HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 26, 23653-23660, June 29, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/26/23653    most recent M009891200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Häkli, M. Articles by Palvimo, J. J. Search for Related Content PubMed PubMed Citation Articles by Häkli, M. Articles by Palvimo, J. J. Social Bookmarking                      What's this?

The RING Finger Protein SNURF Is a Bifunctional Protein Possessing DNA Binding Activity^*

Marika Häkli, Ulla Karvonen, Olli A. Jänne^§, and Jorma J. Palvimo^¶

From the  Biomedicum Helsinki, Institute of Biomedicine (Physiology), ^¶ Institute of Biotechnology, and the ^§ Department of Clinical Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland

Received for publication, October 30, 2000, and in revised form, April 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The small nuclear C₃HC₄ finger protein (SNURF), RNF4, acts as transcriptional coactivator for both steroid-dependent and -independent promoters such as those driven by androgen response elements and GC boxes, respectively. However, SNURF does not possess intrinsic transcription activation function, and the precise molecular mechanism of its action is unknown. We have studied herein the interaction of SNURF with DNA in vitro. SNURF binds to linear double-stranded DNA with no apparent sequence specificity in a cooperative fashion that is highly dependent on the length of the DNA fragment used. SNURF interacts efficiently with both supercoiled circular and four-way junction DNA, and importantly, it also recognizes nucleosomes. An intact RING structure of SNURF is not mandatory for DNA binding, whereas mutations of specific positively charged residues in the N terminus (amino acids 8-11) abolish DNA binding. Interestingly, the ability of SNURF to interact with DNA is associated with its capability to enhance transcription from promoters containing GC box elements. Because SNURF can interact with both DNA and protein (transcription) factors, it may promote assembly of nucleoprotein structures.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RING¹ (really interesting new gene) finger is a motif of conserved cysteines and histidines that coordinate two zinc atoms in a "cross-brace" system, a ligation scheme distinct from those of the classical zinc fingers (1, 2). The RING motifs can be classified into two subgroups according to the presence of a cysteine or histidine in the fifth position: C₃HC₄ (RING-HC) and C₃H₂C₃ (RING-H2) fingers. Otherwise their composition and length can vary substantially. The RING finger has been found in a variety of eukaryotic proteins of diverse evolutionary origin that are involved in various cellular processes such as oncogenesis, development, signal transduction, and apoptosis (1-3). RING fingers have been shown to mediate protein-protein interactions and formation of multi-protein complexes. The RING motif of promyelocytic leukemia gene product is important in the assembly of protein complexes linked to SUMO-1 (a small ubiquitin-like modifier protein) modifications (4). RING finger has also been suggested to act as a DNA-binding motif (5). The function of many RING-containing proteins can be mediated through DNA binding or chromatin association. RAG1 is involved in V(D)J recombination complex, and RAD-16 participates in DNA repair (6). RING finger-containing polycomb group proteins Psc, Su(z)2, Bmi-1, and RING1 are involved in the maintenance of the transcriptionally repressed state of genes by regulating chromatin structure (7-9), and Mel-18 is shown to act as a transcriptional repressor via binding to specific DNA sequence (10). Nuclear receptor mediator TIF is tightly associated with euchromatin (11), whereas BRCA1 appears to be associated with the RNA polymerase II holoenzyme (12). However, the RING structures of these latter proteins have not been implicated in mediating their binding to chromatin or DNA.

Recent intriguing results have shown that many RING proteins are able to mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination in vitro (13, 14) and thus act as E3 ubiquitin ligases. However, as pointed out by Lorick et al. (13), only some of these proteins are likely to function primarily as E3s, and the RING finger-mediated ubiquitination probably provides many of these proteins with a self-regulatory function.

The small nuclear RING finger protein SNURF was initially identified as an androgen receptor (AR)-interacting protein by yeast two-hybrid screening. Rat SNURF is a highly hydrophilic protein composed of 194 amino acid residues of which about 30% are charged (15). The charge distribution of SNURF is asymmetrical; two negatively charged regions separate three basic amino acid clusters. The N terminus of SNURF encompasses a bipartite nuclear localization signal, and the C₃HC₄-type RING motif is localized in the C-terminal region. SNURF interacts with AR via its N-terminal region, whereas the RING finger plays a key role in the binding to promoter specificity protein 1 (Sp1) (16). In addition to AR and other steroid receptors, SNURF also enhances the activity of Sp1-regulated transcription. In contrast to many other coactivators, SNURF does not possess a conventional activation domain(s). To get better insight into the biological role of SNURF, we have studied the interaction of SNURF and its mutated forms with DNA. Our results indicate that SNURF possesses a general DNA binding activity that may explain some of its characteristics as a transcription-activating protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The BaculoGold transfection system and pAcG3X baculovirus transfer vector were purchased from PharMingen. Protease inhibitors phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin A, and aprotinin, as well as double-stranded calf thymus DNA cellulose were obtained from Sigma. Glutathione-Sepharose 4B, CM-Sepharose Fast Flow, single-stranded DNA agarose, Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane and ECL detection reagents were from Amersham Pharmacia Biotech. Horseradish peroxidase-conjugated anti-mouse IgG was from Zymed Laboratories Inc. Protease factor Xa was purchased from Roche Molecular Biochemicals. Human Sp1 was from Promega. HMG-1 was purified from calf thymus (17) and histones were from rat thymus (18).

