©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning of an SNF2/SWI2-related Protein That Binds Specifically to the SPH Motifs of the SV40 Enhancer and to the HIV-1 Promoter (*)

(Received for publication, July 19, 1994; and in revised form, October 17, 1994)

Philip L. Sheridan(§)(¶) Marina Schorpp(§)(**) Marianne L. Voz Katherine A. Jones (§§)

From the Salk Institute for Biological Studies, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/initiator region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.


INTRODUCTION

Activation of RNA polymerase II transcription depends upon gene-specific DNA-binding proteins as well as factors that help enhancer-binding proteins to counter the repressive effects of chromatin structure. The Saccharomyces cerevisiae SNF2/SWI2 protein (1, 2) and its human (3, 4, 5, 6) and Drosophila(7, 8) homologues are required for optimal regulated transcription by many gene-specific activators. The yeast SNF2/SWI2 protein controls expression of several classes of inducible genes (for review, see (9) ) and are required for activated transcription by different enhancer-binding proteins, including, for example, several members of the steroid receptor superfamily(3, 5, 10) . The Drosophila BRM protein is thought to act similarly to SNF2/SWI2 by countering the repressive effects of polycomb on homeotic gene transcription(7) . Although SNF2/SWI2 appears to function within a multiprotein complex that is targeted indirectly to promoters, and is not itself a DNA-binding protein(1, 2, 11, 12) , it is capable of activating transcription when tethered directly to a promoter(2, 12) . Interestingly, the local chromatin structure at SNF2/SWI2-responsive promoters is altered in yeast strains carrying mutations in the snf2/swi2 gene(13) , and the snf2/swi2- mutant phenotype is suppressed in yeast strains that carry alterations in the genes encoding histone H2A, H2B, H3, or other chromatin-associated proteins(13, 14, 15) . These studies suggest that SNF2/SWI2 might act to help overcome nucleosomal repression of gene expression, for example, by perturbing chromatin structure to allow access of regulatory factors to their DNA binding sites. Indeed purified fractions of the multisubunit protein complex that contains SNF2/SWI2 have been shown to disrupt nucleosomes in vitro and enhance binding of the chimeric GAL4/VP16 activator to DNA(16, 17) . Taken together, these findings suggest a model in which SNF2/SWI2 actively dissociates nucleosomes to facilitate binding of regulatory proteins to their control regions on the DNA.

Recently, a large family of proteins have been identified that are more distantly related to SNF2/SWI2 in sequence and function. These include Mot1, a negative regulator of basal transcription that is essential for yeast mitotic growth(18, 19) ; STH1, a protein of unknown function that is similarly essential for mitotic growth in yeast(20) ; lodestar, a protein that enters the nucleus in a cell cycle-dependent manner and is important for proper segregation of chromosomes in Drosophila(21) ; ERCC6, which mediates preferential repair of transcriptionally active genes and is disrupted in Cockayne's syndrome(22) ; CHD-1, a putative transcription factor that binds directly to DNA(23) ; and RAD5, RAD16, and RAD54(24, 25, 26) , which play a role in the repair of damaged DNA. These different SNF2/SWI2-related proteins are thus implicated in a wide variety of control processes, including gene expression, DNA repair, and control of chromosome segregation. The region most conserved among the different SNF2/SWI2-related proteins spans seven co-linearly arranged motifs that are characteristically found in ATPases and are distantly related to the ATPase domain of several poxvirus proteins(27) . A recombinant SNF2/SWI2 fusion protein has been shown to possess DNA-dependent ATPase activity in vitro(28) , which is important for both its effects on transcription in vivo(28) as well as for its ability to disrupt nucleosomes in vitro(16, 17) . By contrast, the yeast Mot1 protein behaves as a DNA-independent ATPase in vitro(19) . The mechanism of repression of basal transcription by Mot1 is not tightly coupled to chromatin structure, rather, the protein acts to displace the TATA-binding protein from RNA polymerase II promoters in an ATP-dependent manner and can repress transcription in vitro on naked promoter DNAs(19) . These two examples suggest that the different SNF2/SWI2-related proteins may affect cellular processes in distinct ways, through mechanisms that share in common a requirement for ATP hydrolysis.

By screening a HeLa expression library with sequences from the initiator region of the HIV-1 (^1)promoter, we have isolated a cDNA clone encoding a new member of the SNF2/SWI2 family of proteins. The encoded protein (called HIP116) was also found to bind specifically to the SPH (I + II) motifs of the SV40 enhancer. Both of these binding sites are known to be recognized by other transcription factors; e.g. the SPH repeats are bound by TEF-1 transcription factor in non-lymphoid cells(29) , and by the Oct proteins in B lymphocytes(30, 31) , similarly, this region of the HIV-1 promoter is recognized by LBP-1/CP2, TBP, and TFII-I (for reviews, see (32) and (33) ). Antiserum to HIP116 was used to identify the native HIP116 protein in HeLa nuclear extracts, and the protein was purified by DNA affinity chromatography. We show here that HIP116 binds independently of TEF-1 to the SPH repeats of the SV40 enhancer, and, similarly, that it binds independently of LBP-1 to HIV-1 promoter. In addition, we find that the HeLa HIP116 protein is a DNA-dependent ATPase and that this activity is strongly stimulated by SV40 enhancer DNA and can be inhibited with anti-HIP116 antiserum. These findings suggest that HIP116 could affect transcription by acting as a DNA binding site-specific ATPase.


EXPERIMENTAL PROCEDURES

Isolation of the HIP116 cDNA by Expression Screening

The HIP cDNA was isolated by screening an amplified gt-11 cDNA expression library according to the procedure described by Vinson et al.(34) . The library was derived from poly(A)-selected RNA prepared from phorbol-ester treated HeLa cells and contained 6 times 10^5 independent clones. The HIV-1 DNA oligodeoxynucleotides (5`-GCCTGTACTGGGTGGCCATGGTTAGACCAGATC-3` and 5`-TGGTCTAACCATGGCCACCCAGTACAGGCGATC-3`) were 5` end-labeled with [-P]ATP and T4 polynucleotide kinase, annealed, purified on native gels, and ligated prior to use as a probe for hybridization to the filters. The specific activity of the DNA probe was 2 times 10^8 cpm/µg, and hybridizations contained 8 times 10^6 cpm/filter (150-mm filter), each containing a total of approximately 20,000 plaques. The first cDNA clone that was isolated (called 1.3KB/HIP) was subsequently used as a probe to isolate longer cDNAs of 1.5 kb and 2.7 kb from a cDNA library prepared from Jurkat RNA (35) . All cDNAs were sequenced in their entirety from both DNA strands using the dideoxynucleotide sequencing method.

Expression of a trpE/HIP Fusion Protein in Escherichia coli

To obtain recombinant HIP protein for raising antisera, the entire 1.3KB/HIP cDNA was subcloned in frame to the E. colitrpE gene in a bacterial expression vector. Bacterial cell cultures (500 ml) were induced as described by Angel et al.(36) , pelleted at 6000 times g for 10 min, and suspended in 1.3 ml of buffer R: 40 mM Tris-HCl, pH 7.7, 25% (w/v) sucrose, and 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM sodium metabisulfite. The bacterial pellets were lysed by incubation with 0.5 mg of lysozyme at 4 °C for 1 h, and the extract was treated with volume of 10 M urea for 30 min at 37 °C to solubilize the fusion protein. The mixture was centrifuged at 63,000 times g for 1 h to pellet insoluble proteins, and the supernatant was dialyzed for 2 h against 200 ml of TM 0.1M buffer (50 mM Tris-HCl, pH 7.9, 1 mM EDTA, 1 mM DTT, 12.5 mM MgCl(2), 20% glycerol, 0.1 M KCl) containing 1 M urea and then overnight against TM 0.1M buffer without urea. The recombinant trpE/HIP protein was used to generate antisera (Fig. 4) and assayed for binding activity DNase I footprint experiments (Fig. 3).


