Cell lines, plasmid construction, and gene knockdown
The breast cancer cell lines MCF7 and MDA-MB-231 were maintained in DMEM, and AU565 were maintained in RPMI. These mediums were supplemented with 10%
FBS, non-essential amino acids (HyClone), sodium pyruvate (HyClone), and antibiotics (HyClone). HEK293T cells were co-transfected with packaging plasmid (pCMV-Δ8.91), envelope (pMDG), and hairpin pLKO-RNAi vectors (National RNAi Core Facility, Institute of Molecular Biology/Genomic Research Centre, Academia Sinica, Taiwan) for virus packaging. The specific oligo sequences of shRNA are listed in Table 8.
Virus-containing supernatants were collected at 48 hr post-transfection. Cells were treated with virus plus medium containing polybrene (8 μg/ml) for 16 hr. The infected cells were selected with puromycin (0.5 μg/ml). Plasmids expressing SMYD3WT and methyltransferase-dead SMYD3Y239F were constructed as previously described (15).
ChIP assay.
ChIP assays were performed as described (15). Complexes were immunoprecipitated overnight with 2 μg of antibodies specific for SMYD3 (GTX121945, GeneTex), rabbit IgG (GTX35035, GeneTex), H3 (ab1791, Abcam, Cambridge, MA, USA), H3K4me3 (ab10158, Abcam), and RNA polymerase II CTD repeat YSPTSPS (phosphor Ser5) (ab5131, Abcam). Input samples were processed in parallel. Antibody/protein complexes were collected by 40 μl of protein G-coupled Sepharose beads (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and washed as follows: once with Tris/EDTA-150 mM NaCl, twice with Tris/EDTA-500 mM NaCl, and once with PBS. Immune complexes were eluted with 1% SDS and TE buffer. After
decrosslinking, DNA was purified using a PCR cleanup kit (Qiagen) and analyzed by qRT-PCR. The results were expressed as the percentage of the initial inputs. The primer sets used for the ChIP assay are listed in Table 9.
ChIP-seq assay and data analysis.
ChIP DNA was obtained as described above. For library construction, 3 samples were prepared, including input DNA (3 μg), SMYD3-ChIP DNA (100 ng) and H2A.ZK101me2 (45 ng). The samples were adjusted to equivalent concentration for later steps. Illumina adaptor sequences and barcodes were added in a subsequent limited PCR reaction, and the libraries were sequenced with an Illumina MiSeq platform (Illumina, San Diego, CA, USA) at the Technology Commons in College of Life Science and Center for Systems Biology (National Taiwan University, Taiwan). Three times of paired-end sequencing for each sample to get a total of 30 million reads were conducted at a read length of 150 bp. Raw sequences were obtained using the Illumina GA Pipeline software CASAVA v1.8. The sequences generated were subjected to a filtering process to obtain qualified reads. ConDeTri was implemented to trim or remove reads according to their quality score (71). The read data were aligned to the genomic sequences retrieved from the UCSC database using BWA, and these results were imported into the Partek Genomics Suite (Partek Inc., St. Louis, MO) to identify peaks in the ChIP-seq data using a standard ChIP-seq workflow (72). A zero-truncated negative binomial model was fitted to the sequencing data to identify the enriched
regions as peaks (Partek ChIP-Seq white paper:
www.partek.com/Tutorials/microarray/User_Guides/ChIPSeqPeakDetection.pdf). The p-value from the Mann-Whitney U test and the binomial p-value (p < 0.001) were used
to remove any false peaks. In the ChIP-seq data analysis, we collected the H2A.Z and acetylated-H2A.Z datasets (GEO accession number GSM1059388) from the public domain, and these sequences were mapped to the UCSC human genome (hg19) using the Bowtie aligner (according to the following parameters: -a -m 1 -v 3) (73). We then applied the MACS peak caller to the mapped reads to find the significant peaks (p < categorized by the DAVID v6.8 Gene Ontology program (75,76).
Cell fixation and immunofluorescence assays.
