From the Departments of Microbiology and Cell Biology
and § Biochemistry, Indian Institute of Science, Bangalore,
560 012, India and the ¶ Institute for Genomics & Integrative
Biology, New Delhi, 110007 India
Received for publication, December 20, 2001, and in revised form, October 17, 2002
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ABSTRACT |
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Organisms respond to environmental stress by
adopting changes in gene expression at the transcriptional level. Rpb4,
a nonessential subunit of the core RNA polymerase II has been proposed
to play a role in non-stress-specific transcription and in the
regulation of stress response in yeast. We find that in addition to the
temperature sensitivity of the null mutant of Rpb4, diploid null
mutants are also compromised in sporulation and show morphological
changes associated with nitrogen starvation. Using whole genome
expression analysis, we report here the effects of Rpb4 on expression
of genes during normal growth and following heat shock and nutritional starvation. Our analysis shows that Rpb4 affects expression of a small
yet significant fraction of the genome in both stress and normal
conditions. We found that genes involved in galactose metabolism were
dependent on the presence of Rpb4 irrespective of the environmental
condition. Rpb4 was also found to affect the expression of several
other genes specifically in conditions of nutritional starvation. The
general defect in the absence of Rpb4 is in the expression of metabolic
genes, especially those involved in carbon metabolism and energy
generation. We report that various stresses are affected by
RPB4 and that on overexpression the stress-specific
activators can partially rescue the corresponding defects.
The survival of a cell depends on its ability to respond rapidly
to environmental changes. This involves sensing small changes in
multiple parameters, integrating the signals together, and rapidly
changing the expression profile. Temperature and nutrient levels are
prone to frequent fluctuations in the environment and elicit rapid and
transient genome-wide changes. Although transcriptional changes during
stress response have been studied extensively, relatively little is
known about the contribution of core RNA polymerase II in bringing
about these changes in the transcriptional program of the cell.
The yeast RNA polymerase II is composed of 12 subunits, Rpb1-Rpb12.
Rpb1, Rpb2, and Rpb3/Rpb11 are homologs of the bacterial core RNA
polymerase subunits. Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12 are shared
between the three RNA polymerases, I, II, and III (1). Rpb5 has been
shown to have a role in transcriptional activation (2). Rpb4 and Rpb9
are nonessential for normal growth, but their deletion results in
temperature sensitivity (3, 4). Rpb4 interacts with Rpb7, a smaller
essential subunit, to form a subcomplex, which dissociates from the
polymerase on mild denaturation (5). The stoichiometry of Rpb4 within
the polymerase increases during the stationary phase (6).
Another interesting feature of the Rpb4 subunit is that in its absence
cells exhibit a wide variety of phenotypes associated with stress
conditions. An rpb4 We report here that the stress-associated effects of Rpb4 extend beyond
temperature sensitivity. Heat shock, a short and transient exposure to
high temperature, reveals a transcriptional pattern in
rpb4 Strains and Plasmids--
The rpb4 Sporulation--
The yeast cultures were grown until mid-log
phase at 25 °C in YEPD (13). The cells were harvested, washed with
sterile water, and transferred to sterile sporulation medium (1%
potassium acetate). Sporulation counts were performed using a
hemocytometer after 4 days and are expressed as percentages of total
cells (number of tetrads × 100/number of cells); at least 500 cells/sample were counted. The cells were harvested after 12 h of
incubation in sporulation medium for RNA isolation.
Nitrogen Starvation--
Yeast cultures were grown until mid-log
phase at 25 °C in YEPD (13). The cells were harvested, washed with
sterile water, and transferred to sterile SLAD medium (0.67% yeast
nitrogen base without ammonium sulfate and amino acids, 0.05 mM ammonium sulfate, 2% glucose). The cells were harvested
after 12 h of incubation in SLAD medium for RNA isolation.
RNA Isolation and Microarray Analysis--
A detailed account of
the methodology used is published (12). Briefly, the yeast whole genome
microarray slides were procured from the Microarray Facility of the
Ontario Cancer Centre. RNA was isolated using liquid nitrogen lysis
protocol and labeled using a Micromax TSA kit (PerkinElmer Life Sciences).