Production and Purification of SNURF-- Recombinant viruses for wild-type and RING finger-mutated (C136S/C139S) rat SNURF GST fusion proteins were produced in Spodoptera frugiperda Sf9 cells by using the BaculoGold transfection system (15). Sf9 cells were infected with recombinant virus at multiplicity of infection of 1 plaque-forming unit/cell, and cells were grown for three days. GST-SNURF proteins were extracted at 0 °C by sonication in a buffer containing 50 mM Tris-HCl (pH 7.8), 50 µM ZnCl₂, 10% (v/v) glycerol, 0.1% (v/v) Nonidet P-40, 1% (v/v) Triton X-100, 300 mM NaCl, 2 mM EDTA, 0.5 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 10 µg/ml aprotinin. Extracts were centrifuged at 25,000 × g for 30 min. Recombinant proteins were adsorbed to glutathione-Sepharose 4B beads and eluted in a buffer containing 20 mM reduced glutathione, 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 15% (v/v) glycerol, 0.5 mM dithiothreitol (DTT), 50 µM ZnCl₂, and 0.5 mM PMSF. Alternatively, GST was cleaved off by using Factor Xa (2 µg of protease/mg GST fusion protein) in 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM CaCl₂, and 10% (v/v) glycerol at 4 °C for 2 h. The protease was removed by CM-Sepharose Fast Flow chromatography in loading buffer containing 20 mM Tris-HCl (pH 7.0), 50 mM NaCl, and 1 mM DTT, and SNURF was eluted by using 300 mM NaCl in loading buffer. Factor Xa-cleaved SNURF was analyzed by N-terminal sequencing of polypeptides transferred onto PDVF membrane and by mass spectrometry. Wild-type SNURF and different SNURF mutants C136S/C139S, 1-20, 66-98, 178-194, K8A, R9A, R10A, R11A, and R8-11A/K9A were also produced as GST fusion proteins using pGEX-5X-1 vector (Amersham Pharmacia Biotech) and Epicurian coli BL21 CodonPlus (DE3)-RIL cells (Stratagene). SNURF C136S/C139S, 1-20, 66-98, and 178-194 GST fusion proteins were constructed with the aid of polymerase chain reaction and the corresponding pcDNA3.1(+)-FLAG constructs (16). The GST fusions of SNURF mutants R8-11A/K9A, R8A, K9A, R10A, and R11A were made by using oligonucleotide duplexes flanked by BamHI and EcoRI sites and cloned directly into pGEX-5X-SNURF plasmid. The SNURF R18-25A/K22A mutant was constructed by using oligonucleotide duplexes containing AvaI and EcoRI ends. The bacteria were grown at 37 °C to A_{600 nm} = 0.7 and induced with 0.3 mM isopropyl-1-thio--D-galactopyranoside. Cultures were harvested after 3 h at 30 °C and GST fusion proteins were purified by affinity chromatography on glutathione-Sepharose 4B (15). pcDNA3.1(+)-FLAG-SNURFR8-11A/K9A and pcDNA3.1(+)-FLAG-SNURFR8A were assembled by transferring the BamHI/XhoI fragments from pGEX-5X-1 vectors.

DNA-Cellulose Chromatography-- Proteins (5 µg) were incubated with 20 µl of double-stranded (ds) calf thymus DNA cellulose (containing 3 µg of DNA) in 500 µl of binding buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM NaCl, 0.5 mM DTT, and 10% (v/v) glycerol (19). After incubating at 4 °C for 4 h by rotation and washing four times with 1 ml of binding buffer, bound proteins were eluted stepwise with one volume of 0.1, 0.3, 0.6, and 1.0 M NaCl in binding buffer. Eluted fractions, flow-through, and first wash were analyzed on 15% polyacrylamide gels under denaturing conditions (SDS-PAGE), and gels were stained with GelCode Blue Stain Reagent (Pierce) or Coomassie Brilliant Blue.

Electrophoretic Mobility Shift Assay (EMSA)-- Retardation reactions according to Sheflin et al. (20) containing 0.2 pmol of negatively supercoiled or linearized pGEM-9Zf() (Promega) were incubated with various concentrations of SNURF or HMG-1 for 15 min at 37 °C in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 0.5 mM DTT, 0.1 mM EDTA, and 0.3 µg/ml bovine serum albumin in a total volume of 20 µl. The binding reactions were placed on ice for 30 min and then incubated for 5 min at 37 °C. Samples of the reactions were separated on 0.7% (w/v) agarose gels using 1× Tris-phosphate-EDTA as the running buffer, and the gels were stained with ethidium bromide. In EMSA reactions with shorter DNA fragments, proteins were preincubated with carrier DNA poly(dI-dC)₂ (50 or 100 ng/reaction) on ice for 10 min in a buffer containing 20 mM Hepes (pH 7.9), 50 mM KCl, 1 mM MgCl₂, 10% (v/v) glycerol, 2 mM DTT, 0.35 mM EDTA, 0.025% (v/v) Nonidet P-40, 0.25 mM PMSF, 2 µg/ml pepstatin A, 2 µg/ml leupeptin, and 2 µg/ml aprotinin. After the addition of 2 µl of ³²P-labeled DNA probe yielding a total volume of 20 µl, samples were incubated at 22 °C for 30 min. Protein-DNA complexes were resolved on 4% polyacrylamide gel in 0.25× Tris borate-EDTA at 22 °C. Oligonucleotides 5'-CCCTATACCCCTGCATTGAATTCCAGTCTGATAA-3', 5'-GTAGTCGTGATAGGTGCAGGGGTTATAGG-3', 5'-AACAGTAGCTCTTATTCGAGCTCGCGCCCTATCACGACTA-3', and 5'-TTTATCAGACTGGAATTCAAGCGCGAGCTCGAATAGAGCTACTGT-3' (21) were used to create a four-way junction DNA (4H DNA) according to Hill et al. (22) by dissolving appropriate amounts of each oligonucleotide in annealing buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 10 mM MgCl₂, heating at 95 °C for 1 min, and cooling slowly to room temperature. 4H DNA was labeled with [-³²P]ATP and T4 polynucleotide kinase. ³²P-Labeled HindIII/KpnI fragment (232 bp) of the rat p75 neurotrophin receptor promoter and HindIII/NcoI (33 bp), BglII/NcoI (65 bp), and KpnI/NcoI (135 bp) fragments of Sp1₂-TATA-LUC were used in additional EMSA experiments (15, 23).

