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(Received for publication, December 28,
1994; and in revised form, January 11, 1995) From the
Transcription of the ribosomal RNA genes by RNA polymerase I is
tightly coordinated with the rate of cell growth. The RNA polymerase I
transcription factor, UBF, activates transcription by binding to
elements within the promoter and enhancer elements within the
intergenic spacer but is not required for basal transcription. To
assess the role of UBF in modulating ribosomal DNA transcription, we
studied its expression in NIH3T6 fibroblasts when transcription was
repressed in response to serum starvation and stimulated following
refeeding. Our results demonstrate a correlation between the amounts of
UBF protein and the rates of ribosomal DNA transcription in quiescent
and serum-stimulated cells. Nuclear run-on assays and Northern blot
analyses demonstrated that the UBF gene was a primary response gene,
exhibiting characteristics similar to those of c-myc and SRF. These results suggest that the regulation of
transcription of the UBF gene by polymerase II represents a pathway by
which cells modulate transcription by RNA polymerase I. Mitotic cell growth requires continuous ribosome biogenesis to
ensure that subsequent generations contain the ribosomes necessary to
support protein synthesis. The more rapidly cells progress through the
cell cycle, the more rapidly the process of ribosome biogenesis must
occur(1, 2, 3, 4) . This process is
frequently limited by the rate of transcription of the 45 S rRNA genes
(rDNA), the precursor of 18, 5.8, and 28 S rRNA. Thus, the mechanism by
which mammalian cells regulate transcription by RNA polymerase I is a
question central to our understanding of the process of cellular
growth. The RNA polymerase I transcription factor UBF can activate
transcription from the rDNA promoter by binding to elements within the
promoter and to enhancer elements within the nontranscribed
spacer(1, 2, 3, 4) . One mechanism
by which cells regulate the activity of UBF is by controlling its
degree of phosphorylation(5, 6) . Transcription by
RNA polymerase I is tightly coordinated with the rate of cell growth.
Ribosomal RNA synthesis is significantly down-regulated when cells are
exposed to protein synthesis inhibitors, made quiescent, or exposed to
hormones that repress
growth(3, 9, 10, 11) . It has been
shown that RNA polymerase I isolated from such cells has a severely
diminished ability to initiate specific
transcription(9, 10, 11, 12) . The
purified RNA polymerase I transcription factor(s) that directs this
activity is referred to as either TFIC(13) ,
TIF-IA(14) , or Factor C* (15) by the laboratories
studying this problem. These three factors may represent different
biochemical preparations of the same factor. However, this remains to
be established. In addition to this regulatory mechanism, we have
demonstrated that upon serum deprivation, the phosphorylation of a
second RNA polymerase I transcription factor, UBF, decreases, reducing
its ability to activate rDNA transcription(5) . Further, when
cells are refed serum or presented with other growth stimuli, the
phosphorylation of UBF increases in conjunction with the increase in
rDNA transcription (5) . ( The possibility that various cell types might exhibit
alternative pathways by which UBF activity is regulated was then
considered. To address this question, we measured the rates of rDNA
transcription, the amounts of UBF protein and mRNA, and UBF gene
expression in exponentially growing, quiescent (serum-starved), and
stimulated (cells stimulated by the addition of 10% serum after
starving) NIH3T6 cells. Here we present evidence that the amount of UBF
in NIH3T6 fibroblasts decreases in response to serum starvation and
that upon refeeding the level of UBF returns to original levels.
Furthermore, the rate of accumulation of UBF mRNA when the cells are
stimulated and the resistance of this increase to cycloheximide
demonstrate that UBF is a primary response gene. The results of nuclear
run-on assays confirm this conclusion. The rate of UBF mRNA synthesis
increased 20-fold when cells were stimulated by serum as compared with
the relatively low level observed in quiescent, subconfluent cells. In
conjunction with previous studies(7, 8) , these
results suggest that the regulation of expression of the UBF gene (a
gene transcribed by RNA polymerase II) represents a pathway by which
cells modulate transcription by RNA polymerase I.
These studies were designed to investigate the regulation of
UBF protein and mRNA levels during transitions in the rates of rDNA
transcription and growth. As indicated in Fig. 1A,
nuclear run-on assays confirmed that the rate of rDNA transcription was
depressed in serum-starved, quiescent cells. When the fibroblasts were
stimulated by the addition of serum, the rate of rDNA transcription
increased over a period of approximately 12 h to that exhibited by
exponentially growing cells. This relatively slow return to a normal
rate of transcription suggested that the factor limiting the rate of
rDNA transcription was not one with a rapid turnover. One of the likely
candidates, Factor C*, has a short half-life(12, 15) .
