inhibition of rna polymerase i transcription in...

Inhibition of RNA Polymerase I Transcription in DifferentiatedMyeloid Leukemia Cells by Inactivation ofSelectivity Factor 11

Lucio Comai,2 Yahui Song, Changyao Tan,3 andTiffany BuiDepartment of Molecular Microbiology and Immunology and K. NorrisJr. Comprehensive Cancer Center, University of Southern California,School of Medicine, Los Angeles, California 90033

AbstractTranscription by RNA polymerase I (pol I) regulates therate of ribosome biogenesis and the biosyntheticpotential of the cell; therefore, it plays an importantrole in the control of cell growth. Differentiation of thehuman promyelocytic leukemic cell line U937 isaccompanied by drastic decreases in pol Itranscriptional activity. We have used cell-free extractsprepared from undifferentiated and differentiated U937cells to investigate the molecular mechanismsresponsible for this inhibitory process. Our analysisindicates that the activity of the TATA binding protein(TBP)/TBP-associated factor (TAF) complex selectivityfactor 1 (SL1), one of the factors required for accurateand promoter-specific transcription by RNA pol I, isseverely repressed in differentiated U937 cells.Moreover, the reduction in SL1 activity is not aconsequence of a decrease in SL1, because there is nodetectable difference in the abundance of TBP or TAFsbefore and after U937 cell differentiation. In conclusion,our results indicate that the selectivity factor SL1 is animportant target for the regulation of pol I transcriptionduring cell differentiation.

IntroductionDuring hematopoietic cell differentiation, quantitative andqualitative changes in gene expression occur, and disruptionof the balance between proliferative and antiproliferative sig-nals can lead to the abnormal growth associated with leu-kemia and other neoplastic disorders. The underlying regu-latory mechanisms that are involved in these processes arepoorly understood. The human promyelocytic leukemic cellline U937 is an established model for studying hematopoietic

cell differentiation in vitro (1). These immature cells can beinduced by the phorbol ester TPA4 to differentiate along themonocytic lineage into functionally and morphologically ma-ture nonproliferating cells.

Because a large amount of the cell’s energy and resourcesduring cell growth and cell division are used to make ribo-somes, regulation of rRNA synthesis and ribosomal biogen-esis may provide an important mechanism for controllingthese cellular processes. Thus, molecules that modulate theexpression of the rRNA genes may exert a dual effect on bothcell 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(3–5). At least two factors, UBF and SL1, in addition to RNApol I, are necessary to direct accurate and promoter-specificinitiation of transcription from the rRNA gene promoter (6, 7).Human UBF is a Mr 97,000 polypeptide that recognizes boththe core and upstream control elements of the human rRNApromoter in a sequence-specific manner (8, 9). Human UBFcontains four high mobility group boxes, one of which me-diates DNA binding and has a hyperacidic tail that is neces-sary for transactivation (10, 11). The NH2-terminal region hasbeen found to mediate UBF dimerization (10). The secondessential factor necessary for accurate RNA polymerase Itranscription is the selectivity factor SL1. SL1 is a multisub-unit complex composed of the TBP and three TAFs, TAFI48,TAFI63, and TAFI110 (12–14). SL1 does not bind specificallyto the rRNA promoter. However, in presence of UBF, astrong cooperative DNA binding complex is formed at therRNA promoter that is critical for initiation of transcription (6,8). The recruitment of SL1 to the promoter is mediated by theCOOH-terminal activation domain of UBF and is modulatedby UBF phosphorylation (11). Mutations in UBF that abolishDNA binding, such as the removal of the high mobility groupbox 1, or that impair the interaction between UBF and SL1,such as dephosphorylation or removal of the COOH-terminaldomain, result in a drastic reduction in pol I transcriptionalactivity (10, 11, 15). In addition, a recent study also showedthat the interaction between SL1 and UBF is influenced bythe phosphorylation status of at least one of the TAFIs in theSL1 complex (16). Thus, these findings provide strong evi-dence that the network of interactions among UBF, SL1, andthe rDNA promoter plays a major role in the regulation of polI transcription.

RNA pol I activity is tightly linked to the signals that controlcell growth (3, 4), and a number of physiological and path-

Received 8/16/99; revised 10/1/99; accepted 10/15/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 This work was funded by Research Grant RPG-97-058-01-NP from theAmerican Cancer Society.2 To whom requests for reprints should be addressed, at Department ofMolecular Microbiology and Immunology, School of Medicine, Universityof Southern California, 2011 Zonal Avenue, HMR-509, Los Angeles, CA90033. 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-ace-tate; pol I, RNA polymerase I; UBF, upstream binding factor; SL1, selec-tivity factor 1; TBP, TATA binding protein; TAF, TBP-associated factor;pRb, retinoblastoma protein.

