Cell lines and plasmids
Human erythroleukemic K562 cells were purchased from American Type Culture Collection (ATCC, CCL-243) and cultured in RPMI 1640 medium supplemented with 15% fetal calf serum (Gibco), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Gibco) at 37°C with 5% CO2 in a humidified incubator. Human embryonic kidney 293T cells were purchased from ATCC (CRL-3216) and cultured in Dulbecco’s modification of Eagle medium with 10% fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Gibco) at 37°C with 5% CO2 in a humidified incubator. The mouse FLAG-Trim28 expression plasmid was constructed as described (27). The EcoRI-SalI fragment from FLAG-Trim28 was ligated into pCMV-HA-N (Clontech) for HA-Trim28 expression. The TRIM28 mutants were generated with the Q5 Site-Directed Mutagenesis kit (NEB) and primers shown in Table S4A. HA-ZFP57 was cloned via PCR and the sequence confirmed. The ZNF445-DDK-Myc expression plasmid was purchased from OriGene. The GAL4DBD-KRAB expression vector and 5×GAL4DBS-E1bTATA-luciferase reporter were kindly obtained from Dr. Frank J. Rauscher, III.
CRISPR-mediated KO of TRIM28 and generation of mutant TRIM28-K304Q in K562 cells
TRIM28 KO was performed by nucleofection of a Cas9 ribonucleoprotein (RNP). Cas9 sgRNAs, targeting TRIM28, were designed using the CRISPR Design Tool at www.benchling.com. Only sgRNAs with high predicted off-target scores (more precise) were selected. The sgRNAs were synthesized by in vitro transcription and purified by denaturing PAGE as described (69). Each purified sgRNA was refolded into a functional structure in buffer containing 20 mM HEPES pH 7.5, 150 mM KCl, 10% glycerol, 1 mM 2-mercaptoethanol, and 1 mM MgCl2.
The TRIM28-K304Q mutant was produced by co-nucleofection of Cas9 RNP and a synthetic DNA repair template for homology-directed repair. A 1490-nt wild-type sequence of TRIM28, including the K304 site, were amplified by PCR using the following primer sets: TRIM28 genomic DNA (gDNA) forward primer 5’-CTCTACATCTTCCCAATAAATGGCCCAGTG-3’ and reverse primer 5’-TGTGAACAAAGCAGAACCCTCTGCCTCAGT-3’. The PCR reaction contained 200 ng genomic DNA and Taq polymerase. The PCR DNA fragment was ligated into pGEM-T Easy (Promega) to construct a pGEM-T Easy/hTRIM28 wild-type plasmid.
Site-directed mutagenesis was then performed to introduce the K304Q mutation in the plasmid using the primer set of the Q5 Site-Directed Mutagenesis kit (New England Biolabs) and primer sets (hTRIM28-K304Q site-directed forward 5’-GCTCAATAAGCGGGGCCGTGTG and reverse 5’-TCCTGCATGATCTGCAGGATGGCC). The resulting plasmid, pGEM-T Easy/hTRIM28-K304Q, carried the K304Q (AAG>CAG) mutation as well as a nearby silent mutation encoding a SacI restriction sequence for screening. The DNA repair template was amplified from the plasmid by PCR using the primer sets K304Q homology-directed repair (HDR) template forward 5’-CTACCTAGCCTGACCTGCTGTG and reverse 5’-CTCACCCCAGACAGCATCATCA. Recombinant Cas9 was purified as described (DOI: 10.1002/cpmb.43). Cas9 RNP was prepared by incubating purified recombinant Cas9 and sgRNA at 1:1.2 molar ratio at 37°C for 10 min. The DNA repair template was then added to the RNP mixture. Nucleofection of human CML K562 cells was performed in a Lonza 4D Nucleofector system using SE Cell Line 4D-NucleofectorTM kit and FF-120 pulse (Lonza). After nucleofection, the cells were incubated at 37°C for 48 h, and single cells were sorted by a BD FACSJazz automated cell sorter at the Flow Cytometry Core Facility of the Institute of Biomedical Sciences, Academia Sinica. The same primer set (TRIM28 gDNA primers) that is complementary to the flanking regions of the HDR region was used to amplify the target region of individual single clones. The co-integrated SacI restriction site was used to screen for the insertion of the K304Q mutation.
