Identification As a Transcription Repressor

MOLECULAR AND CELLULAR BIOLOGY, Sept. 1994, p. 6068-6074 Vol. 14, No. 9 0270-7306/94/$04.00 + 0 Copyright ©D 1994, American Society for Microbiology Purification and Characterization of Nucleolin and Its Identification as a Transcription Repressor TZUNG-HORNG YANG,' WEN-HAI TSAI,' YU-MAY LEE,2 HUAN-YAO LEI,3 MING-YANG LAT, DING-SHINN CHEN,4 NING-HSING YEH,s AND SHENG-CHUNG LEE' 2* Institute of Molecular Medicine, College of Medicine,' and Department of Intemal Medicine,4 National Taiwan University, Institute of Biological Chemistry, Academia Sinica,2 and Institute of Microbiology and Immunology, National Yang Ming Medical College,s Taipei, and Department of Microbiology, National Cheng Kung University, Tainan,3 Taiwan Received 8 April 1994/Returned for modification 11 May 1994/Accepted 7 June 1994

Expression of the acute-phase response genes, such as that for alpha-i acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor.

The initiation of transcription in eukaryotes is an intricately reaction, AGP/EBP is up-regulated, while factor B is down- controlled process. Short sequence motifs in the promoter regulated (24). The cloning and characterization of these regions of genes interact in a specific manner with DNA- positive and negative factors will hold the key to further binding transcription factors. These bound factors interact with understanding of the transcriptional regulation of acute-phase general transcription factors and thereby result in gene tran- genes in general and the AGP gene in particular. scription. Not only transcriptional activators but also repres- Recently, a number of nuclear proteins with RNA-binding sors are important in the controlled regulation of gene expres- activities have been identified, cloned, and characterized (1, 2, sion. For a given gene, the combinations of cis elements and 16, 23, 33). Apart from their ability to bind to RNA, their the trans-acting factors are major determinants of transcrip- biological functions are relatively poorly understood. Among tional activity. Protein-protein interactions and posttransla- these proteins, nucleolin is known to be a ubiquitously ex- tional modifications are important for regulating the activities pressed multifunctional protein involved in ribosomal biogen- of these factors. An array of transcriptional activators and esis (5), transcriptional regulation of pre-rRNA (3, 5), and repressors have been identified and characterized (9, 12, 14, nucleolar translocation of ribosomal proteins (4, 22, 27). 15, 18, 24, 35, 37). We have previously shown that the binding activity of factor Tissue injury and infection produce significant alterations of B decreases during the acute-phase response (24). In order to the host metabolic and immune homeostasis (19). It has understand more about this negative regulation, we have become increasingly clear that many of these changes result attempted the purification and characterization of this factor from a complex cascade of mononuclear phagocyte-derived from rat liver as well as from a mouse hepatoma cell line, 129p. endogenous mediators, in particular, cytokines. Injection of The purified B-motif-binding protein we obtained has all purified lipopolysaccharide (LPS) into laboratory animals characteristics of nucleolin, including amino acid sequence leads to the development of many biological activities with homology and serological cross-reactivity. In this report, we similarities to those that follow tissue injury and infection. present evidence showing that nucleolin in general functions as These can range from an acute-phase response to shock with a transcriptional repressor for the AGP gene. We are able to lethal outcome. The well-studied biological activities of LPS- show that purified as well as recombinant nucleolin recognizes induced liver gene expression are mediated by multiple cyto- the negative cis element (i.e., B motif) in the AGP promoter kines, including interleukins 1, 6, and 11, leukemia inhibitory region in a sequence-specific manner. factor, and tumor necrosis factor alpha (13, 19, 30, 31). We used LPS-induced transcription of the alpha-1 acid glycoprotein (AGP) gene as a model for studying the regulation of gene MATERUILS AND METHODS expression during the acute-phase response (9, 24). Transcrip- Preparation of nuclear extract from 129p cells. Mouse 129p tion of the AGP gene in response to LPS treatment is cells were inoculated into C3H/HeJ mice and grown as ascites regulated by both a positive factor, AGP/EBP (C/EBP-P), and cells. Cells were isolated from the ascites fluid, washed several a negative factor, factor B (24). During the acute-phase times with phosphate-buffered saline (PBS), and spun down at 1,200 x g. Cells were then resuspended in 5 volumes of buffer A (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethane- * Corresponding author. Mailing address: Institute of Molecular sulfonic acid] [pH 7.9], 15 mM KCl, 0.1 mM EDTA, 1 mM Medicine, College of Medicine, National Taiwan University, Taipei, dithiothreitol [DTT], and 1 mM phenylmethyl sulfonyl fluo- Taiwan. Fax: 886-2-321-0977. ride) and incubated for 10 min on ice. Afterwards, the cells 6068 VOL. 14, 1994 NUCLEOLIN FUNCTIONS AS A TRANSCRIPTION REPRESSOR 6069