Thus, it is unlikely that the activity of this transcription initiation
factor is the only component modulating the rate of rDNA transcription
in response to serum stimulation.
Figure 1:
Comparison of ribosomal DNA
transcription and UBF protein levels in exponentially growing,
quiescent, and serum-stimulated fibroblasts. A, ribosomal DNA
transcription in nuclei isolated from exponentially growing (Exp.), quiescent, and serum-stimulated fibroblasts. Nuclei
were isolated, and nuclear run-on assays were carried out using equal
numbers of nuclei as described(20) . The radioactivity
hybridized to the rDNA was detected by autoradiography and quantitated
by either liquid scintillation spectrophotometry of the blots or laser
densitometry of the autoradiographs. The lack of hybridization to
control slots containing vector sequences, not shown, demonstrated the
specificity of the hybridization reactions. The autoradiograph
represents the results of a typical experiment. The bargraph depicts the average results of three separate
determinations. B, UBF levels in exponentially growing,
quiescent, and serum-stimulated fibroblasts. UBF1 and UBF2 protein
levels were detected in cell lysates from exponentially growing,
quiescent, and serum-stimulated fibroblasts, prepared as
described(20, 21) , following SDS-PAGE of cell lysates
(10 µg of protein) on 10% polyacrylamide gels by Western blot
analysis using UBF-specific antibodies (21) and goat,
anti-rabbit IgG coupled to alkaline phosphatase as
described(20) . The experiment was repeated five
times.
On the other hand, a second
candidate, UBF, has been reported to have an apparent half-life of 24 h
in L6 myoblasts(7) . This latter observation led us to examine
the possibility of a correlation between the amount of UBF present in
the fibroblasts and the rate of rDNA transcription. Western blot
analyses demonstrated that the amount of UBF protein present in
fibroblasts decreased significantly following the removal of serum from
the culture medium (Fig. 1B, lanes1 and 2). Furthermore, when quiescent, subconfluent 3T6
cells were stimulated by the addition of serum, the amount of UBF
protein increased to nearly normal levels after only 5 h (Fig. 1B, lane3). Twelve h after
refeeding, the level of UBF was 50% greater than that found in
exponentially growing cells (Fig. 1B, lane4). The results from the Western blots and the nuclear
run-on assays demonstrated that the rate of rDNA transcription
paralleled the changes in the mass of UBF after stimulation of growth
in response to serum. Northern blot analyses were performed to
determine whether alterations in the cellular level of UBF mRNA could
account for the changes in UBF protein that occurred in response to
serum stimulation (Fig. 2). Analysis of the levels of UBF mRNA
in serum-starved cells demonstrated a decrease in UBF mRNA (Fig. 2, lanes1 and 2). Following
serum stimulation, the level of UBF mRNA increased relatively rapidly.
Three h after serum stimulation, the amount of mRNA increased 4-fold
compared with the level observed in quiescent cells (Fig. 2A, lanes 7-9). The level of UBF
mRNA in the stimulated cells peaked by 6 h and was greater than that
observed in exponentially growing cells. Twenty-four h after serum
stimulation, the level of UBF mRNA returned to that found in
exponentially growing cells (Fig. 2, compare the first and lastlanes). Interestingly, the increase in
UBF mRNA did not require de novo protein synthesis as revealed
by its resistance to cycloheximide treatment (Fig. 2A, lane6).
Figure 2:
UBF
mRNA levels change in response to serum. A, RNA was isolated
from exponentially growing (Exp.), quiescent (Qui.),
and stimulated fibroblasts following addition of serum at the times
indicated and from refed fibroblasts treated with cycloheximide (Chx, 5 µg/ml) and serum (10%) for 1 or 2 h. RNA samples
(20 µg) isolated from exponentially growing, quiescent, and
stimulated fibroblasts following addition of serum at the times
indicated were denatured, separated by agarose-formaldehyde gel
electrophoresis, blotted to Zeta-Probe filters, hybridized to the
probes for the indicated mRNAs, and subjected to autoradiography. B and C, comparison of the levels of UBF mRNA, c-myc mRNA, and c-fos mRNA with
time in response to changes in serum concentration and cycloheximide (Cyclo., 5 µg/ml). Culture conditions were as described
under ``Materials and Methods.'' RNA was isolated at the
times indicated from cells treated as indicated, and the levels of the
mRNAs for UBF, c-myc, and c-fos were detected as
described.
