Biochemische Zellphysiologie (B0200) and Intelligente Bioinformatiksysteme (H0900), Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
* Author for correspondence (e-mail: w.pyerin{at}dkfz-heidelberg.de)
Accepted 7 January 2003
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Summary |
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Key words: Protein kinase CK2, Saccharomyces cerevisiae, Cell cycle, Gene expression, Chromatin remodeling
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Introduction |
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Despite being one of the earliest protein kinases discovered
(Burnett and Kennedy, 1954),
the biological role of CK2 is still far from being completely characterized.
This is mainly a consequence of CK2's marked pleiotropic nature. So far, more
than 300 substrates have been identified
(Pinna, 2002
). The nature of
these substrates predominantly proteins related to transcription and
transcription-directed signaling has led to the assumption that CK2
plays a global role in cell regulation, with roles particularly in gene
expression and signal transduction. Many CK2 functions appear to be
individually dispensable, but their collective loss is not. On the basis of
its vital importance and known interactions with components of stress
signaling, growth signaling and survival signaling pathways, a role for CK2 as
a survival factor has been proposed (Ahmed
et al., 2002
).
In S. cerevisiae, CK2 is required for G2/M and G1 phases of the
cell cycle (Glover, 1998).
Previously, we have shown that the cell cycle entry of human cells from G0 and
the subsequent early progression through G1 also requires CK2; its
perturbation in cultured human cells at either the nucleic acid level (by
antisense oligonucleotides) or the protein level (by microinjection of
antibodies or substrate peptide analogs) significantly inhibits this process
(Pepperkok et al., 1993
;
Pepperkok et al., 1994
;
Lorenz et al., 1993
;
Lorenz et al., 1999
).
Concomitantly, expression of immediate early genes such as fos is
suppressed (Pepperkok et al.,
1993
). The immediate early gene products, for their part, trigger
waves of gene expression, reflecting phase-specific hierarchical transcription
programs (Iyer et al., 1999
).
When cells re-enter the cell cycle, they either resume proliferation or go
into apoptosis. Because cells are able to remain in G0 for any length of
period, re-entry may, in a cell sociological context, have important effects,
including cancer development. CK2 has been linked to such pathophysiological
processes (ole-MoiYoi, 1995
;
Seldin and Leder, 1995
;
Landesmann-Bollag et al.,
2001
).
CK2 may carry out its global role in gene expression through common transcriptional features and/or through higher-order structural processes such as nucleosomal organization. The mechanism CK2 uses is not known, but it should be disclosed by comprehensive comparison of CK2-affected genes. We examined the situation at cell cycle entry by using a comparative genome-wide expression analysis of S. cerevisiae CK2 subunit deletion strains. The data not only provide a comprehensive overview of CK2-linked gene expression, but also reveal specific contributions of the individual CK2 subunits and identify functional connections. Several CK2-affected genes encode metabolic pathway and spindle pole body (SPB) components as well as cell cycle control proteins such as cyclins and are differentially affected by the respective CK2 subunits. Strikingly, most CK2-linked genes lack common control features, and a significant proportion of temporarily altered genes encode chromatin-remodeling and modification proteins. Together with available data on CK2's association with and phosphorylation of diverse chromatin components and modifiers, our results strongly suggest a global role for protein kinase CK2 in nucleosomal remodeling processes that are particularly important at transition points such as cell cycle (re-)entry.
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Materials and Methods |
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-Factor-based synchronization
Pheromone-based synchronization was performed as previously described
(Spellman et al., 1998).
Briefly, yeast cells were grown to early log phase (OD600 0.2) and
arrested by the addition of
-factor (12 µg/ml), followed by 2 hours
of cultivation. After centrifugation, arrest release was effected by
resuspension of the cell pellet in fresh (pheromone-free) medium to an
OD600 of 0.18. During further cultivation samples were taken 0, 7
and 14 minutes after release by adding culture aliquots to sterile ice. Cells
were immediately collected by centrifugation, and pellets were flash-frozen in
liquid nitrogen. Synchrony was verified by FACS analysis as previously
described (Nash et al.,
1988
).
Sample preparation, hybridization and scanning procedure
The following procedures were performed as previously described
(Ackermann et al., 2001).