Nuclear Extract TABLE 1. Purification of the B-element-binding negative Crude transcription factor 1 Sepharose Heparin-- Total Activity' Fold Yield 0.3 M 0.4 M 0.5 M Fraction protein Total Specific purifi- (%o) ,,Q-Sepharose (mg) (U) (U/mg) Crude nuclear extract 1,129 112,900 100 1.0 100 0.45 M 0.6 M Heparin-Sepharose 82 28,510 347 3.5 7.26 I MONO-S 1 Q-Sepharose 37 18,974 513 5.1 3.28 0.35 M 0.35 M Mono-S 2.4 17,106 7,128 71.3 0.21 DNA-specific affinity 0.12 9,408 78,400 784 0.01 C - Affinity a One unit of activity is arbitrarily defined as the binding of 7.3 fmol of B probe Column by 10 ,ug of nuclear extract. Binding was quantitated by excising the protein-DNA complex from the dried polyacrylamide gel and measuring the amount of radioactivity. Flowthrough Flowthrouigh I B - Affinity Column wise Elution ml; equilibrated with NDB buffer [25 mM HEPES, pH 7.9, 40 Stepl mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsul- FIG. 1. Purification scheme for the factor (from nuclear extracts of fonyl fluoride, and 10% glycerol]) and eluted with NDB buffer mouse hepatoma cell line 129p) that binds to the oligonucleotide B containing 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 1.0 M NaCl. Active sequence of the AGP gene promoter. fractions were pooled, concentrated, dialyzed, and passed over a Q-Sepharose column (1.6 by 5 cm; 10-ml bed volume) equilibrated with NDB buffer containing 0.15 M NaCl. The were centrifuged at 2,500 x g for 5 min, resuspended in 2 column was eluted with NDB buffer containing a 0.15 to 1.0 M volumes of buffer A, and homogenized in a Dounce homoge- NaCl linear gradient. Active fractions were pooled again, nizer equipped with a B-type pestle (usually 10 strokes). The concentrated, dialyzed, and passed over a Mono-S column in a homogenate was centrifuged at 6,700 x g for 10 min, and the fast protein liquid chromatography system. The column was crude nuclear pellet was resuspended in 3 ml of buffer A per eluted with NDB buffer containing 0.15 to 0.8 M NaCl. Active 109 cells. Ammonium sulfate was added to the solution to a fractions were again pooled, dialyzed, and passed through a final concentration of 300 mM. The nuclei were lysed by gentle nonspecific oligonucleotide affinity column. The flowthrough shaking at 4°C for 30 min. The chromatin was sedimented by fractions from the nonspecific affinity column were pooled and centrifugation at 20,000 x g for 30 min. Proteins from the loaded onto a B oligonucleotide affinity column which had supernatant were precipitated with ammonium sulfate (0.3 been prepared according to the procedure of Wu et al. (36). g/ml). The pellets were collected by centrifugation, dissolved in The nonspecific and specific oligonucleotide sequences for buffer C (50 mM HEPES [pH 7.9], 50 mM KCl, 0.1 mM affinity columns are as follows: EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and nonspecific, 5' -GATCCGAAGGGGCTGGTGAGATTGTGCCACAGCTCTAC-3' 10% glycerol), and dialyzed against buffer C. 3'-GACCACTCTAACACGGTGTCGAGATG-5' About 1.1 of from nuclear Protein purification. g proteins specific, 5' -GAAACGTAAGCACTGTCCCTGGCTTCAGTCCCATGCCCT-3' extracts was used for sequential purification over heparin- 3'-GACAGGGACCGAAGTCAGGGTACGGGA-5' Sepharose, Q-Sepharose, Mono-S, and oligonucleotide affinity columns. Briefly, aliquots of nuclear extracts were loaded onto Southwestern (DNA-protein) blot analysis. Southwestern the heparin-Sepharose column (5 by 5 cm) (bed volume, 100 blot analysis was based on a method described by Vinson et al.