Reconstitution of Mononucleosomes-- An XbaI/HincII fragment (164 bp, 1.5 pmol) from pGL3-basic (Promega) was end-labeled with [-³²P]dCTP and Klenow fragment and was mixed with donor chromatin from rat liver (a gift from Dr. Örjan Wrange, Karolinska Institute, Stockholm, Sweden) to a final concentration of 0.1 mg/ml in a buffer containing 1 M NaCl, 15 mM Tris-HCl (pH 7.5), 0.2 mM EDTA, and 0.13 mM PMSF. Nucleosomes were reconstituted by high salt exchange method and purified by 7 to 30% glycerol gradient centrifugation (24). Increasing concentrations of GST-SNURF or the GST-SNURF1-20 mutant were incubated with a constant amount of reconstituted nucleosomes, and EMSAs were performed as described for naked DNA.

SNURF-Histone Interactions in Vitro-- GST-SNURF or GST adsorbed onto glutathione-Sepharose 4B beads was first washed with 0.2% (v/w) N-lauroylsarcosine (Sarkosyl) in a binding buffer containing 20 mM Tris-HCl (pH 7.8), 50 mM NaCl, 0.05% (v/v) Nonidet P-40, 50 µM ZnCl₂, 0.1 mM DTT, 0.2 mM EDTA (pH 8.0), 0.5 mM PMSF, 5 µg/ml pepstatin A, and 10 µg/ml aprotinin and then with binding buffer without Sarkosyl. Sepharose beads containing 20 µg GST or GST-SNURF were incubated alone or with histones extracted from rat thymus in 500 µl of binding buffer for 4 h at 4 °C and subsequently washed four times with 1 ml of binding buffer containing 400 mM NaCl. Bound histones were eluted by 0.2% (v/w) Sarkosyl containing binding buffer, samples were analyzed on 15% polyacrylamide gels under denaturing conditions, and the gels were stained with GelCode Blue stain reagent.

Cell Culture and Transfections-- COS-1 cells were obtained from American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 25 units/ml of streptomycin and penicillin. For transactivation assays, 5 × 10⁴ cells were seeded on 12-well plates 24 h prior to transfection using the FuGENE reagent (Roche Molecular Biochemicals). Four hours before the addition of DNA, the cells received fresh medium with 10% fetal bovine serum. After 18 h, the medium was changed to Dulbecco's modified Eagle's medium supplied with 2% fetal bovine serum. Luciferase and -galactosidase activities and the concentration of soluble cell proteins were assayed as previously described (15). For immunolocalization of SNURF mutants in COS-1 cells, cells grown on glass coverslips (1.6 × 10⁵ cells on 6-well plates) were transfected with 750 ng of pcDNA-FLAG-SNURF or mutated SNURF forms in the same vector (25). Cells were processed, and immunofluorescence labeling and microscopy were performed as described in (25).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Purification of Recombinant SNURF-- Recombinant GST-SNURF produced in Sf9 cells was adsorbed onto glutathione-Sepharose and cleaved with Factor Xa to remove the GST tail. In addition to the full-length SNURF migrating at ~34 kDa on SDS-PAGE (11), protease digestion produced smaller amounts of shorter polypeptides of ~26 kDa and ~16 kDa in size (Fig. 1B). N-terminal sequencing and mass spectrometric analysis indicated that the 34-kDa product corresponds to the full-length SNURF. The two smaller polypeptides were identified as secondary cleavage products of SNURF by Factor Xa (26). Internal cleavages at Arg⁸⁵ and at Arg¹⁸¹ produced an N-terminal SNURF fragment containing two stretches of basic amino acid residues and a C-terminal fragment encompassing the RING finger motif, respectively (Fig. 2). The proportion of the full-length SNURF in GST-cleaved preparation was ~60% of the total protein. In addition to SNURF fragments, the preparation did not contain other proteins in detectable amounts.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   SNURF adheres to DNA cellulose. DNA binding activity of SNURF was compared with that of HMG-1 by using double-stranded calf thymus DNA cellulose. GST-SNURF (A), SNURF lacking the GST tail (B), HMG-1 (C), or GST alone (D) were incubated with the matrix, washed, and eluted stepwise with the indicated NaCl concentrations as described under "Experimental Procedures." Entire eluted fractions, 5% of flow-through (F) and 5% of washes (W) were subjected to 15% SDS-PAGE, and the gel was stained with GelCode Blue stain reagent. Input (I) samples correspond to 20% of the amount of the proteins incubated with matrices.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Sequence characteristics of SNURF protein. The charge distribution of SNURF is asymmetrical; positively and negatively charged amino acids are indicated by bolding and underlining, respectively. Cysteine and histidine residues of C3HC4 type RING finger motif (boxed) are depicted by asterisks, and amino acid residues of potential nuclear localization signals are shown by italics. Arrowheads depict the internal cleavage sites by Factor Xa.