63Vol. 11, 63–70, January 2000 Cell Growth & Differentiation

ological stimuli affect the rate of RNA pol I transcription(17–21). Recently, it has been proposed that the retinoblas-toma tumor suppressor gene product (pRb) may be involvedin 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 cellsU937 begin to differentiate, there is an accumulation of pRbin the nucleoli and a sharp decrease in rRNA synthesis (22,23). In vitro experiments with mouse extracts and recombi-nant pRb suggested that the binding of pRb to UBF inhibitsthe DNA binding activity of this transcription factor (24).Despite these studies, it has not been proven that the inter-action between pRb and UBF is indeed responsible for therepression of pol I transcription in differentiated U937. Todefine the mechanism of pol I transcription inhibition in dif-ferentiated cells, we have analyzed the transcriptional prop-erties of extracts from U937 cells that were induced to dif-ferentiate by treatment with TPA. In this study, we found thatthe 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 immunopre-cipitation assays, we were able to determine that there wasno significant difference in the abundance of the SL1 factorbetween mock and TPA-induced cells. Taken together, theseresults suggest that inhibition of SL1 activity, most likely

through a posttranslational modification of one or more of itscomponents, is at the basis of the pol I transcription repres-sion in differentiated hematopoietic cells.

ResultsrRNA Transcription Is Drastically Reduced after Differ-entiation of U937 Cells. To determine the level of rRNAsynthesis before and after differentiation, total RNA wasextracted from undifferentiated U937 cells and from cellsthat were induced to differentiate by treatment with TPA for19 h. Control cells were treated with the same volume ofethanol, the solvent used to solubilize TPA. The level of59-precursor rRNA transcript in undifferentiated and differ-entiated cells was determined by S1 nuclease protectionassays. As shown in Fig. 1, there is a dramatic difference inthe abundance of 59 rRNA between TPA-treated (Lanes 1and 2) and mock-treated (Lanes 3 and 4) U937 cells. Quan-titation analysis indicated that the level of pre-rRNA is re-duced ;6–8-fold after cell differentiation. b-actin mRNAexpression showed no variation between mock-and TPA-treated cells.5 Because the 59 end of the precursor rRNA israpidly degraded, the level of the 59-end transcript faithfullyreflects the rate of initiation of transcription (25). Therefore,these results provide further evidence that pol I transcriptionis repressed after U937 cell differentiation (22).

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

Fig. 1. RNA pol I transcription decreases after U937 cell differentiation.S1 analysis of total RNA (5 mg) isolated from TPA-induced (Lanes 1 and 2)and mock-induced (Lanes 3 and 4) U937 cells. Two independent prepa-rations of total RNA from mock- and TPA-induced cells were tested. TheRNAs were hybridized with a 59-end, 32P-labeled 60 bases oligonucleo-tide complementary to the nucleotide 220 and 40 of the human ribosomalDNA gene. Quantitation by PhosphorImager indicates that transcription is;8-folds lower in differentiated (TPA-treated) cells (not shown).

Fig. 2. Extracts from TPA-treated cells are deficient in pol I transcrip-tional activity. A human rDNA template (prHu3) was transcribed in vitrousing 4 mg (Lanes 1 and 3) or 6 mg (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 de-scribed in “Materials and Methods.” Transcription data were quantitatedusing a PhosphorImager, and relative activities are as indicated.

64 Repression of pol I Transcription in Differentiated U937

Extracts of Differentiated U937 Cells Are Impaired forpol I Transcription. To dissect the molecular events re-sponsible for the down-regulation of transcription by RNApol I in differentiated U937 myeloid cells, we then establishedan in vitro transcription system. Extracts were prepared fromU937 cells that were either induced to differentiate with TPAat a concentration of 100 nM or mock-induced with an eth-anol solution without TPA. Equal amounts of extracts weretested in an in vitro transcription assay using a plasmidcontaining the human ribosomal DNA promoter as template.Transcription assays were performed under the standardconditions, and rRNA transcripts were detected using the S1nuclease assay (26). As shown in Fig. 2, the RNA pol Itranscriptional activity is ;6-fold lower in the TPA-inducedU937 cell extract (Lanes 3 and 4), as compared with theextract prepared from mock-induced cells (Lanes 1 and 2).These results demonstrate that the repression of rRNA syn-thesis upon TPA-induced differentiation of hematopoieticcells is faithfully reproduced in an in vitro transcription sys-tem.