(A) CNE H Q S AFI BSA AFII (B1) CNE H Q S AFI kDa kDa 200- t 200- r w

116- .... 116- -.:;z.4...... 4... ;: :., 80- 97- -._~~~~~~~~~~~~~~.__ *Ay:-v -4 - 50-

.- 67- w .m*~~IAM 35- 55-

18- 35-

FIG. 2. Biochemical analysis of the purified factor. (A) Coomassie blue staining patterns of SDS-PAGE analysis of various chromatographic fractions (10 ,ul per loading). CNE, crude nuclear extract; H, heparin-Sepharose fraction; Q, Q-Sepharose fraction; S, Mono-S fraction; AFI and AFII, DNA affinity fractions of Q-Sepharose-derived 0.45 and 0.6 M NaCl fractions, respectively; BSA, carrier protein (bovine serum albumin alone). (B) Southwestern blot analysis of various chromatographic fractions. In each fraction, a band with an apparent molecular mass of 97 kDa was detected (arrows). 6070 YANG ET AL. MOL. CELL. BIOL.

(A) BSA Li 129P AF

competitor - b/c b/c sp1 gcf

...... ;ai"ii M_ ,,, ..:......

*.... .:::......

probe B :.,4.::: (B) b/c m* b/c CNE AF CNE AF kDa 200- .z :4 ,Wzis AW.- " 1% ,: t. 116- 7. .%s

.4 80- 50- 35-

FIG. 3. DNA sequence-binding specificity of the purified 97-kDa protein. (A) Gel mobility shift assay in the presence of a IOOX excess of various competitors. Left lane, control (no competitor); b/c, oligonucleotide B (100 ng); m*b/c, mutant oligonucleotide B; spl, oligonucleotide containing Spl motif; gcf, oligonucleotide containing GCF motif (a GC-rich sequence); right lane, free probe only. (B) South- western blot analysis. Crude nuclear extract (CNE) (20 [Lg) and DNA affinity-purified protein (AF) (10 RlI) were separated by SDS-PAGE, blotted to the PVDF membrane, and incubated with a wild-type oligonucleotide B (b/c) or mutant oligonucleotide B (m* b/c) probe. FIG. 4. DNase I footprinting assays. The end-labeled antisense The 97-kDa protein is indicated by an arrow. strand of an AGP promoter fragment (positions -180 to +10) generated by PCR was used as a probe. BSA, free probe and bovine serum albumin control; Li, liver nuclear extract (20 p.g); 129P AF, DNA affinity column fractions (5 and 10 Ill, respectively). The bracked B indicates the footprinting protection region. (34). Samples were mixed with 2x sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 5 min, and proteins were separated by SDS-10% PAGE. After electrotransfer, the polyvinylidene buffer. Electrophoresis was performed at 150 V at room difluoride (PVDF) membrane was incubated with BLOTTO temperature with buffer circulation. (50 mM Tris-HCl [pH 7.5], 50 mM NaCl, mM EDTA, 1 mM DNase I footprinting was performed by incubating 5 ng of an DTT, 5% nonfat milk) for 30 min at room temperature. The end-labeled DNA fragment (positions -180 to + 10 from the membrane was washed twice (for 10 min each) with binding AGP promoter region, generated by PCR) with the samples to buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, be assayed in 20-plI reaction mixtures containing the same 1 mM DTTI) and incubated with oligonucleotide B probe buffer as in the gel mobility shift assay (with the addition of 1 (5'-TACTGTCCCTGGCTTCAGTCCCATGCCCT-3') (2 x mM MgCl2) for 90 min on ice. DNase I (1 to 3 pld, 30 p.g/ml; 106 dpm/ml) in the presence of 5 p.g of sonicated Escherichia Sigma) freshly diluted in 10 mM MgCl2-5 mM CaCl2 was coli DNA per ml for 60 min at room temperature. The added to the mixture. Samples were digested for 2 to 3 min on membrane was washed three times for 15 min each at 4°C. ice, and the reaction was then stopped by the addition of 80 pl Analysis of protein-DNA interaction. Gel mobility shift and of stop solution containing 75 ,.g of yeast tRNA per ml, 20 mM DNase I footprinting assays were performed as described EDTA, and 0.5% SDS. Samples were extracted with phenol- previously (24). Briefly, an end-labeled oligonucleotide probe chloroform and precipitated with ethanol at -70°C. Pellets (1 to 2 ng, 20,000 cpm) was incubated together with samples in were dried, resuspended in sequencing solution (95% form- a 20-pu reaction mixture containing 50 mM NaCl, 20 mM amide, 1% xylene cyanol FF, and 1% bromphenol blue), Tris-HCl (pH 7.6), 0.2 mM EDTA, 10% glycerol, 5 mM DTT, heated at 95°C for 3 min, and loaded onto a sequencing gel. and 1 p.g of poly(dI-dC) for 20 min at room temperature. The Western blot (immunoblot) analysis. The samples were mixture was loaded onto a 5% polyacrylamide gel (acrylamide- treated as described for Southwestern analysis. The protein- bisacrylamide, 30:1) containing 4% glycerol in Tris-glycine blotted membrane was incubated with PBS containing 5% VOL. 14, 1994 NUCLEOLIN FUNCTIONS AS A TRANSCRIPTION REPRESSOR 6071

