Screening of a human cDNA library by yeast two-hybrid system
In order to identify proteins encoded by the human fetal brain cDNA library and interacting with the hinge domain of SMC3, a bait plasmid was generated by subcloning the SMC3-465/807 polypeptide coding region into the yeast two-hybrid system bait vector pGBKT7 (Clontech) (see fig. for a diagram of the constructs used and their designation). The insert was generated by PCR using Pfu
DNA polymerase and primers terminated with restriction sites that allowed the directional cloning of the products into the accepting vector (see Additional file 1
for a listing of the primers used in this study). Mouse full-lenght SMC3 cDNA was used as template. To identify the gene encoded by the interacting plasmid, the prey insert was sequenced by priming at the Gal4AD site (5'-AATACCACTACAATGGA-3'). The sequences were BLASTed against the nr and human dEST databases. DNA restriction digestions provided information on the size of the clones retrieved.
5' RACE and cloning of the complete Hinderin coding sequence
The total RNA was isolated using TRI-reagent from 293, HepG2 and HeLa cells. The 5' RACE assay was performed using a RLM-RACE Ambion kit. The generated cDNA was used as template for nested PCR. The inner PCR product was cloned into pCRII-Topo vector (Invitrogen) and sequenced using a T7 primer. The sequence matched that of the DKFZp451C1618 clone (AL832625) and extended 5' to the published sequence of KIAA1328 (AB037749). Based on this information, the complete Hinderin coding region was generated by RT-PCR utilizing mRNA extracted from 293 cells, and the product cloned in frame with the tag sequence in pcDNA3.1/V5-His TOPO (Invitrogen). When transfected into 293 cells, the expressed product detected with an anti-V5 monoclonal antibody had size 69 KDa, as expected for a protein encoded by the Hinderin-V5 fusion gene (fig. ).
Mapping of the protein interacting sites
In order to map the SMC3 interacting site(s) a series of truncated constructs were produced in pGBKT7 vector. The inserts required for the SMC3-1/186, SMC3-976/1217, SMC3-552/807 and SMC3-711/807 constructs were produced by PCR. SMC3-465/550 and SMC3-465/716 were generated by introducing stop codons in the sequence of SMC3-465/807 using a QuikChange XL kit (Stratagene). The mutated duplex oligonucleotides used had the forward sequence: 5'-CTTTCTATACTTGTGT AAGTCACTGCTGGTAAC-3' and 5'-GACCAGTTGATGAACTAAATGCAGATAGAG-3', respectively. SMC3-465/643 and SMC3-643/807 were obtained by digesting SMC3-465/807 at the PstI or alternatively the NdeI restriction sites present in the vector multiple cloning site and at the SmaI site of the insert. The resulting linear constructs were blunt-ended and religated. pGADT7-SMC3-465/807 was generated by retriving the insert from the bait vector. SMC1-485/670 encoding the entire SMC1 hinge region, was generated by RT-PCR from 293 cells mRNA and cloned in pGADT7. Hinderin deletion constructs H-64/360 and H-360/578 were generated by restriction digestion of the pACT2-Hinderin-47/578 clone identified in the yeast two-hybrid system screening with NcoI/BglII and BglII/XhoI respectively, followed by religation of the blunt-ended vector. Hinderin in bait pGBKT7 vector was generated by retrieving the H-47/578 insert from the prey clone. H-177/360 and H-1/85 inserts were obtained respectively by PCR and by digestion of the Hinderin pcDNA3.1 expression vector with BamHI/EcoRI. Both were subsequently subcloned in pGADT7. To assess the strenght of interaction between different SMC3 and Hinderin domains, three colonies were randomly selected and grown overnight in 5 ml of selection media. After cell lysis by freeze thawing in 300 ml of 100 mM Na2HPO4 pH 7.0, 10 mM KCl, 1 mM MgSO4, buffer, β-galactosidase activity was assessed using ortho-nitrophenyl-β-D-galactopyranoside as substrate (1 mM) in the presence of 50 mM β-mercaptoethanol. After 2 h incubation at 37°C the color intensity was read at 420 nm.
