Figure 1. ChIP analysis.
(a) SMYD3, H2A.ZK101me2, H2A.Z, and H2A.Zac occupancy around TSSs. SMYD3 and H2A.ZK101me2 ChIP signals were normalized to input sequences. H2A.Z and
H2A.Zac datasets were obtained from the public database (GEO accession number GSM1059388). (b and c) The workflow of ChIP-seq analysis. Details were described in the results, and the materials and methods Excel files were kept in the Lab computer.
Figure 2. SMYD3 may be involved in DNA repair.
(a) 28 genes were identified in the cross-referenced data comparison between whole-genome microarray analysis of RNAs isolated from shLuc vs. shSMYD3 MCF7 cells and SMYD3 ChIP analysis. (b) The confirmation of the microarray analyses for the
expressions of candidate genes in the shSMYD3/shLuc dataset using qRT-PCR. The fold changes of each gene expression in the microarray data were listed in Table 1. **, P <
0.01, ***, P < 0.001 vs. shLuc control. All values in the histograms were means ± SD of triplicates and data were representative of n ≥ 3 for each experiment.
Figure 3. SMYD3 is required for DNA repair machinery.
(a) Whole-genome microarray analysis of RNAs isolated from shLuc vs. shSMYD3 MCF7 cells was conducted. With a cut-off of absolute normalized fold change ≦ 0.5 (log2 normalized ratios < -1), the list of down-regulated genes was further categorized by the DAVID v6.8 Gene Ontology program to reveal their gene ontology process. (b) γH2A.X foci formation at indicated times after 1.67 Gy IR treatment in shLuc or shSMYD3 MCF7 cells. *, P < 0.05. **, P < 0.01. (c) γH2A.X foci formation at indicated times after 1.67 Gy IR treatment in shLuc or shSMYD3 MCF7 cells complemented with the vector control, SMYD3 or mutant SMYD3Y239F. *, P < 0.05. **, P < 0.01. ***, P < 0.001.
Figure 4. SMYD3-depleted cells are hypersensitive to IR stress.
(a-c) Clonogenic survivals of shLuc and shSMYD3 MCF7 (a), MDA-MB-231 (b) and AU565 cells (c) treated with indicated dosages of IR. All values in the diagrams were means ± SD of triplicates and data were representative of n ≥ 3 for each experiment.
Figure 5. SMYD3 deficiency increases the ratio of DNA damage after IR treatment.
shLuc and shSMYD3 MCF7 cells were treated with 1.67 Gy IR and then analyzed for DNA damage by comet assay at indicated times. (a) Representative images with white arrows point to comet tails, which signify DNA damage. (b) Quantification of the percentage of nuclei with comet tails. (c) Quantification of comet tail moment using the
CometScore software. Data are presented as a quantile box plot. The middle lines in boxes indicate the median; upper and lower box edges indicate the 25th and 75th percentiles; and bars indicate the 10th and 90th percentiles. Statistical analysis was performed by one-way analysis variance (35). ***, P < 0.001.
Figure 6. SMYD3 mediates the HR pathway.
(a) Relative HR activity of shLuc, shSMYD3, and shEXO1 MCF7 cells, which was indicated by the percentage of GFP-positive cells relative to the shLuc control. The EXO1 knockdown cells were used as a positive control. ***, P < 0.001 vs. shLuc control. (b) Relative NHEJ activity of shLuc, shSMYD3 and shKu70 cells. NHEJ activity was measured by normalizing the luciferase activity to the renilla activity. The Ku70-knockdown cells were used as a positive control. ***, P < 0.001 vs. shLuc control.
(c) Relative MMEJ activity of shLuc, shSMYD3 and shPOLQ MCF7 cells, which was indicated by the percentage of GFP-positive cells relative to the shLuc control. The POLQ-knockdown cells were used as a positive control. ***, P < 0.001 vs. shLuc control.
Figure 7. The knockdown efficiency of each knockdown clones used.