a) VKLAKAGKTH GEAKKMAPPP KEVEEDSEDE EMSEDEDDSS GEEEVVIPQK KGKKATTTPA (A) (B) (C) b) VKLAKAGKTH GEAKKXAP 01 61 CNE H S AF CNE AFI AFII CNE AFI AFII KKVVVSQTKK AAVPTPAKKA AVTPGKKAVA TPAKKNITPA KVI PTPGKKG AAQAKALVPT kDa kDa 121 220- -200 PGKKGAATPA KGAKNGKNAK KEDSDEDEDE EDEDDSDEDE DDEEEDEEEP PIVKGVKPAK 113- 1Ioosii 116 181 AAPAAPASED EEDDEDEDDE EDDDEEEEDD SEEEVMEITT AKGKKTPAKV VPMKAKSVAE 75- 241 - 50- EEDDEEEDED DEDEDDEEED DEDDDEEEEE EEPVKAAPGK RKKEMTKQKE APEAKKQKVE 42- 301 35- - - GSEPTTPFNL FIGNLNPNKS VNELKFAISE LFAKNDLAVV DVRTGTNRKF GYVDFESAED 35 * 18 - 361 I. LEKALELTGL KVFGNEIKLE KPKGRDSKKV RAARTLLAKN LSFNITEDEL KEVFEDAMEI 421 FIG. 6. Western and Southwestern blot analyses of the purified RLVSQDGKSK GIAYIEFKSE ADAEKNLEEK QGAEIDGRSV SLYYTGEKGQ RQERTGKTST 97-kDa protein. (A) Western blot analysis following each purification 481 step. The loading sequence and abbreviations are the same as in Fig. WSGESKTLVL SNLSYSATKE TLEEVFEKAT FIKVPQNPHG KPKGYAFIEF ASFEDAKEAL 2B. (B and C) Western and Southwestern blots, respectively, of S41 derived from 0.45 and 0.6 M NaCl elutions on NSCNKMEIEG RTIRLELQGS NSRSQPSKTL FVKGLSEDTT EETLKESFEG SVRARIVTDR affinity-purified protein LOGS NSRSQP Q-Sepharose. Crude nuclear extract (CNE) (20 ,ug) and DNA affinity 601 column-purified protein from 0.45 and 0.6 M NaCl elutions (15 ,ul) ETGSSKGFGF VDFNSEEDAK AAKEAMEDGE IDGNKVTLDW AKPKGEGGFG GRGGGRGGFG (AFI and AFII, respectively) were separated by SDS-PAGE, blotted to 661 KKTKFE the membrane, and subjected to Western blot analysis with monoclo- GRGGGRGGRG GFGGRGRGGF GGRGGFRGGR GGGGDFKPQG nal antibody to human nucleolin. The arrows indicate the 97-kDa FIG. 5. Amino acid sequence analysis of the purified factor. Ap- protein. proximately 100 pmol of the purified, PVDF membrane-blotted factor was subjected to N-terminal sequencing. The SDS-PAGE-separated factor was excised and digested with V8 protease, and the resulting fragments were separated again by SDS-PAGE and blotted to the taining AGP promoter (positions -37 to + 10) and chloram- PVDF membrane for sequencing. The amino acid sequence of mouse phenicol acetyltransferase (CAT) (24). nucleolin (a) (7) and the corresponding part of the sequenced factor Cell cultures, DNA transfections, and CAT assays. Baby (b) (boldface) are shown to be identical. hamster kidney (BHK) cells were cultured as a monolayer in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. DNA transfections were performed by the calcium phosphate precipitation technique. For each 6-cm- nonfat milk for at least 30 min at room temperature. Mono- diameter petri dish, 2 ,ug of the target plasmid, AGP-CAT, and clonal antibody to human nucleolin was added, and the 1 to 2 ,ug of pCMV-Nu or pCMV-Nu-Rev were precipitated. incubation was continued for 1 h at 37°C. The monoclonal pGEM3-CMV was used as a carrier to give a total of 4 ,ug of antibody to human nucleolin was prepared by immunization DNA in each transfection assay. Other plasmids were used as with a purified human T-cell nuclear preparation as described described previously (24). Cells were harvested at 48 h post- previously (10). The