Glucose Estimation--
Yeast cells were inoculated from
overnight cultures into synthetic medium at similar densities and grown
in a shaker (250 rpm) at 25 °C (13). Sterile medium was used as
control for the amount of glucose. The samples were recovered at
appropriate times during the growth curve, the cells were removed by
centrifugation, and the supernatant was used for glucose estimation.
100 µl of suitable dilution was mixed with 3,5-dinitrosalicylic acid
(DNSA) reagent and boiled for 10 min. The tubes were cooled to room
temperature, and the solutions were diluted to 1 ml. Absorbance at 540 nm was used to calculate the glucose concentration from a standard
graph. The concentration of glucose at the time of inoculation was
taken as 100%. The percentage glucose utilized at time
t = [amount of glucose at t0 Data Bases and Software Used--
Data from the Munich
Information Centre for Protein Sequences data base, the
Saccharomyces Genome Data Base, and the Yeast Proteome Data
Base were used to classify genes according to their function (14-16).
SCPD (Saccharomyces cerevisiae Promoter Database) was
used to retrieve promoter sequences, and MEME (Multiple Em for
Motif Elicitation) was used to analyze them (17). Clustering of gene
expression data was carried out using CLUSTER (18). Data from published
microarray experiments used to compare with data generated in our
experiments was retrieved using yMGV (Yeast Microarray Global
Viewer) (19).
Rpb4 a nonessential subunit of the RNA polymerase II, is required
for the survival of yeast cells at extreme temperatures above 34 °C
and below 12 °C (3). At room temperature, the rpb4
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant is unable to survive at extreme temperatures (>34 °C and <12 °C) and dies rapidly
during a prolonged stationary phase (3, 6). Recently, several groups have shown that Rpb4 plays an important role in the activation of many
genes (7, 8) but has a milder effect on the basal expression of these
genes. Expression analysis of specific genes showed that RNA polymerase
II from the mutant lacking Rpb4 cannot transcribe some genes (9). Whole
genome expression profiles and two-dimensional gel electrophoresis of
proteins have shown that in the absence of Rpb4, the polymerase is
inactivated at high temperature (37 °C for 45 min to 1 h) (10,
11). Recently, we have reported the initial observations from whole
genome expression analysis of rpb4
mutant before and
after heat shock (12).
that is strikingly different from that of wild type
cells. We have studied whole genome expression profiles of haploid and diploid yeast cells, under conditions of normal growth and starvation. Comparison of expression profiles of rpb4
and wild type
cells show that in the absence of Rpb4, some genes involved in specific pathways of stress response are down-regulated in the corresponding condition. In general, in the absence of Rpb4, the transcription of
many genes involved in key physiological pathways like glycolysis and
energy generation is affected. Nevertheless, Rpb4 is not an essential
gene. Therefore, the aberrant stress response defects shown by yeast
cells in the absence of RPB4 may be a consequence of an underlying
defect in fine tuning the expression of metabolic genes.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
wild type strains (SY10-1: Mat a, his3
-200, ura3-52, leu2-3,
112, lys2-1, rpb4
::HIS3/pPS2; SY10-2:
Mat a, his3
-200, ura3-52, leu2-3, 112, lys2,
rpb4
::HIS3/pNS114) have been described
before (8). The corresponding diploid rpb4
/rpb4
strain
was generated by crossing SY10 (Mat a, his3
-200, ura3-52, leu2-3, 112, lys2-1, rpb4
::HIS3) to its
Mat
sibling. The diploids were also transformed with pPS2 (vector)
and pNS114 (pPS2 carrying RPB4 gene). The CGX19
(MATa/
, ura 3-52/ura 3-52, shr3-102/shr3-102) strain was
a kind gift from Dr. Erica Golemis. PHD1 as well as IME4 open reading frames were PCR-amplified and expressed in
yeast from multicopy plasmids. The PCR primers used for IME4
amplification were IME4f (5'-CGG GAA TTC AAT AAA AGT TGT
AAG CAG GC-3') and IME4r (5'-CCC GCT GCA GTC TTT TTT ATG
ACC A-3'), and those for PHD1 were PHD1f (5'-AGC AGA
TCT ATA TGT ACC ATG TCC C-3') and PHD1r (5'-CCC GAA
TTC ATG ATA ATT CAT TTT TTG C-3'). PCR amplification was carried
out by routine methods as described (13). The media used for routine
growth and manipulation of yeast cultures were made as described
(13).
