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Department of Molecular Microbiology and Immunology and K. Norris Jr. Comprehensive Cancer Center, University of Southern California, School of Medicine, Los Angeles, California 90033
Abstract
Transcription by RNA polymerase I (pol I) regulates the rate of ribosome biogenesis and the biosynthetic potential of the cell; therefore, it plays an important role in the control of cell growth. Differentiation of the human promyelocytic leukemic cell line U937 is accompanied by drastic decreases in pol I transcriptional activity. We have used cell-free extracts prepared from undifferentiated and differentiated U937 cells to investigate the molecular mechanisms responsible for this inhibitory process. Our analysis indicates that the activity of the TATA binding protein (TBP)/TBP-associated factor (TAF) complex selectivity factor 1 (SL1), one of the factors required for accurate and promoter-specific transcription by RNA pol I, is severely repressed in differentiated U937 cells. Moreover, the reduction in SL1 activity is not a consequence of a decrease in SL1, because there is no detectable difference in the abundance of TBP or TAFs before and after U937 cell differentiation. In conclusion, our results indicate that the selectivity factor SL1 is an important target for the regulation of pol I transcription during cell differentiation.
Introduction
During hematopoietic cell differentiation, quantitative and qualitative changes in gene expression occur, and disruption of the balance between proliferative and antiproliferative signals can lead to the abnormal growth associated with leukemia and other neoplastic disorders. The underlying regulatory mechanisms that are involved in these processes are poorly understood. The human promyelocytic leukemic cell line U937 is an established model for studying hematopoietic cell differentiation in vitro (1) . These immature cells can be induced by the phorbol ester TPA4 to differentiate along the monocytic lineage into functionally and morphologically mature nonproliferating cells.
Because a large amount of the cell’s energy and resources during cell growth and cell division are used to make ribosomes, regulation of rRNA synthesis and ribosomal biogenesis may provide an important mechanism for controlling these cellular processes. Thus, molecules that modulate the expression of the rRNA genes may exert a dual effect on both cell growth and cell division (2) .
rRNA genes are transcribed by a specialized polymerase, RNA pol I, which is localized in the nucleoli of eukaryotic cells (3, 4, 5) . At least two factors, UBF and SL1, in addition to RNA pol I, are necessary to direct accurate and promoter-specific initiation of transcription from the rRNA gene promoter (6 , 7) . Human UBF is a Mr 97,000 polypeptide that recognizes both the core and upstream control elements of the human rRNA promoter in a sequence-specific manner (8 , 9) . Human UBF contains four high mobility group boxes, one of which mediates DNA binding and has a hyperacidic tail that is necessary for transactivation (10 , 11) . The NH2-terminal region has been found to mediate UBF dimerization (10) . The second essential factor necessary for accurate RNA polymerase I transcription is the selectivity factor SL1. SL1 is a multisubunit complex composed of the TBP and three TAFs, TAFI48, TAFI63, and TAFI110 (12, 13, 14) . SL1 does not bind specifically to the rRNA promoter. However, in presence of UBF, a strong cooperative DNA binding complex is formed at the rRNA promoter that is critical for initiation of transcription (6 , 8) . The recruitment of SL1 to the promoter is mediated by the COOH-terminal activation domain of UBF and is modulated by UBF phosphorylation (11) . Mutations in UBF that abolish DNA binding, such as the removal of the high mobility group box 1, or that impair the interaction between UBF and SL1, such as dephosphorylation or removal of the COOH-terminal domain, result in a drastic reduction in pol I transcriptional activity (10 , 11 , 15) . In addition, a recent study also showed that the interaction between SL1 and UBF is influenced by the phosphorylation status of at least one of the TAFIs in the SL1 complex (16) . Thus, these findings provide strong evidence that the network of interactions among UBF, SL1, and the rDNA promoter plays a major role in the regulation of pol I transcription.
