(Received for publication, March 2, 1995; and in revised form, June 15, 1995)
From the
Using a biochemical approach, we have detected an activity in Saccharomyces cerevisiae extract that displays the same DNA
binding specificity as the mammalian E2F transcription factor and
interacts with TTTCGCGC promoter elements. Additional studies revealed
that this factor, termed SCELA (S. cerevisiae E2F-like
activity), also binds to the closely related SCB promoter sequences.
SCB sites (consensus: TTTCGTG) are involved in the cell cycle
regulation of several S. cerevisiae cyclin genes and have been
shown to interact with the heterodimeric yeast Swi4-Swi6 complex.
However, genetic studies clearly demonstrate that SCELA is not related
to Swi4 or Swi6. These experiments imply that SCB sites are able to
interact with at least two activities: Swi4-Swi6 and SCELA. Because SCB
sites are critical for the periodic activation of cell cycle genes, we
asked whether SCELA is regulated during yeast cell cycle. Employing a
temperature-sensitive strain, we were able to demonstrate that the DNA
binding activity of SCELA oscillates during the cell cycle and reaches
its maximum at the transition between the G and S phases.
Preliminary studies suggest that this fluctuation is mediated by
phosphorylation/dephosphorylation events. Further characterization of
SCELA by UV cross-linking experiments indicate a molecular mass of 47
kDa for this activity. In addition, we present evidence strongly
suggesting that SCELA is actually the DNA binding moiety of a large
300-kDa protein complex. Together, these studies firmly indicate that
SCELA (as part of a larger complex) plays a critical role in cell cycle
regulation of SCB-containing genes, such as CLN cyclins and HO endonuclease. This hypothesis is consistent with other
studies that conclude that the SCB-mediated cell cycle oscillation of CLN cyclins and HO requires activities that are
distinct from Swi4-Swi6. Finally, it is worth mentioning that the
similarities between SCELA and E2F, which is a crucial component in
mammalian cell cycle regulation, extend well beyond the DNA binding
specificity. In analogy to E2F, SCELA oscillates during the cell cycle,
interacts with other cellular activities, and binds to promoter
elements that are known mediators of cell cycle control. We will
discuss possible functions for SCELA in yeast cell cycle regulation and
its relationship to E2F.
During the past decade, tremendous strides have been made toward identifying the molecular mechanisms that govern the eucaryotic cell cycle. Numerous studies clearly demonstrated that cyclin-dependent kinases, in concert with various cyclins, are key elements of the machinery that controls cell cycle progression (1) . Although cyclin-dependent kinase/cyclin-mediated phosphorylation plays a pivotal role in eucaryotic cell cycle regulation, it is clear that periodic activation of transcription is equally crucial for the proper execution of cell cycle events(2) . Although most genes are expressed at approximately constant rates throughout the cell cycle, a limited number of genes display a high degree of variation in their transcription rate during the cell cycle. Genes that fall into the latter category include those encoding products that control DNA metabolism, structural proteins, and components of the basic cell cycle regulatory machinery. Many of these genes are controlled by promoter-specific transcription factors that are themselves subject to cell cycle regulation. For example, the cell cycle-regulated transcription of the mammalian histone H2B gene is mediated by the Oct-1 transcription factor(3, 4) . Oct-1 activity in turn is modulated during the cell cycle by a complex series of phosphorylation events.
Another well-documented example of
transcription factor fluctuation during the mammalian cell cycle
constitutes the E2F transcription factor(5) . E2F was
originally identified as an E1A-dependent activity that mediates
transcriptional activation of the adenoviral E2 early gene(6) .
Subsequent studies revealed that E2F also modulates the transcription
of several cellular target genes: dihydrofolate reductase, DNA
polymerase , cdc2, B-myb, all of which play an
important role in cell cycle progression and DNA
synthesis(7, 8, 9, 10) . In the case
of dihydrofolate reductase and B-myb, functional E2F binding
sites are essential for the cell cycle regulation of these
genes(7, 10) , establishing a direct link between E2F
and transcriptional oscillation during the mammalian cell cycle.
Furthermore, E2F forms a number of distinct complexes that contain
proteins known to be critical for proper cell cycle progression. Among
these proteins are the retinoblastoma anti-oncogene product, two
related activities (p107 and p130), cyclin A, cyclin E, and
cyclin-dependent kinase 2 (11, 12, 13) .
