Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston,
Massachusetts 02115
Received 22 July 1998/Returned for modification 19 August
1998/Accepted 28 August 1998
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INTRODUCTION |
The differentiation of a diploid
somatic cell into haploid sex cells via a meiotic cell cycle is a
hallmark of eukaryotic organisms. Gene regulation in haploid and
diploid cells is tightly regulated, although in distinct manners.
Haploids need tight control of the intracellular signals and genes that
trigger a meiotic cell cycle, because meiosis is a lethal event. In
contrast, diploids induce a meiotic cell cycle only upon an appropriate
environmental signal(s) or a developmental program.
The meiotic regulatory cascade has been well studied in the budding and
fission yeasts Saccharomyces cerevisiae and
Schizosaccharomyces pombe. In both organisms, there is a
complex pathway that starts from the mating-type loci and involves
meiosis-specific genes and other genes that have multiple cellular
roles (20, 29). Both organisms contain three copies of
mating-type cassettes; one of these is active and two are silent. There
are two mating-type alleles (a and 
for S. cerevisiae and P and M for S. pombe), and each cassette
expresses two distinct proteins. Finally, two of the mating-type
proteins, S. cerevisiae 2 and S. pombe
Pm, contain homeodomains that are 41% identical and are
likely to recognize similar DNA sequences (15, 28).
In S. cerevisiae, cell type control of meiotic gene
regulation is mediated by transcriptional repression (20).
Diploid a/ cells specifically express the
a1-2 heterodimer that directly represses transcription of
RME1 by binding to its promoter (5). Rme1
directly represses transcription of IME1, the key gene whose product directly activates a set of genes that are required for meiosis
(6). Although IME1 transcription is necessary for
meiosis, it is not sufficient to trigger a meiotic cell cycle without
other cellular signals (13, 23). In starved haploid cells,
IME1 transcription is toxic but not lethal (21).
Thus, the S. cerevisiae mating-type loci activate
IME1 transcription and the meiotic pathway by an indirect
mechanism, i.e., by repressing a repressor.
Meiotic regulation in S. pombe can be reduced to the
transcriptional status of just one gene, mei3
(28). Transcription of mei3 is required for
meiosis and is sufficient to trigger meiosis independently of ploidy
and nutritional stress (19). In wild-type cells,
mei3 is transcribed only transiently in nitrogen-starved diploids (19). Mei3 is not homologous to Ime1
(23), and it mediates its effect on meiosis by a different
mechanism. Mei3 binds to the Pat1 (Ran1) kinase and blocks its
autophosphorylation and the phosphorylation of Ste11 and Mei2, the
physiological substrates (17, 25). Pat1 kinase activity is
required for a mitotic cell cycle; the lack of Pat1 kinase activity
leads to a meiotic cell cycle independently of ploidy and nutritional
stress. Because Mei3 is necessary and sufficient to induce meiosis, it
is critical that mei3 transcription is tightly controlled in
haploid cells.
Genetic experiments indicate that mei3 is regulated by the
coexpression of the four mating-type proteins (29). Two of
the mating-type proteins, Mc and Pc, are
expressed in vegetatively growing cells, whereas the other two
proteins, Mm and Pm, are induced specifically
by nitrogen starvation (15). Thus, in wild-type haploids,
mei3 is not expressed, due to the lack of either
Mm or Pm. Nitrogen-starved diploids in which
the pheromone receptor is bound by pheromone are the only cells in
which both Mm and Pm are expressed
(2). In principle, the mating-type proteins could bind
directly to the mei3 promoter and activate transcription. Alternatively, these proteins could indirectly activate mei3
transcription via another, as of yet unidentified, gene(s). By analogy
with meiotic regulation in S. cerevisiae, one possibility is
that the mating-type proteins directly repress transcription of a
currently unidentified repressor which directly represses
mei3 transcription.
To understand the molecular mechanisms of mei3
transcriptional regulation, we performed a detailed mutational analysis
of the mei3 promoter. We define five positive regulatory
elements that synergistically activate and correctly regulate
mei3 transcription but obtain no evidence for negative
control elements. We demonstrate that one of the critical
mei3 promoter elements is a binding site for Pm
and that Pm functions as a direct activator of
mei3 transcription in vivo. In addition, we suggest that
another mei3 promoter element may contain multiple binding
sites for Mc. These observations indicate that the
underlying logic of meiotic transcriptional regulation in S. pombe is very different from that found in S. cerevisiae. In addition, the analysis here represents the first detailed dissection of a promoter from S. pombe.
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MATERIALS AND METHODS |
DNA molecules.