amount of glucose at t] × 100/amount of glucose at
t0 and plotted against time in hours.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant grows slowly and loses viability rapidly in stationary phase (3,
6). We constructed homozygous null mutants at the RPB4 locus
to study the effect of Rpb4 on other stress responses. We found that
the rpb4
/rpb4
strain was unable to
sporulate efficiently compared with a
rpb4
/rpb4
strain carrying a plasmid
expressing RPB4 under the control of its own promoter (Fig.
1A). Overexpression of
RPB4 resulted in a further increase in sporulation levels
(Fig. 1B). We also observed that under nitrogen starvation
conditions, rpb4
/rpb4
cells were more
elongated than the wild type and bud in a unipolar budding pattern.
This pattern resembles pseudohyphae formation. Expression of
RPB4 restored the normal cell shape and budding pattern
(Fig. 1C).
View larger version (46K):
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Fig. 1.
Diploid cells lacking Rpb4 are defective in
sporulation but predisposed to pseudohyphae formation.
A, the rpb4D/rpb4 strain
transformed with a vector (left panel) or a RPB4
gene containing plasmid (right panel) was grown in rich
medium and transferred to sporulation medium. The cells were
photographed (at 800×) after 4 days. B, sporulation
percentage after 4 days was measured and plotted as a percentage of
total cell numbers (at least 500 cells/strain). C, the two
strains used in A were grown in SLAD medium and photographed
(at 400×) after 18 h. The left panel shows
rpb4D/rpb4
cells with vector, whereas the
right panel shows rpb4D/rpb4
cells
with the RPB4 (CEN) plasmid.
Previous studies on the various aspects of transcription in
rpb4 cells have led to the suggestion that RNA polymerase
II is unable to function effectively at high temperatures in the absence of Rpb4 (5, 9-11). To understand the effect of Rpb4 on
transcription during normal and stress conditions, we determined whole
genome expression patterns using microarray analyses under different
stresses. The various strains and conditions used in each experiment
are summarized in Table I. RNA was
isolated from the null mutant of RPB4 carrying either a
vector or a centromeric plasmid bearing RPB4 gene (under the
control of its own promoter) grown under identical conditions.
The data points which showed consistent results in duplicate
spots and reciprocal experiments were used for further analyses. We
normalized the intensity of the signal from each spot to the total
intensity in each channel. Following normalization, the intensity of
the spots corresponding to ACT1 gene (actin) in both
channels was comparable. The genes, which showed more than 2-fold
differences consistently in each condition, were compiled. All further
analysis was done using this data set. The total number of genes
up-regulated and down-regulated in the mutant as compared with the wild
type are tabulated (Table II). Under
normal growth conditions Rpb4 affects the expression of 120 and 121 genes, respectively in haploid and diploid yeast cells. The effect of
Rpb4 is on a similar scale in other conditions of stress such as
sporulation and nitrogen starvation. In all conditions, except heat
shock, the genes affected by Rpb4 amounted to nearly 1.7% of the
genome. Following a short duration of heat shock, the effect of Rpb4 is
more pronounced, extending to 9.2% of the genome (589 genes: 237 up-regulated and 352 down-regulated).
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We examined the effect of Rpb4 in various stress and nonstress
conditions by comparing the overlap between the down-regulated genes in
various conditions. The Venn diagrams in Fig.
2 show that the extent of overlap in the
affected genes is minimal between stress and nonstress conditions.
Overall, the transcriptional effects of RPB4 seem to be
specific for the environmental condition because there are very few
genes that are dependent on Rpb4 under all conditions. The overlap
between the expression profiles under various stress conditions is
marginally higher than that between nonstress and stress
conditions.
