From the Wellcome Trust and Cancer Research Campaign Institute of Cancer and Developmental Biology and the Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, United Kingdom.
Received for publication, March 14, 2001
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ABSTRACT |
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RNA polymerase III (Pol III)
synthesizes various small RNA species, including the tRNAs and
the 5 S ribosomal RNA, which are involved in protein synthesis.
Here, we describe the regulation of human Pol III transcription in
response to sustained cell cycle arrest. The experimental system used
is a cell line in which cell cycle arrest is induced by the regulated
expression of the tumor suppressor protein p53. We show that the
capacity of cells to carry out Pol III transcription from various
promoter types, when tested in vitro, is severely reduced
in response to sustained p53-mediated cell cycle arrest. Furthermore,
this effect does not appear to be due to direct inhibition by p53. By
using complementation assays, we demonstrate that a subcomponent of the
Pol III transcription factor IIIB, which contains the proteins
TATA-binding protein and TAF3B2, is the target of repression. Moreover,
we reveal that TAF3B2 levels are markedly reduced in extracts from cell
cycle-arrested cells because of a decrease in TAF3B2 protein stability.
These findings provide a novel mechanism of Pol III regulation and
yield insights into how cellular biosynthetic capacity and growth
status can be coordinated.
Cellular growth and proliferation are two distinct processes.
Proliferation is defined as the increase in the number of cells, which
occurs when cells progress through a new cell division cycle. Cell
growth, on the other hand, is defined as the increase in mass of an
individual cell. Growth is related to the biosynthetic capacity of a
cell, a reliable indicator of which is the level of protein synthesis
(1). Conceptually, it seems requisite that the processes of growth and
proliferation are linked and that sensitive fine tuning exists between
them. At present, however, very little is known about the molecular
mechanisms that ensure that this crucial cross-regulation is achieved.
After neoplastic transformation of cells, uncontrolled proliferation is
only sustainable if it is linked to deregulated cell growth (2). Major
determinants of protein synthesis are the ribosomes and the machinery
that delivers activated amino acids to the ribosomes during
translation. Growth is therefore dependent on the availability of
adequate supplies of rRNAs and tRNAs. It has been shown that, as cells
enter quiescence, existing ribosomes disaggregate into their subunits,
the levels of rRNAs and tRNAs decrease, and net protein synthesis is
down-regulated. These events are reversed upon mitogenic stimulation
(2). But how are regulation of growth and regulation of proliferation
jointly accomplished? The prevailing model is that tumor suppressor
proteins play a crucial role in eliciting this control (1, 3, 4). For example, the retinoblastoma protein regulates entry into S phase by
controlling E2F, a transcription factor involved in the expression of S
phase-inducing genes (5, 6). It may also affect cell growth potential
by repressing the activities of RNA polymerases I (7) and III (8),
which synthesize rRNAs and tRNAs, respectively. Multi-functional
proteins such as retinoblastoma protein may therefore be vital for
adjusting cellular growth potential to match proliferative activity.
Conversely, as a consequence of losing retinoblastoma protein function,
cells may lose control over both proliferation and growth and be put on
a fast track toward neoplasia (1).
RNA polymerase (Pol)1 III
synthesizes a number of small RNA species, including the 5 S rRNA,
tRNAs, the spliceosomal U6 small nuclear RNA, the 7SL RNA of the
signal recognition particle, and the adenovirus VAI RNA
(9). The promoters of genes transcribed by Pol III are subdivided into
three groups, type 1, type 2, and type 3 promoters, based on promoter
structure and their requirements for basal Pol III transcription
factors (10). TFIIIA is a monomeric protein factor specific for the
promoters of the 5 S rRNA genes (type 1 promoters) (11-13). TFIIIC is
a multimeric protein required for both type 1 and type 2 promoters.
Human TFIIIC consists of at least nine polypeptides (14) and can be
split into two subcomponents, TFIIIC1 and TFIIIC2 (15). For type 3 promoters, only the C1 component of TFIIIC is required as an initiation
factor (16). Like TFIIIC, TFIIIB is an important Pol III transcription
factor involved in initiating transcription on type 1 and type 2 promoters. According to the sequential recruitment model, TFIIIB is
assembled into the preinitiation complex by TFIIIC on type 2 promoters
or by the TFIIIC-TFIIIA complex on type 1 promoters and is positioned upstream of the transcription start site (17). In vitro
studies have shown that TFIIIB then recruits Pol III to the promoter to direct accurate initiation of transcription and can do so even after
TFIIIC has been stripped off the promoter (18). TFIIIB contains the
TATA-binding protein (TBP) and various TBP-associated factors
(TAFs) (19-22). Although the number and identity of TAFs in human
TFIIIB have not been fully established, a TAF that has been
characterized in some detail is TAF3B2 (previously named BRF or
hTFIIIB90) (23, 24). Recently, two additional TAFs that form
part of TFIIIB have been identified: hB", which is the human homologue
of the yeast TFIIIB component, B", and hBRFU/TFIIIB50 (25, 26).