The starting molecule, pWH4, is a derivative
of pBSKS+ (Stratagene) that has a mutated SacI site and
contains the 1.78-kb HindIII ura4 fragment
cloned between the PstI and SmaI sites and a
1.95-kb HindIII-EcoRI mei3
fragment with a translationally silent mutation of the KpnI
site in the coding region. pWH6 is a derivative of pWH4 in which the
coding and 3' untranslated regions of mei3 are replaced by a
1.9-kb SacI-SpeI ade6 fragment
containing the coding region (with an engineered SacI site
at the ATG initiation codon) and 3' untranslated region. pWH8 is a
derivative of pWH4 in which the mei3 coding region was
replaced by a 1.69-kb SacI-PstI fragment
containing the firefly luciferase coding region (7) with an
engineered SacI site at the ATG initiation codon.
mei3 deletion mutant DNAs were generated by standard methods
with exonuclease III or Bal31 nuclease digestion or by PCR
mutagenesis. In general, promoter fragments contain KpnI
sites at the 5' end, SacI sites at the 3' end, and
SalI (in most cases) or MluI sites (in some
cases) at the junction point. Linker-scanning and base pair
substitution mutations were created by PCR-mediated site-directed mutagenesis with appropriate oligonucleotides. Insertion-scanning mutations were created by linearizing linker-scanning derivatives with
the linker restriction site, blunting the ends, and religating or by
inserting oligonucleotide linkers of appropriate size into the linker
restriction site. All mutations were created in pWH4 and were confirmed
by DNA sequencing. To generate ade6 and luciferase fusions,
Asp718-SacI mei3 promoter fragments
were transferred into pWH6 and pWH8, respectively.
To generate altered-specificity derivatives of Pm, an
830-bp DNA fragment that encodes Pm and extends from the
endogenous BamHI site 140 bp upstream of the transcription
start site to an engineered XbaI site 140 bp downstream of
the translational stop codon was generated by PCR amplification of
S. pombe genomic DNA and cloned between the BamHI
and XbaI sites of pBS-KS. Derivatives of this plasmid
containing desired mutations in the putative DNA recognition helix of
Pm were generated by site-directed two-step PCR
mutagenesis. Wild-type and mutant Pm fragments (generated by cleavage with PstI and Ecl136II) were
subcloned between the PstI and SmaI polylinker
sites of pAD2 (1), a high-copy-number plasmid with an
ade6 selectable marker.
Construction of yeast strains.
S. pombe strains used
in this study are listed in Table 1. DNA
molecules containing the mutant mei3 promoter derivatives were linearized with StuI and introduced into appropriate
ura4 mutant strains, as described previously (4).
To confirm proper integration of these DNAs into the ura4
locus, Ura+ strains were analyzed on Southern blots in
which SmaI-digested genomic DNA (10 µg) was hybridized
with a ura4 probe (32P-labeled 1.78-kb
HindIII fragment). The parental strain (or gene convertants) yields a 5.3-kb fragment, whereas correct integrants yield
a single fragment of 12 kb (size varies slightly depending on whether
the DNA was derived from pWH4, pWH6, or pWH8). Therefore, proper
integration could be assayed on a Southern blot by observing a shift of
a single 5.3-kb SmaI ura4 fragment to a single SmaI ura4 fragment of approximately 12 kb. Incorrect or multiple
integrants contain multiple ura4 fragments of various sizes.
Phenotypic assays.
To assay for sporulation, WP16
(h90 leu1-32 ade6-210 ura4-595
mei3::LEU2+) cells containing pWH4
derivatives were grown overnight at 30°C in 2 ml of YEL plus Ade
medium, washed once in H2O and once in 150 mM NaCl, and
resuspended in the 20-µl residual volume of 150 mM NaCl. Twenty
microliters of this cell suspension was spotted on SPA sporulation
plates, incubated for 3 days at room temperature, and exposed to iodine
vapors for 3 min. Iodine stains the polysaccharides that are found in
the ascus walls and in the cell walls of ascospores but not in
vegetative cells. Wild-type cells appear as a dark ring at the edges of
the spots, representing cells that are in contact with the surface of
the plate and which have mated and sporulated. Cells that are not in
contact with the plate surface do not receive enough nutrients to mate
and sporulate; hence, they are unstained. Sporulation phenotypes are
defined as follows: ++, sporulation indistinguishable from the wild
type; +, noticeably less sporulation than the wild type; ±, barely
detectable sporulation; 
, no detectable sporulation (see Fig. 1).
Sporulation phenotypes are consistent among independent
Ura+ transformants containing the same mei3
promoter construct.
The activity of mei3 promoter mutants in nitrogen-sated
haploids was assayed by using WP17, WP45, or WP162 (heterothallic ura4 ade6
) strains containing pWH6 derivatives
(mei3-ade6 fusions). Ade+ phenotypes were tested
by the ability of these strains to grow on glucose minimal medium
lacking adenine and by their color on rich medium (Ade+
strains are white, whereas Ade
strains are red).
For altered-specificity experiments, pAD2 plasmids expressing wild-type
and mutant derivatives of Pm were introduced into WP16
derivatives carrying wild-type or mutant D alleles by selecting for
Ade+ colonies. Plasmid-borne Pm derivatives
were expressed from the natural promoter and hence presumably only
under conditions in which the endogenous Pm was expressed.
Quantitation of mei3 promoter activity by luciferase
assays.
pWH8 derivatives integrated into the ura4 locus
of WP162 (h mei3 ura4 lys1) were mated to
WP151 (h+ mei3 ura4 pro2) on a sporulation plate
overnight, and diploids were subsequently selected on minimal NBA plus
0.01% Ade plates. Diploid (or haploid control) cultures (50 ml) were
grown at 30°C in EMM-2 minimal medium lacking lysine and proline to
mid-log phase (A600 = 0.7) and then divided into
two portions. One portion (35 ml) was washed twice with water,
resuspended in 30 ml of EMM-2 containing amino acids but lacking
NH4Cl, and incubated at 22°C for 4 h with shaking.