Figure 4: Detection of HIP116 in HeLa nuclear extracts by immunoprecipitation and Western blot analysis. A, analysis of anti-HIP immunoprecipitates from extracts of [S]Met-labeled HeLa cells by SDS-PAGE and autoradiography. M, protein size markers; P, extracts incubated with control (preimmune) serum; I, extracts incubated with anti-HIP116 antiserum; IB, extracts in which the immunoprecipitation reactions were blocked by incubation with an excess of recombinant unlabeled HIP116 prior to addition of anti-HIP116 antiserum. The arrow indicates the 116-kDa protein specifically detected in this assay. B, proteins immunoprecipitated from HeLa nuclear extracts treated with preimmune (P) or immune (I) serum were analyzed by Western blotting with anti-HIP116 antiserum. The 116-kDa protein is indicated with an arrow. C, Southwestern blot analysis of protein immunoprecipitated from HeLa nuclear extracts treated with preimmune (P) or anti-HIP116 immune (I) serum. The blot was incubated with labeled HIV-1 promoter DNA sequences spanning the HIP116 binding site (-30 to +4).




Figure 3: Binding of recombinant HIP116 and AP-1 proteins to the SV40 enhancer as analyzed by DNase I footprint experiments. A, binding of recombinant HIP116 to the HIV-1 promoter is shown in the panel on the left. Reactions containing trpE/HIP116 or LBP-1 are indicated above each lane, and the DNA sequence in the TATA/initiator region of the HIV-1 promoter bound by HIP116 (-33 to +3) is shown to the right of the gel. DNA sequence markers are shown in lane G; control bacterial cell extract (uninduced) was added to the reaction in lane marked None; 100 ng of purified HeLa LBP-1 protein was added to lane marked LBP-1; and 10 and 25 µg of bacterial extracted expressing trpE/HIP were added to lanes 3 and 4, respectively, which are marked rHIP. B, binding of recombinant HIP116 to the 72-bp repeats of the SV40 enhancer. Reactions in lanes 1 and 4 contained 25 µg of control (uninduced) bacterial cell extract, and the other reactions included 25 µg of bacterial extract containing the trpE/HIP116 fusion protein (lane 2) or 25 µg of bacterial extract containing a trpE/c-jun fusion protein, which binds to the P motifs of the SV40 enhancer. The HIP116 binding sites on the SV40 enhancer are indicated with brackets, and a single binding site sequence is shown alongside the footprint.



Generation of Rabbit Polyclonal Antiserum

To generate antibodies specific to HIP116, the recombinant trpE/HIP fusion protein was isolated as a gel slice from a preparative SDS-PAGE, emulsified into Freund's adjuvant, and injected into a New Zealand White rabbit. Initial injections contained 200 and 100 µg of trpE/HIP, respectively, followed by subsequent injections of 25-50 µg of soluble trpE/HIP fusion protein, which had been eluted from a gel into Laemmli buffer, denatured in 6 M guanidine HCl, and renatured by exhaustive dialysis into TM 0.1M and then 1 times phosphate buffer saline solution. Preimmune serum obtained before the first injection served as a control for the specificity of the antisera. To purify the antibodies prior to their use in gel shift experiments, a portion of the crude antisera was diluted with an equal volume of 20 mM sodium phosphate, pH 7, and centrifuged at 35,000 times g to remove insoluble protein. The samples were filtered and passed over a protein G-Superose column (HR 16/5, Pharmacia Biotech Inc.) equilibrated in 20 mM sodium phosphate buffer, pH 7, and the bound IgG was eluted with 100 mM glycine HCl buffer, pH 2.7, and subsequently adjusted to neutral pH. The purified antiserum was required for gel shift and immunoprecipitation assays, whereas crude or IgG-purified antisera were found to be equally useful for Western immunoblot experiments.

Preparation of [S]Methionine-labeled HeLa Cell Extracts and Immunoprecipitations

To identify HIP116 protein in extracts from HeLa cells, a 100-mm plate of exponentially growing HeLa-TK(-) cells (1-2 times 10^7 cells) was washed with Tris-buffered saline and incubated at 37 °C in serum-free medium (Dulbecco's modified Eagle's medium) for 15 min. 70 µl of [S]methionine (TranS-label, ICN, 12 µCi/µl) was added to the media, and the cells were incubated for 2 h. The cells were then washed twice with Tris-buffered saline solution and incubated for 10 min on ice in 1 ml of radioimmune precipitation buffer (10 mM sodium phosphate, 100 mM NaCl, 0.1% SDS, 1% deoxycholate, 0.01% aprotonin, 50 µM leupeptin, pH 7.5). The cells were then passed through a 20-gauge needle several times, mixed with 100 µl of Pansorbin cells (Calbiochem), and centrifuged at 12,000 times g for 30 min at 4 °C. The supernatant was adjusted to a volume of 1.5 ml, and a 500-µl aliquot was incubated with 3 µl of the crude preimmune or immune HIP116 antibodies for 2 h at 4 °C. For the blocking reaction, 1 µg of purified recombinant trpE/HIP protein was preincubated with antibody prior to the immunoprecipitation step. 40 µl of protein A beads (Pharmacia; beads were used as a 1:1 suspension with radioimmune precipitation buffer) was added and the protein/antibody/beads slurry was rotated at 4 °C for 1 h. The beads were pelleted at 12,000 times g for 1 min at 4 °C, and the supernatant was removed using a 18-gauge needle. The beads were washed with radioimmune precipitation buffer, and the pelleted beads were resuspended in sample buffer (120 mM Tris, 1% SDS, 500 mM beta-mercaptoethanol, 10% glycerol, 0.01% bromphenol blue), boiled for 3 min, and loaded directly onto an SDS-PAGE.

SDS-PAGE and Southwestern Blotting Experiments

For Western blot experiments, the proteins present in immunoprecipitates from HeLa nuclear extracts were treated with either preimmune or immune sera, resuspended and boiled in SDS sample buffer, and analyzed on an 8% gel (SDS-PAGE). The gels were transferred to nitrocellulose (Schleicher and Schuell, 0.45-µm filters) using a semi-dry electrophoretic transfer apparatus operating at 1 mA/cm^2 for 90 min. Nonspecific sites on the nitrocellulose were blocked with wash buffer (20 mM Tris, 500 mM NaCl, 0.1% Tween 20, pH 7.5) containing 9% powdered milk, for 60 min at room temperature. The nitrocellulose was then incubated overnight at 4 °C with a solution containing a 1:500 dilution of crude HIP116 antibodies in wash buffer plus 5% milk, washed, and then incubated for 2 h at room temperature with a 1:1000 dilution of horseradish-peroxidase conjugated goat anti-rabbit IgG (heavy and light chain specific, Cappel). The nitrocellulose filter was washed, equilibrated in substrate buffer (20 mM Tris, 500 mM NaCl, pH 7.5), and the immunoreactive bands were visualized by incubation with substrate buffer containing 0.5 mg/ml 4-chloro-1-napthol in 15% (v/v) methanol and 0.025% (v/v) H(2)O(2).

For SouthWestern blots, the transferred proteins were denatured by incubation of the filter in Z` buffer (25 mM HEPES, 12.5 mM MgCl(2), 0.1 mM EDTA, 0.1% Nonidet P-40, 2 mM DTT, 0.1 mM PMSF, 10% glycerol, pH 7.8) containing 50 mM KCl and 6 M guanidine HCl, for 5 min at room temperature. The nitrocellulose filter was then incubated with Z` 0.05M buffer to renature the proteins, followed by incubation with Z` 0.05M buffer containing 5% milk at room temperature for 5 min. The filter was washed with Z` 0.05M and placed in hybridization solution (Z` 0.05M, 2 µg/ml salmon sperm DNA, 3 times 10^6 cpm/ml of labeled DNA) containing the [P] end-labeled HIV-1 DNA, and incubated overnight at 4 °C. The filter was then washed with an excess of Z` 0.05M buffer at room temperature prior to autoradiography.