Antibodies, preparation of extracts, co-IP, and western blotting
Cells were harvested and washed one time with PBS and lysed with whole-cell extract buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% (v/v) Triton X-100, 1 mM DTT, protease inhibitor, and phosphatase inhibitor). Cell lysates were shaken at 4°C for 30 min and centrifuged at 12,000×g, 4°C for 5 min. The extracts were pre-cleared with protein G agarose (Sigma Aldrich) for 1 h at 4°C and immunoprecipitated with anti-FLAG M2 agarose (Sigma Aldrich) or anti-HA agarose (Sigma Aldrich) for 2 h at 4°C. Unbound proteins were removed by washing three times with whole-cell extract buffer. SDS-PAGE sample buffer (diluted 1:4) was added to each agarose bead sample, with subsequent heating at 100°C for 5 min, SDS-PAGE, semi-dry transfer to a polyvinylidene difluoride membrane (Millipore), and western blotting with appropriate primary antibodies and horseradish peroxidase–conjugated secondary antibodies. The immunocomplexes were visualized by enhanced chemiluminescence (Western Lightning, PerkinElmer Life Sciences) with subsequent exposure to x-ray film (FUJIFILM Corp.). The primary antibodies used were specific for the following proteins: ZNF445 and HA (Bethyl Laboratories), integrins and β-actin (Cell Signaling Technology), Gal4 (Santa Cruz Biotechnology), FLAG (mouse; Sigma Aldrich), and FLAG (rabbit; (70)). Rabbit anti-human TRIM28 was generated by immunizing rabbits with a peptide encompassing residues 14–43 (anti-TRIM28-N). The antibody was purified with a peptide-agarose affinity column. Monoclonal anti-human TRIM28 (clone 20A1) was produced as described (27) and obtained from Biolegend. The secondary antibodies were goat anti-mouse (KPL, 474-1806) and goat anti-rabbit (KPL, 474-1516).
Identification of associated proteins from immunoprecipitated TRIM28 and TRIM28-K304Q
Peptide identification by MS was performed by the Mass Spectrometry Common Facility at the Institute of Biological Chemistry, Academia Sinica, using a LTQ-Orbitrap Velos system (Thermo Fisher). Data interpretation and correlations between the spectra and amino acid sequences within a human EST database and customized FLAG-Trim28 sequence were analyzed using Mascot (Matrix Science software package).
Total cellular RNA was extracted using TRIzol Reagent (Invitrogen). Total RNA of each sample was quantified with a 2100 Bioanalyzer (Agilent Technologies), NanoDrop (Thermo Fisher Scientific), and 1% agarose gel electrophoresis. Total RNA (1 μg) with a RNA integrity value >6.5 was used for library preparation. Libraries were prepared for next-generation sequencing according to the manufacturer’s protocol (Agilent Technologies). Poly(A) mRNA was isolated using the Poly(A) mRNA Magnetic Isolation Module or rRNA removal kit (New England Biolabs). The mRNA was fragmented and primed using First Strand Synthesis Reaction Buffer (New England Biolabs) and random primers. First-strand cDNA was synthesized using ProtoScript II Reverse Transcriptase (New England Biolabs), and second-strand cDNA was synthesized using the Second Strand Synthesis Enzyme Mix (New England Biolabs). Bead-purified] double-stranded cDNA was then treated with End Prep Enzyme Mix to repair both ends, and dA-tailing was carried out in the same reaction, followed by T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using beads, and fragments of ~420 bp (insert size ~300 bp) were recovered. Each sample was then amplified by PCR for 13 cycles using primers P5 and P7, each of which carried sequences that could anneal with the flowcell to perform bridge PCR; P7 carried a six-base index to allow multiplexing. The PCR products were cleaned up using beads, validated using a Qsep100 (Bioptic), and quantified with a Qubit3.0 fluorometer (Invitrogen). Then, libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument (Illumina). Sequencing was carried out using a 2 × 150-bp paired-end configuration, and image analysis and base calling were conducted with HiSeq Control Software (HCS)+ OLB +GAPipeline-1.6 (Illumina) on the HiSeq instrument.