membrane was washed three times (15 transfection, and 30 ,ig of protein from the cellular extracts min each) with PBS containing 0.1% Tween 20 and incubated was used for the CAT assay. with horseradish peroxidase-labeled goat anti-mouse immuno- globulin. The washed membrane was treated with reagents RESULTS from the ECL detection system (Amersham) according to the manufacturer's protocols. Purification of B-motif-binding protein. We have previously Amino acid sequencing. The purified protein was separated identified factor B as a negative transcription factor for the by SDS-PAGE, electroblotted to a PVDF membrane, and AGP gene enriched in the liver. Factor B can also be detected visualized by Coomassie blue staining. The 97-kDa polypeptide in the C3H mouse hepatoma cell line 129p. We utilized 129p band was excised from the membrane and subjected to N- cells, which can be grown as ascites, to generate the large terminal amino acid sequencing. This sequencing was kindly amounts of nuclear extract needed to purify the factor. Ap- performed by Pao-Miau Yuan of Applied BioSystems, Foster proximate 1.1 g of nuclear extract from 129p cells was purified City, Calif. Subsequently, the SDS-PAGE-separated polypep- successively on heparin-Sepharose, Q-Sepharose, Mono-S, and tide was digested with V8 protease (Sigma) and subjected to oligonucleotide affinity columns. The purification scheme is another round of SDS-PAGE (11). Following blotting onto the outlined in Fig. 1. The factor was purified near to homogeneity PVDF membrane, the peptides were visualized by staining after passage over the affinity column (Fig. 2A). Gel mobility with Coomassie blue. Several bands were excised and sub- shifts and Southwestern blots were performed following each jected to amino acid sequencing with an Applied BioSystems step of purification and are shown in Fig. 2B. These data model 477A sequencer. indicate that a 97-kDa protein was purified. The recovery of Plasmids. Full-length human nucleolin cDNA was cloned the purified factor was estimated to be 0.1%, as shown in Table into the pCMV expression vector, which contains the cytomeg- 1. alovirus immediate-early gene promoter and the simian virus The oligonucleotide-binding specificity of the purified pro- 40 splicing and poly(A) signals. The resulting expression vector tein was investigated by gel mobility shift and Southwestern was named pCMV-Nu. Antisense nucleolin cDNA was also blot assays (Fig. 3). In the gel mobility shift assay, neither the cloned into the expression vector and designated pCMV-Nu- Sp-1 nor the GCF oligonucleotide competed for the binding of Rev. AGP-CAT and various constructs containing mutations the oligonucleotide B probe. The mutated oligonucleotide B were constructed and used as previously described (17, 24). could only partially compete for binding of the wild-type C4-TATA/CAT is derived from the tetrameric C oligonucleo- oligonucleotide B probe (Fig. 3A). Similar results were ob- tide (GATCGATTIGTGTCACAG) ligated to the TATA-contained by Southwestern assays (Fig. 3B). These results suggest 6072 YANG ET AL. MOL. CELL. BIOL.

