkh1p was nuclear by constructing a fusion gene in which the coding region for the GFP was fused immediately downstream of the coding region for the Fkh1p DNA binding domain. This FKH1-GFP fusion functioned as wild-type FKH1 (M. MIELKE, unpublished results). Fluorescence microscopy indicated that the fusion protein localized to the nucleus, suggesting that Fkh1p was a nuclear protein (Fig 9A). To test whether the Fkh1p DNA binding domain was required for Fkh1p function, we constructed a FKH1 gene that contained a precise deletion of the coding region for the FKH1 DNA binding domain (fkh1DBD). This fkh1DBD failed to provide FKH1 function in either silencing or pseudohyphal growth (Fig 9B and Fig C). Importantly, deletion of the DNA binding domain did not reduce the steady-state levels of the mutant protein significantly (Fig 9D). Thus, the Fkh1p was a nuclear protein that required its DNA binding domain for its functions in silencing and pseudohyphal growth.
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The DNA binding domains of Fkh1p and Fkh2p were interchangeable:
One explanation for differences between FKH1 and FKH2 was that the two proteins had different DNA binding specificities in vivo and thus regulated different sets of target genes. In this view, pseudohyphal growth would require that expression of both the Fkh1p and Fkh2p gene targets be disrupted, whereas the silencing phenotypes would be affected differently depending on whether Fkh1p or Fkh2p gene targets were affected. Although the DNA binding domains of Fkh1p and Fkh2p are 75% identical, several of the amino acids that do differ between the domains are proposed to regulate DNA binding specificity and affinity in other Fkh proteins ( HMR-SSa sir1 strain to a level similar to that caused by multicopy expression of wild-type FKH1 (Fig 10A). Furthermore, substitution of the FKH1 gene with the FKH1FKH2DBD hybrid gene at the normal FKH1 chromosomal position provided a level of FKH1 function sufficient to prevent pseudohyphal growth in a strain containing a deletion of FKH2 (Fig 10B). These data suggest that the Fkh1 and Fkh2 proteins bound at least a subset of the same gene targets in vivo that were sufficient to modify both phenotypes associated with these genes. Thus, any differences between FKH1 and FKH2 could not be explained simply by differences in the DNA binding domains of Fkh1p and Fkh2p.
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| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The work presented here was based on the prediction that the function of SIR1 in silencing HMRa could be enhanced or bypassed by the overexpression of a particular gene(s). Since changes in cell-cycle progression can enhance silencing at HMRa in strains containing mutations in SIR1 (
Redundant functions for FKH1 and FKH2 revealed by their effects on yeast cell morphology:
In the absence of both FKH1 and FKH2, yeast cells grew with an elongated morphology and in filaments that failed to separate except under vigorous sonication (M. E. BOSE, unpublished results), were flocculent when grown in liquid culture (C. A. FOX, unpublished results), and penetrated solid agar medium. These observations indicate that FKH1 and FKH2 have