Briefly, total RNA was isolated by applying the hot phenol protocol followed
by mRNA concentration using oligo(dT)-cellulose (Ambion). 3 µg of mRNA were
reverse transcribed using SuperScriptTM Choice System for first and
second strand cDNA synthesis (Gibco BRL); 1 µg cDNA was transcribed in
vitro using biotinylated ribonucleotides (ENZO BioArrayTM High
YieldTM RNA transcript labeling kit, ENZO Diagnostics). After
fragmentation, 15 µg of biotin-labeled cRNA were taken for hybridization to
oligonucleotide arrays (YG-S98 Arrays, Affymetrix) at 45°C, 60 rpm for 16
hours. Staining (performing the three-stain procedure including a signal
amplification step) and washing steps (applying the protocol EukGE-WS2.v4)
were carried out on a GeneChip Fluidics Station 400 (Affymetrix). Arrays were
scanned on a HP GeneArrayTM scanner (Affymetrix). The resulting image
data were analyzed using the GeneChip Expression Analysis software (Micoarray
Suite version 4.0; Affymetrix).
Data processing and correspondence analysis
For data analysis, expression values corresponding to an `absent call' or
to a negative value were set to one, followed by uploading data in M-CHIPS
[multi-conditional hybridization intensity processing system
(Fellenberg et al., 2002)].
This MATLAB-based tool was used for further analysis (normalization and
filtering).
For normalization, loglinear regression accounting for affine-linear
deviations among the different hybridizations
(Beissbarth et al., 2000) was
applied. Each hybridization experiment was normalized with respect to the
gene-wise median of the control condition (time point 0 of the corresponding
wildtype), which was subsequently referred to as the standard. To correct for
distortions, the 5% quantile of each hybridization was subtracted initially.
As the transcription levels of the majority of genes were unaltered in the
different conditions, normalization factors were calculated on the basis of
the majority of the spots. In contrast to Beissbarth et al.
(Beissbarth et al., 2000
),
low-intensity signals were kept, and hybridizations were shifted additively
back to a more natural range, that is, to the level of the standard
(Fellenberg et al., 2001
).
To visualize interdependencies among the high-dimensional data received in
two-way contingency tables with rows representing genes and columns
representing hybridizations, singular value decomposition
(Alter et al., 2000) was used
for reducing dimensionality, and the so-called chi-squared distance among the
data points was approximated from below. Afterwards, genes and hybridizations
were plotted together in a two-dimensional space, where expression
specificities are indicated by the distances from the center.
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Results |
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We found various interdependencies between gene expression profiles and CK2
perturbations. By applying correspondence analysis to the data
(Fellenberg et al., 2001), the
association of individual genes with a specific condition (yeast strain and
time point of interest) was determined from their distance from the center and
location; the further the distance from the center, the more pronounced the
association of the gene with the considered condition
(Fig. 1). For instance,
SLK19, a G1 phase gene whose product is involved in spindle dynamics
control, is modestly repressed in the ckb1
ckb2
strain but strongly repressed in strains
cka1
and cka2
and thus only slightly
(Fig. 1A) or significantly
(Fig. 1B,C) shifted to
wild-type areas, respectively. In addition to such strain-specific transcript
deviations, time-point-dependent deviations are also indicated for each gene:
transcript levels of YGP1, for example, a M/G1 phase gene encoding a
starvation-induced glycoprotein, were elevated at all time points in strains
ckb1
ckb2
and cka2
but not in
cka1
so that the gene is localized at a significant distance
from the center along the 7 minutes mutant axis
(Fig. 1A,C) or within the
center (Fig. 1B), respectively.
By contrast, TSM1, a G2/M phase gene encoding a TATA-binding
protein-associated factor (TAF150), increased transcription at the later time
points in strains cka1
and cka2
but not
ckb1
ckb2
, positioning TSM1 farther
from the center, between the 7 and 14 minutes mutant axes
(Fig. 1B,C), or within the
center, respectively. In summary, correspondence analysis shows CK2-linked
expression of various genes at cell cycle entry and indicates specific
contributions by the individual CK2 subunits.
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By considering the greater than two-fold transcript deviations in at least
one of the CK2-deficient strains at one of the time points as significant, we
identified 283 genes of the 900 characterized as cell-cycle-regulated in
the literature (Spellman et al.,
1998
; Cho et al.,
1998
) as linked to CK2 in their expression
(Fig. 2A). Most transcriptional
alterations (140) were observed in the regulatory subunit double mutant.