Since the levels of UBF mRNA increased
relatively rapidly and occurred in the presence of cycloheximide, we
examined the possible correlations between the behavior of UBF and
other primary response genes such as c-myc and c-fos. RNA was isolated from fibroblasts
that had been serum-stimulated for short periods of time. These samples
were then probed for c-myc and c-fos both to confirm that the fibroblasts displayed the classic primary
response to serum deprivation and stimulation and to compare the
magnitude and kinetics of their induction with that of UBF mRNA (Fig. 2B). The time course and magnitude of UBF mRNA
induction more closely paralleled the pattern observed for c-myc mRNA than the pattern for c-fos (Fig. 2C). However, the maximum induction of UBF
mRNA occurred 3-6 h after serum stimulation by which time the
levels of both c-fos and c-myc had returned to that found in exponentially growing cells. The
rate with which the level of UBF mRNA increased upon serum stimulation
suggested that its accumulation resulted from an elevated rate of UBF
gene transcription. We therefore carried out nuclear run-on assays to
establish the relative rates of UBF mRNA synthesis in exponentially
growing, quiescent, and serum-stimulated cells (Fig. 3). These
assays revealed that in response to the withdrawal of serum the rate of
synthesis of UBF mRNA decreased to 15% of that observed in
exponentially growing cells. Furthermore, nuclei isolated from cells
that had been stimulated by the addition of serum for 2 h synthesized
UBF mRNA at a rate 20-fold greater than that observed in nuclei
isolated from quiescent, subconfluent cells. These observations
demonstrated that the increased cellular content of UBF observed in
serum-stimulated cells resulted from increased rates of transcription
of the UBF gene.
Figure 3:
Transcription of the UBF gene in nuclei
isolated from exponentially growing (Exp.), quiescent (Quies.), and serum-stimulated fibroblasts after 2 and 4 h.
Cells were cultured and nuclear run-on assays carried out as described
under ``Materials and Methods.'' The autoradiograph presents
the results of a typical experiment. The bargraph depicts the average results of three separate determinations. The
radioactivity hybridized to p405 rUBF was detected by autoradiography
and quantitated by either liquid scintillation spectrophotometry or
laser densitometry. The lack of hybridization to control slots
containing vector sequences demonstrated the specificity of the
hybridization reactions.
Primary response genes are rapidly induced when
quiescent, subconfluent cells are stimulated by the addition of
growth-promoting factors, and this induction does not require de
novo protein synthesis. The observation that the rise in UBF mRNA
is an early event following serum stimulation and is resistant to
cycloheximide supports the hypothesis that the UBF gene is a primary
response gene(16) . Moreover, the pattern of regulation of UBF
mRNA is similar to those patterns demonstrated by other primary
response genes such as c-myc, SRF (serum
response factor), and fra-1(16, 17) . While
these genes are down-regulated in quiescent cells and transiently
induced in response to serum, they are constitutively expressed in
proliferating cells. The pattern of regulation of UBF mRNA reported
here is most similar to that of SRF mRNA with regard to the degree of
induction by serum and the effect of cycloheximide on this
response(18) . Interestingly, the continued accumulation of UBF
mRNA after refeeding in the presence of cycloheximide, when rDNA
transcription is turned off, suggests that the UBF gene is responding
to the signal to resume rDNA transcription and not as a consequence of
rDNA transcription. A consideration of these results argues that the
increased rate of rDNA transcription occurring following serum
stimulation is modulated by UBF rather than (or in addition to) Factor
C* as has been suggested in other reports. The relatively slow recovery
of rDNA transcription suggests that one or more of the components
required for transcription must be accumulating during this period.
Tower and Sollner-Webb (12) have demonstrated that Factor C*
activity recovers rapidly, within 1 h, after being down-regulated by
cycloheximide. While this response may differ from the recovery of
Factor C* activity after serum starvation and refeeding, our results
suggest that it is unlikely that the rate of the rise in rDNA
transcription upon serum stimulation solely reflects the accumulation
of Factor C*. The parallel increases in UBF protein and rDNA
transcription suggest a cause and effect relationship. This
interpretation is consistent with previous in vitro(24) and in vivo observations(8, 19) . Our results suggest that at
least one mechanism by which cells mediate transcription by RNA
polymerase I involves the regulation of transcription of the UBF gene
by RNA polymerase II. On the other hand, when Chinese hamster ovary
cells are serum-starved and refed the amount of UBF does not vary.