The remaining portion was diluted into 45 ml of EMM-2 lacking lysine
and proline and grown for 3.5 h at 30°C. After measuring the
A600, cultures were washed once with ice-cold
H2O, resuspended in 0.9 ml of buffer (100 mM
KPO4 [pH 7.8], 1 mM dithiothreitol) on ice, and divided
into three samples (approximately 0.4 ml) to which 0.2 ml of
acid-washed glass beads were added. Cells were disrupted by vortexing
in a multitube vortexer on high at 4°C for 12 min, and insoluble
material was removed by microcentrifugation for 5 min at 4°C and
placed on ice. Luciferase activity was measured by adding 100 µl of
each sample to a glass vial containing 200 µl of assay buffer (25 mM
Tricine, 15 mM MgCl2, 5 mM ATP, 500 µg of bovine serum
albumin per ml) and 100 µl of 0.5 µM luciferin that had been placed
in a luminometer. Photon emission was detected for 10 s, and
luciferase activity was normalized to the number of cells. To minimize
experimental variation, luciferase activities of the various strains
were normalized to that of the strain containing the intact
mei3 promoter (502 nucleotides of mei3 promoter
sequence) by using samples that were prepared and analyzed in parallel,
and assays were carried out on independent occasions. The experimental
error is ±30% (except for strains that express very low luciferase
activity, where the error is higher).
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RESULTS |
Defining the minimal mei3 promoter by sequential 5'
deletions.
The mei3 promoter region (defined here as
sequences 502 bp upstream of the mRNA initiation site) contains a
putative TATA element (TATAAG) located between 33 and 28
with respect to the mapped transcription start site (19).
This location is in excellent accord with in vitro transcription
reactions reconstituted with S. pombe components (9,
18), and TATAAG is a moderately functional TATA
element (27). In the vicinity of the mRNA initiation site, the mei3 promoter contains a sequence (GCATCCA,
located between 2 and +5) that weakly resembles a eukaryotic
initiator element (consensus PyPyAN[T/A]PyPy) (14).
To assess whether the isolated mei3 promoter (to 502) is
sufficient to confer nitrogen-starvation-dependent transcriptional induction, a mei3 segment (502 to +94) was fused
translationally to the luciferase structural gene. A stable diploid
strain containing this DNA integrated at the ura4 locus was
grown to mid-log phase in nitrogen-rich medium and shifted to medium
lacking nitrogen. A peak of luciferase activity was observed 3 h
after nitrogen starvation (data not shown), a result in accord with the
observation that mei3 mRNA levels are maximal between 2.5 and 4 h after nitrogen starvation (19). Thus, the
information necessary for proper mei3 transcription resides
in the 502 nucleotides of mei3 promoter and 5' untranslated region.
To define the minimal mei3 promoter region, mei3
DNAs successively deleted for sequences upstream of the mRNA initiation
site were integrated in single copy at the ura4 locus of a
mei3 deletion strain (WP16), and the resulting transformants
were tested for sporulation efficiency. Deletions that contain 502 and
327 bp upstream of the mRNA initiation site (1 and 2,
respectively) have sporulation phenotypes indistinguishable from a
wild-type strain, whereas a deletion retaining 278 bp (3) has
markedly reduced sporulation efficiency (Fig.
1). These results suggest that sequences
downstream of 328 are necessary and sufficient to confer proper
mei3 expression and that the region between 502 and 327
is not essential.
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FIG. 1.
Definition of two positive regulatory regions by
deletion analysis of the mei3 promoter. For each
mei3 deletion mutant, solid bars indicate regions that are
present and blank spaces indicate regions that are deleted; nucleotide
positions at the boundaries indicate the last residue that is present
and are defined with respect to the +1 transcriptional initiation site.
Sporulation phenotypes (assessed by iodine staining) conferred by these
mei3 derivatives are defined as follows: ++,
indistinguishable from a wild-type strain; +, noticeably reduced from
wild-type levels; ±, barely detectable; , no detectable sporulation.
Sporulation assays that indicate the phenotypic range are shown for
selected mei3 derivatives (1, 6, 4, and 5). The
approximate locations of regions I and II are indicated (see text).
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Crude internal deletions define two positive regulatory
regions.
To define elements necessary for mei3
transcription, we analyzed the sporulation phenotypes conferred by a
set of internal deletions within the mei3 promoter region.
Two separable promoter regions are identified from the crude deletion
analysis shown in Fig. 1. Region I (327 to 259) is defined by the
observation that strains containing 7 sporulate normally whereas
strains containing the more extensive 5 fail to sporulate. A
deletion that removes much of region I (6) is partially defective
for sporulation. For region II (158 to 66), the distal end is
defined by 7, which sporulates normally, and the proximal end is
loosely defined by 15, which sporulates with a reduced efficiency.
Most internal deletions within region II are completely unable to
sporulate. However, a small deletion at the distal end, 9,
sporulates with reduced efficiency, suggesting that this derivative
only partially disrupts a positive element in region II.
Dissection of region I into elements A and B.
