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Cell Growth & Differentiation Vol. 11, 63-70, January 2000
© 2000 American Association for Cancer Research


Articles

Inhibition of RNA Polymerase I Transcription in Differentiated Myeloid Leukemia Cells by Inactivation of Selectivity Factor 11

Lucio Comai2, Yahui Song, Changyao Tan3 and Tiffany Bui

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. 1Citation , 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) .



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Fig. 1. RNA pol I transcription decreases after U937 cell differentiation. S1 analysis of total RNA (5 µg) isolated from TPA-induced (Lanes 1 and 2) and mock-induced (Lanes 3 and 4) U937 cells. Two independent preparations of total RNA from mock- and TPA-induced cells were tested. The RNAs were hybridized with a 5'-end, 32P-labeled 60 bases oligonucleotide complementary to the nucleotide -20 and 40 of the human ribosomal DNA gene. Quantitation by PhosphorImager indicates that transcription is ~8-folds lower in differentiated (TPA-treated) cells (not shown).

 
Extracts of Differentiated U937 Cells Are Impaired for pol I Transcription.
To dissect the molecular events responsible for the down-regulation of transcription by RNA pol I in differentiated U937 myeloid cells, we then established an in vitro transcription system. Extracts were prepared from U937 cells that were either induced to differentiate with TPA at a concentration of 100 nM or mock-induced with an ethanol solution without TPA. Equal amounts of extracts were tested in an in vitro transcription assay using a plasmid containing the human ribosomal DNA promoter as template. Transcription assays were performed under the standard conditions, and rRNA transcripts were detected using the S1 nuclease assay (26) . As shown in Fig. 2Citation , 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.



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Fig. 2. Extracts from TPA-treated cells are deficient in pol I transcriptional activity. A human rDNA template (prHu3) was transcribed in vitro using 4 µg (Lanes 1 and 3) or 6 µg (Lanes 2 and 4) of whole-cell extracts (WCE) from undifferentiated (Lanes 1 and 2) or differentiated (TPA-treated; Lanes 3 and 4) U937 cells. Whole-cell extracts were prepared as described in "Materials and Methods." Transcription data were quantitated using a PhosphorImager, and relative activities are as indicated.

 
Extracts from Differentiated U937 Cells Do Not Contain a Soluble Repressor of pol I Transcription.
To determine whether the decreased transcriptional activity was attributable to a soluble repressor found in the extracts from differentiated cells, we performed a mixing experiment. A constant amount of extract from undifferentiated U937 cells was mixed with increasing amounts of extracts from TPA-induced cells, incubated on ice for 30 min, and then tested in transcription reactions. As shown in Fig. 3Citation , 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.



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Fig. 3. Differentiated U937 cell extract does not contain a soluble repressor. A human rDNA template was transcribed in vitro using 5 µg of undifferentiated (mock-U937; Lanes 1–5) or differentiated (TPA-U937; Lanes 6 and 7) U937 whole-cell extract. In Lanes 3–5, increasing amounts (5, 10, and 15 µg, respectively) of extracts from differentiated cells were added to the reaction mixtures containing extracts (5 µg) from undifferentiated cells and the rDNA template and incubated at 30oC for 15 min before the addition of nucleotides. In Lane 1, the rDNA template was omitted.

 
SL1 Activity Is Specifically Diminished in Differentiated U937 Cell Extracts.
To determine whether the defect in transcription was attributable to an inhibition of the RNA pol I itself, we measured the pol I enzymatic activity in differentiated and undifferentiated U937 cell extracts. For this purpose, we used an assay that measures the ability of RNA pol I to randomly initiate transcription on nicked DNA. Several matched pair samples were tested, and in none of them was there any significant difference in the level of nucleotide polymerization by pol I between undifferentiated and differentiated extracts (Fig. 4)Citation .



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Fig. 4. Undifferentiated and differentiated U937 cell extracts display similar levels of pol I polymerization activity. Nonspecific RNA pol I assays were carried out as described in "Materials and Methods" using HeLa, undifferentiated U937, and differentiated U937 whole-cell extracts. Incorporated [3H]UTP was counted in scintillation fluid with a Beckman scintillation spectrometer. Bars, SDs calculated from three independent experiments.