SNURF Binds to DNA-- Double-stranded calf thymus DNA cellulose was used as an affinity matrix to examine the DNA binding activity of SNURF. Proteins were incubated with dsDNA cellulose in low salt, and the adsorbed proteins were eluted using a stepwise gradient of NaCl. Most of GST-SNURF bound to DNA and was eluted between 0.3 and 0.6 M NaCl (Fig. 1A). The full-length SNURF lacking the GST tail was eluted at a salt concentration of 0.6-1.0 M NaCl. The N-terminal SNURF fragment (26 kDa) eluted together with full-length SNURF, whereas the C-terminal fragment containing the RING finger motif (16 kDa) was present in the flow-through fraction (Fig. 1B). High mobility group protein-1 (HMG-1) was used as a representative of DNA-binding proteins that display little or no specificity for the target DNA sequence (27). Interestingly, the adherence of HMG-1 to DNA was more sensitive to the ionic strength than that of SNURF in that most of the HMG-1 eluted from dsDNA cellulose at a salt concentration of 0.3-0.6 M NaCl (Fig. 1C). GST did not adhere to DNA under identical conditions (Fig. 1D). Similar to HMG-1 (28), SNURF also binds to single-stranded DNA agarose (results not shown).

Because dsDNA cellulose experiments indicated that SNURF binds to DNA, it was important to assess whether there is nucleotide sequence specificity in SNURF binding. To this end, a selection and amplification binding assay, a polymerase chain reaction-based random oligonucleotide selection technique, was employed (29, 30). SNURF was incubated with a 60-bp oligonucleotide containing a 20-bp central region of degenerate nucleotide sequence. However, these experiments failed to show any apparent sequence preference for binding of SNURF to DNA.

SNURF Binds Efficiently to Various Types of DNA-- SNURF binding capacity toward supercoiled and linearized plasmid DNA was compared by using a gel retardation assay. GST-SNURF was incubated with supercoiled and linear pGEM-9Zf() DNA (2.9 kb) at various protein:DNA molar ratios, and protein-DNA interactions were monitored by agarose gel electrophoresis and ethidium bromide staining. SNURF bound to all the supercoiled DNA, and the mobility of SNURF-DNA complexes was progressively retarded with increasing SNURF concentrations (Fig. 3A), a phenomenon typical of general DNA-binding proteins (20). Similar mobility shift changes are not usually seen with sequence-specific DNA-binding proteins. SNURF retarded the mobility of both DNA types more efficiently than HMG-1. However, comparison of the interaction of SNURF between supercoiled and corresponding linear DNA did not reveal a clear preference for either DNA type (Fig. 3). GST alone did not interact with DNA.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Binding of SNURF to supercoiled and linear DNA as analyzed by electrophoretic mobility shift assay. Indicated molar protein:DNA ratios of GST-SNURF, HMG-1, or GST were incubated with negatively supercoiled pGEM-9Zf()(A) or with the corresponding linearized DNA (B) as described under "Experimental Procedures." Protein-DNA complexes were resolved by electrophoresis on 0.7% agarose gels containing 1× Tris-phosphate-EDTA and visualized by ethidium bromide staining.

To study further the DNA binding characteristics of SNURF, the interaction of SNURF with 4H DNA was examined and compared with that of HMG-1 (31, 32). EMSAs on parallel polyacrylamide gels were used to compare the binding of SNURF and HMG-1 to 4H DNA and linear DNA (a 232-bp restriction fragment). ³²P-Labeled DNA fragments were incubated with increasing concentrations of SNURF or HMG-1. As shown in Fig. 4A, SNURF bound to 4H DNA and formed a weak DNA complex already at <10 nM protein concentration (lane 2). Most of the probe was up-shifted at ~40 nM SNURF (lane 4), and an increase in the amount of protein up-shifted the complex further (lanes 5 and 6). Interestingly, HMG-1 bound to 4H DNA less efficiently than SNURF, and the complexes of HMG-1 were less stable during electrophoresis than those formed by SNURF. With linear DNA, progressive retardation of DNA was observed when the amount of SNURF was increased (Fig. 4B), indicating that, as in the case of 4H DNA, more than one SNURF molecule can interact with the same DNA molecule simultaneously. Comparable DNA binding pattern was obtained with GST-SNURF (results not shown), ruling out that the retardation pattern was due to SNURF fragments in protein preparations.

View larger version (53K): [in this window] [in a new window]   Fig. 4.   SNURF can interact with four-way junction DNA. A, increasing concentrations of SNURF (6-140 nM) or HMG-1 (20-550 nM) were preincubated with 50 ng of poly(dI-dC)2 for 10 min at 0 °C prior to the addition of 32P-labeled 4H DNA, and the incubation was continued for 30 min at 22 °C. B, 32P-labeled linear 232-bp DNA was used instead of 4H DNA under identical conditions. Protein-DNA complexes were separated on 4% nondenaturing polyacrylamide gels containing 0.25× Tris borate-EDTA followed by autoradiography. F, free DNA.