The aim of the work presented here was to investigate how Pol III
transcription is regulated during changes in cell growth and
proliferation. Toward this end, we used the human fibroblast cell line
TR9-7, derived from a patient with Li-Fraumeni syndrome, in which both
copies of the endogenous gene for p53 are inactivated (27). TR9-7
cells are stably transfected with a recombinant construct that allows
for the inducible expression of p53 from a tetracycline-controlled
promoter. Thus, whereas p53 expression is barely detectable in TR9-7
cells grown in the presence of 1 µg/ml of tetracycline, decreasing
the tetracycline concentration in the growth medium results in an
increase in both p53 expression and function, as determined by the
induction of the p53-responsive p21/Waf1 gene (27). Furthermore, the
levels of p53 expression in TR9-7 cells upon tetracycline withdrawal
are comparable with those induced in a wild-type cell line in response
to DNA damage (27). This degree of p53 induction in the TR9-7 cells
results in a slow onset, reversible cell cycle arrest in both the
G1 and G2/M stages of the cell cycle. Complete
cell cycle arrest is achieved 4-6 days after the onset of p53
induction. TR9-7 cells can be maintained in this arrested state for up
to 20 days and yet can still return to a normal cycling state afterward
(27). The TR9-7 cell line therefore provides a versatile experimental
system in which cell cycle arrest can be manipulated. As discussed
below, by use of this system, we have gained insights into the
mechanisms by which sustained cell cycle arrest leads to a reduced
capacity for Pol III transcription.
Plasmids--
The following plasmids were used as templates for
Pol III in vitro transcription reactions. pGlu6 contains a
HindIII fragment including a human tRNAGlu gene
inserted into pAT153 (28). pHu5S3.1 contains a 638-base pair
BamHI-SacI fragment from human genomic DNA
including a 5 S rRNA gene, inserted into pBluescript SK+ from
Stratagene (29). pBSK-Leu was constructed by subcloning a 240-base pair
EcoRI-HindIII fragment containing a human
tRNALeu gene (30) into pBluescript SK+.
pBSK-VAI was constructed by subcloning a 232-base pair
XbaI-KpnI fragment including the adenoviral VAI gene into pBluescript SK+. The VAI promoter
from this construct contains a point mutation in the A block (G-23
changed to A), which brings it closer to the conserved A block
consensus found in eukaryotic tRNA genes and increases the promoter's
strength (31, 32).
The following plasmids were used as templates for the synthesis of RNA
probes for RNase protection analysis. pGEM-TAF3B2 contains a sequence
comprising nucleotides 97-999 of the coding sequence of the human
TAF3B2 cDNA (24) cloned into pGEM-T (Promega); a clone was selected
in which the 5'-end of the insert was oriented toward the bacteriophage
T7 promoter. pGEM-ACT contains a sequence comprising the first 217 base
pairs of the third expressed exon of the Western Blotting--
Western immunoblot analyses were performed
as described by Coligan et al. (33). Primary antibodies were
mouse monoclonal anti- Tissue Culture--
The TR9-7 cell line was a gift from George
Stark (Department of Molecular Biology, Cleveland Clinic Foundation,
Cleveland, OH) and were grown as described previously (27). To suppress p53 expression, cells were grown in the presence of 1 µg/ml
tetracycline. To induce p53 expression, cells at about 80% confluence
were replated at a 1:6 dilution, grown in the presence of tetracycline
for 1 day, and then washed twice with 1× phosphate-buffered saline. Growth was then continued for various periods of time in the absence of
tetracycline before harvesting. Proteasome inhibition in TR9-7 cells
was induced by adding the calpain/proteasome inhibitor
N-acetyl-Leu-Leu-norleucinal (LLnL; Sigma) to the growth
medium at a final concentration of 20 µM, as described by
Shieh et al. (35). Cells were grown in the presence of LLnL
for 1 h before harvesting.
Extract Preparation--
TR9-7 cells were washed in 1×
phosphate-buffered saline and incubated with 2.5 ml/dish of 0.5×
trypsin/EDTA solution (Sigma) for 5 min at 37 °C. Detached cells
were collected, transferred into a 15-ml centrifugation tube,
centrifuged for 5 min at 500 × g, and washed twice in
1× phosphate-buffered saline. To prepare whole cell extracts, cell
pellets were resuspended in an equal volume of freshly prepared whole
cell extraction buffer (50 mM NaF, 20 mM HEPES,
pH 7.8, 450 mM NaCl, 25% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin,
0.5 µg/ml protease inhibitor, 1.0 µg/ml trypsin inhibitor, 0.5 µg/ml aprotinin, 40 µg/ml bestatin). Cells were subjected to three
cycles of freeze-thawing (quick freezing on dry ice and thawing at
30 °C) and then centrifuged in a microcentrifuge at top speed for 7 min at 4 °C. The supernatant containing the protein extract was
divided into samples, frozen on dry ice, and stored at Protein Fractionation--
HeLa cell nuclear extracts were
fractionated on Whatman P11 phosphocellulose according to Segall
et al. (37). Chromatography was done at 4 °C in buffer
D-100 (20% (v/v) glycerol, 20 mM HEPES, 100 mM
KCl, 5 mM MgCl2, 0.2 mM EDTA, 20 µM ZnSO4, 2 mM dithiothreitol, 1 mM sodium metabisulfite, and 0.5 mM
phenylmethylsulfonyl fluoride; adjusted to pH 7.9) at flow rates of
1.0-1.5 column volumes/h. 150-200 mg of nuclear extract were loaded
on 15-20 ml of equilibrated phosphocellulose resin (binding capacity
of 10 mg of protein/ml of resin). The flow-through fraction was
collected as phosphocellulose fraction (PC) A. Proteins were eluted in
a stepwise fashion with buffer D containing increasing concentrations
of KCl: with buffers D-350, D-600, and D-1000 containing KCl at 350, 600, and 1000 mM, respectively. Fractions that eluted
between 100 and 350 mM KCl were collected as PC-B
fractions, those between 350 and 600 mM KCl were collected
as PC-C, and those between 600 and 1000 mM were collected
as PC-D. For each elution, fractions with the highest protein
concentrations were pooled and dialyzed against buffer D-100. Pooled
PC-B and PC-C fractions used for transcription assays and further
purification had protein concentrations of approximately 1.5 and 0.5 mg/ml, respectively. The PC-B fraction was further purified and
fractionated as described by Lobo et al. (20), except that a
BioCAD Sprint perfusion chromatography system (PerSeptive Biosystems)
was used. The Poros HQ resin (strong anion exchange matrix;
quarternized polyethyleneimine) and the Poros HS resin (strong cation
exchange matrix; sulfopropyl) were from Roche Molecular Biochemicals.