(A) (B) (C) Ni XL1 Ni XL1 competitor b/c sp 1 gcf kDa - 220 - -113 - .-- C - 89 - - 75 - , -...... ~~~~~~~ -42 - uw _ ~~~~~~~~~~~~~~~~~...... - 35 - probe

FIG. 7. Binding specificity of the recombinant nucleolin. (A) Recombinant nucleolin (amino acids 225 to 706) was recognized by monoclonal antibody to human nucleolin (Ni). XL1, control (E. coli lysates). The arrow indicates the recombinant nucleolin, which is about 41 kDa. (B) Southwestern analysis of recombinant nucleolin. Ni, oligonucleotide B; XL1, E. coli lysates. (C) Gel mobility shift assay, demonstrating that recombinant nucleolin recognizes oligonucleotide B specifically. Left lane, free probe oligonucleotide B; second lane from left, no competitor. The following lanes contain 25X and SOx excesses of competitor oligonucleotide B (b/c), Spl oligonucleotide competitor (spl), and GCF oligonucleotide competitor (gcf). that the purified 97-kDa protein can bind to the B motif in a amino acid 225 to 706) was expressed in E. coli XL.1 bacteria sequence-specific manner. by using a Bluescript vector (Stratagene, La Jolla, Calif.). This During protein purification by Q-Sepharose column chro- recombinant protein was used for Southwestern blot, Western matography, the factor eluted heterogeneously. Two main blot, and gel mobility shift analyses. It could be recognized by peaks of oligonucleotide B-binding activity were eluted, at 0.45 monoclonal antibodies to nucleolin (Fig. 7A) and could bind to and 0.6 M NaCl, respectively. Both fractions contained the the oligonucleotide B probe (Fig. 7B). Furthermore, it could 97-kDa protein and had similar oligonucleotide B-binding also recognize the B motif in a sequence-specific manner (Fig. activities as assessed by Southwestern and gel mobility shift 7C). Taken together, these data suggest that the recombinant assays (see Fig. 6C). Surprisingly, only the fraction that eluted behaves similarly to the purified factor B according to several at 0.45 M NaCl possessed the DNase I footprinting activity biochemical criteria. (Fig. 4). The biochemical basis for this difference in specifici- Identification of ties remains to be studied. the function of nucleolin in the promoter activity of AGP. To identify the functional role of nucleolin in Amino acid sequence analysis of the purified factor. To facilitate N-terminal sequencing of the affinity column-purified the promoter activity of AGP, we performed several transfec- factor, we transferred the SDS-PAGE-separated protein to a tion experiments with pCMV-Nu, pCMV-Nu-Rev, the wild- PVDF membrane. Approximately 100 pmol of the purified type AGP promoter, or the B-motif-deleted AGP promoter factor was used. A stretch of 18 amino acids, VKLAKAGK linked to a CAT reporter gene (C4-TATA/CAT). We found a THGEAKKMAP, was determined with the Applied Biosys- dose-dependent inhibition of wild-type AGP promoter activity tems model 477A automatic sequencer (sequencing was kindly by pCMV-Nu. The inhibitory effect on the B-motif-deleted performed by P. M. Yuan). Comparison of this sequence with mutant (i.e., C4-TATA/CAT) promoter is diminished (Table the GenBank database showed it to be 100% homologous to 2). pCMV-Nu-Rev, used as a control, has no inhibitory effect mouse nucleolin (Fig. 5). on the AGP promoter (Table 2). These results indicate that To unequivocally establish that the purified 97-kDa factor is nucleolin functions as a repressor for the AGP gene and that indeed nucleolin, the SDS-PAGE-separated protein band was this repression is dependent on the B motif. excised, treated with V8 protease, and separated by SDS- PAGE again. The separated polypeptides were transferred to a PVDF membrane and visualized by staining with Coomassie blue. Approximately 20 pmol was used for sequence analysis. An additional sequence, LQGSNSRSQP, was determined. This sequence corresponds to residues 557 to 566 of nucleolin TABLE 2. Transcriptional repression by nucleolin of the (Fig. 5). Taken together, these sequence data clearly suggest AGP gene in BHK cells that the purified factor is closely related or identical to nucleolin. % conversion of CAT activity Biochemical characterization of the purified factor B. Fur- Effector AMt (1pg)a (mean + SD) with reporter: ther characterizations were performed to define the properties AGP/CAT C4-TATA/CAT* of the purified factor. Monoclonal antibodies to nucleolin were CMV-Nu 0 27 3 89 8 used for Western blot analysis. As shown in Fig. 6A and B, the 1 12 2 86 8 oligonucleotide B-binding activity following each step of puri- 2 6±0.5 71 14 fication could be correlated with the recognition of monoclonal antibodies to nucleolin. Thus, we demonstrated the serological CMV-Nu-Rev 1 20 + 5 107 ± 17 cross-reactivity of the purified factor and nucleolin. However, 2 19 0.1 100 2 expression of full-length recombinant nucleolin in E. coli was aAmount of effector used in transfection experiment. unsuccessful; truncated recombinant human nucleolin (from b B-motif-deleted AGP promoter used as a control. VOL. 14, 1994 NUCLEOLIN FUNCTIONS AS A TRANSCRIPTION REPRESSOR 6073