Region I was
dissected by smaller internal deletions and linker-scanning mutations
(Fig. 2). Elements A and B are defined by
nonoverlapping portions of region I that confer increased sporulation over that conferred by 4, which is completely deleted for region I. Element A (327 to 295) is defined by 6 and 19, which confer partial sporulation activity in the absence of element B. This location
is consistent with the phenotypic distinction between 2 and 3
(Fig. 1). However, deletion of element A has no significant phenotypic
effect if element B is present (e.g., 16 and 17). Element B
(273 to 256) is defined by the comparison of 4, which has an
extremely weak sporulation phenotype, and 17, which confers wild-type sporulation. Sequences between 262 and 273 are important for element B, because their removal leads to reduced promoter activity
(compare 18 and 19). Consistent with this observation, linker-scanning mutations in element B significantly reduce (20) or
eliminate (21) sporulation under conditions where element A is
deleted. However, linker-scanning mutations in element B (ls1 to ls4)
do not affect sporulation in the context of the intact mei3
promoter region, presumably due to compensation by element A and
perhaps by sequences between 273 and 295. In this regard, the
downstream endpoint of element A is poorly defined and may extend
beyond 295. Thus, region I contains at least two partially redundant
positive elements that contribute to mei3 transcription.
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FIG. 2.
Definition of elements A and B by deletion analysis of
region I. The structures and sporulation phenotypes of the indicated
mei3 derivatives are shown as described in the legend to
Fig. 1, with the addition that solid lines indicate the location of
linker-scanning mutations. The approximate locations of elements A and
B are indicated (see text).
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Identification of elements D and E in region II.
Region II was
initially dissected by insertion-scanning mutations that replace 10 bp
of the mei3 promoter region with 12 or 14 bp of heterologous
sequence (Table 2). At least one positive element is located between 158 and 86, because all
insertion-scanning mutations in this region are severely or completely
defective for sporulation whereas 22, 51, and 52 are fully
functional. In addition, there is a distinct element (termed E) that is
defined by a small internal deletion (14 [Fig. 1]) which removes
residues 73 to 60. Element E is clearly separable from the further
upstream elements by virtue of insertion-scanning mutations 51 and
52, which do not detectably affect sporulation; these derivatives also establish the upstream boundary of element E at 72.
Because insertion-scanning mutations affect both nucleotide sequence
and spacing, we further dissected region II with 10-bp linker-scanning
mutations covering sequences from 167 to 27 (Fig.
3). In striking contrast to the result
with the comparable insertion-scanning mutations, nearly all of the
linker-scanning mutations confer wild-type sporulation. The two
exceptions, ls13 (92 to 83) and ls15 (74 to 65), are completely
defective for mei3 promoter function, respectively defining
two positive elements, D and E. Sporulation is reduced, but not
eliminated, by ls19, a linker-scanning mutation that destroys the
putative TATA element. The fact that the ls15 mutation eliminates
sporulation indicates that element E is defined by a specific sequence,
not by an effect on spacing. Further, residues 72 to 67
(TCCGTG) are particularly important for element E function
because linker-scanning mutations on either side of this region, ls14
(82 to 73) and ls16 (66 to 57), confer normal sporulation (Fig.
4).
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FIG. 3.
Definition of elements D and E by deletion analysis of
linker-scanning mutations. The structures and sporulation phenotypes of
the indicated mei3 derivatives are shown as described in the
legend to Fig. 1, except that linker-scanning mutations are defined by
the regions that are mutated; e.g., ls13 substitutes 10 bp for
nucleotides 92 to 83 inclusive. The locations of elements D, E, and
C, which is defined primarily by insertion-scanning mutations (see
Table 3), are also indicated.
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FIG. 4.
Nucleotide sequences of elements C, D, and E and
additional alleles of element D. Element C, which is defined primarily
by insertion-scanning mutations (see Table 3), is a large region that
contains six sequences (boldface letters with arrows indicating
orientation) that strongly resemble consensus recognition sites for
Mc and other DNA-binding domains that contain the HMG
structural motif (8, 10); underlined bases deviate from the
consensus. The most critical bases of elements D and E are shown in
boldface letters and are bracketed by horizontal lines. Shown below are
the structures (mutated base pairs are indicated in small letters) and
phenotypes of the relevant mei3 derivatives used to define
these elements. Derivatives d1 to d4 were designed to assess whether
element D is recognized by Pm in vivo (see Fig. 6).
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Element D is defined by the 92 to 83 linker-scanning mutation
(ls13), and residues 92 to 87 (TTACAC) are particularly important because a disruption of sequences further downstream (51)
does not affect sporulation (Fig. 4). The spacing between elements D
and E is not critical because insertion-scanning mutations (51 and
52) between them have minimal phenotypic effects. Element D
coincides with a sequence (CTTTACACG, located between 86
and 94) that has eight of nine matches with the consensus half-site, A(A/T)NTACAPyPu, recognized by the S. cerevisiae 2
homeodomain (28).
Mating-type protein Pm interacts with element D in
vivo.
Given the element D sequence and the strong similarity
between the 2 and Pm homeodomains (28), we
hypothesized that Pm might activate mei3
transcription in vivo by directly binding to element D of the promoter.
This hypothesis predicts the existence of altered-specificity
derivatives of Pm that function at mei3 promoters containing specifically mutated versions of element D (Fig.
5A). Based on DNA-binding specificity experiments involving the bicoid
and antennapedia homeodomains (11, 12), we generated a
bicoid-like derivative of Pm (S155K) and the following four alleles of element D: a bicoid-like site
(TAATCCC [d3]),
antennapedia-like binding sites (TTATTAG [d2]
and TAATTAG [d4]), and a mutant
site (TTGAGCG [d1]) that presumptively would
not interact with homeodomains (underlining indicates differences from
wild-type mei3 element D).
