NMR and Fluorescence Studies of DNA Binding Domain of INI1/hSNF5

Abstract

INtegrase Interactor 1 protein (INI1/hSNF5) or BRG1-associated factor 47 (BAF47) is a SWI/SNF-related matrix associated actin dependent regulator of chromatin subfamily B member. DNA binding domain of INI1/hSNF5 is cloned into E.coli expression vectors, pET32a and purified as a monomer using size exclusion chromatography. NMR data show that $INI1^{DBD}$ has folded state with high population of ${\alpha}$-helices. By fluorescence-quenching experiments, binding affinities between $INI1^{DBD}$ and two double stranded DNA fragments were determined as $29.9{\pm}2.6{\mu}M$ (GAL4_1) and $258.7{\pm}5.8$ (GAL4_2) ${\mu}M$, respectively. Our data revealed that DNA binding domain of INI1/hSNF5 binds to transcriptional DNA sequences, and it could play an important role as a transcriptional regulator.

Introduction

Integrase interactor 1 protein (INI1)/BRG-1 associated factor 47 (BAF47) is known as a hSNF5 or SMARCB1 and it is a core subunit of ATP-dependent SWI/SNF chromatin remodeling complexes.1-3 Chromatin remodeling complexes promote gene expression, transcriptional repression.4-6 INI1/ hSNF5 regulates expression of numerous eukaryotic genes by altering DNA-histone interactions and it is the first host protein identified as an IN-interacting factor by two-hybrid screenings.2 In previous studies, INI1/hSNF5 interacts with virus proteins, cellular factors such as human immunodeficiency virus type 1 integrase,7 human papillomavirus E1,8 Kaposi’s sarcoma-associated herpesvirus K8,9 Epstein-Barr virus nuclear antigen,10-12 c-MYC(cheng), ALL1, GADD34 and p53.13-15 INI1/hSNF5 is involved in regulation of cell proliferation and cell cycle by prohibiting activation of E2Fdependent genes through p16ink41-CDK4/cyclin D-Rb pathway.16In addition, INI1/hSNF5 plays a role as a tumor suppressor because mutations of INI1 gene lead to aggressive paediatric atypical teratoid and malignant rhabdoid tumors.17

INI1/hSNF5 has functional and conserved regions which are two direct imperfect repeats (repeat 1 and repeat 2) and C-terminal coiled-coiled domain, and a homology region 3 (HR3).18 The functional domains of INI1/hSNF5 are not well characterized except for Rpt1 and Rpt2 motifs, which interact with proteins such as viral or cellular proteins.18 Previous reports show that INI1/hSNF5 interacts with the minor groove of double-stranded DNA and then, conforms positive supercoils in relaxed plasmid DNA. It implies that SWI/SNF complex plays an important role in chromatin remodeling through protein-protein interaction. It has been proposed that INI1/hSNF5 could bind DNA at the minor groove while hBRG1 has been reported to possess an AThook motif which appears to play a role in DNA binding, although its biological functions of DNA binding domain are still unknown.18,19 Here, we performed high-yield protein purification and isotope-labeling of DNA binding domain of INI1/hSNF5 (INI1DBD) to initiate structural study by NMR spectroscopy. Data from fluorescence spectrophotometry supports that INI1DBD binds double stranded GAL4 DNA sequences.

 

Materials and Methods

Cloning and Over-expression of INI1DBD. Human INI1/ hSNF5 was amplified to INI1DBD fragment by polymerase chain reaction (PCR). XhoI/BamHI (NEB) cut each side of amplified fragment. Mixture was incubated 8 h in 37 ℃ heat block. Sub-cloning into modified expression vector pET32a (Novagen Inc.) contains an N-terminal hexa-histidine [(His)6] affinity tag, Thioredoxin (TRX), tobacco etch virus (TEV) protease recognition sites, which were transformed into Escherichia coli (E. coli) (Strain BL21 DE3) (Novagen Inc). In order to obtain INI1DBD, cells were grown on LB broth media at 37 ℃ until optical density reached around 0.6 at 600 nm and then, added 1 mM isopropyl β-D-thiogalactoside (IPTG) from 1 M solution stock. Cells were incubated for 20 hours at 25 ℃, 160 rpm shaker incubator and harvested by centrifugation at 6000 × g for 30 minutes. Cell pellets were stored at −80 ℃ freezer.