Eighty of these were ckb1
ckb2
specific, the
rest was also altered in the catalytic subunit mutants. In the latter, fewer
genes were concerned; 56 and 47 were cka1
and
cka2
specific, respectively. Regarding time point
distribution, the total number of affected genes were comparable at 0 and 7
minutes (157 and 155 genes, respectively) but lower at 14 minutes (128 genes),
suggesting stronger CK2 perturbation effects at earlier cell cycle stages
(Fig. 2B).
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CK2-linked genes relate to various cell cycle phases
Surprisingly, the 283 genes affected by CK2 perturbation at cell cycle
(re-)entry do not represent a collection of early genes as one might expect
from observations with human cultured cells
(Lorenz et al., 1993;
Pepperkok et al., 1993
;
Pepperkok et al., 1994
).
Rather, according to their peak expression
(Spellman et al., 1998
;
Cho et al., 1998
), the genes
can be assigned to various cell cycle stages. Aside from affected early genes
(37 M/G1 phase and 95 G1 phase genes), genes ascribed to S phase (30 genes),
S/G2 phase (44 genes), G2/M phase (71 genes) and diverse phases (6 genes) also
exhibited altered transcriptional levels
(Fig. 3A). For all of these
groups, the number of genes deviating in expression remained more or less the
same over time, except for G1 phase genes. Their number decreased steadily so
that less than half of the alterations at 0 minutes were seen at 14 minutes
(data not shown), suggesting particularly strong CK2 requirement for early
G1-specific gene expression.
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When the CK2 mutant strains were grown as asynchronous permanently cycling cultures (early log phase, OD600 0.46), the transcript levels of 106 cell-cycle-regulated genes were altered. This is a significantly smaller number than that obtained for the cell cycle (re-)entry cultures above. Strikingly, only 23 G1-specific genes were altered (Fig. 3B), indicating that CK2 perturbation in asynchronous cultures does not primarily affect G1-specific genes. G2/M-specific genes form the largest group of affected genes (28 genes); the number of M/G1 genes (21 genes) was similar to that of G1 genes, and 14 and 15 expression alterations were found for S- and S/G2-specific genes, respectively.
By comparing the results from cells synchronized at cell cycle entry with
those in permanently cycling cultures, 58 were identified that were altered in
both cultures. 40 of these were found in the regulatory CK2 subunit mutant, 16
being ckb1 ckb2
specific. In the catalytic CK2
subunit mutants, 29 and 25 genes were altered, and 5 and 6 were
cka1
and cka2
specific, respectively
(Fig. 4). Thus, deletion of the
regulatory CK2 subunits not only affects expression of more cell cycle genes
than deleting a catalytic subunit, it is also characterized by a significantly
stronger persistence. The names, expression and cell cycle phase-association
of these 58 persistently altered CK2-linked genes, together with the
physiological roles of their respective gene products, are given in
Table 1.
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More than two thirds of the persistently altered genes exhibited the same
deviation characteristics in terms of transcription elevation/repression owing
to a certain CK2 mutation in both asynchronous and early cell cycle cultures.
For instance, the expression of PCL2, a gene encoding a G1 cyclin,
was elevated predominantly in the cka2 mutant, whereas
MIF2, encoding a centromer protein (the human CENP-C homologue), was
strongly repressed in the ckb1
ckb2
mutant. A
particularly interesting situation was provided by the S phase genes. The gene
products of all seven continuously altered S phase genes (MET6, MET10,
MET14, STR3, ECM17, ICY2, SAM3) are involved in methionine
biosynthesis and belong to a 20-gene `MET' cluster peaking coordinately during
the cell cycle (Spellman et al.,
1998
). Remarkably, although repressed in the ckb1
ckb2
and cka2
mutants, significantly altered
MET genes exhibited exclusively elevated transcript levels in the
cka1
mutant. We also noticed this CK2-subunit- and
isoform-dependence for the S/G2-specific MET cluster gene MET16. In
summary, the MET genes represent an excellent example of subunit- and
isoform-specific CK2 perturbation effects that are apparently not restricted
to early cell cycle expression but rather persist throughout the cell
cycle.
ECA39, the mammalian homologue of the S/G2 gene BAT1, is regulated
by c-Myc and promotes apoptosis in murine cells
(Eden and Benvenisty, 1999).
Yeast BAT1, encoding a branched-chain amino acid transaminase, is
probably involved in the regulation of the G1/S transition, as
bat1
strains exhibit elevated growth rates owing to a
shortened G1 phase and higher rates of UV-induced mutations
(Schuldiner et al., 1996
). The
persistent BAT1 repression in the ckb1
ckb2
mutant suggests an apoptosis-inhibiting CK2 function.