Chromatography was done at 4 °C, and fractions were eluted with a
linear KCl gradient from 100 to 600 mM in buffer Q (same as
buffer D except that it contained additional protease inhibitors:
aprotinin, pepstatin A, and leupeptin all at 1 µg/ml and benzamidine
at 1 mM). 5 ml of PC-B (7.5 mg of protein) were loaded on
the HQ resin, and fractions of 750 µl were collected. The eluted
fractions were dialyzed against buffer Q-100. The fractions containing
peak amounts of the TBP and TAF3B2 were pooled and loaded on the HS
resin. Chromatography on the HS resin was done as described for the HQ resin.
Immunodepletion of Extracts--
Antibodies were adsorbed onto
protein G-Sepharose beads (Amersham Pharmacia Biotech) according to
Harlow and Lane (38). For TBP depletions, 400 µl of beads were
incubated with 1 ml of tissue culture supernatant of MBP-6 cells, a
hybridoma cell line expressing a mouse monoclonal anti-human TBP
antibody (34). 300 µl of antibody-coated beads were equilibrated with
Pol III transcription buffer (20% (v/v) glycerol, 20 mM
HEPES, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 20 µM ZnSO4, and 2 mM dithiothreitol; adjusted to pH 7.9) and incubated with
the same volume of a PC-C fraction for immunodepletion of
TBP-containing protein complexes from PC-C. For p53, 10 µg of DO-1
antibody were linked to 100 µl of protein G-Sepharose beads, and
antibody-coated beads were equilibrated with 1× phosphate-buffered
saline. 70 µg of whole cell extract from TR9-7 cells were depleted
with 10 µl of antibody-coated beads.
Transcription Assays--
Transcriptional activity was assayed
in vitro by measuring the direct incorporation of a
radiolabeled nucleotide into synthesized RNA. The reactions were
carried out in a final volume of 25 µl. As a source of transcription
factors and Pol III, we used whole cell extract from TR9-7 cells
(generally 0.8-1.2 µl; corresponding to 20 µg of protein) and/or
partly purified factors that had been dialyzed into buffer D-100. The
reactions were adjusted with buffer D-100 and other reagents such that
the final salt concentrations were equivalent to 60% of those of
buffer D-100. Reactions also contained 20 units of RNase inhibitor, 0.5 mM each of ATP, GTP, and CTP, 10 µM UTP, 1 µCi of [ Random Polymerase Initiation Assay--
Random RNA
polymerization assays were performed as described by Roeder (39) in a
volume of 20 µl containing 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM dithiothreitol, 2.5 mM MnCl2, 100 mM (NH4)2SO4, 32 µg of whole cell
protein extract, 5 µg of poly(dA-dT), 20 units of RNase inhibitor, 1 mM ATP, 0.5 mM UTP, and 1 µCi of [ RNase Protection--
TR9-7 cells at ~50% confluence were
harvested from four 140-mm-diameter tissue culture plates by
trypsination and pelleted by centrifugation at 500 × g
for 5 min at 4 °C. Cell pellets were resuspended in ice-cold
Dulbecco's modified Eagle's medium, and the cell density was measured
by microscopy. 5 × 106 cells were then collected by
centrifugation, and total cellular RNA was isolated from the cell
pellets using the RNeasy Mini Kit (Qiagen). RNA was eluted in 70 µl
of diethyl procarbonate-treated water. RNA yields were typically
in the range of 120-200 µg. Analyses of TAF3B2 and Pol III Transcription Is Repressed during Sustained Cell Cycle
Arrest--
After withdrawal of tetracycline from the growth medium of
TR9-7 cells, we found that they stopped proliferating with similar kinetics to those published previously (27) and became enlarged and
flattened compared with cells grown in the continual presence of
tetracycline (data not shown). To investigate the expression of p53 and
p21/Waf1 in response to tetracycline withdrawal over a 7-day period, we
carried out Western blot analysis. As shown in Fig.
1, p53 and p21 were barely detectable
when TR9-7 cells were grown in the presence of tetracycline. Their
levels, however, increased markedly when tetracycline was withdrawn,
reaching a peak after about 1 day and then slowly declining (blots were
also probed for
To assay for Pol III activity, transcription assays were carried out
in vitro with TR9-7 whole cell extracts and DNA templates containing the promoter regions and coding sequences of various Pol III
target genes, including the human 5 S rRNA gene (type 1 promoter)
and the human tRNALeu, tRNAGlu6, and the
adenoviral VAI genes (all type 2 promoters). Fig.
2 (A-D) shows that all four
Pol III promoters were transcribed efficiently by extracts from
proliferating TR9-7 cells (lanes P). Analysis of the
assays performed at 1 or 200 µg/ml of
To assess in more detail the kinetics of repression of Pol III
transcription, we analyzed the transcriptional capacity of extracts
generated from cells every day over a 7-day period of tetracycline
withdrawal (Fig. 3A). These
studies revealed that levels of VAI transcription declined
progressively throughout the time course, with virtually no
transcription being observed in days 6 and 7. Similar results were
obtained using the human tRNALeu promoter (Fig.
3B).