The knockdown efficiency was validated by qRT-PCR six days after selected with puromycin. (a) Knockdown efficiency of SMYD3 and EXO1 in MCF7/DR-GFP cells. (b) Knockdown efficiency of SMYD3 and Ku70 in MCF7 cells. (c) Knockdown efficiency of SMYD3 and POLQ in MCF7 cells. ***, P < 0.001 vs. shLuc control. All values in the histograms were means ± SD of triplicates and data were representative of n ≥ 3 for each experiment.
Figure 8. Complementation of SMYD3 in shSMYD3 cells is able to recover its HR deficiency.
(a) Western blotting of cells subjected to Luc or SMYD3 knockdown and complemented with vector control or Myc-tagged SMYD3 or mutant SMYD3Y239F. (b) Exogenous expression of SMYD3, but not the mutant SMYD3Y239F, restored the HR activity. ***, P < 0.01 vs. shLuc with the expression of the vector plasmid control. N.S.:
not significant.
Figure 9. SMYD3 knockdown downregulates HR gene expressions.
The confirmation of the microarray analyses for the expressions of candidate genes in the shSMYD3/shLuc dataset using qRT-PCR. The fold changes of each gene expression in the microarray data were listed below. *, P < 0.05, **, P < 0.01, ***, P < 0.001 vs.
shLuc control. All values in the histograms were means ± SD of triplicates and data were representative of n ≥ 3 for each experiment.
Figure 10. SMYD3 knockdown downregulates the expression of DNA repair foci.
(a and b) DNA repair foci formation of MDC1 foci (a) and BRCA1 foci (b) formation at indicated times after 1.67 Gy IR treatment in shLuc or shSMYD3 MCF7 cells. *, P <
0.05. **, P < 0.01. ***, P < 0.001. All values in the histograms were means ± SD of triplicates and data were representative of n ≥ 3 for each experiment.
Figure 11. SMYD3 regulates the expression of MDC1 through methylating histone H3K4.
(a) ChIP assay was performed in MCF7 cells using specific antibodies. The examined positions at the MDC1 locus were indicated, in which region S3 and region TA for the predicted SMYD3 and TATA box binding sites, respectively. (b-e) ChIP assays were performed with SMYD3-repressed MCF7 cells using specific antibodies shown at the top of the histogram. Fold enrichment of each antibody compared with IgG was shown.
(f) Ratios of H3K4me3/H3 ChIP signals were displayed. In (b-f), immunoprecipitated chromatin was quantified by qRT-PCR. *, P < 0.05. **, P < 0.01. All values in the histograms were means ± SD of triplicates and data were representative of n ≥ 3 for each experiment.
Figure 12. SMYD3 activates the expression of EXO1 and RAD54B through methylating histone H3K4.
(a and g) ChIP assays were performed in MCF7 cells using specific antibodies. The examined positions at EXO1 and RAD54B loci were indicated, in which region S3 and region TA are predicted SMYD3 and TATA box binding sites, respectively. (b-e and h-k) ChIP assays were performed with SMYD3-repressed MCF7 cells using specific
antibodies indicated at the top of the histogram. Fold enrichment of each antibody compared with IgG was shown. (f and l) Ratios of H3K4me3/H3 ChIP signals were shown. In b-f and h-l, immunoprecipitated chromatin was quantified by qRT-PCR. *, P
< 0.05. **, P < 0.01. All values in the histograms were means ± SD of triplicates and data were representative of n ≥ 3 for each experiment.
Figure 13. SMYD3 methylates histone and non-histone substrates to regulate different pathways that are important for hallmarks of cancer.
SMYD3 directly methylates proteins that are involved in cell proliferation and angiogenesis. Moreover, SMYD3 methylates histones that are widely spread on chromatin to facilitate transcription of target genes. Hence, the high level of SMYD3 stimulates cancer development.
Chapter 6. Table.
Table 1. Down-regulated genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences) cross -referenced with SMYD3 ChIP-seq data.