Evidence for a role of CK2 in apoptotic cellular processes not only via gene
regulation but also at the protein level is increasing. For instance, Li et
al. have recently shown that CK2-mediated phosphorylation is required for
activation of a caspase-inhibiting protein in order to prevent apoptosis
(2002
).
Other S/G2 genes as well as G2/M and M/G1 genes with similar deviation characteristics in early cell cycle and asynchronous cultures are involved in amino acid metabolism and nutrition supply. These include genes for the amino acid permease Gap1, the aromatic amino acid aminotransferase Aro9, the acid phosphatase Pho5 and the propionate metabolism-associated protein Pdh1.
The remaining third of permanently altered genes exhibited diverse
deviation characteristics. Several of the genes encode proteins involved in
the pheromone signaling pathway, including FUS1, KAR4 and
SST2, which encode a MAP kinase, a transcription factor, and a
GTPase-activating protein, respectively. In the ckb1
ckb2
mutant, the genes were strongly repressed during the
early cell cycle but elevated in asynchronous culture, whereas in the
cka2
mutant they were unaltered in the early cell cycle and
repressed in asynchronous culture.
In contrast to genes associated with the pheromone pathway, MET cluster and
nutrition supply, various gene groups that constitute considerable portions of
the CK2 perturbation-affected genes at cell cycle (re-)entry (see below) were
significantly underrepresented among the genes deviating in permanently
cycling CK2 mutant cultures. For example, of 30 genes linked to chromatin
remodeling (CHR) and/or spindle pole body (SPB) organization that exhibited
expression deviations at cell cycle (re-)entry, only two genes (MIF2
and ICY2) were altered in asynchronous cultures. Similarly, of at
least 15 genes deviating at cell cycle (re-)entry whose products are involved
in DNA replication and cell cycle control, only PCL2 and
NDD1 showed altered transcript levels in asynchronous cultures. This
contrast clearly indicates that, although CK2 perturbation considerably
affects early cell cycle expression of genes crucial for the (re-)entry, yeast
cells are obviously able to compensate for these transcriptional deviations
during the course of cell cycle progression.
CK2-linked genes at cell cycle (re-)entry
After subtraction of the 58 permanently altered genes, the remaining 225
genes should more closely reflect CK2-linked genes specifically related to
cell cycle (re-)entry. Below, we present all the genes falling into this
category, grouped according to when their expression peaked during the cell
cycle. These data and the physiological roles of their respective gene
products are presented in Table
2,Table
2,Table
2,Table 2.
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CK2-linked G1 phase genes
84 cell-cycle-regulated genes are significantly affected by CK2
perturbation at cell cycle (re-)entry peak during G1. Generally, if deviation
tendencies are also considered, we found that 90% of altered G1 genes were
altered in at least two of the three analyzed CK2 subunit deletion strains.
About two thirds of these 84 genes exhibited similar deviation characteristics
(repression, elevation) in the CK2 mutants. The rest included genes, such as
HSN1 or YJR154W, whose expression was oppositely affected,
providing further evidence for a functional specialisation of the catalytic
CK2 isoforms and autonomous roles for the regulatory subunits, as already
noted for mammalian cells (Lorenz et al.,
1999; Vilk et al.,
1999
). Most of the significantly altered G1-specific genes were
repressed. One group of such genes is represented by four members of the
11-gene TOS family (TOS6, 7, 10, 11). TOS originally comes from a
multicopy suppressor of DNA topoisomerase II (TopII) mutants, named
TOS1 (Thomas et al.,
1991
). TopII is regulated by CK2-mediated phosphorylation
(Alghisi et al., 1994
);
however, as long as the biological function of the TOS gene family is not
fully understood, a possible connection to CK2 remains speculative.