Repression of Pol III Transcription in TR9-7 Extracts Is Not
Directly Mediated by p53--
Full repression of Pol III transcription
in TR9-7 cell extracts only takes place 5-6 days after p53 levels
have been fully induced (Figs. 1B and 3), suggesting that
p53 is unlikely to be directly mediating the repression. To see whether
this was indeed the case, we carried out several types of analysis.
First, we tested whether transcription in extracts from proliferating
TR9-7 cells was sensitive to exogenously added recombinant p53 that had been expressed and purified from baculovirus-infected insect cells
(Fig. 4A). Two different
amounts of p53, 150 and 450 ng, were added to 10 µg of protein of
whole cell extract from proliferating TR9-7 cells
(second and third P lanes, respectively),
and levels of transcription were then compared with those of extracts
in which no p53 had been added (first P lane) and of
extracts from cell cycle-arrested TR9-7 cells (lane A) in
which endogenous p53 had been induced by incubation in the absence of
tetracycline for 6 days. Notably, 150 ng of recombinant p53, equating
to >1% of the total protein in the assay, had no significant effect
on transcription from the VAI promoter by proliferating
TR9-7 cell extract (Fig. 4A, first and
second lanes). By contrast, transcription was greatly
reduced in extracts from cell cycle-arrested TR9-7 cells (Fig.
4A, lane 4), even though only 0.5 ng of
endogenous p53 or less was present per 10 µg of cellular protein (for
rough estimate of endogenous p53 levels, see Fig. 1A). The
endogenous p53 in such reactions thereby constitutes roughly 0.005% of
the total protein, which is 200-300 times less than the amount of the
recombinant p53 used in the above experiment. Nevertheless, the
addition of 450 ng of p53 (~4.5% of the total protein in the reaction) led to repression of VAI transcription in TR9-7
extracts (Fig. 4A, lane 3). One possibility is
that this is due to a "squelching" effect, similar to that observed
by Mack et al. (42) when studying the
p53-dependent repression of Pol II transcription at high
levels of exogenously added p53.
As a second approach to assessing the mechanism of p53 repression in
the TR9-7 system, we tested whether repression of Pol III
transcription could be sustained after selectively removing p53 from
extracts of growth-arrested cells. Cairns and White (43) reported that
RNA Pol III preinitiation complexes are resistant to the repressive
effects of p53 when they are preassembled on the promoter before adding
p53 but not when p53 is added to extracts before the DNA template. We
therefore reasoned that if p53 was indeed responsible for Pol III
repression in TR9-7 cells, extracts should regain their
transcriptional activity if p53 was removed by immunodepletion before
carrying out transcription assays. To see whether this was the case, we
coupled the p53-specific mouse monoclonal antibody DO-1 to protein
G-Sepharose beads and incubated these with extracts from TR9-7 cells.
Fig. 4B shows that p53 was efficiently removed from extracts
by use of this procedure. Notably, and as shown in Fig. 4C,
extracts from growth-arrested cells (lanes A) did not regain
transcriptional activity when p53 was depleted (compare lanes
2 and 4). As a control for these experiments, the immunodepletion procedure did not impair the transcriptional capacity of extracts from proliferating cells (lanes P; compare
lanes 1 and 3). Taken together with the other
data described below, these findings strongly suggest that repression
of transcription upon sustained p53-mediated cell cycle arrest in
TR9-7 cells is not caused directly by p53.
A Subunit of TFIIIB Is a Target for Transcriptional
Repression--
The next question we investigated was which of the
factors involved in Pol III transcription is the target for repression in cell cycle-arrested TR9-7 cells. First, we tested for the activity of the Pol III enzyme itself to see whether it might become inactivated upon cell cycle arrest. The method used for these experiments was a
random Pol III initiation assay on a synthetic template consisting only
of adenine and thymidine (poly(dA-dT); Ref. 39). The assay was carried
out in the presence of different concentrations of
We then determined whether one of the basal Pol III transcription
factors was deficient in response to sustained cell cycle arrest. To do
this, we generated partially purified fractions of the various factors
and tested whether the addition of these fractions to extracts
of arrested cells was able to restore Pol III transcriptional capacity.
The transcription factors required for RNA Pol III transcription can be
fractionated by chromatography of nuclear extracts from HeLa cells on
phosphocellulose (37), and transcription from the VAI
promoter can be reconstituted in vitro with the PC-B and
PC-C fractions, which supply transcription factors TFIIIB and TFIIIC,
respectively, in addition to the RNA polymerase III enzyme that is
present in both fractions. These are the only fractions required for
efficient VAI transcription in vitro (20, 44).
Some reports, however, have concluded that the PC-C fraction can
contain a subcomponent of TFIIIB, as judged by the presence of TBP,
which is part of TFIIIB (20, 44). Some TBP was indeed found in our PC-C
fractions (data not shown), and we therefore removed TBP (and possibly
any TBP-associated factors) by immunodepletion with an anti-TBP
antibody. This TBP-depleted PC-C fraction (PC-C(TBP
Notably, and as shown in Fig.
5A, the addition of increasing
amounts of PC-B to extracts from cell cycle-arrested TR9-7 cells strongly increased transcription from the VAI template. By
contrast, the addition of PC-C(TBP
To verify that it was indeed TFIIIB that conferred complementation to
the cell cycle-arrested TR9-7 cells, we took advantage of the fact
that it can be further fractionated into two components, 0.38M-TFIIIB
and 0.48M-TFIIIB, both of which are required to reconstitute full
TFIIIB activity (24). The 0.38M-TFIIIB component contains TBP and at
least one TAF, named TAF3B2, whereas the 0.48M-TFIIIB component is
devoid of TBP and contains additional Pol III basal factors (24). We
therefore purified the 0.38M-TFIIIB subcomponent first by anion and
then by cation exchange chromatography. After verifying that TBP and
TAF3B2 co-fractionated over both columns (Fig.