DISCUSSION REFERENCES 1. Anderson, J. T., M. R. Paddy, and M. S. Swanson. 1993. PUBI is Recently, the genes for a number of nuclear proteins with a major nuclear and cytoplasmic polyadenylated RNA-binding have been identified and and protein in Saccharomyces cerevisiae. Mol. Cell. Biol. 13:6102-6113. RNA-binding activity cloned, 2. Anderson, J. T., S. M. Wilson, K. V. Datar, and M. S. Swanson. their proteins have been characterized (1, 2, 6-8, 16, 23, 28, 1993. NAB2: a yeast nuclear polyadenylated RNA-binding protein 33). Apart from their RNA-binding activities, the biological essential for cell viability. Mol. Cell. Biol. 13:2730-2741. functions of these proteins are poorly understood. Among 3. Belenguer, P., V. Baldin, C. Mathieu, H. Prats, M. Bensaid, G. these proteins, nucleolin is a ubiquitous, multifunctional pro- Bouche, and F. Amalric. 1989. Protein kinase NII and the regula- tein thought to be involved in ribosome biogenesis (8, 21), tion of rDNA transcription in mammalian cells. Nucleic Acids regulation of pre-rRNA transcription (3, 5, 20), and shuttling Res. 17:6625-6635. of ribosomal proteins to the nucleolus (4, 22, 25, 26, 32). 4. Borer, R. A., C. F. Lehner, H. M. Eppenberger, and E. A. Nigg. no studies 1989. Major nucleolar proteins shuttle between nucleus and However, previous have identified nucleolin as a cytoplasm. Cell 56:379-390. transcriptional repressor. 5. Bouche, G., M. Caizergues-Ferrer, B. Bugler, and F. Amairic. In this paper, we presented several lines of evidence indi- 1984. Interrelations between the maturation of a 100 kDa nucle- cating that nucleolin indeed functions as a transcriptional olar protein and pre-rRNA synthesis in CHO cells. Nucleic Acid repressor for the AGP gene. (i) The purified oligonucleotide Res. 12:3025-3035. B-binding factor has been unequivocally identified as nucleolin 6. Bourbon, H. M., and F. Amalric. 1990. Nucleolin gene organiza- by amino acid sequencing analysis. (ii) The purified factor can tion in rodents: highly conserved sequences within three of the 13 be a monoclonal to introns. Gene 88:187-196. recognized by antibody human nucleolin. 7. Bourbon, H. M., B. Lapeyre, and F. Amairic. 1988. Structure of the (iii) Nucleolin can recognize the B motif in a DNA fragment mouse nucleolin gene: The complete sequence reveals that each spanning from position -180 to + 10 of the AGP promoter as RNA binding domain is encoded by two independent exons. J. identified by DNase I footprinting assays. (iv) Recombinant Mol. Biol. 200:627-638. nucleolin shares antigenic properties and sequence specificity 8. Bugler, B., H. Bourbon, B. Lapeyre, M. 0. Wallace, J. H. Chang, with the purified factor. (v) Recombinantly expressed nucleo- F. Amalric, and M. 0. J. Olson. 1987. RNA binding fragments lin can function as a repressor for AGP gene expression as from nucleolin contain the ribonucleoprotein consensus sequence. demonstrated by transfection assays. (vi) In agreement with J. Biol. Chem. 262:10922-10925. the 9. Chang, C. J., T. T. Chen, H. Y. Lei, D. S. Chen, and S. C. Lee. 1990. results of biochemical characterizations, the repressor Molecular cloning of a transcription facyor, AGP/EBP, that function of nucleolin depends on the B motif of the AGP belongs to members of the C/EBP family. Mol. Cell. Biol. 10:6642- promoter. 