Because of their temporal coregulation with CLN2 transcription, 76
G1-specific genes peaking at mid-G1 have been categorized into a CLN2 cluster
(Spellman et al., 1998), which
contains many genes involved in DNA replication and other important cell cycle
functions. Collectively, we found 14 altered CLN2 cluster genes, including
CDC45, RNR1 and CLN2 itself. Several CLN2 cluster genes are
induced by the transcription factors MBF and/or SBF
(Spellman et al., 1998
). Six
of the 21 known MBF-regulated genes and 10 of the 24 SBF-regulated genes, as
determined by Simon et al. (Simon et al.,
2001
), were altered; however, we did not observe a common
deviation pattern. The related heterodimers SBF (Swi4/Swi6) and MBF
(Mbp1/Swi6) provide crucial G1-specific transcriptional regulation in the
yeast cell cycle. One of their main functions is the transcriptional
activation of the G1/S cyclin genes CLN1 and CLN2. SBF and
MBF clearly do not work alone; the list of factors that influence SBF and MBF
activities or cooperate with them is growing. Cdc68, a general transcriptional
regulator, is an activator of their components Swi4 and Swi6
(Lycan et al., 1994
) and has
been identified by Glover as a high probability CK2 substrate relevant to cell
cycle progression (Glover,
1998
). Recently, Cdc68 has been found to be associated with all
four CK2 subunits in yeast (Gavin et al.,
2002
; Ho et al.,
2002
). Possible phosphorylation of Cdc68 by protein kinase CK2
might explain the polymorphic deviation patterns for SBF/MBF-regulated genes
in the CK2 mutants, because mutations in CDC68 can repress or
activate the regulation of transcription (Wittmeyer et al., 1997).
During G1, the SPB replicates and expression of several SPB components
consistently peaks at this stage (Spellman
et al., 1998). A remarkably high number of G1 genes affected in
our study encode proteins required for SPB assembly or spindle movement
control: the SPB component genes NUF1 and CNM67 exhibited
divergent expressional alteration characteristics in the diverse CK2 mutants,
whereas HCM1 and PAC11, whose gene products are needed for
SPB assembly, were primarily repressed. Repression was also observed for
SLK19, IPL1 and KAR3, which encode proteins participating in
spindle dynamic control. By contrast, transcript levels of SMC1 and
CSM2, whose products are also directly or indirectly involved in
chromosome segregation processes (CSM2 in meiosis), were exclusively
elevated. Thus, in the early cell cycle, CK2 perturbation causes complex
expressional changes in spindle-function-associated genes.
Three of the affected G1 genes are involved in chromatin remodeling:
CAC2, encoding a chromatin assembly factor subunit, had elevated
expression in the cka2 strain, whereas ESC4 and
ASF1, encoding proteins with chromatin silencing and anti-silencing
functions, respectively, exhibited divergent expression deviations depending
on the CK2 subunit(s) deleted. Recently, ASF1 deletion in yeast has
been reported to induce cell death with apoptotic features
(Yamaki et al., 2001
). An
interpretation of the ASF1 transcript profiles (slightly repressed in
ckb1
ckb2
; significantly elevated in
cka1
) in this context seems difficult.
CK2-linked S phase genes
In common with the G1 genes, we found that most of the 23 S phase genes
that show significant deviation in expression are repressed. In the
ckb1 ckb2
strain, only a single gene,
STU2, whose product may play a role in organization of microtubule
ends at SPBs, exhibited significantly elevated transcript levels. In the
catalytic subunit mutants, the repression:elevation distribution was more
balanced. In addition to STU2, another microtubule/SPB-associated S
phase gene, DAM1, which encodes a protein required for spindle
integrity and kinetochore function, was altered (elevated at 0 minutes in both
cka1
and cka2
strains). This is consistent
with the diverse expression deviations of spindle-function-linked G1 genes
(see above). In common with the G1 group, certain S phase genes that are
significantly affected by CK2 perturbation, such as SWI1 and
TEL2, are associated with chromatin-remodeling processes. In addition
to the eight MET cluster genes also altered in asynchronous cultures (see
above), we identified another three MET genes (MET13, SER33 and
MET28) exhibiting virtually the same isoform/subunit-specific
expression deviations (repression in ckb1
ckb2
and cka2
, elevation in cka1
). For these genes,
the corresponding deviation tendencies were also observed in the asynchronous
culture (data not shown).
CK2-linked S/G2 phase genes
35 S/G2-specific genes exhibited CK2-perturbation-linked expression
deviations at cell cycle (re-)entry and again repression was the major
deviation. As before, genes linked to SPB/microtubule organization, such as
KIP3 (encoding a kinesin-related protein) and CIK1 (encoding
a SPB-associated protein), and genes whose products are involved in chromatin
remodeling, such as HOS3 (encoding a histone deacetylase) and
CSE4 (encoding a CENP-A homolog), exhibited diverse deviation
characteristics. In this context, expression of FKH1, encoding a
forkhead transcription factor involved in cell cycle control, was continuously
elevated in the ckb1 ckb2
strain, but
unaltered in cka1
and slightly elevated (only at 0 minutes) in
cka2
strains, respectively. Fkh1 target gene expression is
high in virtually all cell cycle phases but primarily in G2/M
(Simon et al., 2001
).