Multicopy plasmids expressing the Pm derivatives from the
natural promoter were introduced into wild-type strains containing single integrated copies of the mei3 alleles containing
element D mutations (Fig. 5B). As
expected, promoters containing the element D mutations are extremely
defective in the ability to support sporulation in the presence of
normal amounts of Pm (vector only). However, the
bicoid-like S155K derivative confers efficient (and perhaps greater
than wild-type) levels of sporulation (and hence mei3
expression) in combination with the bicoid-like d3 allele but not with
any of the other mutations of element D. In addition, sporulation is
observed when the S155K derivative is introduced into a strain
containing the wild-type promoter but lacking Pm (data not
shown). Thus, the S155K derivative has relaxed DNA-binding specificity
in that it interacts with wild-type and bicoid-like versions of element
D but not with antennapedia-like or mutated forms of this element. The
ability of the S155K derivative of Pm to suppress a
specific mutation of element D in a manner consistent with the known
DNA-binding specificity of bicoid (11, 12) indicates that
Pm interacts with element D in vivo. Consistent with this
conclusion, overexpression of wild-type Pm weakly
suppresses the sporulation defect conferred by the d2, d3, and d4
alleles.
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FIG. 5.
Pm interacts with element D in vivo. (A)
Wild-type Pm interacts with wild-type element D
(TTACACG) but not with the bicoid-like d3 allele
(TAATCCC), indicating that the Pm-element D
interaction is critical for sporulation. An altered-specificity
derivative of Pm (S155K) that interacts with the d3 allele
should effectively recreate the Pm-element D interaction
and permit sporulation. (B) Sporulation phenotypes (assayed by iodine
staining) for strains containing the indicated alleles of element D
(WP20, WP434, WP495, WP496, and WP499) and plasmids overexpressing the
wild type or S155K derivative of Pm. All strains, including
the control containing the pAD2 vector, contain the chromosomal copy of
Pm.
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Unusual properties of element C.
Element C is defined by the
observation that sporulation is severely or completely defective in
strains containing insertion-scanning mutations that locally disrupt
any small region between 158 and 92 (Table 2). Although the
boundaries of elements C and D are not precisely defined (and may
overlap), element C is distinct from element D because its function
cannot be eliminated by linker scanning. Mutations that disrupt the
distal (9, 23, 24) or proximal (47) end of element C confer
some mei3 promoter function, whereas centrally located
mutations are generally nonfunctional. This observation might reflect
disruption of redundant subelements at the ends of element C or the
importance of centrally located residues for element C function.
The unusual feature of element C is that 10-bp linker-scanning
mutations anywhere within this large region do not significantly affect
sporulation (Fig. 3), whereas insertion-scanning mutations at the same
positions drastically reduce mei3 promoter function (Table
2). It is unlikely that the linker sequences themselves encode positive
elements that compensate for the disruption of element C. In all cases
tested, changing the linker sequence does not affect the sporulation
phenotype. In addition, several of the internal deletions that disrupt
element C function have the same junction sequences as the
linker-scanning mutations that have no phenotypic effect. Finally, in
all cases tested, mei3 function is disrupted by small
deletions within element C that decrease spacing by 1, 5, 9, and 18 bp
or by insertion mutations that increase spacing by 1, 2, 4, 8, 9, 10, 12, 14, and 20 bp (Table 2). In the most dramatic example, ls11 and
46 differ only by the deletion of a single base pair yet have
completely different sporulation phenotypes. Taken together, these
observations suggest that any change in spacing drastically affects
element C function whereas a change in the nucleotide sequence has much less effect.
We also analyzed very long linker-scanning mutations in which 27- or
28-bp regions were replaced with heterologous DNA (Fig. 3). Two such
mutations (ls20 and ls22) significantly reduce, but do not completely
eliminate, mei3 expression. However, the 149 to 121
mutation (ls21) has substantial overlap with the above mutations yet
confers wild-type levels of sporulation. These apparently contradictory
observations are suggestive of redundancy in element C (see
Discussion). As expected, a large linker-scanning mutation that also
disrupts element D (ls23) completely eliminates mei3 promoter function.
Quantitation of mei3 promoter activity in mutant
strains.
To quantitate mei3 promoter activity,
mei3 derivatives with a range of sporulation phenotypes were
fused to the luciferase reporter gene and integrated into the
ura4 locus. Luciferase activity was assayed in uninduced
(nitrogen sated) and induced (nitrogen starved for 4 h)
mei3 diploids (Table 3).
Interestingly, all derivatives with noticeable defects in sporulation
(i.e., +, ±, and phenotypes) show <10% of the wild-type
levels of luciferase activity. This observation suggests that a limited
amount of mei3 transcription is sufficient to confer
significant (perhaps even wild-type) levels of sporulation.
Importantly, although experimental errors in the luciferase
measurements make it difficult to discriminate among the classes of
sporulation-defective mutants, it is clear that any visible reduction
of sporulation reflects a severe decrease in mei3 promoter
function. For this reason, we assayed only a limited number of
derivatives with non-wild-type sporulation properties.