These E2F-containing complexes fluctuate during the cell cycle, again
suggesting that E2F plays an important role in this
process(13, 14) . E2F regulation is not limited to E2F
complexes, but it has been reported that the E2F-1 gene transcription
is induced during the late G
phase of the cell
cycle(15) . Finally, a recent report describes that
microinjected E2F-1 protein is capable of driving quiescent cells back
into the S phase(16) . Together, these data establish E2F as a
pivotal player in cell cycle progression that links the cell cycle
machinery to the transcription apparatus. It is likely that E2F is also
involved in the cessation of cell growth that accompanies
differentiation, because E2F aggregates are regulated during P19 cell
differentiation along the neuroectodermal cell lineage (17) .
Mature neurons exit the cell cycle, and it is conceivable that the
observed fluctuation of E2F complexes participates in this event.
A large body of evidence clearly shows that major aspects of cell cycle control are conserved between mammals and yeast. For example, cyclin-dependent kinases and cyclins are present in both species, and the human cdc2 gene was cloned based on its ability to complement a fission yeast cdc2 mutant(18) . Furthermore, it is now clear that many transcription factors are conserved throughout evolution(19, 20) . These observations prompted us to ask whether there exists a yeast activity that resembles E2F and carries out a similar cell cycle-related function. An additional reason for selecting the yeast system is its suitability for cell cycle investigations because of its powerful genetic tools, vast array of cell cycle mutants, and almost effortless accumulation of extracts for biochemical studies. Although approaches to identify E2F-like factors in yeast based on sequence similarity have not been successful(21) , we decided to take a biochemical approach. This led to the identification of a 30-kDa fission yeast protein that exhibits a number of E2F-like properties, including DNA binding specificity and transcriptional activation(22) . However, this factor is not cell cycle-regulated.
We have now continued our studies in budding yeast, Saccharomyces cerevisiae. Using gel shift analysis, we have detected an activity that displays a striking similarity to the mammalian E2F transcription factor. That is, it recognizes the same DNA sequence, it interacts with other cellular proteins, and it oscillates during the cell cycle. The potential role of this activity in yeast cell cycle regulation is discussed.
Figure 1: Binding specificity of SCELA. Gel shift reactions were initiated with 1 ng of radioactive E2F oligonucleotide and 30 µg of heparin-agarose-purified yeast extract in the presence of 1 µg of herring sperm DNA and 100-fold molar excess of unlabeled oligonucleotides. The following unlabeled oligonucleotides were used: lane 1, E2F (5` ACTAGTTTCGCGCCCTTTCT 3`); lane 2, SCB (5` ACTAGTTTCGTGCCCTTTCT 3`); lane 3, MCB (5` ACTAGTGACGCGTCCTTTCT 3`); lane 4, none. The position of the shifted band is indicated by the arrow. In order to improve resolution of the DNA-protein complex, the free probe was electrophoresed out of the gel.
During the course of our studies, we noticed that the
utilized E2F site (TTTCGCGC) strongly resembles the consensus sequence
(TTTCGTC) of the SCB element family that are important promoter sites
involved in the cell cycle regulation of certain S. cerevisiae genes and that interact with the heterodimeric Swi4-Swi6
complex(25, 26) . In order to investigate whether
SCELA is also able to recognize SCB sites, we performed competition
experiments. As can be seen in Fig. 1, lane 2, the
DNA-binding protein identified in our laboratory not only interacts
with an authentic E2F site, but also recognizes an SCB element
(TTTCGTG). We have extended these DNA binding studies and summarized
the results in Table 1. As expected, the yeast E2F-like factor
interacts with oligonucleotides MYC and E2F, both of which contain
binding sites for the mammalian E2F transcription factor (TTTCCCGC and
TTTCGCGC, respectively). Furthermore, the factor is competed by
oligonucleotides SCB and SCB*, both of which contain well established
binding sites (TTTCGTG and TTTCGAG, respectively) for
Swi4-Swi6(27) . In addition, our yeast activity also binds to
oligonucleotides SCB and SCB3 (contains three contiguous
SCB sites), both of which contain perfect SCB sites but have flanking
sequences that differ from the ones present in SCB and
SCB*(27) . We repeated these studies numerous times and always
detected competition with SCB site-containing oligonucleotides.
Together, this clearly shows that certain members of the SCB site
family are recognized by SCELA as well as Swi4-Swi6. However, we have
identified one authentic SCB site (27) , TTTCGTT (oligo SCB-1),
that does not interact with SCELA (Table 1). Another native SCB
site(27) , TTTCGTA (oligo SCB-2), recognizes SCELA only poorly (Table 1). In contrast, both SCB-1 and SCB-2 bind Swi4-Swi6 with
high affinity ( Table 1and (27) ). Conversely, two
mutated SCB family members that do not recognize Swi4-Swi6 (TTGCGTG in
oligo SCBm1 and TTTTGTG in oligo SCBm2) clearly bind SCELA ( Table 1and (27) ). These experiments unequivocally show
that although SCELA is able to recognize several members of the SCB
site family, its DNA binding specificity differs from the one displayed
by Swi4-Swi6. This is corroborated by the fact that Swi4-Swi6 is not
able to interact with an oligonucleotide that contains only a single
SCB site (27) . (
)SCELA, on the other hand, binds to
single as well as contiguous SCB sites (Table 1). Furthermore,
studies carried out by Andrews and Moore demonstrated that some SCB
family members are virtually identical to the E2F sequence (TTTCGCGC)
and are recognized by Swi4-Swi6 ( Table 1and (27) ).