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We classified the known genes according to their functional roles as
annotated in the Munich Information Centre for Protein Sequences data
base (Table III). In all experiments, a
substantial number of the affected genes were of unknown
functions, and the largest number of genes affected by the absence of
Rpb4 were involved in metabolism and energy generation. Hence, the
general defect associated with rpb4 seems to be its
inability to express metabolic genes properly. in addition to this
defect in the expression of basic physiological pathways, specific
defects associated with each condition studied are summarized
below.
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Heat Shock-specific Defects--
Rpb4 has a pronounced effect on
gene expression following heat shock. In comparison with other stress
and nonstress conditions, a larger number of genes are affected
following heat shock. Functional classification of these genes revealed
that they are involved in basic metabolic pathways. A striking feature
of the transcriptional profile was that rpb4 cells after
heat shock showed higher levels of transcripts of genes involved in
protein synthesis. These include genes, which code for 97 ribosomal
proteins (of 132), proteins of the cap-binding complex, translation
initiation factors, aminoacyl tRNA synthetases, components of the
ribosome associated complex, and proteins involved in processing and
transport of rRNA. It is well known that yeast cells transiently
repress genes involved in protein synthesis following heat shock (20).
The increase in expression of these genes is probably a reflection of a
defect in this repression.
Sporulation-specific Defects--
Wild type and
rpb4/rpb4
strains were grown in rich medium
at permissive conditions until mid-log phase. The cells were then transferred to sporulation medium. Following 12 h of incubation in
sporulation medium, 67 genes showed a more than 2-fold decrease in
expression in rpb4
/rpb4
cells when compared
with wild type cells under identical conditions. Interestingly, 22 of
these 67 genes (33%) are on the right arm of the second chromosome. A
majority of these genes are involved in carbon metabolism, as is the
case in other conditions. RIM4, a regulatory gene, and
SPS1, SPS2, SPS4, and
SPS100 are down-regulated in
rpb4
/rpb4
. The SPS genes are
involved in spore wall synthesis and are usually induced during the
late stages of sporulation (21). They require the master regulator of
meiosis, Ime1, for their expression. This agrees well with our earlier
observations, using 4,6-diamidino-2-phenylindole staining,
electron microscopic analyses, and Northern analyses, that
rpb4
/rpb4
cells are arrested in the early
steps of meiosis and are unable to express early meiotic genes (data
not shown). None of the 12 genes up-regulated under this condition is
known to play any role in sporulation.
Defects Associated with Nitrogen Starvation
Conditions--
Despite the difference in morphology of
rpb4/rpb4
cells, we did not detect any
general induction of genes known to be involved in pseudohyphae
formation in these strains following nitrogen starvation.
IRA1, BEM1, AXL1, and ADH1
are the only genes differentially expressed in rpb4
known
to be important in pseudohyphae formation (22).
We clustered genes according to the ratio of their expression in mutant
rpb4 (or rpb4
/rpb4
) to wild
type in the five conditions tested (room temperature, heat shock, rich
medium, sporulation condition, and pseudohyphal growth condition) as
listed in Table I. Details of some interesting clusters are shown in
Fig. 3. A more extensive table with the
ratios of expression values of all of the differentially expressed
genes is available as supplementary data (Table SI).
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Many genes that code for hexose transporters of the major facilitator
class were found to be down-regulated in the mutant lacking Rpb4. We
therefore checked the rate of glucose uptake in these strains. We found
that in agreement with the expression profile, haploid and diploid
RPB4 deletion mutants consumed glucose slowly compared with
the wild type. At mid-log phase, haploid and diploid wild type cells
had exhausted 68 and 61% of the glucose provided in the medium,
respectively. But in RPB4 deletion mutants these numbers
dropped to 3 and 24%, respectively (Fig.
4). The homozygous diploid mutant
compared with the haploid strain lacking Rpb4 seems to be less
defective at high temperature and in glucose uptake, although the
reasons for this difference are not clear. In normal and stress
conditions rpb4 showed a distinctly different transcriptional profile compared with that of wild type cells. We
compared protein profiles of the rpb4
cells to wild type
under conditions in which we had found transcriptional differences. We
found that there were gross changes in protein profiles reflecting the
differences in transcription profiles (results not shown). Even under
normal conditions of growth there are substantial differences in the
levels of many proteins in rpb4
. Thus, the absence of Rpb4 resulted in altered transcriptional and protein profiles in normal
and different stress conditions.