Interestingly, Fkh1 binds to promoters of various
chromatin-structure-associated genes, including the S-specific TEL2
gene (see above) and HOS3 (Simon
et al., 2001
). Deviation characteristics of these genes do not
allow us to ascribe these changes directly to altered FKH1
expression; however, the association of Fkh1 with all four CK2 subunits
(Ho et al., 2002
) suggests
that its transcriptional activity might be affected by CK2.
CK2-linked G2/M phase genes
Genes that peak at G2/M phase form the second largest group of altered
genes. Transcription of 54 genes was affected, with repression and elevation
occurring equally frequently. Again, chromatin-structure-linked genes, such as
SRI1 (repressed), whose product interacts with the Swi/SNF and RSC
remodeling complex, and AHC1 (elevated), encoding a Ada histone
acetyltransferase complex component, exhibited expression deviations.
Remarkably, transcript levels of the G1 cyclin Cln3, whose cooperation with
Cdc28 (also named Cdk1) triggers the first step of a new cell cycle in yeast,
was repressed at the early time points in the catalytic subunit mutants,
suggesting a possible cell cycle (re-)entry delay compared with the
wildtype.
Spellman et al. defined a MCM cluster comprising 34 genes peaking at about
the M/G1 boundary that is primarily involved in initiation of DNA replication
(Spellman et al., 1998).
Transcriptional levels of 15 MCM cluster genes classified as G2/M specific
were altered in one or more of the CK2 mutants. Two interesting MCM cluster
genes exhibiting significantly different deviation characteristics between the
regulatory subunit double mutant and the catalytic subunit deletion strains
are represented by SPO12 and TSM1, which encode a putative
positive regulator of mitosis and TAF150 (see
Fig. 1), respectively.
TSM1 expression was significantly elevated at all time points in the
catalytic subunit mutants (except cka1
at 0 minutes) but only
showed an elevation in the ckb1
ckb2
strain at
7 minutes. Conversely, SPO12 was found continuously repressed in the
regulatory subunit double mutant but virtually unaltered in the catalytic
subunit deletion strains. Most MCM cluster genes contain binding sites for the
transcription factor Mcm1 in their promoter regions
(Spellman et al., 1998
). We
detected expression deviations at cell cycle (re-)entry for 10 out of the 27
genes found inside and outside of the MCM cluster identified as Mcm1-regulated
by Simon et al. (Simon et al.,
2001
). Mcm1 is an essential, multifunctional cell cycle regulator
that is highly homologous to human serum response factor
(Kuo et al., 1997
).
Differential phosphorylation of Mcm1 may modulate its transcriptional activity
on specific cofactors. Kuo et al. have identified more than eight differently
phosphorylated Mcm1 isoforms (Kuo et al.,
1997
), including one that is necessary for the osmotic stress
response. There is strong evidence that this specific Mcm1 phosphorylation is
provided by protein kinase CK2, which has also been demonstrated to be
involved in salt tolerance pathways
(Glover, 1998
). It is
conceivable that CK2-mediated phosphorylation of Mcm1 possibly at
additional sites causes expression deviations of Mcm1-regulated genes
related to further physiological and cell cycle functions.
CK2-linked M/G1 phase gene
Transcription levels of 26 M/G1-specific genes were significantly affected
by CK2 perturbation at cell cycle entry. Generally, repression was dominant
(it was observed for two thirds of the altered M/G1 genes) and was most
prominent in the regulatory subunit deletion strain. Several primarily
repressed genes are associated with metabolic pathways and nutritional supply.
Examples include the phosphate transporter gene PHO89 and the
long-chain fatty acid CoA-ligase gene FAA1, which exhibit various
degrees of repression in the three CK2 mutants. The gene products of
FAA1, encoded on chromosome 15, and its putative duplication on
chromosome 8, FAA4, which, by contrast, showed elevated expression in
the catalytic CK2 subunit mutants, are involved in fatty acid import
(Faergeman et al., 2001).
Remarkably, YGP1, which encodes a glycoprotein synthesized in
response to starvation conditions, exhibited elevated transcription levels in
the ckb1
ckb2
and cka2
strains, suggesting an impaired nutrient supply owing to CK2 perturbation.