Purification of INI1DBD. Harvested cells were suspended in lysis buffer [25 mM Sodium Phosphate, 300 mM NaCl, 5 mM β-mercaptoethanol, protease inhibitor cocktail-(Roche) pH 7.5] and lysed by sonicator [90% amplify, 1s pulse, 10 cycle]. The cell lysates were centrifuged for 30 minutes, 14000 × g at 4 ℃. After centrifugation, Ni2+ affinity chromatography was used for protein binding (Amersham Pharmacia Biotech, Uppsala, Sweden). The proteins were further washed with 60 mM imidazole washing buffer and eluted with 500 mM imidazole elution buffer. TRX-tag was cleaved with Tobacco etch virus (TEV) protease at 25 ℃.20 Final purification was performed by size exclusion column chromatography using a HiLoad Superdex 75 column in 10 mM HEPES, 100 mM NaCl, 10 mM Dithiothreitol (DTT), 0.01% NaN3 solution at pH 7.0.

Size exclusion chromatography with four reference proteins [albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome C (12.4 kDa) and aprotinin (6.5 kDa)] was used to confirm the molecular weight of INI1DBD by the following equation: log Y = −1.2177 X + 6.263 (X = elution fraction/ 45.85 mL, R2 = 0.9942). Purified protein was concentrated to 1.0 mM using an Amicon Ultra centrifugal filter device (Millipore).

NMR Spectroscopy. For hetero-nuclear NMR experiments, we prepared isotope labeled proteins. Cells were cultured in M9 minimal medium containing 15N labeled NH4Cl (15N, 99%, Cambridge Isotope Laboratories, Inc.). Protein sample was prepared in NMR buffer solution (10 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% NaN3, at pH 7.0 containing 90%H2O/10%D2O). All NMR experiments were performed at 25 ℃ on a Bruker DRX900 spectrometer equipped with a z-shielded gradient triple-resonance cryo-probe. The 1H chemical shifts were referenced to internal sodium 4,4- dimethyl-4-silapentane-1-sulfonate (DSS). The 1H-15N HSQC experiment was acquired using a uniformly 15N-labeled sample. Pulsed-field gradient (PFG) techniques with a WATERGATE pulse sequence were used for all H2O experiments, resulting in a good suppression of the solvent signal.21 All NMR data were processed using NMRpipe and NMRDraw software.22

Fluorescence Experiments. Two double-stranded oligonucleotides, (GAL4_1 (5'-CGATGATGAAGATACC-3'), GAL4_2 (5'-GTGCACGATGCACAG-3)) were designed based on data from Morozov et al.18 and purified using polyacrylamide gel electrophoresis (PAGE) (Operon). Binding affinities between GAL4 DNA and INI1DBD were measured on a model RF-5301PC spectro-fluorophotometer (Shimadzu, Kyoto, Japan). INI1DBD was titrated up to a molar ratio of 1:2.5 (INI1DBD: GAL4 DNA) using a 2 mL thermostat cuvette. Samples were excited at 280 nm for sidechain of the aromatic residues of INI1DBD, and emission spectra were recorded for light scattering effects from 270 to 500 nm.23 The Kd values were calculated by the equation log (F0 − F / F) = log (1/Kd) + n log [ligand], where F0 and F represent fluorescence intensity of the protein at 355 nm in the absence and presence of DNA,24 respectively. The number n represents the ligand binding site of the protein.

 

Results and Discussion

Cloning and Expression of INI1DBD. DNA binding domain of INI1/hSNF5 corresponding residues from 106 to 183 was successfully cloned by PCR amplification and subcloned into pET32a (Novagen Inc.) at the BamHI and XhoI site (Fig. 1(a)). INI1/hSNF5 consists of three structural domains which are DNA binding, Repeats1 and Repeats2 domain (Fig. 1(b)). The construct contains TRX, hexahistidine [(His)6] affinity tag with TEV protease recognition sequence (ENLYFQG) in the N-terminus (Fig. 1(b)). INI1DBD protein was successfully expressed in both 250 mL LB and M9 media using 1 mM IPTG at 25 ℃.

Figure 1.Schematic representation of the vector map and structural domains of INI1/hSNF5. (a) The vector map of INI1DBD expressed in E. coli with TEV cleavage site is shown. (b) Structural domains of INI1/hSNF5 are drawn by data from previous studies.18 The vector consists of a Thioredoxin, (His)6 tag and TEV enzyme cleavage site (ENLYFQG). (c) Secondary structures and primary sequence of INI1DBD were shown. Secondary structures were predicted by psi-pred server (http://bioinf.cs.ucl.ac.uk/psipred/).