Cells were seeded on glass coverslips coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA) and allowed to attach for 48 hr followed by 1.67 Gy IR treatment (IBL 637, CIS Bio International, Gif-sur-Yvette, France). After washed with phosphate buffered saline (PBS), cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. The fixed cells were then permeabilized with 0.1%
Triton X-100 in PBS for 10 min. After 30 min of blocking with 1% BSA in PBS, cells were washed in PBS and incubated with primary antibodies for 3 hr. After three times washed in PBS containing 0.05% Triton-X for 5 min, the cells were incubated with
secondary antibodies for 1 hr. Finally, cells were washed three times with PBS containing 0.05% Triton-X and embedded in 1 μg/ml DAPI (Sigma-Aldrich) containing mounting solution on glass slides. The cells were visualized with an Olympus fluorescence microscope. Images were captured using a Spot advanced imaging system.
The primary antibodies used were γH2A.X (1:200, 05-636, Millipore-Upstate, Temecula, CA, USA), BRCA1 (1:100, sc-6954, Santa Cruz Biotechnology, CA, USA), 53BP1 (1:100, sc-22760, Santa Cruz Biotechnology) and MDC1 (1:200, A300-053A, Bethyl Laboratories, Montgomery, TX, USA).
Colony formation assay.
For the colony formation assay, control (shLuc) or knockdown (shSMYD3) cells were seeded (5,000 cells for shLuc cells, and 20,000 and 10,000 cells for shSMYD3#1 and shSMYD3#2 cells, respectively) in 6-cm dishes two days before IR (0, 1.67, 3.34, 5.01 Gy) treatment. Cells were incubated for 15 days, fixed in 4% paraformaldehyde for 5 min, washed once with PBS, stained with 0.1% crystal violet, and then washed with distilled water. The survival rate was calculated by comparing the colonies numbers with the non-irradiated cells in each group.
Nuclear/cytosol fractionation. washed twice with 1 ml cytoplasmic lysis buffer. Nuclei were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1% sodium
dodecyl sulfate (SDS), 1 mM DTT, protease inhibitor, 1 mM PMSF, 1 mM EDTA) and lysed completely by sonication. Nuclei and cytosolic extracts were then subjected to Western blot analysis.
Western blot analysis.
Western blot analysis was performed as described(15). The primary antibodies used were SMYD3 (1:3000, GTX121945, Genetex, San Antonio, TX, USA), MDC1
(1:1000, GTX102673, GeneTex), EXO1 (1:1000, GTX109891, GeneTex), RAD54B (1:1000, GTX103291, GeneTex), Tubulin (1:5000, GTX112141, GeneTex), and nuclear matrix protein p84 (1:10000, NB100-174, Novus, Littleton, CO, USA). The quantification of protein expression was performed using ImageJ software (Image Processing and Analysis in Java). All protein expression levels were normalized against the corresponding control protein levels as indicated. Images were representatives of n ≥ 3 for each experiment.
Comet assay.
DNA strand breaks were evaluated using alkaline single cell gel electrophoresis (comet) assay following the procedure of Olive and Banath (77). In brief, 2×105 cells with 4-day SMYD3 knockdown were seeded in 6-well tissue-culture plates and incubate for 24 h for cell attachment. Cells were treated with 1.67 Gy IR and harvested for indicated time. Cells were washed with PBS twice and resuspended in 1 ml PBS. About 50 µL of the resuspended cells was mixed with 100 µL of low melting point agarose at 45 °C and spread the suspension over the well with the pipette tip. The slides were placed at 4 °C in the dark until gelling occurred and then immersed in pre-chilled lysis buffer at 4 °C for 30 min. After that, the buffer was aspirated and replaced with a pre-chilled alkaline solution for 40 min at 4 °C. After lysis and unwinding, the slides
were placed in a horizontal electrophoresis tank filled with freshly prepared alkaline electrophoresis buffer. The electrophoresis was run for 15 min at 15 V and 25 mA. After electrophoresis, the slides were rinsed with PBS and placed in PBS until filming. The slides were added with 100 µL of 10000X diluted SYBR gold (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 1 min for DNA staining. DNA migration was observed using fluorescence microscope at a magnification of 20X (Carl Zeiss Apo Tome, Germany). For each condition, at least 100 randomly selected cells were analyzed. The quantification of the tail moment, which is a representation of the fluorescence intensity in the tail relative to the head, was performed using CometScore (Autocomet.com).