RNA pol I activity is tightly linked to the signals that control cell growth (3 , 4) , and a number of physiological and pathological stimuli affect the rate of RNA pol I transcription (17, 18, 19, 20, 21) . Recently, it has been proposed that the retinoblastoma tumor suppressor gene product (pRb) may be involved in the regulation of RNA pol I transcription in human cells, which are induced to differentiate by the addition of TPA (22) . These studies showed that as soon as human myeloid cells U937 begin to differentiate, there is an accumulation of pRb in the nucleoli and a sharp decrease in rRNA synthesis (22 , 23) . In vitro experiments with mouse extracts and recombinant pRb suggested that the binding of pRb to UBF inhibits the DNA binding activity of this transcription factor (24) . Despite these studies, it has not been proven that the interaction between pRb and UBF is indeed responsible for the repression of pol I transcription in differentiated U937. To define the mechanism of pol I transcription inhibition in differentiated cells, we have analyzed the transcriptional properties of extracts from U937 cells that were induced to differentiate by treatment with TPA. In this study, we found that the activity of the SL1 factor was severely inhibited in TPA-treated U937 cells, whereas UBF activity was not affected. Interestingly, using Western blot analysis and immunoprecipitation assays, we were able to determine that there was no significant difference in the abundance of the SL1 factor between mock and TPA-induced cells. Taken together, these results suggest that inhibition of SL1 activity, most likely through a posttranslational modification of one or more of its components, is at the basis of the pol I transcription repression in differentiated hematopoietic cells.
Results
rRNA Transcription Is Drastically Reduced after Differentiation of
U937 Cells.
To determine the level of rRNA synthesis before and after
differentiation, total RNA was extracted from undifferentiated U937
cells and from cells that were induced to differentiate by treatment
with TPA for 19 h. Control cells were treated with the same volume
of ethanol, the solvent used to solubilize TPA. The level of
5'-precursor rRNA transcript in undifferentiated and differentiated
cells was determined by S1 nuclease protection assays. As shown in Fig. 1
, there is a dramatic difference in the abundance of 5' rRNA between
TPA-treated (Lanes 1 and 2) and mock-treated
(Lanes 3 and 4) U937 cells. Quantitation analysis
indicated that the level of pre-rRNA is reduced 
6–8-fold after cell
differentiation. ß-actin mRNA expression showed no variation between
mock-and TPA-treated
cells.5
Because the 5' end of the precursor rRNA is rapidly degraded, the level
of the 5'-end transcript faithfully reflects the rate of initiation of
transcription (25)
. Therefore, these results provide
further evidence that pol I transcription is repressed after U937 cell
differentiation (22)
.
|
, the RNA pol I transcriptional activity is 6-fold lower in the
TPA-induced U937 cell extract (Lanes 3 and 4), as
compared with the extract prepared from mock-induced cells (Lanes
1 and 2). These results demonstrate that the repression
of rRNA synthesis upon TPA-induced differentiation of hematopoietic
cells is faithfully reproduced in an in vitro transcription
system.
|
, the titration of increasing amounts of extracts from differentiated
U937 cells (Lanes 3–5) did not affect the transcriptional
activity of the extracts from undifferentiated cells. These results
exclude the possibility that the extracts from TPA-induced cells
contain a soluble inhibitor of transcription and suggest that the
inhibition of rRNA synthesis is most likely attributable to a specific
decrease in the activity of one or more components of the pol I
transcriptional machinery.
|
.
|
. To exclude the possibility that UBF
added to the transcription reactions was nonfunctional, the
transcriptional activity of HeLa-purified UBF, using in
vitro reconstituted transcription assays with purified SL1 and RNA
pol I, is shown in Fig. 5C
. Thus, differentiated U937 cell
extracts appear to be primarily deficient in SL1 activity.
Interestingly, the addition of purified SL1 could also stimulate the
transcriptional activity of undifferentiated U937 cell extract,
suggesting that SL1 is a limiting factor for pol I transcription in
U937 cells.5
|
A, the SL1 fraction from differentiated (TPA-induced) U937
cells (Lanes 5–7) is several folds less active than the
respective SL1 fraction from the undifferentiated cells (Lanes
2–4).
|
. UBF activity from both
undifferentiated and differentiated U937 extracts is quite low compared
with the activity of HeLa-purified UBF. Interestingly, the partially
purified UBF fraction from differentiated U937 cells was more active
than the corresponding fraction from undifferentiated cells, as
determined in both assays condition. These experiments have been
carried out in the presence of limiting amounts of SL1 (1.0 ng) to
maximize UBF response. These results indicate that UBF is not
down-regulated upon U937 cell differentiation and further support the
concept that a reduction in the activity of the SL1 factor is the major
cause for the inhibition of rRNA synthesis in differentiated U937
cells.