Extracts from Differentiated U937 Cells Do Not Containa Soluble Repressor of pol I Transcription. To determinewhether the decreased transcriptional activity was attribut-able to a soluble repressor found in the extracts from differ-entiated cells, we performed a mixing experiment. A constantamount of extract from undifferentiated U937 cells wasmixed with increasing amounts of extracts from TPA-in-duced cells, incubated on ice for 30 min, and then tested in

transcription reactions. As shown in Fig. 3, the titration ofincreasing amounts of extracts from differentiated U937 cells(Lanes 3–5) did not affect the transcriptional activity of theextracts from undifferentiated cells. These results excludethe possibility that the extracts from TPA-induced cells con-tain a soluble inhibitor of transcription and suggest that theinhibition of rRNA synthesis is most likely attributable to aspecific decrease in the activity of one or more componentsof the pol I transcriptional machinery.

SL1 Activity Is Specifically Diminished in DifferentiatedU937 Cell Extracts. To determine whether the defect intranscription was attributable to an inhibition of the RNA polI itself, we measured the pol I enzymatic activity in differen-tiated and undifferentiated U937 cell extracts. For this pur-pose, we used an assay that measures the ability of RNA polI to randomly initiate transcription on nicked DNA. Severalmatched pair samples were tested, and in none of them wasthere any significant difference in the level of nucleotidepolymerization by pol I between undifferentiated and differ-entiated extracts (Fig. 4).

In addition to RNA pol I, two additional factors, UBF andSL1, are required for accurate transcription of human rRNAgenes. Regulation of either of these two factors may accountfor the observed repression of pol I transcription after differ-entiation. To test this hypothesis, we determined whether theaddition of exogenous SL1 or UBF was able to rescue pol Iactivity in differentiated U937. SL1 or UBF fractions purifiedfrom HeLa cells were added to in vitro transcription reactionscontaining extracts from TPA-induced cell. The results ofthese experiments indicated that SL1, but not UBF, was ableto fully rescue RNA pol I activity (Fig. 5, A and B). To exclude

Fig. 3. Differentiated U937 cell extract does not contain a soluble re-pressor. A human rDNA template was transcribed in vitro using 5 mg ofundifferentiated (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 mg, respectively) of extracts from differentiated cells wereadded to the reaction mixtures containing extracts (5 mg) from undiffer-entiated cells and the rDNA template and incubated at 30oC for 15 minbefore the addition of nucleotides. In Lane 1, the rDNA template wasomitted.

Fig. 4. Undifferentiated and differentiated U937 cell extracts displaysimilar levels of pol I polymerization activity. Nonspecific RNA pol I assayswere carried out as described in “Materials and Methods” using HeLa,undifferentiated U937, and differentiated U937 whole-cell extracts. Incor-porated [3H]UTP was counted in scintillation fluid with a Beckman scin-tillation spectrometer. Bars, SDs calculated from three independent ex-periments.

65Cell Growth & Differentiation

the possibility that UBF added to the transcription reactionswas nonfunctional, the transcriptional activity of HeLa-puri-fied UBF, using in vitro reconstituted transcription assayswith purified SL1 and RNA pol I, is shown in Fig. 5C. Thus,differentiated U937 cell extracts appear to be primarily defi-cient in SL1 activity. Interestingly, the addition of purified SL1could also stimulate the transcriptional activity of undifferen-tiated U937 cell extract, suggesting that SL1 is a limitingfactor for pol I transcription in U937 cells.5

To confirm the deficiency of SL1 activity in TPA-inducedU937 cells, we partially purified SL1 from undifferentiatedand differentiated U937 extracts using a well-establishedfractionation procedure. The purified SL1 fractions were thentested in in vitro reconstituted transcription reactions in thepresence of HeLa-purified pol I and UBF. Importantly, thepartially purified SL1 fraction did not contain any detectablepol I and UBF activities (see “Materials and Methods”). Asshown in Fig. 6A, the SL1 fraction from differentiated (TPA-induced) U937 cells (Lanes 5–7) is several folds less activethan the respective SL1 fraction from the undifferentiatedcells (Lanes 2–4).

