©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The RNA Polymerase I Promoter-activating Factor CPBF Is Functionally and Immunologically Related to the Basic Helix-Loop-Helix-Zipper DNA-binding Protein USF (*)

Prasun K. Datta (§) , Asish K. Ghosh (§) , Samson T. Jacob (¶)

From the (1) Department of Pharmacology and Molecular Biology, Chicago Medical School, North Chicago, Illinois 60064

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Analysis of the core promoter sequence of the mammalian ribosomal RNA genes revealed an E-box-like sequence (CACGCTG) to which the upstream stimulatory factor (USF) binds. Because the core promoter binding factor (CPBF) binds specifically to the core promoter sequence of the ribosomal RNA gene (Liu, Z., and Jacob, S. T. (1994) J. Biol. Chem. 269, 16618-16626) and resembles USF in some respects, we explored the potential relationship between USF and CPBF. Competition electrophoretic mobility shift assay using labeled core promoter probe and several unlabeled competitor oligonucleotides showed that USF can indeed bind to the core promoter and that only those oligonucleotides with an E-box sequence could compete in the promoter-protein complex formation characteristic of CPBF. This complex formation was thermostable, a unique property of USF. Furthermore, antibodies raised against USF cross-reacted with the 44-kDa component of rat CPBF. To prove further the relationship between CPBF and USF, we examined the effects of the unlabeled USF and ribosomal gene core promoter oligonucleotides on polymerase (pol) I and pol II transcription. Both oligonucleotides inhibited rDNA transcription as well as transcription from the adenovirus major late promoter. Only the oligonucleotides that contain the E-box sequence competed in the transcription. These data indicate that the promoter sequence of mammalian ribosomal RNA gene contains an USF-binding site, that the 44-kDa polypeptide of CPBF is related to the 44/43-kDa polypeptide of human USF, and that USF or USF-related protein can transactivate both pol I and pol II promoters.


INTRODUCTION

Transcription of ribosomal RNA genes (rDNA) is controlled by cis-acting elements and trans-acting factors (usually proteins) that interact with the specific cis-elements (for reviews, see Refs. 1-3). There are three important cis-acting elements on rDNA, namely promoters, enhancers, and terminators. The core promoter region of most species lies between -40 and +10 with respect to the initiation site. The enhancer element in rat rDNA consists of a repeat element (11.5 times repeat of a 130-base pair sequence) (4, 5) located proximal to the core promoter and a non-repeat element far upstream (approximately 2 kilobase pairs from the +1 site) of the core promoter (4) . The repeat element is pol I()-specific, whereas the non-repeat element can enhance transcription from both pol I and pol II promoters in vitro and in vivo (5, 6) . A third cis-acting element, which is rather unique to ribosomal RNA gene, is a terminator sequence located at the 3` end of pre-ribosomal RNA coding region and just upstream of the promoter (7) .

Until 1990, only limited success was achieved in the characterization of all the crucial trans-acting factors that interact with the various cis-elements on rDNA and regulate rDNA transcription initiation. In the past 3 years, however, significant achievements have been made in this regard. In addition to RNA polymerase I, initiation of rDNA transcription requires TATA-binding factor (TBP) and TBP-associated pol-specific factors called TAFs, present in a complex called SL1 (8) . The species specificity of pol I transcription, which is one of the hallmarks of pol I transcription, appears to reside in one or more of TAFs (9) . Human or frog SL1 does not interact with respective core promoter element unless another factor, upstream binding factor (UBF), is also present (10, 11) . The rodent SL1, however, can independently bind to the core promoter (12) .

A factor from the rat mammary adenocarcinoma ascites cells designated EBF (enhancer 1-binding factor) that binds to the enhancer and promoters of rat rDNA was characterized in our laboratory (13) . Subsequent study showed that this protein is related to the human Ku autoantigen (14) and that it is involved in the initiation of rDNA transcription, particularly in the preinitiation complex formation (15) . Recently, we have characterized another protein from the rat cells, called core promoter-binding factor (CPBF) (16) . This protein consists of two polypeptides with molecular masses of 44 and 39 kDa. Reconstitution transcription assay showed that it is essential for rDNA transcription. Immunoprecipitation experiments using antibodies against the human Ku protein revealed that rat EBF can cross-react with these antibodies and that it can interact physically and functionally with CPBF (17) . Because association between EBF and CPBF enhances DNase I footprinting of the core promoter()and stimulates rDNA transcription in a synergistic manner, it was of interest to characterize CPBF further with respect to the nature of its functional interaction with EBF/Ku. In the course of this study, we found that CPBF, the core promoter-binding factor, is immunologically and functionally related to USF, the upstream stimulatory factor.