Six of the altered M/G1 genes belong to a SIC1 cluster, which comprises 27
genes peaking in late M or at the M/G1 boundary
(Spellman et al., 1998). There
was no common expressional deviation pattern for these genes; instead,
characteristics were quite divergent with respect to the direction of
alteration (repression or elevation) and to the CK2 mutants concerned. A
further four SIC1 cluster genes (CTS1, YER124C, YHR143W and
SCW11) affected were classified by Spellman et al. as G1 specific
(see above) (Spellman et al.,
1998
). Similarly, two altered MCM cluster genes (HST4 and
YGP1) have been characterized as M/G1 specific
(Spellman et al., 1998
). The
fact that temporally coregulated genes are categorized into the same cluster
but assigned to different cell cycle stages demonstrates that phasing of
cell-cycle-regulated genes can only be crude
(Spellman et al., 1998
) and
includes many borderline cases.
CK2-linked genes without phase categorization
Another three cell-cycle-regulated genes that peak at multiple phases
(Cho et al., 1998) exhibited
significant deviations in expression. Among these genes whose transcript
levels were primarily altered in the regulatory subunit double mutant were the
DNA helicase gene RRM3 (repressed) and BUD2 (elevated),
which encodes a GTPase-activating protein involved in bud site selection that
is necessary for viability in the absence of the G1 cyclin Cln3
(Benton et al., 1993
).
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Discussion |
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Our analysis shows that the expression levels of a relatively high
proportion of genes, roughly a quarter of the 900 estimated to be
cell-cycle-regulated in yeast (Spellman et
al., 1998; Cho et al.,
1998
), are affected by protein kinase CK2 and are related to cell
cycle (re-)entry from a G0-like state (pheromone arrest). In addition, 58
genes are also altered in permanently cycling cells (asynchronous cultures),
and these represent rather persistent perturbation effects. According to the
expression peaks, both groups comprise genes from various cell cycle phases.
While not surprising for permanently cycling cells, it is quite remarkable for
cells (re-)entering the cell cycle as these cells were expected to depend on
early genes. Genes with persistently altered expression are primarily involved
in metabolic pathways, nutritional supply and the pheromone response. This
group, therefore, is not specific for cell cycle entry per se. These genes
have various expression features in common. For instance, all significantly
affected MET genes, members of a gene cluster responsible for methionine
synthesis, exhibit the same CK2 subunit- and isoform-specific expression
alteration, that is, repression in ckb1
ckb2
and cka2
strains but elevation in the cka1
strain. Similarly, transcription of the PHO genes, a gene cluster
essential for phosphate supply, is strongly repressed in the ckb1
ckb2
strain, and the same is true for their common
transcriptional activator Pho4. As this activator is subject to the control of
a cell-cycle-linked regulatory complex whose components are CK2-independently
expressed, CK2 perturbation uncouples PHO gene expression from its
regulator (Barz et al., 2003
).
It should be noted that as phosphate supply is vital for all cells, this
observation supports the survival factor hypothesis of CK2
(Ahmed et al., 2002
).
The most interesting genes related to cell cycle (re-)entry fall into two
groups. The first group comprises, predominantly, genes linked to cell cycle
progression and exit, including genes associated with cell cycle engine
control as well as cell division and apoptotic machineries
(Fig. 5). The genes linked to
cell cycle engine control include cyclin-encoding genes and genes encoding
cyclin destruction proteins. For instance, CLN3, which encodes the
cell cycle start cyclin in S. cerevisiae, is repressed in
cka strains, indicating a requirement for CK2 phosphorylation
function for expression. CLN2, which encodes a cyclin active at G1/S
transition and is induced by the Cln3-avtivated central yeast CDK Cdc28, is
repressed in ckb
strains, indicating a requirement for the
regulatory CK2 subunits. Further, we find a Cka1-linked increase in expression
in CDC20, which encodes a subunit of the anaphase-promoting complex,
which is involved in proteolysis of M phase cyclins. Another exit-related gene
is SPO12, which encodes a putative positive exit regulator that
requires Ckb1/Ckb2 for expression. Prominent among the genes related to the
cell division machinery are genes encoding SPB components and SPB interaction
partners. These include HCM1, which encodes a transcription factor
involved in the regulation of SPB assembly, the SPB component gene
NUF1, and CIK1, which encodes another SPB-associated
protein. Remarkably, these SPB genes exhibit high CK2-subunit- and
isoform-specific expression dependencies.