In addition, to determine whether the activity of UBF alsochanged upon differentiation, we tested the partially purifiedUBF fraction from undifferentiated and differentiated cells inan in vitro reconstituted assay with SL1 and RNA pol I puri-fied from either HeLa or U937 cells (Fig. 6B). UBF activityfrom both undifferentiated and differentiated U937 extractsis quite low compared with the activity of HeLa-purified UBF.Interestingly, the partially purified UBF fraction from differ-entiated U937 cells was more active than the corresponding

fraction from undifferentiated cells, as determined in bothassays condition. These experiments have been carried outin the presence of limiting amounts of SL1 (;1.0 ng) tomaximize UBF response. These results indicate that UBF isnot down-regulated upon U937 cell differentiation and fur-ther support the concept that a reduction in the activity of theSL1 factor is the major cause for the inhibition of rRNAsynthesis in differentiated U937 cells.

The Abundance of SL1 Does Not Change after CellDifferentiation. Human SL1 is a multiprotein complex com-prised of TBP and three associated factors, TAFI48, TAFI63,and TAFI110. Several studies have indicated that each of thesefactors is necessary for the assembly of a functional SL1. Todetermine whether the reduction in SL1 activity in differentiatedcells was attributable to a decrease in the abundance of one ormore of its components, we carried out a series of Western blotanalyses. Whole-cell extracts from undifferentiated and differ-entiated U937 cells were resolved on SDS-PAGE gels, and theabundance of each of the four components of SL1 before andafter differentiation was determined using antibodies raisedagainst each protein. As shown in Fig. 7A, there was no de-tectable change in the level of TBP, TAFI48, TAFI63, andTAFI110 proteins after cell differentiation. In addition, the abun-dance of the UBF factor also did not change between undiffer-entiated and differentiated cells. The results strongly suggestthat differentiation of U937 cells and inhibition of pol I transcrip-tion is not accompanied by a specific decrease in the level ofany of the SL1 subunits. In addition, we observed an additionalslower migrating band in the anti-TBP Western blot with differ-entiated extracts. To determine whether this band was a phos-

Fig. 5. A, exogenous SL1 can stimulate pol I transcription in differentiated U937 cell extract. In vitro transcription reaction were carried out using 4 mg ofwhole-cell extracts from HeLa (Lanes 1 and 2), undifferentiated U937 (Lanes 3 and 4), and differentiated U937 (TPA-treated; Lanes 5–11). Reactions in Lanes6, 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 resultshave been reproduced in several independent preparations. In addition, identical results have been obtained using nuclear extracts. Arrow, the position ofthe 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 mg; Lanes 2, 5, and 8: 9 mg; Lanes 3, 6,and 9: 12 mg) and no SL1 (Lanes 1–3), 1 ml (10 ng/ml) of SL1 (Lanes 4–6), or 2 ml 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. Transcriptionassays 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 usedin the experiments shown in A.


phorylated form of TBP, we performed the Western blot anal-ysis using extracts that were pretreated with alkalinephosphatase. As shown in Fig. 7B, the slower migrating TBPband disappears on phosphatase treatment, suggesting thatthe diminished SL1 activity in differentiated cells may indeedresult from posttranslational modifications of a SL1 subunit,such as TBP.

TAFs and TBP Are Stably Associated in an SL1 Com-plex in Differentiated U937 Cells. Although each of thecomponents of SL1 was present in similar quantities beforeand after differentiation, the previous experiment did notaddress whether SL1 was still present as a stable multipro-tein complex after cell differentiation. For this purpose, SL1was immunoprecipitated from cell extracts prepared from an

Fig. 6. A, SL1 activity is repressed in differ-entiated U937 cell extracts. SL1 (0.8 M KClfraction) was purified by heparin-agarose col-umn chromatography from HeLa (Lane 1, 50ng), undifferentiated U937 (Mock-U937; Lanes2–4, 50, 100, and 200 ng, respectively), ordifferentiated U937 cells (TPA-U937; Lanes5–7, 50, 100, and 200 ng, respectively), andused in transcription reactions with 100 ng ofan HeLa fraction containing pol I and UBF. InLanes 8 and 9, SL1 from undifferentiated U937(Lane 8) or differentiated U937 (Lane 9) wasused in transcription reactions in the absenceof pol I and UBF. Transcripts were quantitatedusing a PhosphorImager, and relative activitiesare as indicated. B, UBF activity in U937 cellextracts. Top panel, UBF (0.4 M KCl fraction)from undifferentiated (Lanes 2, 3, and 6) ordifferentiated (Lanes 4, 5, and 7) U937 cellswas used in reconstituted transcription assayswith pol I and SL1 purified from HeLa cells. InLane 1, the HeLa-purified pol I and SL1 wereused in the absence of UBF. Arrow, the posi-tion of the protected oligonucleotide product.Transcripts were quantitated using a Phos-phorImager, and relative activities are as indi-cated. Bottom, RNA pol I (0.3 M KCl fraction; 6mg/reaction) and UBF fractions [0.4 M KCl frac-tion; 80 ng (Lanes 8 and 10) and 250 ng (Lanes9 and 11)] from either undifferentiated (Lanes 8and 9) or differentiated (Lanes 10 and 11) U937extracts were used in reconstituted transcrip-tion assays in the presence of HeLa-purifiedSL1. In Lane 10, transcription was carried outwith a HeLa-purified fraction containing pol Iand UBF and HeLa SL1.