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Studies of Activator Overexpression in Rescue of Stress Response
Phenotypes--
From previously reported studies and our results
reported here, rpb4 mutants show: 1) defects in survival
under extreme temperatures, 2) defects in sporulation, and 3)
pseudohyphae-like morphology. We have previously reported that the
activation defect of rpb4
strain can be partially rescued
by overexpression of the cognate transcriptional activator (8). Msn2
(transcriptional activator in heat shock response) and some other
proteins (that are not transcriptional activators) have been shown to
partially rescue the temperature sensitivity of rpb4
cells (7). Because our focus in this manuscript is on other stress
responses, we report here the effect of overexpression of specific
transcriptional activators under each condition. We observed that in
rpb4
cells the transcriptional activator IME4,
of early meiosis gene IME1 (23), was not induced at all
compared with the wild type cells, which showed a strong peak of
induction at 0.5 h in sporulation medium (Fig.
5A). We used the inducible
promoter PCUP1 to overexpress IME4 at different
times during pre growth in the rich medium and after transfer of cells
in the sporulation medium. We observed that the sporulation defect was
partially rescued when IME4 was overexpressed after transfer to
sporulation medium (Fig. 5B).
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There are many candidate transcriptional activators regulating
pseudohyphae formation in Saccharomyces cerevisiae. We chose Phd1, a transcriptional activator that exaggerates pseudohyphae formation in strains predisposed to forming pseudohyphae (24). We
overexpressed the Phd1 protein in the
shr3-102/shr3-102 background (CGX19) as well as
in rpb4 and the corresponding wild type strain. The
overexpression in the CGX19 strain served as a control for overexpression of Phd1 because the CGX19 strain is also predisposed to
pseudohyphae formation and shows an exaggerated pseudohyphal response
(24). Overexpression of Phd1 resulted in exaggeration of pseudohyphae
formation in rpb4
mutants as compared with wild type
(Fig. 5C).
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DISCUSSION |
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Previous studies have conclusively shown that Rpb4 can affect the
transcription of many promoters in vitro (5, 7, 9). Studies
using promoter reporter constructs have also shown that various
unrelated genes are affected to different extents in rpb4 (8). On the other hand, the stress-related phenotypes of
rpb4
cells like temperature sensitivity and lethality
during the stationary phase point toward a stress-specific role for
RPB4 in transcription. It was proposed that in the absence
of Rpb4, the polymerase is unstable at 37 °C or above (1, 7, 9, 10).
Instability of the polymerase lacking Rpb4 following exposure to high
temperature is conceivable, but a similar mechanism may not fully
explain the role of Rpb4 in other stresses. In addition to the known
roles in survival at high temperature and stationary phase, we found that rpb4
/rpb4
cells were also defective in
sporulation, a response to extreme starvation, and showed altered
morphology associated with pseudohyphae formation.
We observed that Rpb4 affects (up-regulated and down-regulated) 1.9%
of the total number of genes whose expression pattern was detected
under normal conditions. Consistent with our previous report using
promoter-reporter fusions, we see that the endogenous expression of
GAL1 and INO1 genes is compromised in
rpb4 cells (12). Interestingly, following a short
exposure to nonpermissive temperature, 9.2% of the total genes
detected show changes dependent on Rpb4. Only 9.5% of the genes
dependent on Rpb4 after heat shock are similarly affected during normal
growth. This implies that the role of Rpb4 in transcription immediately
following heat shock is significantly different and more drastic from
that in other stress conditions. It has a less conspicuous and probably
different role during normal conditions of growth. Gasch et
al. (25) and Causton et al. (26) have recently reported
the genome-wide expression pattern during heat shock in yeast cells.
Both reports show that genes involved in carbon metabolism,
mitochondrial function, and glucose transport are up-regulated after
heat shock, and genes involved in protein synthesis are down-regulated.