Log ratio Ratio Gene Title/Gene Symbol
-1.0 0.50 Anillin, Actin Binding Protein/ANLN
-1.0 0.50 DNA (Cytosine-5-)-Methyltransferase 3 Beta/DNMT3B -1.0 0.50 Geminin, DNA replication inhibitor/GMNN
-1.0 0.50 5'-nucleotidase, cytosolic II/NT5C2 -1.0 0.50 POC1 centriolar protein A/POC1A
-1.0 0.50 RAD54 homolog B (S. cerevisiae)/RAD54B
-1.0 0.50 T-cell lymphoma invasion and metastasis 1/TIAM1 -1.1 0.47 Exonuclease 1/EXO1
-1.1 0.47 Fanconi anemia, complementation group I/FANCI
-1.1 0.47 IQ motif containing GTPase activating protein 3/IQGAP3 -1.1 0.47 Opa interacting protein 5/OIP5
-1.1 0.47 Polymerase (DNA directed), alpha 2, accessory subunit/POLA2 -1.1 0.47 Polymerase (DNA directed), theta/POLQ
-1.2 0.44 Low density lipoprotein receptor-related protein 1/LRP1
-1.2 0.44 Protein kinase, cAMP-dependent, regulatory, type II, beta/PRKAR2B -1.2 0.44 Ret proto-oncogene/RET
-1.2 0.44 Solute carrier family 7 (amino acid transporter light chain, L system), member 5/SLC7A5
-1.3 0.41 ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 2/
ST6GALNAC2
-1.4 0.38 Integrin, alpha 9/ITGA9
-1.4 0.38 V-myb avian myeloblastosis viral oncogene homolog-like 2/MYBL2 -1.5 0.35 Glutathione peroxidase 2 (gastrointestinal)/GPX2
-1.5 0.35 Mediator of DNA-damage checkpoint 1/MDC1
-1.5 0.35 Nuclear receptor subfamily 5, group A, member 2/NR5A2 -1.6 0.33 Cytochrome B Reductase 1/CYBRD1
-1.6 0.33 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 12 (GalNAc-T12)/GALNT12 -1.7 0.31 Glutamate decarboxylase 2 (pancreatic islets and brain, 65kDa)/GAD2
-1.8 0.29 EGF containing fibulin-like extracellular matrix protein 1/EFEMP1
-1.8 0.29 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1/SERPINA1
Table 2. Up-regulated genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences) cross -referenced with SMYD3 ChIP-seq data.
Log ratio Ratio Gene Title/Gene Symbol
3.6 12.13 DnaJ (Hsp40) homolog, subfamily B, member 8/DNAJB8
2.8 6.96 Glycerophosphodiester phosphodiesterase domain containing 1/GDPD1 2.7 6.50 Cytoplasmic polyadenylation element binding protein 1/CPEB1
2.6 6.06 Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A/SEMA6A 2.5 5.66 Hydroxysteroid (17-beta) dehydrogenase 14/HSD17B14
1.9 3.73 Tumor necrosis factor (ligand) superfamily, member 10/TNFSF10 1.8 3.48 RAR-related orphan receptor C/RORC
1.5 2.83 CD36 molecule (thrombospondin receptor)/CD36 1.4 2.64 HCLS1 binding protein 3/HS1BP3
1.4 2.64 Ring finger protein 43/RNF43
1.3 2.46 Osteopetrosis associated transmembrane protein 1/OSTM1
1.2 2.30 Protein kinase (cAMP-dependent, catalytic) inhibitor gamma/PKIG 1.2 2.30 RAB7, member RAS oncogene family-like 1/RAB7L1
1.2 2.30 Yippee-like 3 (Drosophila)/YPEL3
1.2 2.30 Zinc finger, DHHC-type containing 11/ZDHHC11