6653. Intact nucleolin has a molecular mass ranging from 91 to 105 10. Chen, C. M., S. Y. Chiang, and N. H. Yeh. 1991. Increased stability kDa (5, 10). However, nucleolin has been shown to be de- of nucleolin in proliferating cells by inhibition of self-cleaving graded autocatalytically as well as by granzyme A (10, 17, 29). activity. J. Biol. Chem. 266:7754-7758. The 105-kDa molecule is the major species in actively dividing 11. Cleveland, D. W. 1983. Peptide mapping in one dimension by cells, while degraded forms with various molecular sizes are limited proteolysis of sodium dodecyl sulfate-solubilized proteins. predominantly expressed in nondividing cells (10, 21). More- Methods Enzymol. 96:222-229. the 12. Costa, R. H., D. R. Grayson, K. G. Xanthopoulos, and J. E. over, self-cleaving activity of nucleolin can be inhibited by Darnell. 1988. A liver-specific DNA-binding protein recognizes nuclear extracts prepared from proliferating cells (10). Our multiple nucleotide sites in regulatory regions of transthyretin, results support these observations. The major nucleolin species alpha-1-antitrypsin, albumin and simian virus 40 genes. Proc. Natl. in hepatoma cells has a molecular mass of about 97 kDa, while Acad. Sci. USA 85:3840-3844. hepatocytes contain several lower-molecular-mass forms of 13. Darlington, G., D. R. Wilson, and L. B. Lachman. 1986. Monocyte- nucleolin which can be detected with antinucleolin monoclonal conditioned medium, interleukin-1, and tumor necrosis factor antibodies (data not shown). The critical questions are (i) stimulate the acute phase response in human hepatoma cells in whether the regulation of nucleolin degradation is linked to its vitro. J. Cell Biol. 103:787-793. function and whether different forms of nucleolin function 14. Das, H. K., T. Leff, and J. L. Breslow. 1988. Cell type-specific (ii) expression of the human apoA gene is controlled by two cis-acting differently. Our studies on the transcriptional repression of the regulatory regions. J. Biol. Chem. 163:1452-1458. AGP gene mediated by the B motif of the AGP promoter and 15. Dobbeling, U., K. Ross, L. Klein-Hitpass, C. Morley, U. Wagner, negative trans-acting hepatic factors may offer some clues. The and G. U. Ryffel. 1988. A cell-specific activator in the Xenopus A2 factor we purified from liver cells specifically recognizes the B vitellogenin gene: promoter elements functioning with rat liver motif as demonstrated by DNase I footprinting analysis (24). nuclear extracts. EMBO J. 7:2495-2501. These results suggest that the liver factor (factor B) and 16. Ellis, E. M., and G. A. Reid. 1993. The Saccharomyces cerevisiae nucleolin share DNA sequence-binding specificity and possibly MTS1 gene encodes a putative RNA-binding protein involved in antigenicity. Whether the intact nucleolin or alternative post- mitochondrial protein targeting. Gene 132:175-183. translationally modified derivatives can function as "factor B" 17. Fang, S. H., and N. H. Yeh. 1993. The self-cleaving activity of in the AGP nucleolin determines its molecular dynamics in relation to cell regulating gene expression in liver remains to be proliferation. Exp. Cell Res. 208:48-53. clarified. 18. Foulkes, N. S., and P. Sassone-Corsi. 1992. 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