equal number of mock-induced and TPA-induced cells. Af-finity-purified antibodies raised against either TAFI63 orTAFI110 were used to immunoprecipitate SL1. The immuno-precipitation products were then resolved on a SDS-PAGEgel, and SL1 was detected using affinity-purified anti-TBPantibodies. As shown in Fig. 8, no significant differences inthe amount of TBP can be seen in the immunoprecipitationreactions, suggesting that SL1 remains associated as a sta-ble complex after differentiation. These results provide fur-ther evidence that the decrease in SL1 specific activity islikely attributable to a posttranslational modification of one ormore of its subunits. Of course, we cannot rule out that thedecrease in SL1 activity may be caused by the absence ofthe other SL1 subunits, TAFI48, within the complex. Becauseof the low titer and poor quality of the antibodies againstTAFI48, we were not be able to obtain interpretable data in

anti-TAFI48 immunoprecipitation reactions. Thus, we cannotstrictly rule out that TAFI48 may be excluded from the SL1complex after cell differentiation.

DiscussionTPA treatment of U937 human myeloid leukemia cells ini-tiates a cascade of cellular events that profoundly influencethe expression of a variety of genes and ultimately lead to celldifferentiation. Differentiation of U937 cells is associated witha significant decrease in rRNA transcription. The TPA-in-duced repression of rRNA transcription is of particular inter-est because it directly affects ribosome production and pro-tein synthesis and, therefore, provides a direct mechanism tocontrol cell growth (2, 27). To elucidate the molecular mech-anisms underlying this repression of pol I transcription, wehave analyzed the components of the transcriptional ma-chinery before and after differentiation. Our studies indicatethat the decrease in transcription reflects a drastic reductionin the activity of the selectivity factor SL1. Complementationassays and fractionation experiments reproducibly show thatSL1 activity is between 8 and 14 folds lower in differentiatedthan undifferentiated U937 cells. This is a significant findingthat implies that regulation of SL1 activity has an in importantrole in the modulation of pol I transcription. Interestingly,Western blot analysis of SL1 from extracts prepared fromundifferentiated and differentiated U937 cells showed noappreciable difference in the abundance of any of the SL1subunits. Immunoprecipitation assays also show that in ei-ther extract, SL1 is found as a stable multiprotein complex.These results suggest that SL1 activity is most likely modu-lated by a posttranslational event induced by the differenti-ation process. The mouse orthologue of SL1, TIF-IB, hasbeen shown recently to be the target of regulation duringmouse F9 embryonal carcinoma stem cell differentiation, andthe reduction in TIF-IB activity appeared to be associatedwith a decrease in the abundance of two mouse TAFs,mTAFI48 and mTAFI95 (28). On the other hand, recent stud-ies have indicated that mitotic inactivation of human rRNAsynthesis is mediated by phosphorylation of TAFI110, one ofthe subunits of SL1 (24). It is therefore tempting to speculatethat the mechanism of human SL1 inactivation in differenti-ated U937 cells resembles the mitotic process. It is currentlyunclear whether phosphorylation or dephosphorylation ofany of the SL1 subunits is involved in down-regulation of SL1activity in differentiated U937 cells. Preliminary experimentsindicate that a slower migrating form of TBP, present in theextracts from differentiated U937 cells, disappears upontreatment with the alkaline phosphatase. These results sug-gest that phosphorylation of TBP may play a role in down-regulation of SL1 activity. In addition, we attempted to ana-lyze the phosphorylation state of SL1 before and afterdifferentiation of U937 cells using in vivo labeling experi-ments. Unfortunately, these experiments were inconclusivebecause of the inability to detect SL1, an extremely lowabundance factor in the cell, in immunoprecipitates from[32P]Pi-labeled cell extracts.