The Abundance of SL1 Does Not Change after Cell Differentiation.
Human SL1 is a multiprotein complex comprised of TBP and three
associated factors, TAFI48,
TAFI63, and TAFI110.
Several studies have indicated that each of these factors is necessary
for the assembly of a functional SL1. To determine whether the
reduction in SL1 activity in differentiated cells was attributable to a
decrease in the abundance of one or more of its components, we carried
out a series of Western blot analyses. Whole-cell extracts from
undifferentiated and differentiated U937 cells were resolved on
SDS-PAGE gels, and the abundance of each of the four components of SL1
before and after differentiation was determined using antibodies raised
against each protein. As shown in Fig. 7
A, there was no detectable change in the level of TBP,
TAFI48, TAFI63, and
TAFI110 proteins after cell differentiation. In
addition, the abundance of the UBF factor also did not change between
undifferentiated and differentiated cells. The results strongly suggest
that differentiation of U937 cells and inhibition of pol I
transcription is not accompanied by a specific decrease in the level of
any of the SL1 subunits. In addition, we observed an additional slower
migrating band in the anti-TBP Western blot with differentiated
extracts. To determine whether this band was a phosphorylated form of
TBP, we performed the Western blot analysis using extracts that were
pretreated with alkaline phosphatase. As shown in Fig. 7
B,
the slower migrating TBP band disappears on phosphatase treatment,
suggesting that the diminished SL1 activity in differentiated cells may
indeed result from posttranslational modifications of a SL1 subunit,
such as TBP.
|
, no significant differences in the amount of TBP can be seen in the
immunoprecipitation reactions, suggesting that SL1 remains associated
as a stable complex after differentiation. These results provide
further evidence that the decrease in SL1 specific activity is likely
attributable to a posttranslational modification of one or more of its
subunits. Of course, we cannot rule out that the decrease in SL1
activity may be caused by the absence of the other SL1 subunits,
TAFI48, within the complex. Because of the low
titer and poor quality of the antibodies against
TAFI48, we were not be able to obtain
interpretable data in anti-TAFI48
immunoprecipitation reactions. Thus, we cannot strictly rule out that
TAFI48 may be excluded from the SL1 complex after
cell differentiation.
|
TPA treatment of U937 human myeloid leukemia cells initiates a cascade of cellular events that profoundly influence the expression of a variety of genes and ultimately lead to cell differentiation. Differentiation of U937 cells is associated with a significant decrease in rRNA transcription. The TPA-induced repression of rRNA transcription is of particular interest because it directly affects ribosome production and protein synthesis and, therefore, provides a direct mechanism to control cell growth (2 , 27) . To elucidate the molecular mechanisms underlying this repression of pol I transcription, we have analyzed the components of the transcriptional machinery before and after differentiation. Our studies indicate that the decrease in transcription reflects a drastic reduction in the activity of the selectivity factor SL1. Complementation assays and fractionation experiments reproducibly show that SL1 activity is between 8 and 14 folds lower in differentiated than undifferentiated U937 cells. This is a significant finding that implies that regulation of SL1 activity has an in important role in the modulation of pol I transcription. Interestingly, Western blot analysis of SL1 from extracts prepared from undifferentiated and differentiated U937 cells showed no appreciable difference in the abundance of any of the SL1 subunits. Immunoprecipitation assays also show that in either extract, SL1 is found as a stable multiprotein complex. These results suggest that SL1 activity is most likely modulated by a posttranslational event induced by the differentiation process. The mouse orthologue of SL1, TIF-IB, has been shown recently to be the target of regulation during mouse F9 embryonal carcinoma stem cell differentiation, and the reduction in TIF-IB activity appeared to be associated with a decrease in the abundance of two mouse TAFs, mTAFI48 and mTAFI95 (28) . On the other hand, recent studies have indicated that mitotic inactivation of human rRNA synthesis is mediated by phosphorylation of TAFI110, one of the subunits of SL1 (24) . It is therefore tempting to speculate that the mechanism of human SL1 inactivation in differentiated U937 cells resembles the mitotic process. It is currently unclear whether phosphorylation or dephosphorylation of any of the SL1 subunits is involved in down-regulation of SL1 activity in differentiated U937 cells. Preliminary experiments indicate that a slower migrating form of TBP, present in the extracts from differentiated U937 cells, disappears upon treatment with the alkaline phosphatase. These results suggest that phosphorylation of TBP may play a role in down-regulation of SL1 activity. In addition, we attempted to analyze the phosphorylation state of SL1 before and after differentiation of U937 cells using in vivo labeling experiments. Unfortunately, these experiments were inconclusive because of the inability to detect SL1, an extremely low abundance factor in the cell, in immunoprecipitates from [32P]Pi-labeled cell extracts.