The Cooperativity of SNURF DNA Binding Is Influenced by the Length of the DNA Fragment-- To study the influence of the length of target DNA sequence on the SNURF-DNA interaction, the ability of SNURF to bind to DNA fragments of varying lengths was compared by using ³²P-labeled linear DNA probes in EMSA. Fixed amounts of labeled DNA of 33, 65, and 135 bp in length were incubated with increasing amounts of SNURF, and DNA-protein complexes were analyzed with EMSA. When the 33-bp DNA fragment was used, only 40% of the probe was up-shifted at 42 nM SNURF concentration, and the position of the complexes suggested presence of one or maximally two SNURF molecules bound to DNA (Fig. 5, lane 2). In contrast, the 65-bp and 135-bp DNA fragments were completely shifted at the same protein concentration (Fig. 5, lanes 6 and 10) and progressive retardation DNA was observed when 200 nM SNURF was used (Fig. 5, lanes 7 and 11), indicating that multiple SNURF molecules can interact with DNA fragments longer than ~60 bp. However, the SNURF-DNA complexes formed with the 135-bp DNA appeared to be more stable than those generated with the 65-bp fragment.

View larger version (79K): [in this window] [in a new window]   Fig. 5.   DNA binding of SNURF is dependent on the length of the target DNA fragment. Indicated concentrations of SNURF were first incubated with 100 ng of poly(dI-dC)2 and subsequently with 32P-labeled double-stranded DNA of 33 bp (lanes 1-3), 65 bp (lanes 5-7), or 135 bp (lanes 9-11) in length as described under "Experimental Procedures." Protein-DNA complexes were resolved by electrophoresis on 4% polyacrylamide gels under nondenaturing conditions and visualized by autoradiography.

The N-terminal Basic Amino Acid Cluster of SNURF Is Critical for DNA Binding-- To elucidate the regions of SNURF mandatory for the interaction with DNA, EMSAs were performed with mutated SNURF proteins expressed in Epicurian coli and purified as GST fusion proteins. GST-SNURF(C136S/C139S), in which two of the N-terminal cysteines of the RING finger (Fig. 2) were converted to serines, interacted with the ³²P-labeled 232-bp DNA fragment as efficiently as wild-type GST-SNURF (Fig. 6), indicating that an intact zinc-coordinated RING structure is not mandatory for the interaction of SNURF with DNA. The deletion mutant lacking the second basic amino acid cluster (SNURF66-98) bound to DNA as efficiently as full-length SNURF, whereas a C-terminal deletion mutant of SNURF (178-194) lacking the third basic stretch formed slightly more labile DNA complexes (Fig. 6). In contrast, the deletion of amino acids 1-20, including most of the N-terminal basic amino acid cluster, blunted the ability of SNURF to interact with DNA.

View larger version (81K): [in this window] [in a new window]   Fig. 6.   The positively charged amino acids in the N terminus of SNURF are required for DNA binding. A, GST-SNURF together with GST fusions of SNURF mutants C136S/C139S, 1-20, 66-98, 178-194, R8-11A/K9A, R8A, K9A, R10A, and R11A (100 nM each) produced in Epicurian coli were compared for their ability to bind to 32P-labeled linear 232-bp DNA under conditions described in Fig. 4. B, SDS-PAGE analysis of the purified GST fusion proteins used in the EMSA experiment. Proteins (1 µg) were detected by Coomassie Brilliant Blue staining. F, free DNA.

To assess the role of this latter basic amino acid-containing stretch in DNA binding, arginines and lysines in the region were individually or together converted to alanines, and the corresponding GST fusion proteins were analyzed by EMSA. As shown in Fig. 6, mutation of neither Lys⁹, Arg¹⁰, nor Arg¹¹ alone reduced significantly the DNA complex formation (lanes 10-12), whereas conversion of Arg⁸ to Ala resulted in clearly more labile DNA complexes. When Arg⁸, Lys⁹, Arg¹⁰, and Arg¹¹ were all changed to alanines, the compound mutant (R8-11A/ K9A) bound to DNA as poorly as the deletion mutant SNURF1-20. Arginine 18, 23, and 25 as well as lysine 22 do not appear to play a critical role in DNA binding because a SNURF mutant having these amino acid residues mutated to alanines was still capable of binding to DNA². Taken together, these data indicate that amino acids 8-11 are the principal residues responsible for contacting DNA and that Arg⁸ plays a special role in the DNA binding of SNURF.

SNURF Binds to Nucleosomes-- To examine whether SNURF can also interact with nucleosomes, EMSAs were performed with increasing amounts of GST-SNURF or SNURF1-20 and a constant concentration of nucleosomes that were reconstituted with a ³²P-labeled 164-bp DNA fragment. Although the mononucleosome fraction was purified by glycerol gradient centrifugation, some free DNA was present in the preparation, which is depicted as the band migrating faster than that of mononucleosomes (Fig. 7A, lane 1). This minor contamination of the mononucleosome fraction with free DNA was, in fact, beneficial as it permitted comparison of the binding of SNURF to naked DNA and to nucleosomes in the same assay. As illustrated in Fig. 7, GST-SNURF retarded efficiently the mobility of the reconstituted nucleosome complex. However, as shown by the disappearance (upshift) of free DNA at a somewhat lower SNURF concentration than that of mononucleosomes (cf. lanes 4 and 6), SNURF appears to bind slightly more avidly to naked DNA than to nucleosomes. As was the case with free DNA, the N-terminally truncated SNURF1-20 was practically unable to retard nucleosome particles (Fig. 7A, lanes 9-15).

View larger version (47K): [in this window] [in a new window]   Fig. 7.   SNURF binds to nucleosomes. A, 32P-labeled in vitro reconstituted mononucleosomes were incubated with increasing concentrations (nM) of GST-SNURF (lanes 2-8), or the SNURF1-20 mutant (lanes 9-15). Samples were analyzed on a 4% nondenaturing polyacrylamide gel at 4 °C and visualized by autoradiography. Arrow and arrowhead depict the position of unbound nucleosomes and free DNA, respectively. B, GST-SNURF or GST immobilized onto glutathione-Sepharose was incubated with or without rat thymus histones for 4 h at 4 °C, and the matrices were washed extensively with buffer containing 0.4 M NaCl as described under "Experimental Procedures." Bound histones were eluted with 0.2% Sarkosyl and resolved by electrophoresis on 15% SDS-PAGE. Lane 1, 10% of the input histones; lanes 2 and 4, histones eluted from GST-SNURF and GST matrices, respectively; and lanes 3 and 5, elutions from GST-SNURF and GST matrices not incubated with histones, respectively.