In response to heat shock, rpb4
cells have lower
transcript levels of genes involved in carbon metabolism and
mitochondrial function and higher transcript levels of genes of
ribosomal proteins as compared with wild type. It appears that
rpb4
cells are incapable of adopting the normal
transcriptional response to heat shock. Recently, genome-wide expression analyses done after exposing rpb4
cells to
high temperatures for a relatively longer time than reported here has
confirmed that most of the transcription in the cell is eventually shut down in rpb4
cells (10). Taken together, it appears that
immediately following temperature stress, rpb4
cells do
not adopt a normal heat shock-associated transcriptional response
(repression of ribosomal proteins and up-regulation of mitochondrial
genes, hexose transporters, and galactose metabolism genes). With
prolonged incubation at high temperature, the polymerase lacking Rpb4
becomes incapable of transcribing 98% of the genome, the reach of this inactive polymerase being as wide as that of rpb1-1, a
conditional mutant of the largest subunit of the polymerase (10).
The genes up-regulated and down-regulated after heat shock were grouped
based on the factors (proteins, environmental factors, other inducers
of gene expression) to which they are known to respond. These
transcriptional regulators are probably either functionally correlated
to Rpb4 or dependent on Rpb4 for their function. The maximum number of
genes down-regulated in rpb4 after heat shock are known
to be under the control of Msn2, the transcriptional activator of
stress response element (STRE)-regulated genes (27). This agrees
with the observation reported earlier that Msn2 overexpression can
partially complement the temperature sensitivity of rpb4
cells (7). We tested the effect of overexpression of some of the
transcriptional activators known to affect sporulation and pseudohyphae
formation. It is difficult to choose the activators to be tested
because the stress responses are complex phenotypes, and many
regulators play important roles at various stages in the given
response. We decided to test the effect of IME4
overexpression on sporulation because it is known to regulate
IME1, which is one of the early positive regulators of
meiotic genes. We have seen that rpb4
/rpb4
cells fail to induce endogenous IME4 under these conditions
(Fig. 5A). Because the inducible CUP1 promoter is
unaffected by the absence of Rpb4 (8), using the CUP1
promoter we induced the expression of IME4 in
rpb4
/rpb4
cells in a manner similar to its
expression in the wild type cells to overexpress IME4. The
observation that the sporulation defect was rescued significantly only
after induction of the activator supported our earlier observation.
Similarly the overexpression of PHD1, a transcriptional
activator known to enhance the pseudohyphal phenotype in strains
predisposed to forming pseudohyphae, clearly enhanced the pseudohyphal
morphology of rpb4
/rpb4
strain. This again
supports the notion that the defect in transcriptional activation in
rpb4
/rpb4
mutant is specific for certain
sets of genes we proposed earlier (8).
The genes affected under heat shock conditions involved in
mitochondrial function were seen to be under the control of Hap4 and
Gcn5 (28), both of which are also down-regulated in rpb4. Therefore the effect on mitochondrial function is probably due to Hap4
expression being compromised. A similar analysis of the up-regulated
genes (during heat shock) revealed that most of these genes (47%) are
co-regulated in the presence of rapamycin. This agrees well with
previous reports from a high throughput screen that rpb4
is one of the mutations that confers rapamycin resistance (29). The
deletion of RPB4 appears to have dual effects following heat
shock: an immediate effect on many pathways critical to the physiology
of the cell and an additional effect on the stability of the
polymerase. In the absence of Rpb4, following prolonged exposure to
high temperatures, the polymerase is rendered unstable leading to a
defect in transcription as severe as in inactivation of the polymerase
in an rpb1-1 mutant.
YGP1, a gene known to be highly induced in various stress responses
(30) is among the most severely down-regulated genes in
rpb4/rpb4
under stress conditions. Mutants
of IRA1 are known to constitutively express the
cAMP-dependent PKA pathway, which regulates pseudohyphae
formation (22). The down-regulation of IRA1 in
rpb4
/rpb4
cells specifically following
nitrogen depletion may be responsible for their tendency to form
pseudohyphae. During sporulation, as mentioned above, the spore wall
synthesis genes SPS1, SPS2, SPS4, and
SPS100 were all down-regulated in
rpb4
/rpb4
cells at 12 h post-induction
of sporulation. These "late" genes are normally induced in wild
type at this stage during sporulation (21). Ime1, the master regulator
of sporulation, regulates all four genes. Ime4, a positive regulator of
sporulation, which is required for Ime1 expression, is not induced in
rpb4
/rpb4
cells (Fig. 5A). The
inability to initiate transcription of early meiotic genes agrees with
the down-regulation of downstream late genes.