In addition to determine the activity of SL1, we have alsoanalyzed the transcriptional properties of the partially puri-fied UBF fractions. The results of the in vitro reconstituted

Fig. 7. A, Western blot analysis of TBP, TAFs, and UBF from undiffer-entiated and differentiated U937 cells. Forty mg of mock-treated andTPA-treated U937 extracts were resolved on a 10% SDS-PAGE andanalyzed by Western blot with antibodies as indicated. An SL1 fractionfrom HeLa cells was used as a control (SL1). All antibodies used wereaffinity purified except for anti-TAFI48. B, Western blot analysis of TBPfrom alkaline phosphatase-treated U937 extracts. Fifteen mg of mock-treated and TPA-treated U937 extracts were preincubated with 0.5 ml(1000 units/ml) of calf alkaline phosphatase (Lanes 1 and 3) or 0.5 ml ofphosphatase 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.


transcription assays indicate that UBF activity in differenti-ated cells is higher that in undifferentiated cells. The signif-icance of these findings is currently unclear and requiresfurther investigation. Nevertheless, these results further sup-port our finding that down-regulation of SL1 is at the basis ofpol I repression upon U937 cell differentiation.

Previous studies indicated that differentiation of U937 isaccompanied 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, pRbcan repress pol I transcription by directly binding to UBF (23,24). However, it has never been shown that the activity ofUBF is affected upon U937 cell differentiation. On the otherhand, our biochemical studies strongly suggest that down-regulation of SL1 activity is at the basis of the repression ofpol I transcription in differentiated U937 cells. In addition, ourdata show that UBF is more active in the differentiated cells.The difference between our findings and the published datamay reflect differences in the experimental approach. It ispossible that in the reconstituted transcription assays usingmouse extracts, recombinant pRb is sufficient for the inhibi-tion of pol I transcription by binding, stoichiometrically, toUBF. However, it is unclear if there is a stoichiometric inter-action between pRb and UBF in differentiated U937 cells.5

Conversely, our analysis has been performed on endoge-nous factors using U937 cell extracts; therefore, it may betterrecapitulate the process that occurs in vivo. Our study can-not rule out that the binding of pRb to UBF is still required forrepression of pol I transcription in differentiated U937 cells. Itis conceivable that the interaction between UBF and pRbmay represent one step in a process that ultimately leads tothe down-regulation of SL1 activity and repression of pol Itranscription. In analogy to the recently proposed mecha-nism of pRb repression of class II genes, pRb may functionin the recruitment of other factors to the rDNA promoter thatfacilitate or directly catalyze the inactivation of SL1 (29). In

conclusion, our study underscores the role of SL1 as acritical target for regulation of pol I transcription in differen-tiated hematopoietic cells. The precise role of pRb in thisprocess remains to be elucidated, and future studies willaddress the link between SL1, pRb, and repression of pol Itranscription in differentiated hematopoietic cells.

Materials and MethodsCell Culture. U937 cells were grown and subcultured every 2 days inRPMI 1640 supplemented with 10% heat-inactivated FCS at 37oC, 5%CO2 in humidified atmosphere. After seeding the cells in fresh growthmedium at an initial density of 2–3 3 105 cells/ml, TPA was added for 19 has a stock solution in ethanol to achieve the final concentration of 100 nM.Control cells (mock-induced) received an equal volume of ethanol. HeLaS3 cells were grown in suspension in MEM supplemented with 5% new-born calf serum.

Preparation of Cell Extracts. For the preparation of cell extracts fromtranscription assays, Western blots, and immunoprecipitations, we har-vested each time ;4–6 3 108 cells [;12–15 (150-mm) plates]. Whole-cellextracts were prepared accordingly to the method developed by Manleyet al. (30). Nuclear extracts were prepared as described in Zhai et al. (21).Whole-cell extracts were used as starting material for the fractionationstudies. Partially purified SL1 was prepared by chromatography on PorosHE1 (heparin-agarose). Briefly, whole-cell extracts were loaded onto aPoros 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 washedextensively with TM/0.1, and it was then step-eluted with TM buffercontaining 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. Transcriptionassays and Western blot analyses were used to identify fractions con-taining RNA pol I, UBF, and SL1. Peak fractions for each activity werepooled and dialyzed into TM containing 0.1 M KCl and 0.1% NP40. HeLaRNA 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 outas described previously using either 30 or 100 ng of rDNA gene templatein the presence of 100 mg/ml of a-amanitin. Quantitation analysis wasperformed using a PhosphorImager (Molecular Dynamics). In the extractmixing experiment, the reaction mixture was incubated at 30oC for 15 minbefore the addition of all four ribonucleotides.