Micronuclei counts.
For micronuclei analysis (46,47), cells were seeded and fixed as described in the immunofluorescence assays. The cells were then incubated with SYBR gold (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 1 min, washed twice with PBS, and mounted for microscopy. Objects were defined as micronuclei if they were clearly separated from the nuclei, were round- to oval-shaped with distinct borders, had an area of less than a quarter of the area of a nucleus, and showed staining characteristics similar to those of nuclei.
RNA analysis and quantitative real-time polymerase chain reaction (qRT-PCR).
Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA, USA). RNA was reverse-transcribed into first-strand cDNA using AMV reverse transcriptase (Promega, Madison, WI, USA). cDNA was amplified with KAPA SYBR Fast PCR Mix (KAPA Biosystems, Woburn, MA, USA) and subjected to analysis using a CFX Connect Real-Time System thermal cycler (Bio-Rad, Hercules, CA, USA). RPL30 mRNA, which encodes the ribosomal protein L30, was used as an internal control. The
relative abundance of mRNA was calculated after normalization with RPL30 mRNA using the CT equation. For verifying candidate genes in microarray data, primers were either designed based on the coding sequence of each genes using Primer3Plus(78) or directly retrieved from Origene website (http://www.origene.com/). The primers used are listed in Table 9.
HR assay.
HR efficiency was measured in MCF7/DR-GFP cells, according to the previous report (79). The MCF7 DR-GFP cells harbor GFP-based chromosomal reporters. The stable cells possess two differential GFP mutant genes oriented as direct repeats and separated by a drug selection marker, the puromycin N-acetyltransferase gene. Transient expression of the I-SceI enzyme produces a DSB in one of the two GFP mutant genes.
The DSB can be repaired by HR between the two GFP mutant genes, resulting in the restoration of a functional GFP gene and the expression of GFP proteins. After knockdown of target genes for three days, cells were transfected with pCASce to express the I-SceI protein. GFP-positive cells were measured by flow cytometry (BD FACSCalibur, BD Biosciences, Miami, FL, USA) in 48 hr. Ds-Red was transfected in a parallel group as a control for transfection efficiency.
Plasmid based end-joining assay.
Plasmid end-joining assay was conducted as previously described (80). The pGL3-promoter plasmid (Promega), which harbors a luciferase reporter gene, was linearized by HindIII and confirmed by agarose gel electrophoresis. The linearized DNA was purified by gel extraction kit (Qiagen), dissolved in sterilized water, and transfected into cells after knockdown of target genes for three days. Luciferase protein was expressed when the cutting sites were repaired by end-joining. The luciferase activity
was assayed by Luciferase Assay System (Promega). A Renilla plasmid was co-transfected as a control.
Plasmid based MMEJ assay.
MMEJ efficiency was measured according to the previous report (81). The pSV40-MMEJ plasmid, which harbors a GFP reporter gene, was a gift from Dr. Nicolas Mermod. Plasmids were linearized by I-SceI, purified by gel extraction kit (Qiagen), and transfected into cells after knockdown of target genes for three days. GFP protein was expressed when the cutting sites were repaired by MMEJ. The pGK-GFP plasmid was transfected in parallel as a transfection efficiency control. Expression of GFP was measured by flow cytometry (FACSCalibur, BD BioSciences) after 48 hr of transfection.
Statistical analysis.
Each experiment was repeated at least three times with comparable results. Results are expressed as mean ± SD. All statistical analyses were performed using Excel 2010 (Microsoft; Redmond, WA). The p-values for all experiments were obtained using Student’s t tests or one-way analysis of variance (35). A value of P < 0.05 was considered to be statistically significant.