It is evident from our analysis that a few genes are dependent on Rpb4
for their expression irrespective of stress. These are mainly involved
in galactose metabolism and glucose uptake. Interestingly, Kap104, a
-karyopherin required for survival at high temperatures is also
consistently affected (31). KAP104 is present adjacent to
GAL7 on the genome and shows an expression pattern similar
to the GAL gene cluster. Promoter analysis of these genes using
MEME software revealed the presence of a putative regulatory
site upstream of these genes. The motif
(C/T)GGAG(A/C/G)(A/C)CTG(C/T)(T/C)G(A/C)(C/G)CG, which is 60%
similar to the Gal4-binding site, is present in all of the
galactose-regulated genes as expected. In addition to this site, a
15-nucleotide-long T-rich segment is also present upstream of all these
genes. When the entire intergenic region between KAP104 and
GAL7 (~700 bp) was considered, an additional element (TGC(C/G)(A/T)(T/C/G)(T/G)(A/C/T)(G/C/T)T(T/C)TT(T/G)(T/G)A(A/G)(A/C)(C/G/T)(T/C/T)(A/T)T(T/A)(T/A/C)(C/T)T(G/C)G(G/T)(G/A/T)(G/T)(A/C/T)(A/C)(T/C/G)(T/A)(A/C/G)(A/T)T(C/G)A(G/A)CG(A/G)AG(C/G/T)(G/C)) present in the GAL gene cluster and KAP104 was
identified. The significance of these elements is being studied.
We have compared the transcriptional defects of Rpb4 with other
subunits of the transcription machinery. If the genes affected by Rpb4
form a significant subset of genes affected by some other component of
the holoenzyme, we might gain insights into the mechanism by which Rpb4
regulates transcription. Because most other components of the
holoenzyme are essential, their transcriptional effects have been
studied using conditional mutants at restrictive temperatures (32).
Under these conditions Rpb4 affects 9.2% of the genome; this forms a
distinct subset with minimal overlaps with the transcriptome of some
components of the holoenzyme like Srb4, Med6, etc. Table IV summarizes the extent of overlap
between the footprints of Rpb4 and all other well studied
components of the holoenzyme on the transcriptome. More than 50% of
the genes affected in rpb4 in each condition constitute a
subset of genes affected by the CTD kinase, Kin28. Recently, the
Schizosaccharomyces pombe homolog of Rpb4 has been shown to
interact with Fcp1, a CTD phosphatase (33). Thus, the mechanism of
transcriptional regulation by Rpb4 may be linked to CTD
phosphorylation. In conclusion, Rpb4 affects central physiological
processes like glucose uptake and carbon and energy metabolism, which
in turn can regulate various phenotypes. Thus, the diverse stress
response defects seen in rpb4
strains may be a
consequence of a general defect in optimal expression of basic
metabolic pathways.
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ACKNOWLEDGEMENTS |
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We hereby thank all members of our laboratories at the Indian Institute of Science and the Centre of Biochemical Technology. The microarray facility at Centre for Biochemical Technology was used for the work presented here. We especially acknowledge Dr. Ramachandran and his laboratory members (Centre of Biochemical Technology), Dr. Rambodhkar (Centre of Biochemical Technology), and Dr. Raja Mugasimangalam (Genotypic Technologies) for help.
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FOOTNOTES |
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* This work was supported by funds from the Council for Scientific and Industrial Research (to P. P. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental table
(Table SI).
To whom correspondence should be addressed. Tel.:
91-80-394-2292; Fax: 91-80-360-2697; E-mail:
pps@mcbl.iisc.ernet.in.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M112180200
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