1.1 2.14 BCL2/adenovirus E1B 19kD interacting protein like/BNIPL 1.1 2.14 Cytochrome c oxidase subunit VIIb/COX7B
1.1 2.14 DENN/MADD domain containing 1B/DENND1B 1.1 2.14 Fibrosin-like 1/FBRSL1
1.1 2.14 Radial spoke head 1 homolog (Chlamydomonas)/RSPH1
1.1 2.14 TM2 domain containing 1/TM2D1 1.1 2.14 Zinc finger protein 44/ZNF44
1.0 2.00 Aminoadipate-semialdehyde synthase/AASS
1.0 2.00 B-cell CLL/lymphoma 11A (zinc finger protein)/BCL11A 1.0 2.00 Diacylglycerol O-acyltransferase 1/DGAT1
1.0 2.00 ORAI calcium release-activated calcium modulator 3/ORAI3 1.0 2.00 Peroxisomal biogenesis factor 19/PEX19
1.0 2.00 Retinol saturase (all-trans-retinol 13,14-reductase)/RETSAT 1.0 2.00 SUMO1/sentrin/SMT3 specific peptidase 2/SENP2
1.0 2.00 Sbiquitin-conjugating enzyme E2I/UBE2I
Table 3. Down-regulated genes in H2A.Z.1WT/H2A.Z.1K101Q array data (genes with < 0.5 fold differences) cross-referenced with H2A.Z.1K101me2 ChIP-seq data.
Log ratio Ratio Gene Title/Gene Symbol -1.1 0.47 Cadherin 18, type 2/CDH18
-1.3 0.41 Heat shock 22kDa protein 8/HSPB8
-2.3 0.20 DnaJ (Hsp40) homolog, subfamily C, member 2/DNAJC2 -2.3 0.20 Hemoglobin, alpha 1/HBA1
-2.4 0.19 ELL associated factor 2/EAF2
-3.3 0.10 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1/APOBEC1 -3.4 0.09 Sperm associated antigen 17/SPAG17
-3.6 0.08 Lysophosphatidic acid receptor 5/LPAR5
Table 4. Down-regulated genes in H2A.Z.1WT/H2A.Z.1K101Q array data (genes with < 0.5 fold differences) cross-referenced with H2A.Z.1K101me2 ChIP-seq data.
Log ratio Ratio Gene Title/Gene Symbol
4.3 19.70 Reversion-inducing-cysteine-rich protein with kazal motifs/RECK 3.5 11.31 Zyg-11 related, cell cycle regulator/ZER1
3.0 8.00 Developmental pluripotency associated 2/DPPA2 2.9 7.46 SRSF protein kinase 2/SRPK2
2.3 4.92 Reticulon 1/RTN1
2.2 4.59 B-cell CLL/lymphoma 9/BCL9 1.4 2.64 Ankyrin repeat domain 45/ANKRD45 1.4 2.64 Proline-rich coiled-coil 1/PRRC1
1.3 2.46 Alanyl (membrane) aminopeptidase/ANPEP 1.3 2.46 Tripartite motif containing 16-like/TRIM16L
1.3 2.46 Xenotropic and polytropic retrovirus receptor 1/XPR1 1.2 2.30 ARFGEF family member 3/KIAA1244 (ARFGEF3) 1.1 2.14 Karyopherin alpha 5 (importin alpha 6)/KPNA5 1.0 2.00 Carbonic anhydrase VA, mitochondrial/CA5A 1.0 2.00 Early growth response 1/EGR1
Table 5. GO analysis of down-regulated genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences) cross -referenced with SMYD3 ChIP-seq data.
Term Genes Count p-value Benjamini
DNA metabolic process DNMT3B, FANCI, RAD54B, EXO1, MDC1, POLA2, POLQ 7 2.0E-4 7.6E-2
DNA repair MDC1, EXO1, RAD54B, POLQ, FANC, 5 1.5E-3 2.5E-1
Cell cycle FANCI, OIP5, RAD54B, ANLN, EXO1, GMNN, MDC1 7 1.9E-3 2.2E-1
M phase OIP5, RAD54B, ANLN, EXO1 4 2.0E-2 7.3E-1
Table 6. Down-regulated DNA damage stimulus response genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences).