Close inspection of the genes that are regulated by Swi4-Swi6 led to
the identification of two SCB sites in the HCS26 promoter and
one in the HO promoter that are perfect matches of the E2F
recognition site(27) . This suggests that the E2F binding site
is actually a member of the SCB element family and interacts with SCELA
as well as Swi4-Swi6.
Finally, our data demonstrate that SCELA does not interact with the MCB element (MluI cell cycle box, consensus: TGACGCGT) ( Table 1and Fig. 1, lane 3). This promoter element is involved in the cell cycle regulation of a set of S. cerevisiae genes controlled by the heterodimeric Mbp1-Swi6 complex(28) . Our experiments suggest that binding of SCELA is specific for the SCB-dependent genes.
Figure 2: SCELA is not related to Swi4 or Swi6. Gel shift reactions were performed as outlined in the legend to Fig. 1. Extracts were prepared from the following strains: BJ1991 (wild type, lane 1), JO34 (wild type, lane 2), JO57-6B (devoid of Swi4, lane 3), and JO23 (devoid of Swi 6, lane 4). The position of the gel shift is indicated by the arrow. wt, wild type.
Figure 3:
Characterization of SCELA and associated
activities. A, gel shift reactions containing
heparin-agarose-purified material and labeled E2F oligonucleotide (5`
ACTAGTTTCGCGCCCTTTCT 3`) were subjected to UV light irradiation as
described under ``Experimental Procedures.'' Subsequently,
the material was analyzed on a 10% SDS-polyacrylamide gel. Competitions
were performed with 100-fold molar excess of unlabeled E2F
oligonucleotide (lane 3) or 100-fold molar excess of MCB
oligonucleotide (5` ACTAGTGACGCGTCCTTTCT 3`) (lane 2). Lane 1 shows the reaction without competitor. The positions of
protein markers are shown on the right. B, yeast
extract purified by sequential application of heparin-agarose and MonoQ
chromatography was layered onto a 15-45% (v/v) glycerol gradient.
Following centrifugation, fractions were collected and assayed by gel
shift as described in the legend to Fig. 1. The positions of
protein markers analyzed in parallel are depicted at the top.
The following markers were used: -amylase (200kD) and
apoferritin (440kD). C, glycerol
gradient-fractionated material was loaded onto a 2-ml DNA affinity
column. The column material consisted of ligated double-stranded E2F
oligonucleotides (5` ACTAGTTTCGCGCCCTTTCT 3`) covalently linked to an
agarose matrix. After washing, bound proteins were eluted with buffer
containing 0.5 M NaCl and analyzed on a 10% SDS-polyacrylamide
gel. The positions of protein markers are indicated on the left.
Figure 4: Cell cycle fluctuation of SCELA. A, a S. cerevisiae temperature-sensitive cdc15 mutant strain (W303-cdc15) was used to synchronize cells at mid-anaphase by cultivating the cells at 37 °C for 5 h. Subsequently, cells were shifted to the permissive temperature (30 °C), which releases the M phase block. Thereafter, aliquots of cells were removed at 15-min intervals. Gel shifts were carried out as described in the legend to Fig. 1with equal amounts of extracts prepared from different time points. Budding analysis was performed in parallel. B, yeast extract derived from heparin-agarose chromatography was pretreated with agarose-immobilized calf intestinal alkaline phosphatase (CIAP) for 30 min at 37 °C (lanes 2 and 5). After removal of the immobilized enzyme by centrifugation, gel shift reactions were initiated as outlined under ``Experimental Procedures.'' The action of the phosphatase can be blocked by addition of EDTA (lane 3). DNA binding of the complex is partially restored by addition of the catalytic subunit of kinase A (lane 6). Lanes 1 and 4 depict reactions performed with phosphatase buffer alone.