Purification of HIP116 from HeLa Cell Nuclear Extracts

Nuclear extracts prepared from 72 liters (4 times 10 cells) of HeLa S-3 spinner cells were pooled, desalted on an 800-ml P-10 column (medium grade, Bio-Rad), and fractionated over a 20-ml phosphocellulose (Whatman; P-11) column in HEPES buffer (25 mM HEPES, pH 8, 10% glycerol, 2 mM DTT) containing 100 mM KCl (H 0.1 M buffer). The column was washed with H 0.1M buffer and eluted with a salt gradient from 100 to 600 mM KCl followed by a 1 M step fraction. 1.5-ml fractions were collected and assayed directly for DNA binding activity in gel mobility shift experiments. Active fractions were pooled and dialyzed into TM 0.1M buffer (50 mM Tris-HCl, pH 7.9, 12.5 mM MgCl(2), 1 mM EDTA, 20% glycerol, 2 mM DTT, 100 mM KCl) and passed over an 8-ml heparin-agarose column. The column washed with 2 column volumes of Z` 0.1M (25 mM HEPES, pH 7.8, 12.5 mM MgCl(2), 1% Nonidet P-40, 10% glycerol, 2 mM DTT), and bound proteins were eluted with a 100-600 mM KCl gradient followed by a Z` 1.0M step fraction. The fractions were assayed for DNA binding activity or for HIP116 protein directly by gel shift and Western immunoblot assays, respectively.

Fractions containing HIP116 activity were pooled, dialyzed into Z` 0.1M buffer, and passed over DNA affinity resins containing multimerized copies of either the SV40 SPH DNA (used for the first pass) or the HIV-1 initiator DNA (used on the second and third pass DNA affinity columns). The columns were washed with Z` 0.1M, and bound proteins were eluted with four successive steps of Z` 1.0M buffer. Fractions were assayed for HIP116 protein by SDS-PAGE, DNA binding activity, and Western blot experiments. For DNA affinity chromatography, the HIP116 fraction was incubated for 10 min on ice (with 15 and 2 µg of poly(dI-dC), for first and subsequent affinity column runs, respectively) before loading on the resins. In some cases, carbonic anhydrase was added (final concentration of 0.5 µg/µl) to the affinity-purified HIP116 fractions to stabilize its activity, although it was subsequently determined that this was not required. All column buffers included the following protease inhibitors: PMSF (0.1 mM), benzamidine (2 mg/ml), pepstatin A (1 µg/ml), leupeptin (4 µg/ml), aprotonin (10 µg/ml), and soybean trypsin inhibitor (20 µg/ml).

Expression and Purification of His(6)/HIP from Recombinant Baculovirus-infected Sf9 Cells

The expression construct His(6)/HIP, containing a histidine tag at the amino terminus of the full-length HIP116 protein, was constructed by isolating the Asp718-HindIII fragment from the vector pGEMEX/3.2KBHIP, blunting the fragment by treatment with Klenow enzyme, and inserting it into a blunted NcoI site and the HindIII site of the baculovirus recombination vector pBlueBacHis-C (Invitrogen). The recombinant baculovirus, called His(6)/HIP, was isolated by co-transfection of Sf9 cells with linearized wild type AcMNPV and the His(6)/HIP construct using the cationic liposome transfection method (Invitrogen) and plaque-purified through three rounds. The recombinant protein His/HIP116 protein was isolated from a whole cell extract of Sf9 insect cells infected with the recombinant baculovirus for 3 days at 27 °C. The recombinant protein was purified using the Ni-NTA-agarose (Qiagen) without addition of DTT. Specifically, 40 ml of whole cell extract was isolated from 10^9 infected cells and passed over a 0.5-ml Ni column. The column was washed sequentially with buffer D (10 mM HEPES, pH 7.8, 5 mM MgCl(2), 0.1 mM EDTA, 50 mM NaCl, 5 mM beta-mercaptoethanol, 17% glycerol, 0.1 mM PMSF, 10 mM sodium fluoride) containing 0.8 and 8 mM imidizole and eluted with successive steps of buffer D containing 40, 80, or 300 mM imidazole. The purity of the His(6)/HIP protein was determined by SDS-PAGE with silver staining and immunoblot assays, and purified fractions were concentrated by binding to a 0.5-ml heparin-agarose column, eluted with TM 0.4M and dialyzed into TM 0.1M buffer before use in gel shift assays (Fig. 8).


Figure 8: Binding of HIP116 and TEF-1 to the SV40 enhancer SPH motifs. A, binding of HIP116 protein fractions from the second DNA affinity column (In, input or first pass fraction; Ft, flow-through; W1, first wash; W2, second wash; E1, first elution; E2, second elution; see Fig. 7B) to SV40 SPH DNA (lanes 1-12) or to the SV40 GT-IIC site (lanes 13 and 14) in gel mobility shift assays. HIP116 antisera was added to lanes 8, 10, 12, and 14. B, binding of TEF-1 fractions to wild type and mutant SV40 SPH DNAs. C, analysis of the binding of recombinant HIP116 protein purified from baculovirus-infected Sf9 cells to wild type and mutant SV40 SPH DNAs. In B and C, reactions containing HIP116 antisera (alphabeta) are indicated above the appropriate lanes.




Figure 7: Purification of HIP116 from HeLa cells and from recombinant baculovirus-infected Sf9 cells. A, SDS-PAGE analysis of HeLa nuclear proteins eluted from the first pass SV40 SPH DNA affinity resin, visualized by silver staining. M(r), protein molecular weight standards; I, input to the column; F, flow-through fraction; W, wash fraction; E1-E4, sequential step elutions from the first pass (SV40) DNA affinity column at 1.0 M KCl; Bc, recombinant His/HIP116 protein purified from baculovirus-infected cells by Ni affinity chromatography. Arrows indicate the HIP116 and TEF-1 proteins. B, the first pass column elutions (E1-E4) were pooled and applied to a second pass DNA affinity resin on HIV-1 sequence resin (lane I, input fraction; lane F, flow-through fraction; lane W1, first wash; W2, second wash; E, 1.0 M KCl step elution). Western blot analysis of HIP116 protein in the various protein fractions (C, lane I, input to the second pass HIV-1 DNA affinity column; lane F, flow-through fraction; lanes E1 and E2, sequential elutions of second pass purified HIP116; lane Bc, purified recombinant His/HIP116).



Gel Mobility Shift Assays

Binding reactions (12 µl total volume) contained 30 pg of P-end-labeled DNA (1.5 times 10^4 cpm/reaction; 7.5 times 10^8 cpm/µg) mixed with 1 µg of poly(dI-dC) in binding buffer: 25 mM Tris-HCl, pH 7.9, 6.25 mM MgCl(2), 0.5 mM EDTA, 1 mM DTT, 5% glycerol. The reactions were prepared on ice, and 4-µg aliquots of the individual phosphocellulose column fractions were added. The reactions were incubated on ice for 20 min, mixed with 3 µl of dye buffer (10 mM HEPES, 0.01% bromphenol blue, 50% glycerol), and loaded directly onto a 4 or 7% nondenaturing polyacrylamide gels containing 0.5 times TBE, 0.05% Nonidet P-40, and 5% glycerol. The gels were run at 14 V/cm at room temperature for 90 min. DNA affinity-purified fractions of HIP116 or TEF-1 contained 25-50 ng of protein, and no competitor DNA was added. For the supershift experiments with the various antibodies, the binding reactions were incubated for 10 min on ice prior to adding 2 µl (0.1 µg) of HIP116 or control hLEF antiserum. The reactions were incubated for 20 min on ice before loading on the gel. For the DNA competition experiments, the unlabeled competitor DNAs were added to the reaction mix before the protein. Sequences of the competitor DNA sites were: HIV-1, 5`-GATCCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTC-3`; SV40 SPH (I + II) motifs, 5`-GATCAGAAGTATGCAAAGCATGCATCTC-3`; OCT(-), 5`-GATCAGAAGTATTCAAAGCATTATCTC-3`; dpm 2, 5`-GATCAGAAGTATGCAAAGCAAGGATCTC-3`; 0-Sph5, 5`-GATCAGCCTTATGCAAAGCATTACTCTC-3`.