Function Gene Title/Gene Symbol
Antioxidant Thioredoxin domain containing 12/TXNDC12 Base excision repair Nei like DNA glycosylase 3/NEIL3
Base excision repair Nudix hydrolase 1/NUDT1
Base excision repair DNA polymerase delta 1, catalytic subunit/POLD1
Cell cycle Cyclin A2/CCNA2
Cell cycle Cyclin dependent kinase 1/CDK1
Checkpoint effector G2 and S-phase expressed 1/GTSE1 DNA replication Thymidylate synthetase/TYMS
DNA replication Minichromosome maintenance complex component 7/MCM7 DNA replication DNA polymerase epsilon 2, accessory subunit/POLE2
DNA replication and repair Topoisomerase (DNA) II alpha/TOP2A
G2/M checkpoint Denticleless E3 ubiquitin protein ligase homolog/DTL
HR BRCA1 associated RING domain 1/BARD1
HR RAD54 homolog B (S. cerevisiae)/RAD54B
HR RAD18, E3 ubiquitin protein ligase/RAD18
HR Ubiquitin like with PHD And Ring finger domains 1/UHRF1
HR RAD51 recombinase/RAD51
HR BRCA2, DNA repair associated/BRCA2
HR Checkpoint kinase 1/CHEK1
HR Exonuclease 1/EXO1
HR RAD51 associated protein 1/RAD51AP1
HR DNA ligase 1/LIG1
HR, NHEJ Mediator Of DNA damage checkpoint 1/MDC1
HR, mitotic check point Thyroid hormone receptor interactor 13/TRIP13 HR, interstrand cross-links Fanconi anemia complementation group D2/FANCD2 Interstrand cross-links DNA cross-link repair 1B/DCLRE1B
Interstrand cross-links Fanconi anemia complementation group I/FANCI Interstrand cross-links Fanconi anemia complementation group B/FANCB
MMEJ DNA polymerase theta/POLQ
NHEJ Pituitary tumor-transforming 1/PTTG1
Postreplication repair Ubiquitin conjugating enzyme E2 V2/UBE2V2
Sister chromatid cohesion Establishment of sister chromatid cohesion N-acetyltransferase 2/ESCO2
Table 7. Down-regulated HR genes in shSMYD3/shLuc array data (genes with < 0.5 fold differences).
Log ratio Ratio Gene Title/Gene Symbol
-1.6 0.33 SET and MYND domain containing 3/SMYD3 -1.6 0.33 Thyroid hormone receptor interactor 13/TRIP13 -1.5 0.35 Mediator of DNA-damage checkpoint 1/MDC1 -1.4 0.38 BRCA1 associated RING domain 1/BARD1
-1.3 0.41 Ubiquitin-like with PHD and ring finger domains 1/UHRF1 -1.2 0.44 BRCA1 associated RING domain 1/BARD1
-1.1 0.47 Fanconi anemia, complementation group D2/FANCD2 -1.1 0.47 Breast cancer 2, early onset/BRCA2
-1.1 0.47 Exonuclease 1/EXO1
-1.1 0.47 RAD18 homolog (S. cerevisiae)/RAD18
-1.1 0.47 RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)/RAD51 -1.0 0.50 CHK1 checkpoint homolog (S. pombe)/CHEK1