It is well established that eucaryotic cells frequently use phosphorylation to execute cell cycle control(1) . In a first attempt to determine the mechanism of fluctuation for SCELA, we therefore investigated whether it is sensitive to dephosphorylation. As can be seen in Fig. 4B, pretreatment of yeast extract with calf intestinal alkaline phosphatase results in almost complete loss of the DNA binding activity of SCELA (lanes 2 and 5). DNA binding activity is partially restored by incubating calf intestinal alkaline phosphatase-treated extract with the catalytic subunit of kinase A (lane 6). We have repeated this experiment numerous times and have always observed an increase of DNA binding activity upon kinase A treatment. Furthermore, calf intestinal alkaline phosphatase treatment in the presence of EDTA, which chelates zinc ions essential for calf intestinal alkaline phosphatase activity, does not interfere with DNA binding (lane 3). We have also shown that phosphatase buffer alone has no effect on DNA binding (lanes 1 and 4). These control reactions clearly demonstrate that the loss of DNA binding is a result of phosphatase action.
In summary, we have identified and characterized a novel DNA binding activity, termed SCELA, that is able to interact with several members of the SCB site family in S. cerevisiae. It has been demonstrated that in S. cerevisiae, SCB sites also interact with the heterodimeric Swi4-Swi6 activity(25, 26) . Our data clearly demonstrate that SCELA is distinct from these activities and suggest that SCB sequences can bind to at least two factors: Swi4-Swi6 and SCELA.
Several lines of study strongly
implicate SCELA in the cell cycle regulation of yeast genes. Firstly,
the DNA binding activity of SCELA fluctuates during the cell cycle and
reaches its zenith in late G or early S phase. It is likely
that this fluctuation is mediated by phosphorylation/dephosphorylation
events (cf.Fig. 4B). Secondly, SCELA binds to cis-acting promoter elements that are critical for cell cycle
fluctuation. The sites recognized by SCELA belong to the SCB element
family and reside in the promoters of cyclins (CLN1, CLN2, HCS26) and the HO gene(27) .
It is well established that mutation of the SCB sites disrupts cell
cycle regulation of the above genes. Furthermore, the three cyclins and
the HO gene attain their highest level around the G
phase, which is consistent with SCELA being involved in their
cell cycle control. Thirdly, in the absence of Swi4 and/or Swi6, cell
cycle regulation of SCB-containing genes is impaired but not
eliminated(29, 30) . This observation has led to the
hypothesis that SCB-containing genes are not controlled only by
Swi4-Swi6, but another unidentified regulator is involved. It is
obvious that SCELA fulfills all the requirements for this putative
regulator. We therefore propose that the cell cycle fluctuation of CLN1, CLN2, HCS26, and HO is
controlled by at least two activities: Swi4-Swi6 and SCELA. According
to our results, the DNA binding specificities of SCELA and Swi4-Swi6
are not identical, and SCELA interacts only with a subset of the SCB
sites that are recognized by Swi4-Swi6 (cf.Table 1).
However, all the SCB elements that bind SCELA are found in the
promoters of the four above mentioned genes. This suggests that SCELA
and Swi4-Swi6 are not merely redundant activities but that they mediate
slightly different aspects of cell cycle regulation. Genetic studies
firmly indicate that the cell cycle regulation of CLN1, CLN2, and HO consists of at least three levels, two
of which are controlled by Swi4 and Swi6(30) . It is therefore
likely that SCELA is involved in the third level of cell cycle
fluctuation.
It is noteworthy that SCELA (S. cerevisiae E2F-like activity) exhibits an extraordinary degree of similarity to the mammalian E2F transcription factor: 1) SCELA interacts with several promoter elements that are also recognized by E2F. E2F sites are transcriptionally active in S. cerevisiae, further supporting our hypothesis that SCELA has transcriptional potential (32) . 2) In analogy to mammalian E2F, the DNA binding activity of SCELA oscillates during the cell cycle and is affected by its phosphorylation state(5, 33) . 3) Like E2F, SCELA interacts with several cellular activities and forms a 300-kDa complex in S. cerevisiae. E2F is known to interact with cyclins, cyclin-dependent kinases, and members of the retinoblastoma gene family(11, 12, 13) . At present, it is not clear whether the 300-kDa SCELA-containing complex contains yeast homologues of these mammalian proteins. However, the detection of such activities bound to SCELA would considerably strengthen our notion that SCELA carries out an E2F-like function in yeast. Experiments that will provide answers to these questions and illuminate the role of SCELA in cell cycle control have been initiated. Finally, a previous report describes the identification of a 12-kDa activity in S. cerevisiae that also interacts with E2F elements(32) . This 12-kDa factor does not fluctuate during the cell cycle and is not associated with any other yeast proteins(32) . These results, combined with the molecular mass discrepancy, clearly demonstrate that SCELA is distinct from the 12-kDa protein.