6A and data not shown), we
used these fractions in complementation assays. Notably, the fractions
eluted from the cation exchange column that contained peak levels of
TBP and TAF3B2 (fractions 14 and 15) also contained peak levels of the
activity that reconstituted transcription to extracts from cell
cycle-arrested TR9-7 cells (Fig. 6B; this activity also
co-fractionated with both TBP and TAF3B2 on the anion exchange column;
data not shown). None of the other fractions, including those that
contained 0.48M-TFIIIB, contained any reconstituting transcriptional
activity in the complementation assays (data not shown). The
co-fractionation over three columns of the activity that reconstitutes
transcription to cell cycle-arrested TR9-7 extracts with TFIIIB
activity, and in particular the 0.38M-TFIIIB component, therefore
suggests strongly that this is the factor whose activity is regulated
in response to cell cycle arrest.
The TAF3B2 Subunit of TFIIIB Is Destabilized during Sustained Cell
Cycle Arrest--
We next determined which component of 0.38M-TFIIIB
(TBP or TAF3B2) might be regulated by investigating the levels of
expression of these proteins in TR9-7 cells both before and after the
induction of cell cycle arrest. Western blot analysis of whole cell
extracts from proliferating and cell cycle-arrested cells revealed that TAF3B2 levels were greatly decreased in response to proliferation arrest, whereas TBP levels were not markedly affected (Fig.
7; the identity of the additional band
seen when probing with the TAF3B2 antibody is not known, but this
serves as a useful loading control for the two cell extracts). Because
TAF3B2 is essential for TFIIIB activity and for Pol III transcription
of the VAI promoter being tested (23, 24), the decrease in
TAF3B2 levels could thus at least partly account for the reduction in
Pol III transcription capacity in extracts of cell cycle-arrested
TR9-7 cells.
To gain insights into the level(s) at which TAF3B2 was being regulated,
we first carried out an RNase protection analysis of the TAF3B2
mRNA (Fig. 8). This indicated that
TAF3B2 mRNA levels were not significantly reduced in extracts from
cell cycle-arrested TR9-7 cells, meaning that modulation of RNA levels
were unlikely to account for the dramatic reduction at the protein
level. We wondered therefore whether TAF3B2 levels might be controlled
at the level of protein stability. In this regard, we noted that a PEST
sequence (a sequence rich in Pro, Glu, Ser, and Thr) exists toward the
extreme C-terminal end of TAF3B2 (data not shown). PEST sequences are a
common feature of many proteins that can be rapidly degraded by the
ubiquitin-dependent proteasome pathway (45, 46).
Strikingly, addition of the proteasome inhibitor LLnL to the growth
medium of cell cycle-arrested TR9-7 cells led to a dramatic increase
in TAF3B2 protein levels within 1 h (Fig. 9A). Moreover, when we carried
out transcription assays with extracts from LLnL-treated cell
cycle-arrested cells, we found that transcriptional activity was fully
restored to levels observed with extracts from proliferating cells
(Fig. 9B). These data therefore support the conclusion that
loss of Pol III transcription capacity in cell cycle-arrested TR9-7
cells is mediated primarily by destabilization of TAF3B2.
Because LLnL has previously been used by others to rescue uninduced p53
protein from proteasome-mediated degradation and thereby bring about
its stabilization (35), we thought it unlikely that it would lead to
loss of p53 from the cell cycle-arrested TR9-7 cells. Indeed, as shown
in Fig. 9A, p53 remains stable and is present at high levels
in cell cycle-arrested TR9-7 cells following incubation with LLnL.
Therefore, Pol III transcription is restored in this system (Fig.
9B) despite high p53 levels. Taken together, these data
provide strong additional support for the conclusion that repression of
transcription in cell cycle-arrested TR9-7 cells is brought about by
loss of TAF3B2 rather than via p53 directly inhibiting the Pol III
transcription apparatus.
The aim of this study was to investigate whether RNA Pol III
transcription is regulated in response to sustained cell cycle arrest.
RNA Pol III transcription is responsible for the synthesis of various
small RNA species involved in maintaining cellular biosynthetic
capacity. Under conditions of prolonged cell cycle arrest, when cells
do not require high levels of protein synthesis, it might therefore be
assumed that cells can afford to down-regulate Pol III transcriptional
activity. We tested this hypothesis in the human TR9-7 tissue culture
cell line in which cell cycle arrest can be initiated by the inducible
expression of p53 (27). These studies revealed that Pol III capacity
for a range of type 1 and type 2 Pol III promoters is indeed reduced
greatly as a consequence of sustained cell cycle arrest. Furthermore,
we showed that this was not the result of decreased activity of the RNA
polymerase III enzyme itself nor decreased TFIIIC activity. Instead, we
showed that TFIIIB activity was the target for repression and that it was the 0.38M-TFIIIB component but not the 0.48M-TFIIIB component, of
this factor that was specifically repressed. Finally, we revealed that
this repression was accompanied by a dramatic destabilization of the
0.38M-TFIIIB component TAF3B2 and showed that reversing this
destabilization by treating cells with proteasome inhibitors led to
restored Pol III transcriptional capacity. Because TAF3B2 is essential
for Pol III transcription (23, 24), we conclude that its
destabilization is likely to play a major role in effecting the loss of
Pol III capacity in the TR9-7 system. However, it should be noted that
not all human TFIIIB subunits have so far been cloned; it therefore
remains possible that down-regulation of another TFIIIB component may
also contribute to the reduced Pol III transcription that we observe.