-1.0 0.50 Fanconi anemia, complementation group D2/FANCD2 -1.0 0.50 Ligase I, DNA, ATP-dependent/LIG1
-1.0 0.50 RAD51 associated protein 1/RAD51AP1 -1.0 0.50 RAD54 homolog B (S. cerevisiae)/RAD54B
Table 8. Oligo sequences for shRNA-mediated gene knockdown
Clone ID Gene Symbol Target Sequence Region
TRCN0000123290 SMYD3 GCTTCCCGATATCAACATCTA CDS
TRCN0000123291 SMYD3 CAACTCTTTCACCATCTGTAA CDS
TRCN0000331178 EXO1 TGCAGACTGCTGCAAAGCTTT 3’UTR
TRCN0000010332 EXO1 AATGCAGACTGCTGCAAAGCT CDS
TRCN0000332901 XRCC6 (KU70) GATGAGTCATAAGAGGATCAT CDS
TRCN0000332902 XRCC6 (KU70) CCCAAGGTTGAAGCAATGAAT CDS
TRCN0000290546 POLQ CCTTCAATCTTGCTTGCGAAA CDS
TRCN0000290546 POLQ GCTGACCAAGATTTGCTATAT CDS
Table 9. Primers used in this study
Genes Forward sequence Reverse sequence Assay
SMYD3 TTACTGCGAGCAGTCCGAGACA TTGTCCTGGGTTTGGCAACGGA SYBR green qPCR
TRIP13 CAGCAGCACTGCAAAGAAAG AAATCGATGGGCTGTGAGTC SYBR green qPCR
MDC1 GCAAGATGCCACCTGCTGAGAA GCTTCAGGTACTGTAGGAGGCA SYBR green qPCR
BARD1 TGTGGTTTAGCCCTCGAAGT GCCCTCTCAGAAACATCTGC SYBR green qPCR
UHRF1 TGTGGACCATGGGAATTTTT GGGAGCAAAGCAGTTGAGAG SYBR green qPCR
FANCD2 TTCCAGGATGCCTTCGTAGTGG GCAGGAGGTTTATGGCAATCCC SYBR green qPCR
BRCA2 GGCTTCAAAAAGCACTCCAGATG GGATTCTGTATCTCT TGACG TTCC SYBR green qPCR EXO1 TCGGATCTCCTAGCTTTTGGCTG AGCTGTCTGC ACATT CCTAG CC SYBR green qPCR RAD18 GGATTGTCCTGTTTGCGGGGTT GTTTTGGGCA GCGGC TTCCT TT SYBR green qPCR RAD51 TCTCTGGCAGTGATGTCCTGGA TAAAGGGCGG TGGCA CTGTC TA SYBR green qPCR CHEK1 GTGTCAGAGTCTCCCAGTGGAT GTTCTGGCTG AGAAC TGGAG TAC SYBR green qPCR
LIG1 TCACAGAGGCTGAAGTGGC TCAGGCTCTG AAACG CTTTC CG SYBR green qPCR
RAD51AP1 CTTCTGGAAGGCAGTGATGGTG AGAGAAGTCTTCGTCATTATCCTC SYBR green qPCR RAD54B TCATGATCTG CTTGA CTGTG AG TTTTTCCAACGAATCACCTGT SYBR green qPCR
KU70 TGCCACAGGA AGAAG AGTTG CTCTG GAGTT GCCAT GATTT SYBR green qPCR
POLQ CTTGTGGCAT CTCCT TGGAG CA AATCC CTTGG CTGGT CTCCA TC SYBR green qPCR
MDC1 CCTCTCAAAGTGGTGGGATT AATTGCTTGAACCCAGAAGG Region S3: -530~-377 bp
MDC1 AGGAGAATCGCTTGAACCTG CTTAAAGGCTGTCCCCACCT Region TA: -234~-49 bp
EXO1 TCACCTGAGGTTGGGAGTTC ACTGCAACCTCTGCCTCCT Region S3: -2278~-2114 bp
EXO1 AAGGCCCATTTTCAAGGTCT ATTCAGTTCACGCTGGGTTC Region TA: -386~-237 bp
RAD54B AGACCTCCCCAGATGATTCC CCCGAATAGCTGGGACTACA Region S3: -5262~-5049 bp RAD54B TTCGTTTCTATATTCCCAGAACCT ATGATTCGGTGTGTGCGATA Region TA: -398~-247 bp