ATPase Assays

The standard 30-µl reaction contained 50-100 µg of DNA affinity-purified HIP116 in Z` 0.1M buffer containing 50 µM ATP, 12.5 µl [-P]ATP, and, where indicated, 1 µg of SV40 SPH DNA. Reactions were preincubated with or without DNA for 5 min on ice followed by the addition of [-P]ATP and were incubated at 37 °C for 45 min. Nonhydrolyzed ATP was removed by adding 500 µl of 7% activated charcoal (in 50 mM HCl, 250 mM Na(2)HPO(4)) and centrifugation at 15,000 times g for 15 min. The supernatant was transferred to a fresh tube and centrifuged again, and a 10-µl aliquot was used for Cerenkov counting. In cases where antibody inhibition was assayed, antibodies (0.5-30 µg of IgG-purified antisera specific to HIP116 or the control hLEF protein was used, as indicated in the figure legend) were incubated with the HIP116 protein fraction for 10 min on ice prior to the addition of DNA.


RESULTS

The HIP116 cDNA Clone Encodes a SNF2/SWI2-related Protein Containing a RING Finger

The original HIP116 cDNA clone was isolated by screening a gt-11 HeLa cDNA expression library (36) with a labeled, multimerized DNA probe containing HIV-1 (ARV-2) promoter sequences from -10 to +23 (derived from pLTR/CAT and pLTR/CAT+4/+8; (37) ). This region of the HIV-1 promoter includes the RNA initiation site and the DNA-binding site for the LBP-1 transcription factor(37) , but lacks the HIV-1 TATA box. Following a screen of 8.5 times 10^5 plaques, three recombinant phage were identified that bound specifically to the HIV-1 oligonucleotide probe and not to a control probe containing multimerized AP-1/c-jun DNA-binding sites. Restriction mapping and partial sequence analysis of the phage inserts revealed that the three cDNA clones contained overlapping sequences derived from the same gene. Sequence analysis of the largest cDNA clone (called 1.3KB/HIP) revealed the presence of a partial cDNA that contained a single long open reading frame (ORF) and lacked both initiator and stop codons. The insert from the 1.3KB/HIP clone was then used as a probe to isolate two longer overlapping cDNAs from a Jurkat cDNA library(35) , which provided the remainder of the ORF as well as a significant portion of both 5`- and 3`-untranslated leader sequences. The sequence of the complete open reading frame and flanking untranslated region, a total of 3295 bp, was determined in its entirety on both DNA strands by dideoxynucleotide sequencing and is presented in Fig. 1A.



Figure 1: Sequence analysis of the HIP116 cDNA. A, sequence of the full-length cDNA clone and the predicted human HIP116 protein. Numbers to the right of the sequence refer to amino acid numbers, starting from the first possible initiator methionine codon. The longest cDNA we isolated contained 172 bp of 5`-untranslated leader sequence. The RING finger (C(3)HC(4)) domain is boxed. B, homology between HIP116 and other proteins: MOT1(18) , SNF2/SWI2(11) , STH1 (20) , BRM(7) ,RAD5(25) , RAD16(26) . For brevity, homology with other SNF2/SWI2-related proteins, including the human SNF2/SWI2 homologues (4, 5, 6) , the Drosophila lodestar protein(21) , the yeast RAD54 protein(24) , the human ERCC6 protein(22) , and the human CHD-1 protein (23) is not shown. Amino acids are grouped by chemical similarity (G/A, S/T, D/E, K/R, I/L/V/M, F/Y/I). Dashes and asterisks in the sequence indicate sequence spacing changes that were introduced to maintain optimum alignment between the sequences (the asterisk indicate position of omitted amino acids).



Inspection of the HIP116 coding sequence reveals two potential initiator methionine codons at the beginning of the open reading frame (amino acids 1 and aa 4) that conform to the Kozak consensus sequence (38) . Assuming that the first AUG in this ORF initiates translation, the encoded protein would contain 1009 amino acids (predicted molecular mass of 114 kDa) and possess notably high levels of both basic (16.4%) and acidic (12.3%) amino acids, as well as a large number of Ser/Thr residues (12.7%). A computer search of recent protein data bases (SWISS-PROT 28.0, PIR 40.0, GenBank 83.0) using BLASTP and FASTA programs revealed that the predicted HIP116 protein contains striking regions of homology to the yeast RAD5 and RAD16 proteins, as well as to all the other members of the SNF2/SWI2-related protein family (Fig. 1B). The primary regions of homology among these proteins have been noted previously by Davis et al.(18) to include seven consecutive motifs (marked I through VI) that are characteristic of ATPases and DNA helicases. These include the domain I motif, GLGKT, which conforms to the Walker A-box of the nucleotide-binding site, and the domain II motif (Walker B-box sequence), DEGH, which forms part of the Mg-binding pocket and is a variant of the DEAD box (39) . The data base searches also revealed significant homology to the vaccinia NTP1 and NTP2 proteins, indicating that HIP116, like the other SNF2/SWI2-related proteins, may belong to this larger superfamily of ATPases(27) .

In addition to the homology to SNF2/SWI2, the HIP116 ORF contains a Cys/His-rich sequence of the C(3)HC(4) type (RING finger; (40) and (41) ), which precisely matches the consensus established for RING fingers: C(X)(2)C(X)CXHXFC(X)(2)C(X)CPXC. The RING finger motif has been identified in a large and diverse family of regulatory proteins that includes transcription factors, proto-oncogenes, and proteins that affect DNA recombination or repair. The HIP116 RING finger is most closely related in sequence to the RING finger of the yeast RAD18 protein, particularly in the first loop sequence (Fig. 2A; (42) ). Overall, however, the structure of HIP116 is more closely related to the yeast RAD5 (25) and RAD16 (26) proteins, since these two yeast DNA repair proteins each contain a single RING finger motif domain inserted within the ATPase domain. We also noted a possible nuclear localization signal (RPKRRKT; amino acids 384-390) with a nearby potential CKII phosphorylation site (Ser; ESSDSEEIETSE) in the central region of the HIP116 ORF. CKII phosphorylation sites are commonly found next to NLS elements and can enhance the rate of nuclear transport(43) .


Figure 2: Domain structure of HIP116 and Northern blot analysis of HIP116 mRNA. A, comparison of the HIP116 RING finger with that of other RING finger proteins(40, 41) . Dashes in the sequence refer to spacings introduced to maintain the sequence alignment, and asterisks internal to the sequence indicate amino acid residues in the putative loops of the zinc finger structure that were omitted. RING finger proteins that also contain SNF2/SWI2-related putative ATPase domains are indicated with an asterisk, and a different subgroup of RING finger proteins that possess additional Cys/His-rich domains and a coiled-coil domain just downstream of the RING finger (41) are marked with a cross. B, apparent domain structure of the human HIP116 protein. The relative location of the seven SNF2/SWI2-related domains, the RING finger motif, and the DNA-binding domain are indicated. Numbers in parentheses refer to amino acid positions. C, Northern blot analysis of HIP116 mRNA in poly(A)-selected RNA from HeLa S3 and Jurkat (T) cell lines, as indicated above each lane. Arrows depict the 4.6- and 5.6-kb HIP116 mRNAs.