It will clearly be of great interest to determine the features of
TAF3B2 that allow it be targeted for degradation upon sustained cell
cycle arrest and to identify and characterize the
trans-acting factors that mediate this control.
Two previous studies reported that p53 can act as a direct repressor of
Pol III transcription through inhibitory interactions with TFIIIB (43,
47). Furthermore, the promoters we used in our study, namely those of
the human 5 S rRNA gene, two different tRNA genes, and the adenoviral
VAI gene, were inhibited directly by p53 in the study of
Cairns and White (43). In light of these findings, we considered the
possibility that the repressive effects on Pol III transcription that
we observed in TR9-7 cells could be mediated directly by p53. However,
several lines of evidence indicate that this is not the case. First,
the kinetics of Pol III repression in TR9-7 cells correlated best with
the onset of cell cycle arrest, not the kinetics of p53 induction.
Second, the addition of recombinant p53 to extracts of proliferating
TR9-7 cells, in amounts similar to those used by Cairns and White
(43), did not reproduce the repression observed in extracts from cell cycle-arrested cells. The amount of exogenous p53 added in these experiments was at least 100-fold higher than the amounts present endogenously in extracts of cell cycle-arrested cells. Third, transcriptional repression was sustained after removing p53 selectively from extracts of cell cycle-arrested cells by immunodepletion. Finally,
and most compelling, we showed that p53 protein was maintained at high
levels when cell cycle-arrested cells were treated with a proteasome
inhibitor, yet there was full recovery of Pol III transcriptional
activity. It therefore seems clear that p53 is not directly responsible
for repression of Pol III transcription capacity following sustained
cell cycle arrest in the TR9-7 cell system.
The data we present here are in agreement with the model that there is
an intimate linkage between cellular biosynthetic capacity and the
activity of RNA Pol III transcription apparatus (1). We describe a
novel pathway by which Pol III transcription may be negatively
regulated during times when cells do not require elevated levels of
active protein synthesis, such as during sustained cell cycle
arrest. Taken together with other work, our results suggest that
down-regulation of Pol III transcription can be elicited by multiple
mechanisms: direct inhibitory interactions between the retinoblastoma
protein and TFIIIB (8), direct inhibition of TFIIIB by the p53 protein
(43), and destabilization of TAF3B2. Remarkably, all three pathways
converge on TFIIIB, which therefore seems to have evolved as a crucial
target in the regulation of Pol III transcription (3). It is tempting
to speculate that these mechanisms represent distinct but complementary
pathways to bring about regulation of Pol III activity in response to
changes in cellular growth potential and proliferative status. These
pathways could operate in different biological contexts or, at least in some circumstances, could co-operate to reinforce the level of transcriptional control. The unraveling of the molecular details of
TAF3B2 destabilization upon sustained cell cycle arrest may provide
further insights into how this important cross-regulation is achieved,
and this might eventually lead to a better understanding of tissue
growth under both physiological and pathological conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin gene cloned into
pGEM-T; the 5'-end of the insert was oriented toward the bacteriophage
T7 promoter of the vector.
-actin antibody (Sigma), mouse monoclonal
anti-p53 antibody (DO-1; Santa Cruz Biotechnology), and mouse
anti-human p21 monoclonal antibody (Pharmingen). Rabbit anti-human
TAF3B2 polyclonal antibody was a gift from Nouria Hernandez (Cold
Spring Harbor Laboratories, Cold Spring Harbor, NY; Ref. 24). Mouse
monoclonal anti-human TBP antibody (MBP-6) was a gift from S. J. Flint (34). The secondary antibodies (goat anti-mouse antibody from
Sigma and goat anti-rabbit antibody from Pierce) were both horseradish
peroxidase conjugates. Bound antibodies were detected by enhanced
chemiluminescence using the ECL system (Amersham Pharmacia Biotech). In
some cases, nitrocellulose membranes were stripped by incubation in
62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS, and 0.14% (v/v)
-mercaptoethanol at 60 °C for 30 min with gentle shaking,
followed by extensive washing, before reprobing.
80 °C.
Nuclear extracts from HeLa cells were prepared according to Dignam
et al. (36).
-32P]UTP (800 Ci/mmol), and 250 ng of
plasmid DNA bearing a Pol III promoter. Reactions were preincubated for
30 min at 30 °C in the absence of nucleotides and, after the
addition of nucleotides, for a further 40 min at 30 °C. Reactions
were stopped by the addition of 200 µl of stop solution (1 M ammonium acetate, 0.1% (w/v) SDS, containing 0.05 µg/µl torula yeast RNA). RNA products were extracted with
phenol/chloroform, precipitated with ethanol, resolved by SDS-polyacrylamide gel electrophoresis, and subjected to
autoradiography to reveal the sizes of the synthesized RNA products.
-32P]UTP (800 Ci/mmol). After incubation at 30 °C
for 20 min, 18 µl of each reaction were transferred onto a Whatman
GF/C glass microfiber filter and dried. The filters were washed with
ice-cold 5% (w/v) trichloroacetic acid containing 20 mM
tetrasodium pyrophosphate in a funnel until no radioactivity could be
detected in the flow-through. The filters were briefly washed in 70%
ethanol and dried. The radioactivity retained on filters was measured
in a Beckmann LS6000 scintillation counter using the 32P
channel. To determine the Pol III activity, the counts obtained from
the RNA products synthesized in the presence of 200 µg/ml
-amanitin (measuring the activity of Pol I only, because at this concentration
-amanitin inhibits both Pol II and III) were
subtracted from the counts obtained in the presence of 1 µg/ml
-amanitin (measuring the activity of both Pol I and III, because at
this concentration
-amanitin only inhibits Pol II) (40, 41).
