(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) . ()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) .
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. ()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.