Rather, the phosphorylation state, viz. activity, of UBF is
increased when serum-starved cells are refed(5) . This would
suggest multiple pathways for the regulation of UBF activity.
Interestingly, these two pathways can coexist in the same cell type. We
have found that primary cultures of neonatal cardiomyocytes utilize one
or the other of these pathways in response to different hypertrophic
stimuli. (
Volume 270,
Number 9,
Issue of March 3, 1995 pp. 4209-4212
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)These results are
consistent with the hypothesis that at least one of the mechanisms by
which cells control rDNA transcription involves regulation of the
activity of UBF. A corollary of this hypothesis is that in some cells
this regulation involves pathways that lead to increases or decreases
in the level as well as activity of UBF. Consistent with this model, we
have noted that when L6 myoblasts were induced to differentiate, there
were simultaneous decreases in the content of UBF and the rate of rDNA
transcription. These were preceded by a decrease in UBF mRNA levels (7) .
Cell Culture
NIH3T6 fibroblasts, 3
10
cells, were plated on 160-mm plastic tissue
culture plates and grown at 37 °C and 5% CO
overnight
in 
-modified Eagle's medium supplemented with 10% fetal
bovine serum. When cells were to be serum-starved, the medium was
replaced with -modified Eagle's medium supplemented with
0.5% serum. After 3 days in low serum medium, quiescent fibroblasts
were serum-stimulated by adding fresh medium containing 10% fetal
bovine serum.Nuclear Run-on Assays
Nuclei were
isolated, and nuclear run-on assays were carried out using equal
numbers of nuclei as described previously(20) . When rDNA
transcription was to be measured, elongation was carried out for only
15 min. In vitro elongated, radiolabeled RNA was purified and
hybridized to 5 µg of the mouse 45 S rRNA gene sequences (22) immobilized on nitrocellulose. When transcription from the
UBF gene was to be measured, the assays included 250 µCi of
[
P]UTP/5 10 nuclei, and
elongation was carried out for 30 min. Transcription of the UBF gene
was detected by hybridizing the transcripts (50 10 cpm/hybridization reaction) to 5 µg of immobilized
p405rUBF(21) . The radioactivity hybridized to the rDNA was
detected by autoradiography and quantitated by either liquid
scintillation spectrophotometry of the blots or laser densitometry of
the autoradiographs. The lack of hybridization to control slots
containing vector sequences (results not shown) demonstrated the
specificity of the hybridization reactions.Western Blot Analysis
UBF1 and UBF2
protein levels were detected in cell lysates from exponentially
growing, quiescent, and serum-stimulated fibroblasts as
described(20, 21) . Following SDS-PAGE ()of
cell lysates (10 µg of protein) on 10% polyacrylamide gels, UBF
protein was visualized by Western blot analysis using UBF-specific
antibodies and goat, anti-rabbit IgG coupled to alkaline phosphatase as
described (20, 21) .Northern Blot Analysis
RNA was isolated
from exponentially growing, quiescent, and stimulated fibroblasts
following addition of serum at the times indicated as described by Xie
and Rothblum(23) . In some instances the refed fibroblasts were
also treated with cycloheximide (5 mg/ml) for the times indicated. RNA
samples (20 µg) were denatured and separated by
agarose-formaldehyde gel electrophoresis and blotted to Zeta-Probe
filters. To visualize the UBF mRNA, the blots were hybridized with a P-labeled probe for UBF, p405rUBF, a cDNA clone for
UBF1(21) , washed, and subjected to autoradiography and
quantitated by laser densitometry. Elongation factor EF-1a mRNA
sequences were detected using pHFBA13 (ATCC 37854). The probes used to
detect c-fos and c-myc mRNAs were
generously provided by Dr. R. Crawford (Howard Florey Institute,
Melbourne, Australia). All probes were generated by random-primed
labeling. The radioactivity associated with each specific mRNA was
detected by autoradiography and quantitated by scanning densitometry.
)Thus, the existence of multiple, non-exclusive
pathways for regulating the activity of UBF provides cells with a means
to fine tune rDNA transcription in response to a wide array of stimuli.
)))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