RNA Purification and Analysis. Total RNA was isolated using a singlestep procedure by guanidinium thiocyanate-acid phenol-chloroform extrac-

Fig. 8. Immunoprecipitation (IP) of SL1 from undifferentiated and differentiated U937 extracts. Equal amounts (;3 mg of proteins) of mock-treated andTPA-treated U937 extracts were precleared with protein A-agarose and then incubated with affinity-purified antibodies against TAFI110 (Lanes 1 and 2) orTAFI63 (Lanes 5 and 6). Immunocomplexes were precipitated by incubation with protein A-Sepharose, resolved by SDS-PAGE, and analyzed by Westernimmunoblotting with anti-TBP affinity-purified antibodies. A partially purified SL1 fraction (Lanes 3, 4, and 10) and recombinant TBP (rTBP; Lane 7) wereused as controls. Rabbit preimmune serum was used for the immunoprecipitations in Lanes 8 and 9. IgG h.c., IgG heavy chain.


tion. Briefly, cells were collected and lysed in denaturing buffer [4 M guani-dinium isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium laurylsarcosine, and 0.15 M 2-mercaptoethanol]. Genomic DNA was sheared bypipetting up and down several times, and total RNA was prepared by theaddition of 2 M sodium acetate (pH 4.0), 1 volume of water-saturated acidicphenol, and one-fifth of chloroform:isoamyl alcohol (24:1). Samples werevortexed and centrifuged at 10,000 3 g, and the RNA-containing aqueousphase was carefully collected. RNA was further precipitated by the additionof 2.5 volumes of ethanol, washed with 70% ethanol, air-dried, and dissolvedin diethyl pyrocarbonate-treated water. After quantitation by spectrophotom-etry, equal amounts of RNA were used for S1 nuclease analysis. S1 nucleaseanalysis was carried out using an oligonucleotide complementary to theregion between 220 and 40 of the human rDNA gene that was labeled with32P at the 59 end. Preliminary experiments were performed to assure that theradiolabeled oligonucleotide used in each reaction was in vast (.10 fold)excess over the target RNA.

RNA pol I Assay. Random RNA polymerization assays were performedas described by Roeder (31). Each reaction mixture contained 5 mg of nickedherring sperm DNA, 100 mg/ml a-amanitin, and 10 mg of protein extract.

Antibodies and Immunoprecipitation Analysis. Rabbit polyclonalantisera raised against recombinant TBP, TAFs, and UBF were affinity-purified accordingly to published procedures. Immunoprecipitation reac-tions were carried out as described by Comai et al. (12). Immunoreactivitywas shown by the alkaline-phosphatase detection method.

AcknowledgmentsWe are grateful to all of the members of our laboratory for helpful sug-gestions and discussions. We thank J. Tuan and A. Nee for technicalassistance. We are also thankful to the University of Southern CaliforniaLiver Center for the use of the PhosphorImager.

References1. Harris, P., and Ralph, P. Human leukemic models of myelomonocyticdevelopment: a review of the HL-60 and U937 cell lines. J. LeukocyteBiol., 37: 407–422, 1985.

2. Polymenis, M., and Schmidt, E. V. Coordination of cell growth with celldivision. Curr. Opin. Genet. Dev., 9: 76–80, 1999.

3. Paule, M. R. Transcription of Ribosomal RNA Genes by EukaryoticRNA Polymerase I. New York: Springer-Verlag, Inc., 1998.

4. Grummt, I. Regulation of mammalian ribosomal gene transcription byRNA polymerase I. Progr. Nucleic Acid Res., 62: 109–154, 1999.

5. Reeder, R. H. Regulation of RNA polymerase I transcription in yeastand vertebrates. Progr. Nucl. Acid. Res., 62: 293–327, 1999.

6. Learned, R. M., Learned, T. K., Haltiner, M. M., and Tjian, R. T. HumanrRNA transcription is modulated by the coordinate binding of two factorsto an upstream control element. Cell, 45: 847–857, 1986.

7. Learned, R. M., Cordes, S., and Tjian, R. Purification and character-ization of a transcription factor that confers promoter specificity to humanRNA polymerase I. Mol. Cell. Biol., 5: 1358–1369, 1986.

8. Bell, S. P., Learned, R. M., Jantzen, H-M., and Tjian, R. Functional coop-erativity between transcription factor UBF1 and SL1 mediates human ribo-somal RNA synthesis. Science (Washington DC), 241: 1192–1197, 1988.

9. Jantzen, H-M., Admon, A., Bell, S. P., and Tjian, R. Nucleolar tran-scription factor hUBF contains a DNA binding motif with homology toHMG proteins. Nature (Lond.), 344: 830–836, 1990.

