Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells

SET and MYND domain containing protein 3 (SMYD3) is a histone methyltransferase, which has been implicated in cell growth and cancer pathogenesis. Increasing evidence suggests that SMYD3 can influence distinct oncogenic processes by acting as a gene-specific transcriptional regulator. However, the mechanistic aspects of SMYD3 transactivation and whether SMYD3 acts in concert with other transcription modulators remain unclear. Here, we show that SMYD3 interacts with the human positive coactivator 4 (PC4) and that such interaction potentiates a group of genes whose expression is linked to cell proliferation and invasion. SMYD3 cooperates functionally with PC4, because PC4 depletion results in the loss of SMYD3-mediated H3K4me3 and target gene expression. Individual depletion of SMYD3 and PC4 diminishes the recruitment of both SMYD3 and PC4, indicating that SMYD3 and PC4 localize at target genes in a mutually dependent manner. Artificial tethering of a SMYD3 mutant incapable of binding to its cognate elements and interacting with PC4 to target genes is sufficient for achieving an active transcriptional state in SMYD3-deficient cells. These observations suggest that PC4 contributes to SMYD3-mediated transactivation primarily by stabilizing SMYD3 occupancy at target genes. Together, these studies define expanded roles for SMYD3 and PC4 in gene regulation and provide an unprecedented documentation of their cooperative functions in stimulating oncogenic transcription.


Figure S1. Effects of SMYD3 knockdown on proliferation and invasion of bladder and colon cancer cells.
(A) Whole cell extracts were prepared from colon normal (CDC-18Co) and cancer (HCT116, CaCO2, and HT29) cells, bladder normal (UROtsa) and cancer (J82, T24 and RT4) cells, breast normal (MCF-10-2A) and cancer (MCF7 and MDA-MB-231) cells, and prostate normal (MLC) and cancer (LNCaP and DU145) cells. Equal amounts of extracts were analyzed by immunoblotting with anti-SMYD3 and anti-Actin antibodies. (B) HCT116, CaCO2, J82 and T24 cancer cells were transfected with lentiviruses expressing control shRNA (Control sh) or SMYD3-specific shRNA (SMYD3 sh), and knockdown efficiency was determined by immunoblotting. (C) HCT116, CaCO2, J82 and T24 cancer cells were transfected with control shRNA or SMYD3 shRNA, and their proliferation rates were determined by MTT colorimetric assays over a period of 4 days. Results represent the means of three independent experiments ± SD. (D) Control or SMYD3-depleted HCT116, CaCO2, J82 and T24 cancer cells were detached and seeded onto the upper chamber coated with Matrigel, and then allowed to invade toward 10% FBS in the lower chamber. The graph depicts the average number of invaded cells per four fields. Results represent the means ± SD of three independent experiments.

Figure S2. Validation of microarray data.
Microarray data were validated by qRT-PCR of seventeen genes whose expression was decreased upon SMYD3 knockdown and which are related to cell growth and invasion, and two unaffected genes. Primer sequences are listed in Table S3. The values are expressed as fold changes from the mRNA levels in control cells after normalization with ACTB. Data represent the means ± SD of three independent experiments. (A) SMYD3-depleted HCT116 colon cancer cells were infected with lentiviruses expressing RNAi-resistant FLAG-SMYD3 wild-type (wt) or enzymatic dead mutant (F183A), and whole cell extracts were analyzed by immunoblotting with the indicated antibodies. (B) ChIP-qPCR experiments were performed as in Figure 1D, but using primers specific for the MFGE8 gene. The vertical red bar represents the putative SMYD3 binding site. Figure S4. Effects of SMYD3 knockdown on target gene expression. Expression levels of the genes down-regulated (FNBP1, MFGE8, PDLIM7, and WNT3A) or unaffected (KRT81 and GAPDH) were analyzed by qRT-PCR on total RNA isolated from SMYD3-depleted CaCO2, J82 and T24 cancer cells. and H3K4me3 enrichment (D) at the four SMYD3 target genes (FNBP1, MFGE8, PDLIM7, WNT3A) and two control genes (KRT81 and GAPDH) in SMYD3-depleted HCT116, CaCO2, J82 and T24 cancer cells using primers depicted in (B).

Figure S6. Interaction of SMYD3 N-terminal deletion mutants with PC4.
GST alone or GST-PC4, immobilized on glutathione Sepharose beads, was incubated with Histagged SMYD3 deletion mutants. After washing with washing buffer, the bound SMYD3 proteins were immunoblotted with His antibody. Of the input proteins, 5% were examined by immunoblotting. Figure S7. Circular dichroism spectra of SMYD3 and PC4. Spectra of SMYD3 (black) and PC4 (red) are shown. In addition, the spectra of the SMYD3-PC4 complex is depicted in green, which matches the sum of the individual SMYD3 and PC4 spectra, shown in blue. Spectra were recorded at protein concentrations of 5 M in 5 mM K 2 HPO 4 /KH 2 PO 4 , pH 7.4, 25 mM KCl at 25 °C using a JASCO J-810 spectropolarimeter. The observed ellipticity in millidegrees, , was converted into the mean residue ellipticity,  MRW .

Figure S8. Interdependent localization of SMYD3 and PC4 at target genes.
(A) SMYD3-depleted HCT116 colon cancer cells were infected with lentiviruses expressing FLAG-tagged SMYD3 wild-type (wt) or K78/D82/R85 mutant (mt) lacking interaction with PC4, and cell lysates were prepared and analyzed by immunoblotting with anti-SMYD3, anti-FLAG, anti-PC4, and anti-Actin antibodies, as indicated. (B) ChIP-qPCR experiments were performed as in Figure 3C, but using primers specific for the MFGE8 gene. (C) Exogenous expression of FLAG-tagged PC4 wild-type (wt) or SMYD interaction-deficient Q65/R75 mutant (mt) in PC4depleted HCT116 colon cancer cells was confirmed by immunoblotting. (D) ChIP-qPCR assays were done with PC4-depleted HCT116 cells complemented with PC4 wild-type (wt) or mutant (mt) as in (B). The vertical red bar in (B) and (D) represents the putative SMYD3 binding site. (A) SMYD3-depleted HCT116 cells were transfected with SMYD3 wild-type (wt) or K78/D82/R85 mutant (mt), and the expression of the known PC4 target gene SMUG1 was determined by qRT-PCR. (B) SMYD3-depleted HCT116 cells were complemented with SMYD3 wild-type (wt) or K78/D82/R85 mutant (mt) as in (A), and the levels of SMYD3, PC4 and H3K4me3 along the SMUG1 gene were determined by ChIP-qPCR. (C) Wild type (wt) or Q65/R75-mutated (mt) PC4 was expressed in PC4-depleted HCT116 cells, and SMUG1 gene expression was determined by qRT-PCR. (D) ChIP signals for SMYD3, PC4 and H3K4me3 along the SMUG1 gene were determined as indicated.

Figure S10. dCas9-based activation of SMYD3 target genes.
qRT-PCR experiments were performed as in Figure 5C, but using sgRNAs specific for the MFGE8 gene.

Figure S11. dCas9-based accumulation of SMYD3 and H3K4me3 at target genes.
ChIP-qPCR experiments were performed as in Figure 6, but using sgRNAs and primers specific for the MFGE8 gene.