EXPERIMENTAL PROCEDURES

Plasmids and Oligodeoxynucleotides

The following plasmids were used for transcriptional analysis: ( a) pB7-2.0 plasmid, which contains rat rDNA region spanning from -167 to +2000; and ( b) pAdML(CAT) plasmid, which contains adenovirus major late promoter spanning -400 to +10 linked to a 390-base pair synthetic DNA fragment containing C, A, and T residues (for details, see Ref. 18).

The oligonucleotides used as double-stranded DNA probes and competitors in the electrophoretic mobility shift assay were ( a) rat core promoter (rCP), ( b) mouse metallothionein-I (MT-I) MLTF, ( c) mouse MT-I MRE-d, ( d) mouse MT-I Sp1, ( e) CTF/NF1, and ( f) Oct 1. Oligonucleotides rCP, MLTF, MRE-d, and Sp1 were synthesized in the in-house facility. The oligonucleotides CTF/NF1 and Oct 1 were obtained from Promega (Madison, WI).

Preparation of CPBF and Extracts for Transcription

Whole cell extract was prepared from rat mammary adenocarcinoma ascites cells and rat hepatoma N1-S1 cells essentially as described by Zhang and Jacob (13) and was fractionated by DEAE-Sephadex chromatography. The fraction eluting with 175 m M (NH)SO, designated DE-B, was used for rDNA transcription (19) . HeLa nuclear extract was prepared following the protocol of Shapiro et al. (20) . CPBF was purified to homogeneity from rat mammary adenocarcinoma ascites cells according to the procedure of Liu and Jacob (16) . The 44- and 39-kDa polypeptides comprising CPBF were resolved on SDS-polyacrylamide gel electrophoresis and detected by silver staining.

Electrophoretic Mobility Shift Assay (EMSA)

Electrophoretic mobility shift assay using labeled rat rDNA core promoter, MLTF, and fraction DE-B was performed as described by Liu and Jacob (16) .

In Vitro Transcription Analysis

In vitro transcription of rat rDNA was performed essentially as described by Kurl et al. (19) . Plasmid pB7-2.0 linearized with XhoI was used as the template. The size of the correct run-off transcript (initiated at the +1 site) must be 635 nucleotides. In vitro transcription of the pAdML(CAT) template was carried out essentially as described by Sawadogo and Roeder (18) . In competition transcription assays, the extracts were preincubated with competitor oligonucleotides for 10 min on ice prior to addition of the templates. RNA was extracted and electrophoresed on 4% polyacrylamide, 7 M urea gels. The transcripts were visualized by autoradiography and quantitated by densitometric scanning using One scanner (Apple Computer, Inc.) and the Scan Analysis Program.

Western Blot Analysis

HeLa nuclear extract, fraction DE-B from rat ascites cell extract, and purified CPBF were subjected to electrophoresis in 10% SDS-polyacrylamide gels. For immunodetection, proteins were transferred to nitrocellulose membranes and blocked with 10% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST). The membranes were probed with anti-USF antibody (1:1000 dilution) and washed three times with TBST. The bound primary antibody was detected using alkaline phosphatase-conjugated anti-rabbit IgG (Promega), and the color was developed using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium solution.


RESULTS

The Core Promoter Regions of Mammalian Ribosomal RNA Gene Contain an E-box-like Sequence

The similarities in the molecular size and other characteristics of the human gene-specific USF and the larger polypeptide of CPBF prompted us to examine closely the rat ribosomal RNA gene promoter for a potential USF-binding site. Analysis of the core promoter sequence of rat, mouse, hamster, and human ribosomal RNA gene revealed the presence of a sequence CACGCTG (located between +6 and +12 with respect to the initiation site) that was similar to the E-box motif (CACGTG) (24) to which USF (upstream stimulatory factor) binds (see Fig. 1). Although the rDNA promoter sequence contained an additional C in the potential USF binding site, it was logical to investigate further the structural and functional relationship between CPBF and USF.