10. Jantzen, H-M., Chow, A. M., King, D. S., and Tjian, R. Multipledomains of the RNA polymerase I activator hUBF interact with the TATA-binding protein complex hSL1 to mediate transcription. Genes Dev., 6:1950–1963, 1992.

11. Tuan, J., Zhai, W., and 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 isregulated by UBF phosphorylation. Mol. Cell. Biol., 19: 2872–2879, 1999.

12. Comai, L., Tanese, N., and Tjian, R. The TATA-binding protein andassociated factors are integral components of the RNA polymerase Itranscription factor, SL1. Cell, 68: 965–976, 1992.

13. Comai, L., Zomerdijk, J. C. B. M., Beckmann, H., Zhou, S., Admon A.,and Tjian, R. Reconstitution of transcription factor SL1: exclusive bindingof TBP by SL1 and TFIID subunits. Science (Washington DC), 266: 1966–1972, 1994.

14. Rudloff, U., Eberhard, D. Tora, L., Stunnenberg, H., and Grummt, J.TBP-associated factors interact with DNA and govern species specificityof RNA polymerase I transcription. EMBO J., 13: 2611–2616, 1994.

15. Voit, R., Kuhn, A., Sander, E. E., and Grummt, I. Activation of mam-malian ribosomal gene transcription requires phosphorylation of the nu-cleolar transcription factor UBF. Nucleic Acids Res., 23: 2593–2599, 1995.

16. Heix, J., Vente, A., Voit, R. Budde, A., Michaelidis, T. M., and Grummt,I. Mitotic silencing of human rRNA synthesis: inactivation of the promoterselectivity factor SL1 by cdc2/cyclin B-mediated phosphorylation. EMBOJ., 17: 7373–7381, 1998.

17. Grummt, I., Smith, A. V., and Grummt, F. Amino acid starvation affectsthe initiation frequency of nucleolar RNA polymerase. Cell, 7: 439–445, 1976.

18. Hammond, L. M., and Bowman H, L. Insulin stimulates the translationof ribosomal proteins and the transcription of rRNA in mouse myoblasts.J. Biol. Chem., 263: 17785–17791, 1988.

19. Cavanaugh, H. A., and Thompson E. A. Hormonal regulation of tran-scription of rDNA. J. Biol. Chem., 261: 12378–12744, 1986.

20. Mishima, Y. Matsui, T., and Muramatsu, M. The mechanism of de-crease in nucleolar RNA synthesis by protein synthesis inhibition. J. Bio-chem., 85: 807–818, 1979.

21. Zhai, W., Tuan, J., and Comai, L. SV40 large T antigen binds to theTBP-TAF complex SL1 and coactivates ribosomal RNA transcription.Genes Dev., 11: 1605–1617, 1997.

22. Cavanaugh, A. H., Hempel, W. M., Taylor, L. J., Rogalsky, V., Todorov,G., and Rothblum, L. I. Activity of RNA polymerase I transcription factor UBFblocked by Rb gene product. Nature (Lond.), 374: 177–180, 1995.

23. Rogalsky, V., Todorov, G., and Moran, D. Translocation of retinoblas-toma protein associated with tumor cell growth inhibition. Biochem. Bio-phys. Res. Commun., 192: 1139–1146, 1993.

24. Voit, R., Schafer, K., and Grummt, I. Mechanism of repression of RNApolymerase I transcription by the retinoblastoma protein. Mol. Cell. Biol.,17: 4230–4237, 1997.

25. Kass, S., Tyc, K., Steitz, J. A., and Sollner-Webb, B. The U3 smallnucleolar ribonucleoprotein functions in the first step of preribosomal RNAprocessing. Cell, 60: 897–908, 1990.

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 retinoblas-toma protein: a mechanism for growth control? Trends Biochem. Sci., 22:77–80, 1997.

28. Alzuherri, H. M., and White, R. J. Regulation of RNA polymerase Itranscription in response to F9 embryonal carcinoma stem cells differen-tiation. J. Biol. Chem., 274: 4328–4334, 1999.

29. Brehm, A., and Kouzarides, T. Retinoblastoma protein meets chro-matin. Trends Biochem. Sci., 24: 142–145, 1999.

30. Manley, J. L., Fire, A., Sharp, P. A., and Gefter, M. L. DNA-dependenttranscription of adenovirus genes in a soluble whole-cell extract. Proc.Natl. Acad. Sci. USA, 77: 3855–3859, 1980.

31. Roeder, R. G. Multiple forms of deoxyribonucleic acid-dependentribonucleic acid polymerase in Xenopus laevis. J. Biol. Chem., 249: 241–248, 1974.


inhibition of rna polymerase i transcription in...

Documents