-actin
mRNAs were carried out using the RNase protection kit (Roche
Molecular Biochemicals). Plasmids pGEM-TAF3B2 and pGEM-ACT were
linearized with restriction enzymes BbsI and
NcoI, respectively, and RNA antisense probes were
synthesized by transcription from the SP6 promoter. For TAF3B2, 20 µg
of total cellular RNA from TR9-7 cells were used for RNase protection,
and for
-actin 0.2 µg of RNA was used, with 3.0 × 105 cpm of the respective radiolabeled probe in each case.
The RNA products were analyzed by polyacrylamide gel electrophoresis
and autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin, which was used as a loading control).
Notably, at peak levels of expression, the concentration of p53 was
less than 1 ng/25 µg of total soluble cellular protein, as judged by the intensity of a Western blot signal to that of 1 ng of recombinant p53 loaded on a separate lane (Fig. 1A).
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Fig. 1.
Regulation of protein expression in TR9-7
cells in response to tetracycline withdrawal. Whole cell extracts
were prepared from cells that had been grown either in the presence of
tetracycline (day 0) or from cells that had been incubated after
tetracycline had been withdrawn for between 2 and 24 h
(A) or between 1 and 7 days (B). 25 µg
of protein extract were loaded and analyzed by Western blotting for
p53, p21, or human -actin as indicated. As a control, 1 ng of
recombinant p53 was loaded into the seventh lane of
(A).
-amanitin (Fig. 2) reveals
that the observed transcripts were indeed generated by Pol III (40,
41). In striking contrast, transcription from the same promoters was
greatly reduced in extracts from TR9-7 cells that had been cell
cycle-arrested for 6 days (lanes A). Indeed,
PhosphorImager analysis revealed that transcription was reduced by
between 86 and 94%; Fig. 2E). Thus, overall Pol III transcription capacity of extracts derived from TR9-7 cells is markedly reduced following tetracycline withdrawal.
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Fig. 2.
Levels of RNA Pol III-dependent
transcription of various promoters in response to cell cycle
arrest. A-D, total protein extracts were prepared from
proliferating cells (lanes P) or from cells arrested for 6 days (lanes A) following the withdrawal of tetracycline.
Equal amounts of protein (20 µg) were used to transcribe various RNA
Pol III promoters (as indicated). Reactions were carried out at 1 or
200 µg/ml -amanitin to verify that the main transcript was a
genuine product of RNA Pol III. The sizes of selected nucleic acid
marker bands that were run alongside the samples are indicated.
E, levels of transcription in A-D were
quantitated by PhosphorImager analysis; in each case, the amount of
transcript from proliferating cells was assigned a value of
100%.
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Fig. 3.
Time course of effects of growth arrest on
Pol III transcription. Extracts were prepared from proliferating
cells (grown in the presence of tetracycline; day 0) or from cells that
had been incubated in the absence of tetracycline for 1-7 days (as
indicated). Transcription assays with 20 µg of protein extract were
carried out with the VAI promoter (A) or the
tRNALeu promoter (B). nt,
nucleotides.
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Fig. 4.
Linkage between p53 and Pol III transcription
capacity in the TR9-7 cell system. A, effect of adding
exogenous p53 to extracts of TR9-7 cells. Whole cell extract (10 µg
of protein) from proliferating TR9-7 cells (lanes P) was
complemented with two different amounts of baculovirus-expressed
purified human p53: 150 ng (second lane) or 450 ng
(third lane). The effect of p53 complementation on
transcription was measured in vitro using the
VAI promoter. For comparison, assays were also carried out
in the absence of exogenous p53 by using extracts from proliferating
(first lane) or cell cycle-arrested cells (lane
A). B, effect on transcription of immunodepletion of
p53 from TR9-7 cell extracts. Extract from cell cycle-arrested cells
was immunodepleted with an anti-p53 antibody (DO-1) coupled to protein
G-Sepharose beads, and the efficiency of depletion was analyzed by
Western blotting (50 µg of total protein loaded in each lane). As a
control, the blot was also probed with an antibody against -actin.
C, 20 µg of protein extract from either proliferating or
cell cycle-arrested cells was used for in vitro
transcription with the VAI promoter. One set of the two
extracts had been immunodepleted with an antibody against p53 as
described above, and the other had not. nt,
nucleotides.
-amanitin to
allow us to distinguish between the activities of the three nuclear RNA
polymerase enzymes (40, 41). Table I
shows that the activities of all three RNA polymerases were very
similar in extracts from proliferating and cell cycle-arrested cells,
suggesting strongly that repression of Pol III transcription in cell
cycle-arrested TR9-7 cells is not due to a deficiency in the activity
of the Pol III enzyme itself.
Activity of RNA polymerase III in TR9-7 extracts
-amanitin was used to distinguish Pol III
(inhibited by 200 µg/ml
-amanitin) from Pol I (insensitive to 200 µg/ml) and Pol II (inhibited by 1 µg/ml). The activity values are
given as counts per minute of incorporated radioactivity.
)) did
not lose any of its TFIIIC activity (data not shown) and was used as
the source of TFIIIC in the studies below.
) had little or no
effect on transcription (Fig. 5B). Thus, it seems that
TFIIIB, and not TFIIIC, becomes specifically deficient in response to
prolonged cell cycle arrest. This conclusion was supported by the
observation that the PC-C fraction prior to TBP immunodepletion also
contained reconstituting activity (data not shown; also see below),
implicating either TBP or a TBP-associated factor within TFIIIB as the
activity that is deficient in cell cycle-arrested TR9-7 cells.