 
In addition to RNA pol I, two additional factors, UBF and SL1, are required for accurate transcription of human rRNA genes. Regulation of either of these two factors may account for the observed repression of pol I transcription after differentiation. To test this hypothesis, we determined whether the addition of exogenous SL1 or UBF was able to rescue pol I activity in differentiated U937. SL1 or UBF fractions purified from HeLa cells were added to in vitro transcription reactions containing extracts from TPA-induced cell. The results of these experiments indicated that SL1, but not UBF, was able to fully rescue RNA pol I activity (Fig. 5, A and B)Citation . 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. 5CCitation . 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



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Fig. 5. A, exogenous SL1 can stimulate pol I transcription in differentiated U937 cell extract. In vitro transcription reaction were carried out using 4 µg of whole-cell extracts from HeLa (Lanes 1 and 2), undifferentiated U937 (Lanes 3 and 4), and differentiated U937 (TPA-treated; Lanes 5–11). Reactions in Lanes 6, 8, 10, and 11 were supplemented with partially purified SL1 (Lanes 6 and 8; 30 and 60 ng, respectively) or pure UBF (Lanes 10 and 11; 10 and 40 ng, respectively). SL1 and UBF were purified from HeLa cells, and their activity was assessed by transcription and footprinting assays (11 , 12) . These results have been reproduced in several independent preparations. In addition, identical results have been obtained using nuclear extracts. Arrow, the position of the protected oligonucleotide product. B, pol I transcription in differentiated U937 nuclear extracts can be stimulated by the addition of purified SL1. Transcription assays were performed using nuclear extracts from differentiated U937 cells (Lanes 1, 4, and 7: 6 µg; Lanes 2, 5, and 8: 9 µg; Lanes 3, 6, and 9: 12 µg) and no SL1 (Lanes 1–3), 1 µl (10 ng/µl) of SL1 (Lanes 4–6), or 2 µl of SL1 (Lanes 7–9). Identical results were obtained using whole-cell extracts. Transcripts were quantitated using a PhosphorImager, and relative activities are as indicated. C, transcriptional activity of HeLa-purified UBF. Transcription assays were performed using pol I and SL1 purified from HeLa cells in the presence (Lanes 2 and 3) or absence (Lanes 1 and 4) of HeLa-purified UBF used in the experiments shown in A.

 
To confirm the deficiency of SL1 activity in TPA-induced U937 cells, we partially purified SL1 from undifferentiated and differentiated U937 extracts using a well-established fractionation procedure. The purified SL1 fractions were then tested in in vitro reconstituted transcription reactions in the presence of HeLa-purified pol I and UBF. Importantly, the partially purified SL1 fraction did not contain any detectable pol I and UBF activities (see "Materials and Methods"). As shown in Fig. 6Citation 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).



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Fig. 6. A, SL1 activity is repressed in differentiated U937 cell extracts. SL1 (0.8 M KCl fraction) was purified by heparin-agarose column chromatography from HeLa (Lane 1, 50 ng), undifferentiated U937 (Mock-U937; Lanes 2–4, 50, 100, and 200 ng, respectively), or differentiated U937 cells (TPA-U937; Lanes 5–7, 50, 100, and 200 ng, respectively), and used in transcription reactions with 100 ng of an HeLa fraction containing pol I and UBF. In Lanes 8 and 9, SL1 from undifferentiated U937 (Lane 8) or differentiated U937 (Lane 9) was used in transcription reactions in the absence of pol I and UBF. Transcripts were quantitated using a PhosphorImager, and relative activities are as indicated. B, UBF activity in U937 cell extracts. Top panel, UBF (0.4 M KCl fraction) from undifferentiated (Lanes 2, 3, and 6) or differentiated (Lanes 4, 5, and 7) U937 cells was used in reconstituted transcription assays with pol I and SL1 purified from HeLa cells. In Lane 1, the HeLa-purified pol I and SL1 were used in the absence of UBF. Arrow, the position of the protected oligonucleotide product. Transcripts were quantitated using a PhosphorImager, and relative activities are as indicated. Bottom, RNA pol I (0.3 M KCl fraction; 6 µg/reaction) and UBF fractions [0.4 M KCl fraction; 80 ng (Lanes 8 and 10) and 250 ng (Lanes 9 and 11)] from either undifferentiated (Lanes 8 and 9) or differentiated (Lanes 10 and 11) U937 extracts were used in reconstituted transcription assays in the presence of HeLa-purified SL1. In Lane 10, transcription was carried out with a HeLa-purified fraction containing pol I and UBF and HeLa SL1.

 
In addition, to determine whether the activity of UBF also changed upon differentiation, we tested the partially purified UBF fraction from undifferentiated and differentiated cells in an in vitro reconstituted assay with SL1 and RNA pol I purified from either HeLa or U937 cells (Fig. 6B)Citation . 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. 7Citation 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. 7Citation 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.