To examine the expression of HIP116 mRNA in different cell types, Northern blot experiments were carried out using RNA derived from several mammalian cell lines. Two HIP116 mRNA species of 5.6 and 4.6 kb were detected in HeLa (cervical carcinoma) and Jurkat (T cell) lines (Fig. 2C), and low levels of HIP116 mRNA were also present in F9 and HEPG2 cell lines. (^2)In contrast, HIP116 expression was not observed in either of two different mature B cell lines (Namalwa and JY cells). These data suggest that HIP116 is not ubiquitously expressed, although further experiments will be required to better define its pattern of expression in different tissues as we were unable to detect the HIP116 mRNAs from murine tissue using the human cDNA as a probe. Southern blot and in situ chromosomal hybridization experiments revealed that HIP116 is a single-copy gene, and its chromosomal location was mapped to human chromosome 3q25.1-3q26 by somatic cell hybrid analysis and in situ hybridization(44) .

Specific Binding of Recombinant HIP116 to the HIV-1 Promoter and the SV40 Enhancer

To analyze the DNA binding properties of HIP116, sequences encoded by the 1.3KB/HIP clone (HIP amino acids 38-453) were fused in frame to the E. coli trpE gene, and the DNA binding activity of the trpE/HIP fusion protein was examined in DNase I footprint experiments (Fig. 3). Weak but specific binding of trpE/HIP was observed over the HIV-1 RNA start site and the TATA-box (-25 to +2), in a region of the promoter that directly overlaps the LBP-1/CP2 transcription factor binding site (Fig. 3A). An apparently stronger binding site was observed over the 72-bp repeats of the SV40 enhancer (Fig. 3B; nt 111-148 and nt 183-221). This latter site spans a 38-bp region of each of the 72-bp repeats and overlaps with both the P and SPH (I + II) motifs, including the AP-1/c-jun binding site in the P motif (Fig. 3B). Binding of the trpE/HIP protein generated a strong hypersensitive site within the SV40 enhancer footprint, and the region between the two binding sites showed altered susceptibility to DNase I cleavage. Identical DNase I footprints were obtained with a different fusion protein in which the 1.3KB/HIP sequences were fused to the beta-galactosidase gene (data not shown).

These findings indicate that the HIP116 DNA-binding domain is located in the amino-terminal third of the HIP116 cDNA clone, which does not include the RING finger. The DNA-binding domain was further mapped by testing the DNA-binding activity in gel shift assays of a panel of COOH-terminal truncation mutants of HIP116 that were expressed in vitro. The results indicated that the region from amino acids 38 to 285 near the amino terminus of HIP116 encompasses a minimal domain that is both necessary and sufficient for specific binding of HIP116 to DNA in vitro (data not shown). The location of this DNA-binding domain relative to the RING finger and the SNF2/SWI2-like ATPase motifs is shown schematically in Fig. 2B.

HIP116 Binds Independently of TEF-1 to the SV40 Enhancer and Independently of LBP-1 to the HIV-1 Promoter

To detect the native HIP116 protein in HeLa nuclear extracts, polyclonal antibodies were raised against purified recombinant trpE/HIP protein for Western blots and gel mobility shift experiments. Immunoprecipitates from nuclear extracts of S metabolically labeled HeLa cells treated with polyclonal anti-HIP antiserum contained a 116-kDa protein (Fig. 4A, lane I, arrow) which was not precipitated from extracts incubated with the preimmune serum (Fig. 4A, lane P). Recovery of the 116-kDa protein was selectively blocked by preincubation of the anti-HIP immune serum with an excess of the purified trpE/HIP fusion protein (Fig. 4A, lane IB), indicating that the protein is HIP116. The 116-kDa protein was also identified by Western blot analysis of proteins immunoprecipitated from HeLa nuclear extracts with the anti-HIP antiserum (Fig. 4B, arrow), was not seen in immunoprecipitates of the control preimmune serum (Fig. 4B, lane P), and was capable of binding to the HIV-1 promoter DNA in a SouthWestern blot (Fig. 4C, arrow). Thus HIP116 is present in HeLa nuclear extracts, and its size corresponds to that predicted from the full-length cDNA sequence.

To assess the ability of the native HIP116 protein to bind to the SV40 enhancer, HeLa nuclear extracts were fractionated by phosphocellulose column chromatography and protein-DNA complexes formed with SPH motif DNA were analyzed by gel mobility shift experiments. As seen in Fig. 5A (top panel), two predominant complexes were observed with SPH motif DNA. The complex eluting in the flow-through (FT) fraction contained Oct-1 (data not shown), whereas the complex that eluted later in the gradient (fractions 31-47; Fig. 5A) contained HIP116 as it could be specifically disrupted with anti-HIP antibodies (see Fig. 6A, lane 2). The effects of the antiserum were specific for HIP116, since neither Oct-1 nor a nonspecific DNA-binding protein present in these fractions (labeled NS) was affected by the HIP116 antiserum (see Fig. 6B, lane 2). We did not observe a complex on the SPH repeats that contains TEF-1 in the phosphocellulose column fractions, and the HIP116-SPH DNA complex did not contain TEF-1, since it could not be competed with the high-affinity TEF-1 binding site from the SV40 GT-IIC motif and was not affected by antiserum to TEF-1 (data not shown). To locate the elution position of TEF-1 in the gradient, individual fractions were tested for binding to the SV40 GT-IIC motif. As shown in the lower panel of Fig. 5A, TEF-1 activity was detected in fractions 27-39, a region that only partially overlaps with HIP116. This complex was affected by antiserum to TEF-1 (data not shown). As expected, binding of TEF-1 to the GT-IIC motif was not affected by the HIP116 antiserum (see Fig. 6C). Since we did not detect significant binding of TEF-1 to the SV40 SPH motifs at this stage of fractionation, it appears that TEF-1 is either considerably less abundant than HIP116 or that it binds with a lower affinity than HIP116 to the SV40 SPH repeats.


Figure 5: Binding of native HeLa HIP116 to the SPH and GT-IIC motifs of the SV40 enhancer and to the HIV-1 promoter. A, HeLa nuclear extracts were fractionated by phosphocellulose column chromatography and monitored for proteins that bind to the SPH repeats (top panel) or the GT-IIC motif (bottom panel) of the SV40 enhancer in gel mobility shift assays. Numbers above each lane refer to phosphocellulose column fraction numbers; IN, input to the column; FT, flow-through from the column. Specific HIP116 and TEF-1 protein-DNA complexes are indicated with arrowheads; NS refers to a nonspecific DNA-binding protein. B, binding of phosphocellulose column fractions to the TATA/initiator region of the HIV-1 promoter (-30 to +4; lanes 1-15). The different salt concentrations of a two-step gradient are indicated above the panel. HIP116 binding activity is seen in lanes 7-11; LBP-1 is detected in lanes 12-15; other complexes were not characterized. Peak HIP116 column fractions were tested for binding to the SPH oligonucleotide (lanes 16 and 17); the HIV-1 -30 to +4 oligonucleotide (lanes 18 and 19) or an oligonucleotide spanning the -6 to +20 region of the HIV-1 promoter (lanes 20 and 21). HIP116 antiserum was added to reactions 17, 19, and 21, as indicated above each lane.