In addition to determine the activity of SL1, we have also analyzed the transcriptional properties of the partially purified UBF fractions. The results of the in vitro reconstituted transcription assays indicate that UBF activity in differentiated cells is higher that in undifferentiated cells. The significance of these findings is currently unclear and requires further investigation. Nevertheless, these results further support our finding that down-regulation of SL1 is at the basis of pol I repression upon U937 cell differentiation.
Previous studies indicated that differentiation of U937 is accompanied by a relocalization of pRb to the nucleolus (22 , 23) . Once in the nucleolus, pRb can bind to UBF (22 , 24) .5 These findings led to the demonstration that, in vitro, pRb can repress pol I transcription by directly binding to UBF (23 , 24) . However, it has never been shown that the activity of UBF is affected upon U937 cell differentiation. On the other hand, our biochemical studies strongly suggest that down-regulation of SL1 activity is at the basis of the repression of pol I transcription in differentiated U937 cells. In addition, our data show that UBF is more active in the differentiated cells. The difference between our findings and the published data may reflect differences in the experimental approach. It is possible that in the reconstituted transcription assays using mouse extracts, recombinant pRb is sufficient for the inhibition of pol I transcription by binding, stoichiometrically, to UBF. However, it is unclear if there is a stoichiometric interaction between pRb and UBF in differentiated U937 cells.5 Conversely, our analysis has been performed on endogenous factors using U937 cell extracts; therefore, it may better recapitulate the process that occurs in vivo. Our study cannot rule out that the binding of pRb to UBF is still required for repression of pol I transcription in differentiated U937 cells. It is conceivable that the interaction between UBF and pRb may represent one step in a process that ultimately leads to the down-regulation of SL1 activity and repression of pol I transcription. In analogy to the recently proposed mechanism of pRb repression of class II genes, pRb may function in the recruitment of other factors to the rDNA promoter that facilitate or directly catalyze the inactivation of SL1 (29) . In conclusion, our study underscores the role of SL1 as a critical target for regulation of pol I transcription in differentiated hematopoietic cells. The precise role of pRb in this process remains to be elucidated, and future studies will address the link between SL1, pRb, and repression of pol I transcription in differentiated hematopoietic cells.
Materials and Methods
Cell Culture.
U937 cells were grown and subcultured every 2 days in RPMI 1640
supplemented with 10% heat-inactivated FCS at
37oC, 5% CO2 in humidified
atmosphere. After seeding the cells in fresh growth medium at an
initial density of 2–3 x 105 cells/ml, TPA
was added for 19 h as a stock solution in ethanol to achieve the
final concentration of 100 nM. Control cells (mock-induced)
received an equal volume of ethanol. HeLa S3 cells were grown in
suspension in MEM supplemented with 5% newborn calf serum.
Preparation of Cell Extracts.
For the preparation of cell extracts from transcription assays, Western
blots, and immunoprecipitations, we harvested each time 4–6 x
108 cells [12–15 (150-mm) plates].
Whole-cell extracts were prepared accordingly to the method developed
by Manley et al. (30)
. Nuclear extracts were
prepared as described in Zhai et al. (21)
.