CPBF and USF, the Basic Helix-Loop-Helix-Zipper DNA-binding Protein, Bind to the Same Sequence within the Rat rDNA Core Promoter

To prove the potential relationship between CPBF and USF, we studied the specificity of the CPBF binding to the rDNA promoter sequence by a competition EMSA. Fraction DE-B prepared from N1-S1 cells was preincubated with unlabeled competitor oligonucleotides before addition of the labeled core promoter. In the absence of cold competitor oligonucleotide, a doublet (indicated by arrows) of CPBF-DNA complex ( C) was observed (Fig. 2 A, lane 2). An oligonucleotide representing the rat core promoter region with a E-box motif (see Fig. 1) efficiently competed for the CPBF binding activity (Fig. 2 A, lane 3). To confirm that CPBF binding activity represents USF or USF-related protein, unlabeled oligonucleotide corresponding to the MLTF/USF binding site on the mouse metallothionein-I gene promoter was used in the EMSA. This oligonucleotide containing the E-box motif also competed as efficiently as the rat rDNA promoter oligonucleotide for the CPBF binding activity (Fig. 2 A, lane 4). The specificity of the competition was assessed by using oligonucleotides that do not contain E-box motif, namely Sp1, MRE-d (derived from the mouse MT-I gene promoter), NF1, and Oct 1. The oligonucleotide Sp1 that binds Sp1 (25) did not compete for CPBF binding activity (Fig. 2 A, lane 5). Similarly, the metal-responsive element MRE-d that binds another transcription factor (26) did not compete for the CPBF binding activity (Fig. 2 A, lane 6). The other two competitor oligonucleotides, NF1 and Oct 1, which bind the transcription factors CTF/NF1 and Oct 1, respectively, also did not compete for CPBF (Fig. 2 A, lanes 7 and 8). The slower migrating bands probably represent the complex between the core promoter and the RNA polymerase I transcription factor EBF (13) .


Figure 1: Nucleotide sequence of rat (21), mouse (22), hamster (23), and human rDNA (22) core promoters (-36 to +14). The boldface letters indicate the E-box consensus sequence (CACGTG).




Figure 2: A, electrophoretic mobility shift assay using rat core promoter oligonucleotide (rCP) and fraction DE-B (4 µg) from rat N1-S1 cells. Lane 1, P-labeled 58-base pair rCP alone; lane 2, rCP and fraction DE-B. In competition assay, fraction DE-B was preincubated with 100 ng each of the oligodeoxynucleotide corresponding to rCP ( lane 3), MLTF ( lane 4), Sp1 ( lane 5), MRE-d ( lane 6), NF1 ( lane 7), and Oct 1 ( lane 8) (see ``ExperimentalProcedures'' for details on the competitors). Each reaction contained 40 ng of poly(dIdC) as nonspecific competitor. F designates the position of the free probe. Arrow indicates the CPBF-DNA complex ( C) and arrowheads EBF-DNA complexes. B, electrophoretic mobility shift assay using mouse MT-I MLTF oligonucleotide probe and fraction DE-B (4 µg) from rat hepatoma N1-S1 cells. Lane 1, P-labeled MLTF alone; lane 2, fraction DE-B incubated with labeled MLTF probe. In competition assay fraction DE-B was preincubated for 10 min. with 100 ng each of unlabeled oligonucleotides corresponding to the binding sequences for rCP ( lane 3), MLTF ( lane 4), MRE-d ( lane 5), Sp1 ( lane 6), and NF1 ( lane 7). Each reaction contained 100 ng of poly(dIdC) as nonspecific competitor. F designates the free probe, C represents the USF protein-MLTF complex, and C1 represents possibly EBF protein interaction.