However, we examined most of the derivatives that displayed wild-type
sporulation phenotypes, given that a low level of mei3 expression might be sufficient for sporulation. In many cases, these
derivatives express luciferase at levels comparable (within a factor of
2) to that of the wild-type control. However, some of these derivatives
are modestly affected in mei3 promoter function, because
they express luciferase at a level of approximately 30 to 50% of the
wild type; these include a subset of the insertion-scanning mutants
within element C, the large deletion between regions I and II (7),
the deletion between elements A and B (18), and the
insertion-scanning mutation at the downstream edge of element D
(51). Although these partially defective mei3 derivatives
affect our description of the mei3 promoter (see
Discussion), elements A to E are defined by derivatives with much more
severe transcriptional and phenotypic effects.
Evidence against negative elements in the mei3 promoter
region.
Expression of mei3 in haploids is sufficient to
trigger meiosis and initiation of the sporulation pathway, even when
cells are grown in nitrogen-rich medium (19). In this
regard, when vector sequences are fused directly to positions 99 and
20, respectively, the resulting strains are extremely unstable and exhibit iodine staining in nitrogen-rich medium that is characteristic of constitutive sporulation and mei3 expression (data not
shown). In contrast, for all of the mei3 derivatives tested
in this study, we have never observed iodine staining in nitrogen-rich
medium or strain instability characteristic of haploid strains
undergoing inappropriate meiosis. This observation suggests that the
mei3 promoter does not contain negative elements that are
required to repress transcription in haploid cells.
As an independent assay for mei3 expression in haploid
cells, we fused most of our mei3 promoter derivatives to the
ade6 reporter gene and assayed the resulting DNAs for the
ability to support growth on medium lacking adenine. In principle,
elimination of a negative element that represses transcription in
haploids should result in an Ade+ phenotype. However, all
of the strains tested were Ade, indicating that the
mei3 promoter derivatives were inactive in nitrogen-rich
medium. In contrast, mei3::ade6 derivatives in which vector sequences were fused to positions 99 or 20 were Ade+. Thus, despite analyzing numerous derivatives, we have
been unable to generate promoter mutants that express mei3
in nitrogen-rich medium. This observation argues against negative
elements in the mei3 promoter region, and it strongly
suggests that meiotic regulation of mei3 expression is under
positive control.
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DISCUSSION |
Evidence that S. pombe does not utilize a repression
mechanism to prevent meiosis in haploids.
It is essential that
eukaryotic organisms have a mechanism to prevent meiosis from occurring
in haploid cells. In S. cerevisiae, this occurs by an active
repression mechanism involving direct binding of the Rme1 repressor to
the key meiosis-inducing gene, IME1 (6, 22). Loss
of Rme1 function, either by mutation or by a1-2
repression, leads to inappropriate meiosis in starved haploid cells. In
contrast, our detailed mutational analysis of the mei3
promoter provides strong evidence that the failure of haploid S. pombe cells to express mei3 and trigger an
inappropriate meiotic cycle is not due to a transcriptional repression
mechanism. In particular, inappropriate mei3 transcription
in haploid cells was not observed with any of the large number and wide
variety of mutations throughout the mei3 promoter region.
This result is significant because promoter analyses of the type
described here have uncovered negative regulatory elements in a wide
variety of eukaryotic promoters. Indeed, in the case of meiotic
regulation in S. cerevisiae, negative elements were easily
identified in the promoters of IME1 (6, 22) and
IME2, a gene that acts downstream of IME1
(3).
It is impossible to prove that a given promoter does not contain a
negative element. It is always possible that a negative element in a
promoter might be obscured by redundant negative elements or by
overlapping positive elements that are required for expression. Thus,
the validity of the argument that a given promoter lacks a negative
element depends on the number and variety of mutations tested; in this
regard, our analysis is extensive. Furthermore, negative elements are
generally easy to uncover, because even a partial loss of repression
can lead to significant levels of transcription (e.g., 50% repression
results in transcription only twofold below wild-type levels). Because
our assays for inappropriate transcription in haploid cells (iodine
staining and Ade+ phenotype) are very sensitive, it is
likely that the various mei3 promoter derivatives are
inactive in haploid cells and that our analysis would uncover mutants
that only partially relieve repression.
The mei3 promoter is complex and contains at least five
positive elements.
The mei3 region between 327 and
the ATG initiation codon is necessary and sufficient to mediate proper
mei3 expression. Within this region, we have defined five
promoter elements by the following criteria. Element E is defined by a
linker-scanning mutation (ls15) and a small deletion (14) that
eliminate sporulation. Element D is defined by linker-scanning
mutations and multiple base pair substitutions that eliminate
sporulation. Element C is defined by insertion-scanning mutations that
map to a large region (158 to 93) and greatly reduce or eliminate
mei3 expression. Element B is defined by 19, a moderately
sized deletion (295 to 262) that significantly reduces
mei3 expression, and by two linker-scanning mutations that
eliminate sporulation when element A is deleted (derivatives 21 and
22). However, linker-scanning mutations in element B (ls3 and ls4)
have no phenotypic effects in an otherwise intact mei3
promoter. Element A is defined loosely as a region (327 to 295)
that is important for mei3 expression when element B is deleted.