Lentivirus-mediated gene knockdown
Lentiviral vector–mediated short hairpin RNA technology was used for ZNF445 knockdown. Two human ZNF445 short hairpin RNA target sequences, namely GCGCTATAAATGTAATCTATG (TRCN0000415362) and ATCAAACTTTACTCGTCATAT (TRCN0000435973), and scrambled control pLKO.1-shLuc (TRCN0000072243) were obtained from the National RNAi Core Facility at Academia Sinica. Viruses were produced using calcium phosphate–mediated transfection. Early subcultures of 293T cells were co-transfected with 14 μg pPGK-GFP, pLKO.1-shLuc, or pLKO.1-shZNF445 and 14 μg pCMVΔR8.91 and 2 μg pMD.G. After 8 h, the medium was replaced with K562 cell maintenance medium to collect virus. Then, K562 cells in a 6-well plate were infected with viral supernatants in the presence of 8 μg/ml polybrene for 48 h and further selected with 3 μg/ml puromycin for 1 week. Knockdown efficiency was determined based on RT-qPCR and western blotting.
Reverse transcription-quantitative PCR
K562 cells in a 6-cm dish were harvested, and total RNA was prepared with TRIzol reagent (1 ml per dish). After quantification by measuring absorbance at 260 and 280, 2 μg RNA was treated with DNase I (Invitrogen) and then reverse transcribed into cDNA using Superscript IV (Invitrogen). The quantitative PCR (qPCR) was performed with the Corbett Research RG-6000 Real Time PCR Thermocycler (Qiagen). The total volume was 20 μl including QuantiNova SYBR Green master mix (Qiagen), 20-fold diluted cDNA, and 0.3 μM each of the forward and reverse primers (Table S4B). The amplification conditions were 60 cycles of 95°C for 10 s and 60°C for 15 s. The results were analyzed by the 2–∆∆Ct relative quantitation method. All experiments were independently repeated three times.
K562 cells were cultured to a density of 2 × 106/ml and harvested for crosslinking with 1% formaldehyde in medium at room temperature for 10 min. Glycine was added (final concentration, 125 mM) to quench unreacted formaldehyde for 5 min. Cells were pelleted by centrifugation, and the pellet was washed with 5 ml cold PBS. Cells (2 × 107) were lysed in 400 μl lysis buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% (v/v) NP-40, and protease inhibitor cocktail) on ice for 15 min, and the cell suspension was mixed gently every 5 min. Nuclei were collected by centrifugation at 9000 ×g for 5 min at 4°C, resuspended in 200 μl nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, and protease inhibitor cocktail), and kept on ice for 10 min. The nuclear lysates were sonicated using the middle setting of an ice-water Bioruptor (Diagenode) for 10–15 min total (30 s on, 30 s off). The resulting sheared chromatin (2 µl) was resolved by electrophoresis through a 2% agarose gel to check that the length of DNA fragments was 200–600 bp. Each sheared chromatin sample was centrifugated at 12,000 ×g for 5 min at 4°C, and the supernatant was diluted 10-fold with buffer containing 20 mM Tris-HCl pH 8.1, 167 mM NaCl, 0.01% SDS, 1% Triton X-100, 1 mM EDTA, and protease inhibitors. Each chromatin complex sample was divided into two equivalent volumes for IP with either normal IgG or anti-TRIM28-N. After incubating with antibody at 4°C overnight, protein A/G magnetic beads (20 µl) were added for an additional 2 h. The beads were washed one time each with 0.5 ml of each of following buffers: low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% (v/v) IGEPAL CA630, 1% deoxycholic acid–sodium salt, 1 mM EDTA, 10 mM Tris pH 8.1), and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Each wash step was done for 5 min on a rotating platform at 4°C. Chromatin complexes were eluted with 100 μl elution buffer (1% SDS, 0.1 M NaHCO3, containing 1 μl proteinase K) for 2 h at 62°C with mixing, with subsequent incubation at 95°C for 10 min to de-crosslink the chromatin complexes. Samples were cooled to room temperature, and the supernatant was transferred to a new tube. After extraction by phenol/chloroform, the DNA was precipitated by ethanol and analyzed by qPCR with specific primers (Table S4C).
All data are presented as the mean ± SD of at least three independent experiments. Statistical significance (*P < 0.05, **P < 0.01 or ***P < 0.001) was determined by the one-tailed Student's t-test.