To test further the potential relationship of CPBF and USF, an electrophoretic mobility shift assay was performed using labeled major late transcription factor (MLTF), which is known to bind USF. Fraction DE-B from N1-S1 cells was preincubated with unlabeled competitor oligonucleotides before the addition of labeled MLTF oligonucleotide. In absence of the cold competitor, a doublet ( C) of USF-DNA complex was observed (Fig. 2 B, lane 2), which is similar to the doublet of CPBF-DNA complex (Fig. 2 A, lane 2). The most noteworthy observation was that the USF-DNA complex formation was efficiently competed by cold oligonucleotide corresponding to the rat rDNA core promoter oligonucleotide (rCP) (Fig. 2 B, lane 3). As expected, MLTF oligonucleotide also competed for this complex formation (Fig. 2 B, lane 4), whereas MRE-d, Sp1, and NF1 oligonucleotides that do not contain E-box motif did not compete for the USF-DNA interaction (Fig. 2 B, lanes 5-7). The complex C1 (Fig. 2 B) probably represents EBF-DNA interaction, which is competed by cold rCP, MLTF, and to some extent by SP1 and NF1 but not by MRE-d.

CPBF Is Immunologically Related to USF

To substantiate further that CPBF is related to USF, an immunoblot analysis was performed using fraction DE-B from rat cells, HeLa nuclear extract, and purified CPBF, and polyclonal antibodies against the 43-kDa USF component. A strong reactivity of a 43-kDa protein and a weak reactivity to the 44-kDa protein was observed in HeLa nuclear extract (Fig. 3 A, lane 2), whereas a strong reactivity to a 44-kDa protein and a weak reactivity to a 43-kDa protein were observed with fraction DE-B from the rat cells (Fig. 3 A, lane 1). The antibody cross-reacted exclusively to the 44-kDa polypeptide of CPBF but not to the 39-kDa polypeptide in the purified preparation (Fig. 3, B and C). The large polypeptide with a molecular mass of approximately 116 kDa that cross-reacts with the anti-USF antibodies in both fraction DE-B and HeLa nuclear extract probably represents TFII-I, which is known to cross-react with the anti-USF antibodies (27) . These results indicate that the 44-kDa polypeptide of CPBF is immunologically related to USF or that rat CPBF is the rat homolog of human USF.


Figure 3: Immunological relationship of rat CPBF to USF. Western blot analysis was performed using anti-HeLa 43-kDa USF-1 antibodies (see ``Experimental Procedures''). Panel A, lane 1, fraction DE-B (100 µg) from rat ascites cells; lane 2, HeLa nuclear extract (100 µg). The solid arrowhead indicates the immune complex formed between the 44-kDa polypeptide of CPBF and anti-USF antibodies. The open arrowhead indicates the immune complex formed between the 43-kDa human USF1 and anti-USF antibodies. Panel B, purified CPBF (100 ng) was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis and silver-stained. The 44- and 39-kDa polypeptides are indicated by arrowheads. Panel C, immune complex formed between the 44-kDa polypeptide of CPBF and anti-USF antibodies. The solid arrowhead indicates the immune complex formed between the 44-kDa polypeptide of purified rat CPBF and anti-USF antibodies. The markers (in kDa) used were Sigma prestained proteins ( panel A) and Amersham prestained proteins ( panels B and C).



RNA Polymerase I-directed Transcription of Rat Ribosomal Gene Can Be Competed by the USF/MLTF Oligonucleotide in Trans

We reasoned that if the ribosomal gene promoter contains USF/MLTF binding site, an oligonucleotide corresponding to this sequence should inhibit pol I transcription. To test this possibility, the plasmid (pB7-2.0) containing the rat rDNA was transcribed in fraction DE-B prepared from the rat hepatoma N1-S1 cells (see ``Experimental Procedures''). This fraction supported efficient and correct transcription of rat rDNA in absence of competitor oligonucleotides (Fig. 4 A, lane 1). Preincubation with increasing amounts of cold rat core promoter (rCP) resulted in drastic reduction (90%) of transcription (Fig. 4 A, lanes 2 and 3). As anticipated, cold MLTF oligonucleotide also inhibited transcription to similar extent (Fig. 4 A, lane 4). By contrast, MRE-d, which does not bind or compete for CPBF or EBF (see Fig. 2 A), did not affect the pol I transcription (Fig. 4 A, lane 5).