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Fig. 7. A, Western blot analysis of TBP, TAFs, and UBF from undifferentiated and differentiated U937 cells. Forty µg of mock-treated and TPA-treated U937 extracts were resolved on a 10% SDS-PAGE and analyzed by Western blot with antibodies as indicated. An SL1 fraction from HeLa cells was used as a control (SL1). All antibodies used were affinity purified except for anti-TAFI48. B, Western blot analysis of TBP from alkaline phosphatase-treated U937 extracts. Fifteen µg of mock-treated and TPA-treated U937 extracts were preincubated with 0.5 µl (1000 units/µl) of calf alkaline phosphatase (Lanes 1 and 3) or 0.5 µl of phosphatase buffer (Lanes 2 and 4) at 30°C for 15 min, resolved on a 10% SDS-PAGE, and then analyzed by Western blot with TBP antibodies. Arrow, slower migrating TBP band.

 
TAFs and TBP Are Stably Associated in an SL1 Complex in Differentiated U937 Cells.
Although each of the components of SL1 was present in similar quantities before and after differentiation, the previous experiment did not address whether SL1 was still present as a stable multiprotein complex after cell differentiation. For this purpose, SL1 was immunoprecipitated from cell extracts prepared from an equal number of mock-induced and TPA-induced cells. Affinity-purified antibodies raised against either TAFI63 or TAFI110 were used to immunoprecipitate SL1. The immunoprecipitation products were then resolved on a SDS-PAGE gel, and SL1 was detected using affinity-purified anti-TBP antibodies. As shown in Fig. 8Citation , 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.



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Fig. 8. Immunoprecipitation (IP) of SL1 from undifferentiated and differentiated U937 extracts. Equal amounts (~3 mg of proteins) of mock-treated and TPA-treated U937 extracts were precleared with protein A-agarose and then incubated with affinity-purified antibodies against TAFI110 (Lanes 1 and 2) or TAFI63 (Lanes 5 and 6). Immunocomplexes were precipitated by incubation with protein A-Sepharose, resolved by SDS-PAGE, and analyzed by Western immunoblotting with anti-TBP affinity-purified antibodies. A partially purified SL1 fraction (Lanes 3, 4, and 10) and recombinant TBP (rTBP; Lane 7) were used as controls. Rabbit preimmune serum was used for the immunoprecipitations in Lanes 8 and 9. IgG h.c., IgG heavy chain.

 
Discussion

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 {alpha}-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 {alpha}-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. Back

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. Back

3 Present address: Chengdu Institute of Biological Products, Chengdu, Sichuan, 610063, People’s Republic of China. Back

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. Back

5 L. Comai, Y. Song, and T. Bui, unpublished results. Back

Received for publication 8/16/99. Revision received 10/ 1/99. Accepted for publication 10/15/99.