To determine whether the binding of SNURF to nucleosomes resulted merely from interaction of this protein with nucleosomal DNA, the ability of SNURF to bind to free histones was also examined. GST-SNURF adsorbed onto glutathione-Sepharose beads was incubated with a mixture of rat thymus histones, and the matrix was then washed extensively with buffer containing either 0.15 or 0.4 M NaCl and eluted with Sarkosyl. SDS-PAGE analysis of the proteins bound to the GST-SNURF matrix revealed that under high salt conditions, only histones H3 and H4 were specifically retained by SNURF, whereas under a physiological salt concentration SNURF was capable of interacting with all core histones as well as histone H1 (Fig. 7B, lane 2, and data not shown). Collectively, these results suggest that the binding of SNURF to nucleosomes does not rely only on the ability of SNURF to interact with DNA.

The Ability of SNURF to Stimulate Transcription Correlates with Its DNA Binding Activity-- We have previously shown that ectopic expression of SNURF activates minimal promoters containing Sp1-binding sites in front of a TATA box (15, 16). SNURF and Sp1 are also able to cooperate on natural promoters containing GC box elements, such as the rat p75 neurotrophin receptor promoter corresponding to the same 232-bp fragment as that used as the target DNA sequence in the preceding EMSA experiments³. To assess further the importance of the N-terminal basic amino acids of SNURF in transcriptional activation, COS-1 cells were transfected with Sp1₂-TATA-driven luciferase (LUC) reporter (15) along with wild-type or mutated SNURF expression plasmids. In accordance with our previous results, the N-terminally truncated SNURF1-20 was practically inactive in this assay, showing 5% of wild-type activity (Fig. 8A). The extent of DNA binding of R8-11A/K9A and that of R8A was in line with their ability to activate Sp1₂-TATA-LUC reporter in that the latter mutant with some DNA binding activity displayed ~25% of wild-type activity in transactivation assays, whereas the behavior of the compound mutant was comparable with that of SNURF1-20 in both analyses. Similar results were obtained with the rat p75 neurotrophin receptor promoter². Together these results strongly suggest that the DNA binding activity of SNURF is essential for the ability of this protein to activate transcription.

View larger version (54K): [in this window] [in a new window]   Fig. 8.   Stimulation of GC box element-driven transcription is associated with DNA binding ability of the SNURF. A, COS-1 cells on 12-well plates were transfected using FuGENE reagent with 160 ng of Sp12-TATA-LUC, 60 ng of -galactosidase expression vector (pCMV), and 300 ng of indicated SNURF expression plasmids in pcDNA3.1(+)-FLAG backbone. After a 40-h culture, the cells were harvested, and luciferase activities in the cell extracts were adjusted to the transfection efficiency using -galactosidase as an internal control. The reporter gene activity in the presence of empty pcDNA3.1(+)-FLAG vector was set as 1. The mean ± S.E. values of at least six experiments is shown. Inset, Immunoblot analysis of wild-type and mutant SNURF proteins in an experiment corresponding to data shown in panel A. SNURF proteins were immunoblotted from the same lysates (pooled triplicate dishes) from which reporter gene activities were measured with an anti-SNURF antibody (15) and using the ECL system. Localization of wild-type SNURF (B), SNURF1-20 (C), SNURFR8-11A/K9A (D), and SNURFR8A (E) in COS-1 cells. Cells grown on glass coverslips on 6-well plates were transfected with 750 ng of pcDNA-FLAG-SNURF or the corresponding vector encoding mutant SNURF forms. After a 40-h culture, cells were fixed with paraformaldehyde (4%) and permeabilized in 0.1% Triton X-100. SNURF antigen was detected by using anti-FLAG M2 monoclonal antibody followed by fluorescein isothiocyanate-conjugated secondary anti-mouse antibody, and immunofluorescence was recorded by using Bio-Rad MRC-1024 confocal laser system connected to Zeiss Axiovert 135 microscope.

Because the amino acid cluster critical for DNA binding overlaps with the potential bipartite nuclear localization signal in SNURF, we also investigated the subcellular localization of N-terminally mutated SNURF forms by immunocytochemical analysis in COS-1 cells. As shown in Fig. 8B, D and E, the subcellular localization of neither SNURFR8A nor SNURFR8-11A/K9A differed from that of the wild-type protein. Although the deletion of the first 20 amino acids of SNURF (the SNURF1-20 mutant) resulted in more cytoplasmic staining than seen with the other SNURF forms, it did not prevent the truncated protein from entering the nuclei (Fig. 8C). In view of these data, the inability of the DNA binding-deficient SNURF forms to activate transcription from Sp1 element-containing promoters is not attributed to their altered subcellular localization.