Figure 4: A, inhibition of rat rRNA gene transcription by MLTF oligonucleotide in trans. Lane 1, rat rDNA (pB7-2.0) (100 ng) was transcribed in fraction DE-B (25 µg) from rat hepatoma N1-S1 cells (see ``Experimental Procedures'' for details). Lanes 2 and 3 represent the transcripts synthesized in the extract that was preincubated with 100 and 200 ng of rCP, respectively. Lanes 4 and 5, transcription after preincubation of fraction DE-B with 200 ng each of MLTF and MRE-d oligonucleotides, respectively, and then pB7-2.0 template was added to the reactions. Arrow indicates the specific 635-nucleotide run-off transcripts. B, inhibition of RNA polymerase II transcription from the adenovirus major late promoter by rat rRNA gene core promoter (rCP) in trans. AdML G-less cassette was transcribed in HeLa nuclear extract (72 µg) in the absence or presence of competitors (for details, see ``Experimental Procedures''). Lane 1, control transcription in absence of competitors; lane 2, HeLa nuclear extract was preincubated with 100 ng of rat rCP oligonucleotide and pAdMLP(CAT) (250 ng) was added to the reaction. HeLa nuclear extract was preincubated with 100 ng each of MLTF oligonucleotide ( lane 3), NF1 oligonucleotide ( lane 4), MRE-d oligonucleotide ( lane 5) before addition of template and then pAdMLP(CAT) was added and transcription was carried out (see ``Experimental Procedures'' for details of transcription assay). Arrow indicates the 400-nucleotide specific transcript.



RNA Polymerase II-directed Transcription from the Adenovirus Major Late Promoter Can Be Competed by Rat Core Promoter Oligonucleotide in Trans

To establish further whether rat ribosomal gene core promoter binds USF or USF-like factors and whether such binding blocks pol II transcription, transcription of AdML promoter G-less cassette was performed in a transcriptionally competent HeLa nuclear extract in presence or absence of competitor oligonucleotides. This extract supported transcription from the AdML promoter and produced the correct 400-nucleotide transcript (Fig. 4 B, lane 1). Addition of competitor oligonucleotides corresponding to rat core promoter (rCP) and major late transcription factor (MLTF) binding site reduced transcription by 50% (Fig. 4 B, lanes 2 and 3, respectively). Addition of competitor oligonucleotide NF1 also reduced transcription (Fig. 4 B, lane 4), as the AdML promoter contains the binding site for CTF/NF1. On the contrary, addition of cold oligonucleotide MRE-d, which binds the transcription factor MTF-1 (26) , failed to inhibit transcription from the AdML promoter (Fig. 4 B, lane 5). To prove further the relationship between the two factors, the effect of exogenous CPBF on transcription from the AdML promoter was studied. Addition of purified CPBF to the cell-free transcription reaction of AdML G-less cassette resulted in stimulation of transcription (data not shown). These results indicate that the rat core promoter region of rat rDNA can compete for USF or MLTF binding activity, which is known to participate in transcriptional activation of the AdML promoter by RNA polymerase II, and are consistent with the existence of a USF/MLTF recognition sequence in the ribosomal gene promoter.


DISCUSSION

The present study has demonstrated that the 44-kDa polypeptide of rat CPBF is immunologically and functionally related to the RNA polymerase II transcription factor USF/MLTF (28) . It is likely that CPBF is the rat homolog of human USF protein. Several lines of evidence presented here support such a notion. First, anti-USF antibodies cross-react with the 44-kDa subunit of CPBF. Second, binding of CPBF to rDNA core promoter can be inhibited by the MLTF/USF oligonucleotide in trans. Third, RNA polymerase I transcription is competed by MLTF oligonucleotide in trans, whereas RNA polymerase II transcription is competed by rat rDNA core promoter oligonucleotide in trans. Fourth, preincubation with an oligonucleotide that does not contain the E-box motif, the USF-binding site, does not block transcription. Finally, as observed for USF, CPBF is also thermostable; the major CPBF-DNA complex is unaltered in extracts incubated up to 55 °C (data not shown).