Figure 6: Binding of HIP116, TEF-1, and OCT-1 to wild type and mutant SV40 SPH DNAs. A, binding reactions contained 4 µg of phosphocellulose fraction 35 (see Fig. 5) and wild type or mutant SV40 SPH DNA probes as indicated above lanes 1-5; lane 6 contained the SV40 GT-IIC DNA probe. Polyclonal antiserum raised against HIP116 was added to lane 2. B, binding reactions contained approximately 4 mg of the flow-through (FT) fraction from the phosphocellulose column and the same DNA probes as indicated in part A. Lane 2 included the anti-HIP polyclonal antiserum. C, binding reactions contained 4 µg of protein from phosphocellulose fraction 37 and the SV40 GT-IIC DNA probe. Lane 2 included HIP116 antiserum. The wild type and mutant SV40 SPH motif sequences are indicated below the panels; the dpm2 mutation has been shown by Herr and Clarke (46) to inhibit SPH motif activity in vivo, and the 0-Sph5 mutation also inhibits SPH activity in vivo, as well as binding of TEF-1 in vitro(45) .



The native HIP116 protein was also found to bind to a region of the HIV-1 promoter spanning the TATA box and initiator region from -30 to +4 (Fig. 5B). A modified gradient was used on this phosphocellulose column to resolve HIP116 (lanes 7-11) from LBP-1 (lanes 12-15), which also binds to this region of the HIV-1 promoter. The peak HIP116 fraction bound both to the SPH oligonucleotide (lane 16) as well as to HIV-1 DNA oligonucleotides from either the TATA/initiator region (-30 to +4; lane 18) or from the proximal downstream region (-6 to +20; lane 20). Antiserum to HIP116 inhibited binding in each case (lanes 17, 19, and 21) and did not affect the binding of LBP-1 to the promoter. Mutation of the -6 to +20 oligodeoxynucleotide to the triple mutation described by Jones et al.(37) was also found to inhibit binding of HIP116 (data not shown). Therefore the native HIP116 protein binds specifically to the same general region of the HIV-1 promoter as LBP-1 and TBP, the TATA-binding protein.

The DNA Binding Specificity of HIP116 Correlates with SPH Motif Activity in Vivo

To assess the DNA contacts for HIP116 on the SPH repeats, we examined the ability of HIP116 to recognize specific mutations in the SPH repeats. Binding of HIP116 was found to be destroyed by two different mutations that affect the SPH repeats (Fig. 6A; mutants dpm2 and 0-Sph5) and was not affected by a mutation of the overlapping octamer element (OCT-DNA; Fig. 6A). By contrast, the nonspecific DNA-binding protein present in these fractions (NS) bound equally well to both wild type and mutant DNAs. Both the 0-Sph5 and dpm2 mutations have been shown to inactivate SPH enhancer activity in vivo(45, 46) , and the latter mutant has also been shown to prevent trans-activation of the SPH motifs by SV40 large T antigen(47) . We conclude that HIP116 binds avidly to the SPH motifs in nuclear extracts and that its binding specificity correlates well with SPH enhancer activity in vivo. As expected, binding of the Oct-1 protein in the flow-through fraction was not affected by the SPH repeat mutants but was destroyed the OCT(-) DNA mutant. (Fig. 6B) Thus the predominant complexes formed in HeLa nuclear extracts with this region of the SV40 enhancer contain Oct-1 bound to the octamer element and HIP116 bound specifically to the SPH repeats.

Purification of HIP116 from HeLa Nuclear Extracts

We next purified the native HIP116 protein from HeLa nuclear extracts to better characterize its DNA binding and ATPase properties. The pooled phosphocellulose fractions were purified by heparin-agarose chromatography and applied to DNA affinity columns containing the SV40 enhancer SPH motif sequence. The input fractions contained both TEF-1 and HIP116, as determined by binding to the GT-IIC and SPH motif DNAs, respectively. Protein fractions from the affinity column were analyzed by silver stain (Fig. 7A). HIP116 was detected in all four eluate fractions from the SPH DNA affinity column (E1-E4; Fig. 7A), whereas the TEF-1 protein and DNA-binding activity was observed principally in the first two elution fractions (E1 and E2). To separate HIP116 from TEF-1, the E1-E4 fractions were pooled and applied to an HIV-1 DNA affinity resin, which does not bind TEF-1. Both HIP116 and TEF-1 proteins were visible in the input fraction by silver staining and Western blot (Fig. 7B, arrows), and the identity of the TEF-1 band was further confirmed by Southwestern blot analysis using SV40 SPH DNA (data not shown). Upon passage of the first pass affinity column fraction over the HIV-1 affinity resin, TEF-1 was found in the flow-through fraction (Fig. 7B, F), whereas HIP116 bound to the column and eluted in a high salt step (Fig. 7B, E). The presence of HIP116 in the eluant fraction was confirmed by Western blot analysis of these fractions (Fig. 7C). The recovery and yield of HIP116 protein from HeLa nuclear extracts is summarized in Table 1. HIP116 appears to be relatively not abundant in HeLa nuclear extracts as only 12 µg of purified protein was recovered from 25 ml of nuclear extract (derived from approximately 10 HeLa cells). Thus although affinity chromatography using the SPH motif enriched for both TEF-1 and HIP116, these two proteins could subsequently be separated by a second pass on the HIV-1 DNA resin, to which HIP116 but not TEF-1 could bind.



The binding properties of the purified HIP116 and TEF-1 proteins were then analyzed by gel shift experiments, shown in Fig. 8A. The first pass fraction from the SPH motif resin was highly enriched for both HIP116 and TEF-1 activity and for the first time revealed evidence of the two complexes that form upon binding of TEF-1 to the SPH motifs (Fig. 7A, E1-E4). The flow-through and wash (W1) fractions from the second pass HIV-1 affinity resin consist principally of TEF-1 protein (Fig. 7B, lanes Ft and W1) and clearly displayed the ``A'' and ``B'' complexes previously ascribed to TEF-1(30) . As expected, TEF-1 in these Ft and W fractions bound strongly to the SV40 GT-IIC motif (Fig. 7B, lanes 13 and 14). The flow-through and wash fractions also contained a small amount of HIP116 protein, which was affected by polyclonal antisera to HIP116, whereas the HIP antibodies did not affect the A and B complexes formed by TEF-1 (Fig. 8A, lanes 7-10). By contrast, the eluant fractions (E1 and E2 from the second pass column contained HIP116 and lacked TEF-1 (Fig. 8A, lanes 11 and 12). Importantly, neither the migration position of the HIP116 protein-DNA complex nor its DNA binding specificity changed upon the purification of HIP116 and its separation from TEF-1 (Fig. 8A; other binding data not shown). Analysis of the binding of TEF-1 to SV40 enhancer mutants revealed that the dpm2 mutation principally disrupts formation of the TEF-A complex, in which TEF-1 is bound to each of the SPH repeats(45) , whereas the 0-Sph5 mutation, which contains alterations in both repeats, affected the formation of both the A and the B complexes (Fig. 8B). By contrast, binding of HIP116 to the SPH motifs was effectively eliminated with either the dpm2 or the 0-Sph5 mutations (Fig. 6A). Consequently, the HIP116 and TEF-1 proteins form similar, but not identical, complexes with the SPH region of the enhancer and binding of both proteins is affected by specific mutations in the SPH repeats that destroy enhancer activity in vivo.

To confirm the binding specificity of HIP116, the full-length HIP116 cDNA was subcloned into a baculovirus expression vector and expressed as a fusion protein with a histidine tag at the amino terminus. The recombinant His/HIP116 protein was partially purified from baculovirus-infected cells using Ni affinity chromatography (Fig. 7A). As expected, the recombinant His/HIP116 protein (labeled Bc) is a slightly larger protein than the native HIP116 and migrates slightly above the native HeLa HIP116 protein in SDS-PAGE (Fig. 7A) and Western blots (Fig. 7C). The mobility and binding specificity of recombinant His/HIP116 was identical to that of the native HIP116 protein (Fig. 8C). Taken together, these results confirm that HIP116 binds directly and specifically to the SV40 SPH motifs in a manner that is independent of TEF-1.