To examine whether SNURF and Sp1 can bind at the same time to the 232-bp rat p75 neurotrophin receptor promoter fragment, the ³²P-labeled DNA was incubated with purified Sp1 alone or together with increasing concentrations of GST-SNURF, and the DNA-protein complexes were separated by EMSA. In the absence of SNURF, Sp1 formed one major and one minor complex with the probe (Fig. 9, lane 1). When a concentration of SNURF that alone bound the probe only weakly (lane 5) was included with Sp1, a third supershifted DNA complex became visible (Fig. 9, lane 2). When higher concentrations of SNURF were used with Sp1, most of the probe was gradually retarded to the position of the latter supershifted DNA-protein complex (Fig. 9, lanes 3 and 4), and the phenomenon was dependent on an intact N terminus of SNURF (Fig. 9, lanes 8-10). In accordance with the restricted ability of SNURF to interact with short DNA fragments, SNURF did not promote Sp1-DNA interaction when the GC box was embedded in a short oligomer². In sum, these results demonstrate that SNURF and Sp1 may interact concomitantly with the same target DNA sequence, although there was no clear cooperative effect by SNURF on the DNA binding of Sp1 under these in vitro conditions. Promotion of Sp1-GC box interaction cannot, however, be ruled out as a possible explanation for the stimulatory action of SNURF on Sp1-dependent transcription in intact cells.

View larger version (64K): [in this window] [in a new window]   Fig. 9.   Concomitant binding of SNURF and Sp1 to the same DNA target. Purified Sp1 alone (lane 1) or together with increasing concentrations of GST-SNURF (lanes 2-4) or GST-SNURF1-20 (lanes 8-10) were preincubated with 50 ng of poly(dI-dC)2 at 0 °C for 10 min. After the addition of a 32P-labeled 232-bp DNA fragment corresponding to the proximal promoter of the rat p75 neurotrophin receptor gene, the incubation was continued for 30 min at 22 °C. DNA-protein complexes were separated on a 4% polyacrylamide gel containing 0.25× Tris borate-EDTA and 0.1% (v/v) Nonidet P-40 and visualized by autoradiography. F, free DNA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The rat RING finger protein SNURF was originally isolated as an androgen receptor-interacting protein in a yeast two-hybrid screen (15). The corresponding protein in human and mouse has been termed RNF4 (33, 34). These sequences are highly conserved, exhibiting 96% identity between rat and mouse protein and 91% identity between rat and human. Interestingly, no obvious SNURF/RNF4 orthologs are found in Drosophila melanogaster, Caenorhabditis elegans, or Saccharomyces cerevisiae. In addition to interacting with steroid receptors and Sp1 (15, 16), RNF4 has been recently shown to associate with Gscl (goosecoid-like) homeodomain transcription factor (34), the activator of stromelysin I gene transcription (SPBP) (35), and a novel member of the BTB/POZ family PATZ (36). Interaction between SNURF and Sp1 or SPBP is mediated through the RING finger (15, 16, 35), whereas AR and Gscl are interacting with a region N-terminal to the RING finger (15, 16, 34).

SNURF is able to act as a transcriptional coactivator for different transcription factors (15, 16, 35), and conversely, to enhance transcriptional repression elicited by PATZ (36). The actual molecular mechanism of transcriptional regulation by SNURF is not known as no clear-cut intrinsic transcription activation or repression function has been found for SNURF. One potential explanation for the effects of SNURF is that it associates with DNA. Our results indicate that SNURF indeed possesses a general DNA binding ability without an apparent nucleotide sequence specificity. Interaction of SNURF with dsDNA was found to be more resistant to ionic strength than that of another non-sequence-specific DNA-binding protein, HMG-1. SNURF was also able to bind to single-stranded DNA. A similar behavior has been previously reported for HMG-1 and HMG-2 as well as for HMG-14 and HMG-17 (37). These HMG proteins are thought to function as architectural proteins that modify the structure of chromatin to generate conformations that facilitate or enhance various other DNA-dependent activities (27). Some non-sequence-specific DNA-binding proteins, such as Ku autoantigen, recognize preferentially DNA termini (38), which was a property, however, not inherent to SNURF. With linear DNA molecules, the cooperative DNA binding mode of SNURF correlated with the length of the DNA fragment in a fashion that resembles the binding characteristics of heterochromatin protein 1 (39). Heterochromatin protein 1, HMG-14, and -17, SNURF also is capable of binding to nucleosomes (27, 37, 39). In this regard, it is interesting that SNURF can recognize four-way junction DNA and that it shows binding preference for core histones H3 and H4. Binding to these DNA structures is a common property of many architectural proteins, and a wide variety of structurally and functionally unrelated DNA-binding proteins have been shown to bind preferentially to four-way junction DNA (40). Intriguingly, HMGI(Y), that favors binding to the four-way junctions, has been reported recently to associate with SNURF/RNF4 (36).

Besides the RING structure and the two nuclear localization signals (a bipartite-type and an SV40-type), no other protein motifs have been identified in SNURF sequence (Fig. 2). Our results show that the intact RING finger motif is not essential for the ability of SNURF to bind to DNA, further supporting the role of this structure as a protein-binding motif (16). Mutational analyses of the basic amino acid cluster (⁸RKRR¹¹), present in the N terminus of SNURF and overlapping with the bipartite nuclear localization signal, revealed that this sequence is primarily responsible for the interaction with DNA and binding to nucleosomes. Of the individual residues, Arg⁸ plays a key role in DNA binding. These results suggest that the SNURF-DNA interaction is mainly electrostatic in nature. Interestingly, this type of Arg-rich cluster in the Drosophila homeodomain protein Bicoid is necessary for the recognition of both DNA and RNA targets (41), and similar motifs are found in a class of RNA-binding proteins (42). It is, therefore, an intriguing possibility that, like the Bicoid, SNURF is capable of recognizing RNA targets as well.