Whole-cell extracts were used as starting material for the
fractionation studies. Partially purified SL1 was prepared by
chromatography on Poros HE1 (heparin-agarose). Briefly, whole-cell
extracts were loaded onto a Poros HE1 column in TM buffer [50
mM Tris (pH 7.9), 12 mM
MgCl2, 1 mM EDTA, and 10%
glycerol] containing 0.1 M KCl. The column was
washed extensively with TM/0.1, and it was then step-eluted with TM
buffer containing 0.3 M KCl, 0.4
M KCl, and 0.8 M KCl. RNA
pol I eluted at 0.3 M KCl, UBF eluted at 0.4
M KCl, and SL1 eluted at 0.7
M KCl. Transcription assays and Western blot
analyses were used to identify fractions containing RNA pol I, UBF, and
SL1. Peak fractions for each activity were pooled and dialyzed into TM
containing 0.1 M KCl and 0.1% NP40. HeLa RNA pol
I, UBF, and SL1 were prepared as described previously (11
, 12)
. Protein concentrations were determined by Bradford assay.
Transcription Assay.
RNA pol I transcription assays were carried out as described previously
using either 30 or 100 ng of rDNA gene template in the presence of 100
µg/ml of 
-amanitin. Quantitation analysis was performed using a
PhosphorImager (Molecular Dynamics). In the extract mixing experiment,
the reaction mixture was incubated at 30oC for 15
min before the addition of all four ribonucleotides.
RNA Purification and Analysis.
Total RNA was isolated using a single step procedure by
guanidinium thiocyanate-acid phenol-chloroform extraction. Briefly,
cells were collected and lysed in denaturing buffer [4 M
guanidinium isothiocyanate, 25 mM sodium citrate (pH 7.0),
0.5% sodium lauryl sarcosine, and 0.15 M
2-mercaptoethanol]. Genomic DNA was sheared by pipetting up and down
several times, and total RNA was prepared by the addition of 2
M sodium acetate (pH 4.0), 1 volume of water-saturated
acidic phenol, and one-fifth of chloroform:isoamyl alcohol (24:1).
Samples were vortexed and centrifuged at 10,000 x g,
and the RNA-containing aqueous phase was carefully collected. RNA was
further precipitated by the addition of 2.5 volumes of ethanol, washed
with 70% ethanol, air-dried, and dissolved in diethyl
pyrocarbonate-treated water. After quantitation by spectrophotometry,
equal amounts of RNA were used for S1 nuclease analysis. S1 nuclease
analysis was carried out using an oligonucleotide complementary to the
region between -20 and 40 of the human rDNA gene that was labeled with
32P at the 5' end. Preliminary experiments were
performed to assure that the radiolabeled oligonucleotide used in each
reaction was in vast (>10 fold) excess over the target RNA.
RNA pol I Assay.
Random RNA polymerization assays were performed as described by
Roeder (31)
. Each reaction mixture contained 5 µg of
nicked herring sperm DNA, 100 µg/ml -amanitin, and 10 µg of
protein extract.
Antibodies and Immunoprecipitation Analysis.
Rabbit polyclonal antisera raised against recombinant TBP, TAFs, and
UBF were affinity-purified accordingly to published procedures.
Immunoprecipitation reactions were carried out as described by Comai
et al. (12)
. Immunoreactivity was shown by the
alkaline-phosphatase detection method.
Acknowledgments
We are grateful to all of the members of our laboratory for helpful suggestions and discussions. We thank J. Tuan and A. Nee for technical assistance. We are also thankful to the University of Southern California Liver Center for the use of the PhosphorImager.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was funded by Research Grant
RPG-97-058-01-NP from the American Cancer Society. 
2 To whom requests for reprints should be
addressed, at Department of Molecular Microbiology and Immunology,
School of Medicine, University of Southern California, 2011 Zonal
Avenue, HMR-509, Los Angeles, CA 90033. Phone: (323) 442-3950; Fax:
(323) 442-1721.
3 Present address: Chengdu Institute of Biological
Products, Chengdu, Sichuan, 610063, People’s Republic of China.
4 The abbreviations used are: TPA,
12-O-tetradecanoylphorbol 13-acetate; pol I, RNA
polymerase I; UBF, upstream binding factor; SL1, selectivity factor 1;
TBP, TATA binding protein; TAF, TBP-associated factor; pRb,
retinoblastoma protein.
5 L. Comai, Y. Song, and T. Bui, unpublished
results.
Received for publication 8/16/99. Revision received 10/ 1/99. Accepted for publication 10/15/99.
References
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