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Fig. 5.
Transcriptional activity of extracts from
cell cycle-arrested TR9-7 cells complemented with partly purified Pol
III transcription factors. Transcriptional activity was tested
with the VAI promoter and 20 µg of extract in each
reaction. A, extracts from cell cycle-arrested cells
(lanes A) were complemented with increasing amounts of
partially purified TFIIIB (PC-B fraction): 0 µl (second
lane), 1 µl (third lane), 2 µl (fourth
lane), or 4 µl (fifth lane). As a control, a reaction
was also carried out with 20 µg of extract from proliferating cells
(lane P). B, the same assay was carried out as
above, except that extracts were complemented with increasing amounts
of partly purified TFIIIC (anti-TBP-depleted PC-C fraction
(PC-C(TBP )) fraction; same amounts as above).
nt, nucleotides.
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Fig. 6.
Complementation of extracts from cell
cycle-arrested TR9-7 cells with 0.38M-TFIIIB purified by cation
exchange chromatography. Partially purified TFIIIB from the PC-B
fraction was further purified and fractionated by anion exchange
chromatography (data not shown) followed by cation exchange
chromatography (shown here). The proteins were eluted with buffer of
increasing KCl concentration (gradient from 100 to 600 mM
KCl), and fractions of 750 µl were collected. A, Western
blot analysis of fractions 12-18 for TAF3B2 and TBP. 19 µl of each
fraction were loaded after dialysis against 50 mM KCl. The
blot was first probed for TBP (lower panel) and then
stripped and probed for TAF3B2 (upper panel). B,
the same fractions were used to complement extracts from cell
cycle-arrested TR9-7 cells (lanes A). Transcription
reactions using the VAI promoter were carried out with cell
extract (20 µg of protein) complemented with 6 µl of fractions
12-18. For comparison, reactions were also carried out with equivalent
amounts (20 µg of protein) of extracts from proliferating cells
(lane P) and with 20 µg of noncomplemented extracts from
arrested cells. nt, nucleotides.
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Fig. 7.
Levels of TAF3B2 and TBP proteins in TR9-7
cell extracts. Whole cell extract (100 µg of protein) from
proliferating or growth-arrested cells was loaded into the indicated
lanes for Western blot analysis. Separate blots were probed for TAF3B2
(A) and TBP (B). As size markers, 20 µl of
partially purified 0.38M-TFIIIB or 1.6 ng of recombinant human TBP were
loaded in the first lane of A and B,
respectively. The band below the TAF3B2-band in A
(asterisk) was consistently present when whole cell extracts
were probed but absent from partially purified preparations. Its
identity is unknown.
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Fig. 8.
RNase protection analysis of TAF3B2 mRNA
from TR9-7 cells. Total RNA was isolated from proliferating and
cell cycle-arrested cells. A, RNase protection of TAF3B2
mRNA. 3 × 105 cpm of a labeled TAF3B2 antisense
probe were combined for RNase protection with 20 µg of total cellular
RNA (lanes 3 and 4). As a control for the
specificity of the probe, the reaction was also carried out with 10 µg of yeast tRNA instead of cellular RNA (lane 5). To
distinguish between the nondigested probe and the RNase protection
product, 2000 cpm of probe were loaded in lane 2. The probe
size was 277 nucleotides; the size of RNase protection product was 190 nucleotides. B, to show that the two preparations contained
equivalent amounts of RNA, the same set of reactions was carried out
with a labeled probe specific for the -actin mRNA. Because of
the abundance of
-actin mRNA, only 0.2 µg of total RNA were
used. The size of the probe was 307 nucleotides; the size of the
product was 217 nucleotides. nt, nucleotides.
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Fig. 9.
Effects of proteasome inhibition on protein
stability and Pol III transcription in cell cycle-arrested TR9-7
cells. Cell cycle-arrested TR9-7 cells (7 days after tetracycline
withdrawal) were incubated for 1 h with the proteasome inhibitor
LLnL (at 20 µM). The cells were harvested, and the
extracts were prepared. The extracts from proliferating cells and from
arrested cells that had not been treated with LLnL were also prepared
at the same time. A, Western blot of the extracts probed for
the TAF3B2 protein (100 µg of each extract was loaded in this
instance) or for p53 and actin (25 µg of extract was used). For the
TAF3B2 analysis, 20 µl of partly purified 0.38M-TFIIIB was also
analyzed alongside. B, transcription assays were carried out
with extracts from proliferating cells (lane P, lane
1), from cell cycle-arrested cells (A) that had not
been treated with proteasome inhibitor (lane 2), and from
cell cycle-arrested cells that had been treated with 20 µM of the inhibitor LLnL (lane 3). In each
case, 20 µg of extract was used with the VAI promoter as
template.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Jane Bradbury and Steve Bell for valuable scientific input. We are also extremely grateful to George R. Stark for allowing us to use the TR9-7 cell line, to Nouria Hernandez for antibodies against TAF3B2, and to S. J. Flint for the MBP-6 cell line and TBP antibodies.
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FOOTNOTES |
---|
* This work was supported by the Cancer Research Campaign.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.
Supported by a fellowship of the Boehringer Ingelheim Fonds,
Stiftung für medizinische Grundlagenforschung. Present address: Magdalen College, Oxford, OX1 4AU, UK.
§ To whom correspondence should be addressed.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M102295200
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ABBREVIATIONS |
---|
The abbreviations used are: Pol, polymerase; TFIII, transcription factor III; TBP, TATA-binding protein; TAF, TBP-associated factor; LLnL, N-acetyl-Leu-Leu-norleucinal; PC-, phosphocellulose fraction.
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