The five promoter elements are separable because regions between them
can be heavily mutated or deleted with no detectable effect on
sporulation and modest or no reduction of mei3 expression (as assayed by luciferase fusions). Thus, elements A and B are separated by 18 (295 to 273), elements B and C are separated by
7 (256 to 158) and the insertion-scanning mutation 22, and
elements D and E are separated by linker-scanning (ls14) and insertion-scanning (51 and 52) mutations. Elements C and D have not been physically separated, but they are functionally distinct because element D can be inactivated by linker-scanning and base pair
substitution mutations whereas element C can be inactivated only by
insertion-scanning mutations. With the notable exception of element C
(see below), the mei3 promoter elements do not require precise spacing relationships with each other.
Several promoter derivatives are modestly affected in mei3
expression, despite displaying a wild-type sporulation phenotype (Table
3). Such partially defective mei3 derivatives can be
interpreted in a variety of ways. First, these derivatives could
inactivate a promoter element whose function is partially redundant
with another element; e.g., the modest effects of linker-scanning
mutations in element C might reflect redundant functions within this
element (see below). Second, these derivatives might partially affect the function of one of the five defined elements by altering residues at the element boundaries. This explanation is likely for 51, which
affects the downstream edge of element D, and for 18, which affects
the poorly defined boundary between elements A and B. Third, in the
case of deletion mutants (particularly the large 7 between regions I
and II), transcriptional defects might arise from altered spacing
between elements. While recognizing that these partially defective
mutants affect our description of the mei3 promoter, we have
defined the five positive elements based on the properties of mutants
with severe phenotypic and transcriptional effects.
It is important to note that our view of the mei3 promoter
region represents a formal description that does not presuppose a
particular molecular mechanism. Furthermore, our description represents
the simplest interpretation that is consistent with all the data. As is
always the case with promoter dissections, we cannot exclude more
complex interpretations. Finally, although we have identified and
localized five elements within the mei3 promoter region, the
precise boundaries of some of these elements (particularly A, B, and C)
have not been defined and the existence of additional positive elements
has not been excluded.
Evidence that Mc binds to element C and acts as an
architectural transcription factor.
Element C is very large (60 bp) and has the unusual property of being inactivated by
insertion-scanning mutations but not by linker-scanning mutations. We
doubt that element C is simply a spacer sequence that maintains the
correct distance between other promoter elements, because
insertion-scanning or deletion mutations that change the spacing
relationship of elements A, B, and E to element C do not have
phenotypic effects. Instead, we suggest that element C contains
subelements that have a precise spacing relationship to each other
and/or to element D. However, the failure of 10-bp linker-scanning
mutations to inactivate element C implies that such putative
subelements would be functionally redundant. Consistent with this
possibility, some linker-scanning mutations within element C reduce
mei3 expression (Table 3), and a large linker-scanning
mutation (ls20) can virtually inactivate element C.
Interestingly, element C contains six sequences
(ATTGTA, ATTATT, TTTGTT,
ATAGTT, TTTGTT, and TTTGAT)
that resemble the consensus recognition sequence,
(A/T)TTGTT, for DNA-binding proteins with an HMG structural
motif (10). One of the mating-type proteins, Mc
(15), contains a region that strongly resembles (32%
identity, 64% similarity over 76 amino acids) the HMG domain of SRY, a
protein that determines male sex in mammals. Furthermore,
Mc behaves like a typical HMG protein; it binds the HMG
consensus sequence with high affinity primarily through interactions
with the minor groove, and it severely bends DNA (8). For
these reasons, we speculate that element C consists of multiple
Mc interaction sites and that Mc binds and
bends DNA to form a highly specific nucleoprotein structure that is
important for mei3 transcription (Fig.
6). This model, in which Mc
functions as an "architectural transcription factor"
(26), could account for the apparent paradox of functional redundancy and severe spacing constraints, particularly if
protein-protein interactions between Mc molecules
energetically compensate for weak protein-DNA interactions due to
linker-scanning mutations.
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FIG. 6.
Different regulatory logic for cell type control of the
key meiosis-inducing genes in fission and budding yeasts. In S. pombe, diploids express two distinct DNA-binding proteins,
Mc, which contains an HMG domain, and Pm, which
contains a homeodomain homologous to 2. Pm binds to
element D of the mei3 promoter, and Mc is likely
(but not directly shown) to bind multiple sites within element C. These
proteins, together with putative proteins interacting with elements E
and A or B, stimulate transcription of mei3, the trigger for
meiosis. Haploids do not undergo meiosis because Pm and
perhaps these other proteins are not present or active. Given the
unusual properties of element C, we speculate that mei3
transcription requires the formation of an enhancesome that depends on
DNA-binding and bending properties of the HMG protein Mc
(see text). In S. cerevisiae, haploids do not undergo
meiosis because they express Rme1, a repressor that binds the promoter
of IME1, the key meiosis-inducing gene. Diploids can undergo
meiosis because they specifically express the a1 and 2
homeodomain proteins, which form a heterodimeric repressor that
directly binds the RME1 promoter and blocks production of
Rme1. Hence, a1-2 indirectly cause the expression of Ime1
by repressing a repressor, whereas Pm and presumably
Mc directly bind the mei3 promoter and activate
transcription. Furthermore, 2 functions as a repressor whereas the
homologous Pm functions as an activator. The key
meiosis-inducing proteins, Mei3 and Ime1, have unrelated molecular
functions, and the pathways downstream of these proteins are completely
different.
|
|
Pm interacts with element D and directly activates
mei3 transcription.