|
The second group comprises genes with a striking link to chromatin
remodeling and modification. Chromatin-modifying complexes are divided into
two main groups: ATP-dependent chromatin-remodeling complexes that use energy
for nucleosome sliding along the DNA fibre and thus disrupt histone-DNA
contacts (Gasser, 2002;
Becker, 2002
); and
histone-modifying complexes, which add or remove covalent modifications from
histone tails that influence histone-DNA binding and thus affect gene
activation and silencing (Cosma,
2002
). An exactly coordinated cooperation between
chromatin-remodeling complexes, histone modifyers, general transcription
factors and specific activators is required for successful transcription of a
certain gene. For each cell cycle phase, we find altered expression of genes
encoding proteins involved in this important process (Fig. 6). They are
involved in silencing mechanisms (ESC4 and TEL2) and
nucleosome assembly (ASF1, CAC2 and CSE4) or represent
chromatin-remodeling components (SWI1 and SRI1), a DNA
helicase (MCM6) and histone (de)acetylases (HOS3, AHC1,
HST4). In common with the SPB genes, their expression dependence on
different CK2 subunits and isoforms is highly heterogenous, and in
contrast to the persistent alterations shared deviation patterns for
functionally related and/or commonly regulated genes (e.g. by identical
transcription factors) could not be detected. Conversely, our promoter
analyses of genes assigned to one cluster according to similar deviation
characteristics did not reveal any potential upstream regulatory sequences
that might indicate shared transcriptional regulation (data not shown). The
indicated participation of CK2 in chromatin remodeling might explain this
apparently `undirected' effect of CK2 perturbation at cell cycle
(re-)entry.
In addition to the indicated involvement of CK2 in the expression of
chromatin-remodeling genes at cell cycle (re-)entry, evidence for CK2
implications in remodeling processes at the protein level is increasing. In
yeast, all CK2 subunits have been discovered in complex with histones and
general chromosomal remodeling factors
(Gavin et al., 2002;
Ho et al., 2002
;
Krogan et al., 2002
).
Phosphorylation of the nucleosome assembly proteins NAP-1 and -2 by CK2 is
supposed to control nuclear-cytoplasmic histone translocation in fly and man,
respectively (Li et al., 1999
;
Rodriguez et al., 2000
).
Furthermore, architectural non-histone chromatin components, such as the high
mobility group proteins (HMGs) and the heterochromatin-associated protein 1
(HP1), are phosphorylated by CK2, affecting their DNA binding and gene
silencing activities as well as their interaction with specific transcription
factors (Wisniewski et al.,
1999
; Zhao et al.,
2001
; Krohn et al.,
2002
). It has also been shown that CK2 interacts with the basic
leucine-zipper domains of several transcription factors such as ATF1, CREB,
c-Fos or c-Jun, and in this way indirectly binds to DNA so that it can
phosphorylate not only its immediate interacting partner but also proteins
bound to nearby elements (Yamaguchi et
al., 1998
). The yeast ATF1/CREB homologue Sko1 regulates
transcription of hyperosmotic-stress-induced genes such as the sodium pump
gene ENA1, whose expression is defective in ckb
strains (Glover, 1998
).
Interestingly, Sko1 is important for recruitment of SWI/SNF nucleosome
remodeling complexes and SAGA histone acetylase to osmotic-inducible promoters
(Proft and Struhl, 2002
).
CK2-mediated phosphorylation of human histone deacetylases 1 and 2 (HDAC1/2)
has been reported to promote complex formation and enzymatic activity
(Pflum et al., 2001
;
Tsai and Seto, 2002
;
Sun et al., 2002
). Moreover,
Guo et al. found that CK2-nucleosome association and transcription initiation
coincide at previously inactive chromatin regions, suggesting that CK2
participates in promoting the transition to a transcriptionally active
conformation (Guo et al.,
1998
). Consistent with this, evidence for a differential
CK2-mediated phosphorylation of nucleosomal proteins depending on the state of
transcriptional activity has been presented
(Guo et al., 1999
).
Collectively the data strongly suggest that, independent from CK2 involvement in the regulation of specific gene groups (such as the PHO or MET genes), there is a global role for protein kinase CK2 in gene expression, which is based on its functions in essential chromatin-remodeling processes. If this was the case, a perturbation of CK2 would have a general, far-reaching and partly undirected effect on gene expression that is particularly pronounced at transition points such as cell cycle (re-)entry, where switching from one gene expression program to another requires significant chromatin reorganization. That this effect is particularly reflected by altered transcript levels of remodeling-associated genes might be a simple result of their preferred induction at cell cycle entry where their gene products are particularly needed.
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Acknowledgments |
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