Northern blot hybridization and semiquantitative PCR
Total mRNA was extracted with TRI-reagent from subconfluent HeLa and HCT116 cells and separated on 1% agarose. After transfection to a nitrocellulose filter, the Hinderin transcript was identified by hybridization to a 335 bp 32P-labeled cDNA probe annealing to the 5'-end region of the gene. In order to examine the expression of Hinderin in different human tissues, semiquantitative RT-PCR was performed by using 0.5 μg of Marathon-ready first strand cDNA (Clontech) from 16 tissues. The PCR reaction was monitored at 20 and 30 cycles and the product analyzed on 2% agarose. In order to normalize for possible differences in mRNA content, the expression of the housekeeping gene G3PDH was analyzed in each sample.
Protein secondary structure prediction and identification of orthologue forms of Hinderin in other species
The human polypeptide sequence was analyzed with the COILS program to predict globular and coiled-coil domains. The scanning window was set at 21. The homology with other known protein family was examined by querying the NCBI Conserved Domain protein database. We used PSORT to scan for nuclear and other localization signal consensus sequences. To identify orthologue forms of Hinderin in other species, the human protein sequence was BLASTed against the translated EST database of m. fascicularis, mouse, rat, cow, sheep, dog, zebrafish, c. elegans, drosophila, and s. cereviasie. When a homologous sequence had been identified in lower organisms, we ran the COILS program to assess whether it displayed the same secondary structure as that of the matching human sequence.
Protein complex immunoprecipitation
293 cells were grown at ~80% confluence in 35 mm cm plates and transfected with 1 μg of Hinderin-V5 expression vector. After 48 h of incubation, the cells were washed in ice-cold phosphate-buffered saline and lysed in 1.2 ml of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% Na-deoxycholate buffer containing 100 mM NaF, 2 mM Na3VO4, and a cocktail of protease inhibitors. The cell lysate was centrifuged at 12,000 g and the recovered supernatant preabsorbed on protein G-agarose. Aliquots (500 μl) of the cell lysate were then incubated overnight at 4°C with either 25 μg of goat anti-human SMC3 antibody or goat anti-human SMC1 antibody (Santa Cruz Biotech) or alternatively with 10 μg mouse anti-V5 antibody (Invitrogen). The immunocomplexes were captured on Protein G-agarose by incubating 1 h at 4°C. After washing in immunoprecipitation buffer containing 300 mM NaCl, the protein immunocomplex was analyzed by SDS-PAGE and the proteins transferred to nitrocellulose membranes by electroblotting. After saturation in 4% dry milk/0.1% Tween 20 in PBS, the filter was incubated 1 h at RT with primary antibody. The anti-SMC1 and anti-SMC3 immunoprecipitate filter blots were incubated with anti-V5 monoclonal antibody (200 ng/ml) whereas the V5 immunoblots were incubated with either anti SMC1 (100 ng/ml) or anti-SMC3 (100 ng/ml) antibodies. After incubation with species-specific anti IgG HRP-conjugated secondary antibody (1:10,000), the immunocomplexes were visualized by enhanced chemiluminescence reaction (ECL).
Mammalian two-hybrid interaction assay
Inserts corresponding to the SMC3-474/702 and SMC1-474/663 hinge domains were generated by PCR and ligated in the pBIND or the pACT vectors (Promega) respectively through ligation at the BamH
I and Sal
I sites. The resulting bait and prey DNA constructs (0.25 μg/ml each) together with the Hinderin-V5 expression vector (either 0.3 or 1 μg/ml or alternatively 1 μg/ml of empty pcDNA3.1 vector) and the reporter plasmid pG5/luc encoding the firefly
luciferase (0.1 μg/ml), were co-transfected into 293 cells using Lipofectamine. Control experiments were conducted using pACT-Id and pBIND-MyoD vectors (Promega) encoding respectively GAL4:Id and VP16:MyoD fusion proteins known to interact in vivo [20
]. After 48 h incubation, cells were lysed and the expressed luciferase activity quantitated using a dual luciferase reporter assay kit (Promega) and a Lumat LB 9501 luminometer. Firefly
luciferase values were corrected for the transfection efficiency using the values of the Renilla
luciferase activity encoded by the pBIND vector under the control of a strong constitutive promoter. In order to assess the effect of Hinderin on the bait and prey expression, total RNA was extracted from a group of transfected cells and the specifc transcript levels quantitated by RT-PCR using primers annealing to the ends of the fusion protein coding sequence.