USF was identified initially by its ability to bind and activate the adenovirus major late promoter (29, 30) . Two different forms of USF were identified in HeLa cells with molecular masses of 44 and 43 kDa (31) . USF can bind and activate several pol II promoters that include those of mouse metallothionein-I (25, 32) and p53 (Ref. 33, and references therein). It can also stimulate pol III transcription of sea urchin U6 small nuclear RNA gene (34) . To our knowledge, the present study is the first report of the involvement of a USF related protein, namely CPBF, in pol I transcription. The strong immunological cross-reactivity of the 43-kDa (USF-1) antibody with the 44-kDa polypeptide of CPBF in fraction DE-B and purified fraction from rodent cells suggests that either the 44-kDa form is the predominant homolog of USF in rodent cells, or the CPBF polypeptide represents a new member of the USF family or the USF related-helix-loop-helix proteins (Refs. 28 and 35, and references therein).

Although CPBF from the rat cells contains a 39-kDa polypeptide, this polypeptide is not immunologically related to the larger polypeptide. The two CPBF polypeptides can bind to the core promoter (16) . It is, however, not certain whether both polypeptides are required for trans-activation of the pol I promoter. The 39-kDa polypeptide does not appear to be a degradation product of the larger polypeptide, as incubation of CPBF at 37 °C did not result in increased level of the smaller polypeptide (data not shown). The smaller polypeptide may be closely associated with CPBF only in rat or mouse. On the other hand, it may have a specific role in rDNA transcription, which is yet to be elucidated.

It is noteworthy that HeLa USF consists of 43- and 44-kDa polypeptides which interacts independently with a specific DNA element (31) . Using a recombinant 43-kDa USF, Pognonec and Roeder (36) have demonstrated that the 43-kDa component exhibits an intrinsic DNA binding and transcriptional activation, and suggested that the tightly controlled interaction between both components may be necessary for the activation of adenovirus major late promoter. Interestingly, CPBF purified from HeLa cells consists of a single 44-kDa polypeptide, which is identical to the molecular size of human USF-2; this protein by itself could stimulate human and rat rDNA transcription as much as 5-fold.()The purification protocol used in our laboratory may have preferentially selected the 44-kDa polypeptide.

The present data demonstrate that the 44-kDa polypeptide of CPBF or USF/MLTF is involved in the initiation of pol I transcription. By contrast, the 43-kDa polypeptide along with the 44-kDa polypeptide is responsible for the trans-activation of pol II promoters. USF has been shown to interact with RNA polymerase II transcription factor complex, TFIID (30, 31) , which is composed of TATA box-binding protein, TBP and TAFs. Interaction of CPBF, the USF-related protein, with the E-box-like sequence in the rDNA core promoter may help recruit TBP and TAF(s). Testing this possibility requires further study. Nevertheless, this study has clearly shown for the first time that a USF-like protein can bind to the pol I core promoter sequences and trans-activate pol I promoter.


FOOTNOTES

*
This study was supported by United States Public Health Service Grant CA 31894 from the NCI, National Institutes of Health (to S. T. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Both authors have contributed equally to this work.

To whom correspondence should be addressed: Dept. of Pharmacology and Molecular Biology, Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064. Tel.: 708-578-3270; Fax: 708-578-3268.

The abbreviations used are: pol I, RNA polymerase I; pol II, RNA polymerase II; pol III, RNA polymerase III; TBP, TATA-binding protein; TAF, TBP-associated factor; CPBF, core promoter binding factor; EBF, enhancer-1 binding factor; TFII-I, transcription factor II-I; USF, upstream stimulatory factor; rCP, rat core promoter; MLTF, major late transcription factor; MRE-d, metal regulatory element d; AdML, adenovirus major late; EMSA, electrophoretic mobility shift assay

H. Niu and S. T. Jacob, unpublished observation.

Z. Liu and S. T. Jacob, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Robert G. Roeder for providing the adenovirus major late promoter (pAdML(CAT)) construct used in the transcription studies and the antiserum against human USF.


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