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 glycopro- tein (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.