DNA Affinity-purified HIP116 Fractions Possess DNA-dependent ATPase Activity

The observation that a SNF2/SWI2-related protein is targeted specifically to a functional domain of the SV40 enhancer raises the question as to whether HIP116 might affect the structure of the DNA or the access or regulatory proteins to the enhancer by functioning as an ATPase. To begin to address this question, DNA affinity-purified fractions of HIP116 were tested for ATPase activity in vitro. First pass affinity-purified HIP116 fractions did not display appreciable ATPase in the absence of DNA (Fig. 9A, HIP), whereas strong ATPase activity was detected upon addition of SV40 DNA (Fig. 9A, HIP/SV40 DNA). Antibody to HIP116 (alpha-HIP) diminished the ATPase activity (Fig. 9A), which was unaffected by control antisera (to the hLEF transcription factor; (35) ). Similarly, ATPase activity was detected in both second and third pass affinity purified fractions of HIP116 and this activity co-eluted with HIP116 protein on SDS-PAGE as well as with HIP116 DNA binding activity (Fig. 9, A and B). In addition, we determined that purified HIP116 protein binds to radiolabeled ATP in a UV-cross-linking assay (data not shown). We conclude that the native HIP116 protein, like the yeast SNF2/SWI2 protein(28) , can function as a DNA-dependent ATPase.


Figure 9: HIP116 is a DNA-dependent ATPase. DNA affinity-purified HIP116 protein fractions were tested for ATPase activity (see ``Experimental Procedures''). A, reactions containing first pass HIP116 DNA affinity column fractions (lanes 4-8; fraction E4 from Fig. 7B was used) or second pass DNA affinity column fractions (lanes 12-20) were analyzed for ATPase activity. In, input to the second pass DNA affinity column; Ft, flow-through fraction from the second pass affinity column; W1, first wash from the second affinity run; E1, first elution of HIP116 from the second pass affinity column. The purity of each of these fractions is shown in Fig. 7B. Control reactions contained either carbonic anhydrase (lanes 1-3) or the hLEF transcription factor (lanes 9-11). Reactions in lanes 3 and 6 contained polyclonal antiserum to HIP116, whereas control polyclonal antiserum to the unrelated hLEF transcription factor was added in lanes 7 and 11. ATPase activity was tested in the absence (lanes 1 and 4) or presence (lanes 2, 3, and 6-15) of SV40 DNA or in the presence of different nucleic acids (lanes 16-20), as indicated above each lane. B, the second pass HIP116 fractions were applied to a third pass HIV-1 DNA affinity resin, and individual fractions were analyzed for ATPase activity lanes 2-11). The peak fraction was assayed in the absence (lane 12) or presence (lanes 13-17) of SV40 DNA. Inhibition was observed with two different amounts of polyclonal antiserum specific to HIP116 (lanes 14 and 15), but not with comparable levels of antiserum to the unrelated hLEF protein (lanes 16 and 17). HIP DNA-binding activity (shown in the bottom panel) was found to peak in fractions 10-18, coincident with the ATPase activity.



To determine the nucleic acid specificity of the ATPase activity, the effects of very high levels (1 µg) of DNA or RNA on ATPase activity were tested (Fig. 9A). Addition of SV40 SPH motif DNA stimulated ATPase activity 7-10-fold and a more modest stimulation was also observed with calf thymus DNA or single-stranded DNA. RNA did not stimulate the ATPase activity (Fig. 9A). These results are qualitatively similar to that reported for the SNF2/SWI2 proteins(17, 27) . The ATPase activity co-eluted with HIP116 protein across a third pass on the DNA affinity column, and activity in the peak fractions could also be inhibited with antibodies to HIP116 in a dose-dependent manner (Fig. 9B). Because it has been suggested that the SNF2/SWI2 proteins may also possess DNA helicase activity, we tested the various purified HIP116 fractions for the ability to unwind DNA in a helicase assay using an M13 plasmid containing a short double-stranded region spanning the HIP116 binding site on the SV40 SPH motifs. DNA helicase activity was detected in first and second pass HIP116 affinity column preparations; however, it was not present in third pass affinity column fractions that retained ATPase activity, and, most importantly, the DNA helicase activity was not preferentially stimulated by SV40 DNA and could not be inhibited specifically by antibodies to HIP116. We conclude that the native HIP116 protein can function as a relatively strong DNA-dependent ATPase in vitro and that the ATPase activity is preferentially stimulated by its specific DNA binding sites.


DISCUSSION

In this paper we describe the isolation of a cDNA clone encoding HIP116, a protein related to SNF2/SWI2 (1, 20) that binds specifically to the SV40 enhancer as well as the HIV-1 promoter. A number of diverse regulatory proteins have recently been shown to belong to this family, including BRM(7) , MOT1(19), STH1(20) , LDSTR(21) , RAD5(25) , RAD16 (26) , RAD54(20) , CHD-1(23) , and ERCC6(22) . Among these various family members, HIP116 is most closely related to the yeast RAD16 and RAD5 proteins (25, 26) that are involved in DNA excision repair, as the domain structure of all three proteins includes a single RING finger motif interspersed between ATPase motifs III and IV in a position that is virtually identical between the three factors. Therefore HIP116 can be classified together with RAD5 and RAD16 as members of a new subclass of proteins that contain both the RING finger and the SNF2/SWI2-related ATPase motifs. Despite this organizational similarity, HIP116 is not the human homologue of either RAD5 or RAD16, since it is not particularly closely related to these proteins outside of the ATPase and RING finger motifs and, in particular, the yeast RAD5 and RAD16 proteins lack sequences homologous to the HIP116 DNA-binding domain (amino acids 38-453; see Fig. 2B). Indeed, the amino acid sequence of the HIP116 RING finger is most closely related to that of the yeast RAD18 protein(42) , particularly within the loop of the first finger (see Fig. 2A). Although RAD18 is not a SNF2/SWI2-related protein, it acts in conjunction with RAD5, and it has been suggested that the two different RING finger proteins might associate directly(42) . The observation that the proteins most closely related to HIP116 are implicated in DNA excision repair does not exclude a possible role of HIP116 in transcription, as other RNA polymerase II transcription factors, notably the DNA helicase subunit of TFII-H, are known to affect both processes(48, 49) . Apart from the RING finger and ATPase motifs, HIP116 is not closely related to any of the other SNF2/SWI2 proteins, and in particular it does not possess the ``bromodomain'' repeats that are found in SNF2/SWI2(1, 2) , BRM(7) , and STH1(20) , nor the ``trico-tetrapeptide'' repeats of MOT1(18) , or the chromodomain of CHD-1(23) . By these criteria, HIP116 is a new member of the SNF2/SWI2 and RING finger families.

Although the structure of the RING finger is relatively well characterized(50) , its role is not clearly defined. The RING finger of the HSV ICP-0 trans-activator mediates binding of the protein to zinc and is necessary for both transcriptional activation as well as virus replication(50, 51) . The RING finger of both the ICP-0 and the PML-1 proto-oncogenes has been implicated in their subnuclear localization to novel organelle-like structures (52, 53, 54) that have been designated as PML oncogenic domains(53) . The PML protein also contains a coiled coil domain downstream of the RING finger motif that is shared with certain other RING finger proteins(42, 53) , but is not found in the HIP116, RAD5, or RAD16 proteins, nor is it present in the ICP-0 protein. Further experiments will be necessary to determine if the HIP116 RING finger serves to translocate the protein to any specific subnuclear structures or whether the HIP116 RING finger may serve an altogether different purpose.