Purification of INI1DBD. Data from Ni-NTA affinity chromatography shows that INI1DBD is soluble in elution buffer. Fusion protein was eluted at imidazole concentration of 500 mM and purified as a purity of > 90% as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2(a)). INI1DBD was obtained after cleavage of Trx tag followed by Ni-NTA column work. Molecular weight of the purified fusion protein was determined as a 27 kDa and INI1DBD is ~9 kDa after cleavage of Trx tag. To increase protein purity and determine oligomeric state of INI1DBD, size-exclusion chromatography was performed using HiLoad 16/60 superdex TM75. Data from size-exclusion chromatography shows that INI1DBD forms a monomer (~9 kDa) in solution (Fig. 2(b)) by comparing reference proteins as a standard molecular marker. The final yields of the INI1DBD protein are 10-15 mg per 250 mL of cultured cell.

Figure 2.SDS page and elution profile of the size exclusion chromatography. (a) The SDS pages identify INI1DBD during each purification steps. Lane 1 and 2 indicate supernatant and pellet of the cell lysate. Lane 3, 4 and 5 show the elution profiles, flowthrough, washing and elution, respectively. Lane 7, 8-11 and 12 indicate profile after tag cleavage, flow-through, washing and elution, respectively. (b) Elution profile of the size exclusion chromatography with SDS page. INI1DBD was eluted from 80 to 90 mL. Molecular weight of INI1DBD was calculated by the equation, log Y = −1.2177 X + 6.263 (R2 = 0.9942, X = elution fraction/45.85 mL).

NMR Spectroscopy. One-dimensional NMR (1D-NMR) was used to confirm folded state of the purified INI1DBD. A number of high-field methyl proton resonances ranged from 0.5 to 1.0 ppm as well as dispersion of amide proton resonances (7.5-9.5 ppm) strongly suggested that INI1DBD has a stable folded structure under our experimental condition (Fig. 3(a)). 2D 1H-15N HSQC spectrum shows that most of backbone NH resonances were spread over the spectrum in NMR solution (10 mM HEPES, 100 mM NaCl, 2 mM DTT and 0.01% NaN3 at pH 7.0) (Fig. 3(b)). Based on chemical shift information of the backbone amide proton and nitrogen resonances, we expect that INI1DBD has high population of α-helices.

Figure 3.One-dimensional NMR and 1H-15N 2D HSQC spectra of INI1DBD. (A) One-dimensional 1H NMR spectrum of INI1DBD is shown. NMR sample was prepared in 10 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% NaN3 solution, at pH 7.0. NMR experiment was performed on a Bruker DRX900 spectrometer. (b) 1H-15N 2D HSQC spectrum was recorded using uniformly 15N labeled INI1DBD.

DNA Binding of INI1DBD. Previous report suggested that DNA binding domain of INI1/hSNF5 could interact with double strand DNA.18 Data from fluorescence-quenching experiments provide that the binding affinities between INI1DBD and two double stranded DNA fragments were determined as 29.9 ± 2.6 μM (GAL4_1) and 258.7 ± 5.8 (GAL4_2) μM, respectively (Fig. 4(a)). The binding stoichiometry between the protein and DNA is calculated as 1:1 based on data from fluorescence spectroscopy. It is of interest to know that GAL4_1 binds 10 times stronger than GAL4_2. Taken together, these results revealed that DNA binding domain of INI1/hSNF5 binds to transcriptional DNA sequences, and INI1DBD could play an important role as a transcriptional regulator. Since it has also been suggested that INI1DBD would mediate protein-protein interaction with HIV-1 integrase,7 INI1/hSNF5 could modulate viral replication of HIV-1 by binding to minor groove of DNA. This hypothesis will be revealed by structural information of INI1/HIV-1 integrase complex which is currently in progress.

Figure 4.Fluoresence spectra of INI1DBD in the presence of double-stranded DNA. (a) Fitting plot of INI1DBD is shown in the presence of difference mixing ratio of double-stranded DNA (GAL4-1) (1:0 (red), 1:0.2 (green), 1:0.4 (purple), 1:0.6 (black), 1:0.8 (orange), 1:1 (blue), 1:2 (magenta)). (b) Fitting plot of INI1DBD with mixing ratio of double-stranded GAL4-2 DNA (1:0 (red), 1:0.2 (green), 1:1 (purple), 1:1.5 (black), 1:2 (orange), 1:2.5 (blue)).

 

Conclusion

DNA binding domain of INI1/hSNF5 is cloned into E. coli expression vectors, pET32a and purified using size exclusion chromatography. INI1DBD was determined as a monomeric conformation in our experimental condition. Data from one-dimensional NMR and 2D 1H-15N HSQC show that INI1DBD has folded state with high population of α-helices. By fluorescence-quenching experiments, binding affinities between INI1DBD and two double stranded DNA fragments were determined as 29.9 ± 2.6 μM (GAL4_1) and 258.7 ± 5.8 (GAL4_2) μM, respectively, indicating INI1DBD could play an important role as a transcriptional regulator.