References

  1. Harris P., Ralph P. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J. Leukocyte Biol., 37: 407-422, 1985.[Abstract]
  2. Polymenis M., Schmidt E. V. Coordination of cell growth with cell division. Curr. Opin. Genet. Dev., 9: 76-80, 1999.[Medline]
  3. Paule, M. R. Transcription of Ribosomal RNA Genes by Eukaryotic RNA Polymerase I. New York: Springer-Verlag, Inc., 1998.
  4. Grummt I. Regulation of mammalian ribosomal gene transcription by RNA polymerase I. Progr. Nucleic Acid Res., 62: 109-154, 1999.[Medline]
  5. Reeder R. H. Regulation of RNA polymerase I transcription in yeast and vertebrates. Progr. Nucl. Acid. Res., 62: 293-327, 1999.
  6. Learned R. M., Learned T. K., Haltiner M. M., Tjian R. T. Human rRNA transcription is modulated by the coordinate binding of two factors to an upstream control element. Cell, 45: 847-857, 1986.[Medline]
  7. Learned R. M., Cordes S., Tjian R. Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I. Mol. Cell. Biol., 5: 1358-1369, 1986.
  8. Bell S. P., Learned R. M., Jantzen H-M., Tjian R. Functional cooperativity between transcription factor UBF1 and SL1 mediates human ribosomal RNA synthesis. Science (Washington DC), 241: 1192-1197, 1988.[Abstract/Free Full Text]
  9. Jantzen H-M., Admon A., Bell S. P., Tjian R. Nucleolar transcription factor hUBF contains a DNA binding motif with homology to HMG proteins. Nature (Lond.), 344: 830-836, 1990.[Medline]
  10. Jantzen H-M., Chow A. M., King D. S., Tjian R. Multiple domains of the RNA polymerase I activator hUBF interact with the TATA-binding protein complex hSL1 to mediate transcription. Genes Dev., 6: 1950-1963, 1992.[Abstract/Free Full Text]
  11. Tuan J., Zhai W., Comai L. Recruitment of TATA-binding protein-TAFI complex SL1 to the human ribosomal DNA promoter is mediated by the carboxy-terminal activation domain of upstream binding factor (UBF) and is regulated by UBF phosphorylation. Mol. Cell. Biol., 19: 2872-2879, 1999.[Abstract/Free Full Text]
  12. Comai L., Tanese N., Tjian R. The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1. Cell, 68: 965-976, 1992.[Medline]
  13. Comai L., Zomerdijk J. C. B. M., Beckmann H., Zhou S., Admon A., Tjian R. Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 and TFIID subunits. Science (Washington DC), 266: 1966-1972, 1994.[Abstract/Free Full Text]
  14. Rudloff U., Eberhard D., Tora L., Stunnenberg H., Grummt J. TBP-associated factors interact with DNA and govern species specificity of RNA polymerase I transcription. EMBO J., 13: 2611-2616, 1994.[Medline]
  15. Voit R., Kuhn A., Sander E. E., Grummt I. Activation of mammalian ribosomal gene transcription requires phosphorylation of the nucleolar transcription factor UBF. Nucleic Acids Res., 23: 2593-2599, 1995.[Abstract/Free Full Text]
  16. Heix J., Vente A., Voit R., Budde A., Michaelidis T. M., Grummt I. Mitotic silencing of human rRNA synthesis: inactivation of the promoter selectivity factor SL1 by cdc2/cyclin B-mediated phosphorylation. EMBO J., 17: 7373-7381, 1998.[Medline]
  17. Grummt I., Smith A. V., Grummt F. Amino acid starvation affects the initiation frequency of nucleolar RNA polymerase. Cell, 7: 439-445, 1976.[Medline]
  18. Hammond L. M., Bowman H. L. Insulin stimulates the translation of ribosomal proteins and the transcription of rRNA in mouse myoblasts. J. Biol. Chem., 263: 17785-17791, 1988.[Abstract/Free Full Text]
  19. Cavanaugh H. A., Thompson E. A. Hormonal regulation of transcription of rDNA. J. Biol. Chem., 261: 12378-12744, 1986.
  20. Mishima Y., Matsui T., Muramatsu M. The mechanism of decrease in nucleolar RNA synthesis by protein synthesis inhibition. J. Biochem., 85: 807-818, 1979.[Abstract/Free Full Text]
  21. Zhai W., Tuan J., Comai L. SV40 large T antigen binds to the TBP-TAF complex SL1 and coactivates ribosomal RNA transcription. Genes Dev., 11: 1605-1617, 1997.[Abstract/Free Full Text]
  22. Cavanaugh A. H., Hempel W. M., Taylor L. J., Rogalsky V., Todorov G., Rothblum L. I. Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product. Nature (Lond.), 374: 177-180, 1995.[Medline]
  23. Rogalsky V., Todorov G., Moran D. Translocation of retinoblastoma protein associated with tumor cell growth inhibition. Biochem. Biophys. Res. Commun., 192: 1139-1146, 1993.[Medline]
  24. Voit R., Schafer K., Grummt I. Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein. Mol. Cell. Biol., 17: 4230-4237, 1997.[Abstract/Free Full Text]
  25. Kass S., Tyc K., Steitz J. A., Sollner-Webb B. The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell, 60: 897-908, 1990.[Medline]
  26. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. Current Protocols in Molecular Biology, pp. 4.6.1–4.10.10. New York: John Wiley and Sons/Greene, 1991.
  27. White R. J. Regulation of RNA polymerase I and III by the retinoblastoma protein: a mechanism for growth control?. Trends Biochem. Sci., 22: 77-80, 1997.[Medline]
  28. Alzuherri H. M., White R. J. Regulation of RNA polymerase I transcription in response to F9 embryonal carcinoma stem cells differentiation. J. Biol. Chem., 274: 4328-4334, 1999.[Abstract/Free Full Text]
  29. Brehm A., Kouzarides T. Retinoblastoma protein meets chromatin. Trends Biochem. Sci., 24: 142-145, 1999.[Medline]
  30. Manley J. L., Fire A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.. Proc. Natl. Acad. Sci. USA, 77: 3855-3859, 1980.[Abstract/Free Full Text]
  31. Roeder R. G. Multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in Xenopus laevis. J. Biol. Chem., 249: 241-248, 1974.[Abstract/Free Full Text]



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