HIP116 differs from the well characterized SNF2/SWI2 protein in that it does not appear to reside within a large multiprotein complex, and it can bind directly to DNA in a sequence-specific manner. We find that HIP116 binds to the HIV-1 promoter and SV40 enhancer SPH repeats through a DNA-binding domain that is located within the amino terminus of the protein, distinct from both the ATPase domains and the RING finger. The minimal DNA-binding domain (amino acids 38-287) does not contain any well characterized DNA-binding motifs, but does encompass a region of limited homology to DNA-binding domains 2.3 and 2.4 of the bacterial vegetative sigma factors. The presence of the HIP116 RING finger in the native protein did not alter the DNA binding activity of the native protein, nor did incubation of the protein with ATP affect the DNA binding affinity or specificity. Only one other SNF2/SWI2-related protein, CHD-1, has been reported to bind directly to DNA(23) , and the CHD-1 DNA-binding domain maps to its COOH terminus and is unrelated in sequence to the HIP116 DNA-binding domain. Although it has not been determined if CHD-1 possesses ATPase activity, it is attractive to speculate that the DNA binding specificity of these proteins allows both proteins to function as binding site-specific ATPases in the cell, and it will be interesting to learn if other SNF2/SWI2 proteins will be identified that are capable of binding specifically to DNA.

Using antibodies specific to HIP116, we detected specific binding of HIP116 to both the HIV-1 promoter and to the SPH motifs of the SV40 enhancer in HeLa nuclear extracts. The binding site for HIP116 on the HIV-1 promoter largely overlaps with the extensive site for LBP-1. Antiserum to HIP116 did not affect the LBP-1 complex, indicating that LBP-1 and HIP116 bind independently to the promoter in this region and may compete with each other. This region of the HIV-1 promoter is complex and contains both positive and negative cis-acting control elements and a more detailed mapping of the HIP116 binding site will be required to assess its possible role in viral transcription. The binding of HIP116 to the HIV-1 TATA region indicates that it could also affect the association of TBP with the promoter. Although in principle HIP116 it could function in a manner similar to that described for Mot1 (19) , there are numerous differences between the two proteins. For example, Mot1 does not bind DNA directly, an appears to be targeted to the TATA region by protein-protein interactions, since it has been reported to loosely associate with the yeast TBP:TAF complex(19) . Moreover, the ATPase activity of Mot1 is not significantly stimulated by DNA(19) , which is unlike that we observe for HIP116. Given the relationship between HIP116, SNF2/SWI2, and Mot1, it will be important to assess the effects of HIP116 on HIV-1 promoter activity in vitro on naked as well as chromatin-reconstituted promoter DNA templates.

We also examined the interaction of HIP116 with the SV40 enhancer and find that it appears to be the predominant protein bound to the SPH repeats in crude extracts. Most interestingly, two different point mutations in the SPH motifs that have been shown previously to eliminate SPH motif activity in vivo without affecting the overlapping octamer motif (dpm2 and 0-Sph5; (45) and (46) ) were found to prevent binding of HIP116 in vitro (Fig. 6A). These findings suggest that HIP116 may play a positive role in SPH motif activity in the cell. Purification of HIP116 and separation from TEF-1 did not affect either the mobility or the DNA binding specificity of the HIP116 protein-DNA complex that was observed in extracts, and recombinant full-length HIP116 protein displayed an identical binding specificity and mobility. Although the protein complex formed with HIP116 formally resembles that described by Davidson et al.(29) for TEF-1, it contains HIP116 and does not contain TEF-1. Thus HIP116 appears to be more abundant than TEF-1, or may bind to the SPH motifs with higher affinity, since we did not detect binding of TEF-1 to the SPH motifs until it had been significantly enriched by DNA affinity chromatography. As described previously, TEF-1 formed two complexes with the SPH motif DNA (A and B, (45) ), whereas HIP116 forms only a single complex. The TEF-A complex migrates only slightly faster than the HIP116 complex in our gel system, and in the system of Davidson et al.(29) , they may have been indistinguishable. The dpm2 mutation principally affects only the TEF-A complex while the 0-Sph5 mutation affects both A and B complexes(45) , whereas either SPH mutation is sufficient to eliminate binding of HIP116. Therefore the binding of both HIP116 and TEF-1 correlates with SPH motif activity in vivo, and it remains to be determined whether any enhancer mutations can be identified that will completely discriminate between the action of these two different factors.

Despite the fact that TEF-1 complexes are less abundant that those containing HIP116, TEF-1 is strongly implicated in regulating the activity of the SPH motifs as its overexpression in HeLa cells lead to specific repression or squelching of SPH enhanson activity as well as repression of the activity of the SV40 GT-IIC motif to which it also binds(45) . However, expression of TEF-1 in B cell lines, which lack endogenous TEF-1, is not sufficient to lead to SPH activity(45) , indicating that it may require a co-activator protein that is also missing in B cells. Although we find that HIP116 is not expressed in some mature B cell lines (Namalwa or JY cells),^2 it is unlikely to be the missing co-activator for TEF-1, since it does not bind to the SV40 GT-IIC motif, which is a preferred motif for TEF-1 binding and activation. Thus TEF-1 is likely to function as a conventional enhancer-binding protein to activate the SPH repeats, potentially in conjunction with a cell type-restricted co-activator. By contrast, HIP116 may act affect SPH motif activity indirectly. We find that purified fractions of HIP116 contain a relatively strong ATPase activity (400 pmol/µg/min) that is preferentially stimulated by SPH motif DNA and can be specifically inhibited by antibodies to HIP116 (Fig. 9). The HIP116 ATPase activity is significantly stronger than that reported for SNF2/SWI2 (28) and similar to that observed with Mot1 in the absence of DNA(19) . Thus HIP116 might function as a site-specific ATPase to modify the structure of the enhancer DNA, or it might act to promote binding of TEF-1 to its site on nucleosomal DNA, through a mechanism akin to that demonstrated for the yeast SNF2/SWI2 protein(17) . Alternatively, HIP116 may utilize its ATPase activity to strip other proteins from the DNA, thereby facilitating binding of TEF-1. As such, the association of HIP116 with enhancer DNA could be transient and precede stable binding of TEF-1 to DNA. By these means, HIP116 could also contribute to the formation of the 400-bp nucleosome-free zone that encompasses the SV40 enhancer(56) . Further studies of the effects of HIP116 on SPH motif enhancer activity in vivo await the development of a functional assay in which to study TEF-1 activation, potentially including isolation of the TEF-1 co-activator, as well as studies on the ability of TEF-1 to recognize its site in the presence of other competing factors or nucleosomal structures.


FOOTNOTES

*
This work was made possible by grant support (to K. J.) from National Institutes of Health Grant GM38166 and the Harold and Leila Y. Mathers Foundation. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L34673[GenBank].

§
These two authors contributed equally to this work.

Supported by a fellowship from the California Tobacco Research Council.

**
Supported by a postdoctoral grant from the Deutscher Akademischer Austauschdienst. Present address: Institute for Genetics and Toxicology, Kernforschungszentrum Karlsruhe, Karlsruhe, Germany.

§§
To whom correspondence should be addressed: Dept. of Regulatory Biology, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-452-1122; Fax: 619-535-8194.

(^1)
The abbreviations used are: HIV, human immunodeficiency virus; kb, kilobase pair(s); DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; bp, base pair(s).

(^2)
M. Schorpp, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to I. Davidson and P. Chambon (at the LGME-U.184, Institut de Chimie Biologique, Strasbourg, France) for providing antiserum to TEF-1 and P. Angel and M. Karin (University of California, San Diego) for the HeLa gt-11 cDNA library and recombinant AP-1. We also thank M. L. Waterman for the Jurkat library and the anti-LEF antibodies and H. Mangalam for help with data base searches.


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