Several lines of evidence indicate
that element D interacts with Pm in vivo. First, element D
strongly resembles an 2 binding site, and the Pm
homeodomain is 41% identical (70% similar) to the 2 homeodomain.
Although the recognition helix of Pm is five residues
shorter than that of 2, all of the residues that contact bases or
phosphates (28) are conserved between 2 and
Pm, including homeodomain residue 50 (S155 in
Pm), which plays an important role in DNA-binding
specificity (11). Second, mutational analysis of element D
indicates that the critical base pairs for sequence recognition by 2
are essential for mei3 expression. Third, overexpression of
Pm partially suppresses the sporulation-defective phenotype conferred by several element D mutations. Fourth, and most convincing, the S155K derivative of Pm strongly suppresses a specific
mutation of element D; it permits mei3 expression from the
d3 allele but not from other mutant forms of element D. Furthermore,
this allele-specific suppression reflects a direct protein-DNA
interaction between Pm and element D because the
combination of compensating mutations is consistent with the known
DNA-binding specificity of bicoid (11, 12). Given that
Pm interacts with element D in vivo and that element D is
positively required for mei3 transcription, we conclude that
Pm functions as a transcriptional activator at the
mei3 promoter.
The mei3 promoter acts as an on-off developmental
switch because transcription requires synergistic activation by
multiple elements.
It is commonly observed that efficient
transcription of eukaryotic genes requires the combinatorial and
synergistic action of distinct elements in the promoter region.
Wild-type levels of mei3 transcription require the
synergistic activation of elements C, D, and E and region I (which
contains elements A and B). Mutations in any of these four components
drastically reduces mei3 expression.
In the context of wild-type S. pombe cells, the requirement
for at least four independent elements restricts mei3
expression to circumstances when the proteins bound to these elements
are functionally active. At a minimum, these circumstances include the
presence of the M and P mating-type loci, the pheromone signal, and the
environmental condition of nitrogen starvation. Given that the
mei3 promoter elements appear to be unrelated in DNA sequence, we presume that Pm, Mc, and two or
three other proteins must be present and active under the specific
conditions that are appropriate for mei3 transcription (Fig.
6). Under other conditions, we presume that one or more of these
proteins are absent or inactive. Thus, the mei3 promoter
constitutes an on-off developmental switch that is responsible for the
tight regulation of mei3 expression that is critical in the
S. pombe life cycle.
In several respects, the complex mei3 promoter resembles the
human beta interferon enhancer. Transcription of the beta interferon gene is restricted to the specific circumstance of virus infection, and
this restriction reflects the requirement for the binding of multiple
and distinct proteins to their cognate elements in the enhancer
(16, 24). In this sense, the beta interferon enhancer
functions as an on-off environmental switch. The function of the beta
interferon enhancer depends on the architectural transcription factor
HMG-I, and it is exquisitely sensitive to spacing changes between
individual elements. Thus, transcription of the beta interferon gene
requires the assembly of a precisely structured multiprotein-DNA complex termed an "enhancesome" (24). By analogy, we
speculate that developmentally regulated expression of mei3
might involve the assembly of an enhancesome (Fig. 6), a structure not
previously described for unicellular eukaryotes. However, the
observation that elements A, B, E, and perhaps D do not require precise
spacing relationships suggests that a putative mei3
enhancesome might be restricted to element C and Mc. In
contrast, the beta interferon enhancesome is composed of distinct
elements that interact with different proteins.
The regulatory logic for cell type control of the key
meiosis-inducing gene differs between fission and budding yeasts.
In many respects, mating-type control of sporulation is similar in
S. cerevisiae and S. pombe. Both organisms
contain one active and two silent mating-type cassettes, each of the
two mating-type alleles expresses two proteins, and S. cerevisiae 2 and S. pombe Pm contain
homologous homeodomains. Nevertheless, in contrast to the S. cerevisiae mating-type proteins, which directly repress the Rme1
repressor of the key meiotic control gene (IME1), the S. pombe mating-type proteins (Pm and probably
Mc) directly activate mei3, the gene that
triggers meiosis (Fig. 6). Furthermore, the homologous homeodomain
proteins regulate transcription of the direct target genes in opposite
manners; i.e., 2 functions as a repressor whereas Pm
functions as an activator. Finally, to deal with the critical issue of
preventing expression of meiosis-inducing genes in haploid cells,
S. cerevisiae uses an active repression mechanism whereas
S. pombe does not appear to do so. Thus, our results
demonstrate that the underlying logic of cell type control of the key
meiosis-inducing gene in fission yeast is fundamentally different from
that in budding yeast.
We are indebted to Vicki Chandler and Jo Ann Wise for permitting
W.J.v.H. to carry out some of these experiments in their laboratories.
We thank Charles Hoffman, Amar Klar, Maureen McLeod, Fred Ponticelli,
Fred Winston, and Jo Ann Wise for plasmids and/or yeast strains. We
thank Fred Ponticelli for technical advice in the early stages of this
work and Brendan Cormack, Mark Lee, and Jo Ann Wise for comments on the
manuscript. This work was supported by a predoctoral fellowship to
W.J.V.H. from the Howard Hughes Medical Institute, a National
Institutes of Health (NIH) postdoctoral fellowship to D.R.D., and
research grants to K.S. from NIH (GM30186 and GM53720).
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Abstract
Introduction