In addition to abolishing the DNA binding of SNURF, mutagenesis of the N-terminal basic amino acids also blunts the ability of the protein to stimulate transcription even though this particular SNURF domain is not centrally involved in heterologous protein interactions (15, 16). The relative DNA binding activity of various SNURF mutants correlated well with their ability to enhance transcription, strongly suggesting that interaction with DNA is essential for SNURF coactivator function. It is also worth pointing out that the mutations in the N terminus of SNURF did not alter nuclear localization of the proteins. Because also the RING finger-disrupted SNURF(C136S/C139S) interacts with DNA but is incapable of activating Sp1-mediated transcription (16), the binding to DNA cannot be the sole mechanism governing the ability of SNURF to activate transcription.

Taken together with our previous results, these data imply that SNURF enhances Sp1 activity through a combinatorial effect, involving protein-protein interaction and DNA binding (15, 16). SNURF may function through a similar mechanism also with other transcription factors such as Gscl (35). These results are reminiscent of the ability of HMG-1 to stimulate many sequence-specific transcription factors including steroid receptors (27, 43). Interestingly, a non-sequence-specific DNA binding activity has been recently shown for many coregulatory proteins implicated in steroid receptor function such as Hap46, TLS/FUS, and C1D/SUN-CoR (44-48). However, we have been unable to detect an unequivocal augmentation of AR DNA binding domain-DNA interaction by SNURF in vitro². This may reflect the requirement of AR regions outside the DNA binding domain for an efficient interaction with SNURF (15). With regard to AR, our additional experiments have shown that SNURF influences nuclear compartmentalization of the receptor (25). The role of DNA binding activity of SNURF in this process remains to be elucidated.

In conclusion, our data suggest that SNURF is a bifunctional protein that can interact both with transcription factors and with DNA/nucleosomes, thereby promoting the assembly of nucleoprotein structures involved in transcriptional control. A similar bifunctional activity may be a feature common to many transcriptional coregulatory proteins.

	ACKNOWLEDGEMENTS

We thank Marc Baumann for peptide sequencing and mass spectrometric analysis, Hetti Poukka for plasmids, Örjan Wrange for help with nucleosomes, and Kati Saastamoinen and Seija Mäki for technical assistance.

	FOOTNOTES

^* This work was supported by grants from the Medical Research Council (Academy of Finland), the Finnish Foundation for Cancer Research, the Sigrid Jusélius Foundation, Biocentrum Helsinki, the Helsinki University Central Hospital, and the Association for the Cure of Cancer of Prostate (CaP Cure).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Biomedicum Helsinki, Inst. of Biomedicine (Physiology), Univ. of Helsinki, P. O. Box 63, FIN-00014 Helsinki, Finland. Tel.: 358-9-19125291; Fax: 358-9-19125302; E-mail: jorma.palvimo@helsinki.fi.

Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M009891200

² M. Häkli, O. A. Jänne, and J. J. Palvimo, unpublished observations.

³ H. Poukka, O. A. Jänne, and J. J. Palvimo, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: RING, really interesting new gene; SNURF, small nuclear RING finger protein; AR, androgen receptor; Sp1, promoter specificity protein 1; PMSF, phenylmethylsulfonyl fluoride; GST, glutathione S-transferase; DTT, dithiothreitol; ds, double-stranded; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; 4H DNA, four-way junction DNA; bp, base pair(s); HMG, high mobility group; LUC, luciferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				H. V. Heemers and D. J. Tindall Androgen Receptor (AR) Coregulators: A Diversity of Functions Converging on and Regulating the AR Transcriptional Complex Endocr. Rev., December 1, 2007; 28(7): 778 - 808. [Abstract] [Full Text] [PDF]

				A. Kosoy, T. M. Calonge, E. A. Outwin, and M. J. O'Connell Fission Yeast Rnf4 Homologs Are Required for DNA Repair J. Biol. Chem., July 13, 2007; 282(28): 20388 - 20394. [Abstract] [Full Text] [PDF]

				S.-M. Wu, W.-C. Kuo, W.-L. Hwu, K.-Y. Hwa, R. Mantovani, and Y.-M. Lee RNF4 Is a Coactivator for Nuclear Factor Y on GTP Cyclohydrolase I Proximal Promoter Mol. Pharmacol., November 1, 2004; 66(5): 1317 - 1324. [Abstract] [Full Text] [PDF]

				D. Curtin, H. A. Ferris, M. Hakli, M. Gibson, O. A. Janne, J. J. Palvimo, and M. A. Shupnik Small Nuclear RING Finger Protein Stimulates the Rat Luteinizing Hormone-{beta} Promoter by Interacting with Sp1 and Steroidogenic Factor-1 and Protects from Androgen Suppression Mol. Endocrinol., May 1, 2004; 18(5): 1263 - 1276. [Abstract] [Full Text] [PDF]

				R. Pero, F. Lembo, E. A. Palmieri, C. Vitiello, M. Fedele, A. Fusco, C. B. Bruni, and L. Chiariotti PATZ Attenuates the RNF4-mediated Enhancement of Androgen Receptor-dependent Transcription J. Biol. Chem., January 25, 2002; 277(5): 3280 - 3285. [Abstract] [Full Text] [PDF]

				B. Saville, H. Poukka, M. Wormke, O. A. Janne, J. J. Palvimo, M. Stoner, I. Samudio, and S. Safe Cooperative Coactivation of Estrogen Receptor alpha in ZR-75 Human Breast Cancer Cells by SNURF and TATA-binding Protein J. Biol. Chem., January 18, 2002; 277(4): 2485 - 2497. [Abstract] [Full Text] [PDF]

This Article Abstract