Requirement of histone methyltransferase SMYD3 for estrogen receptor-mediated transcription

Abstract

SMYD3 is a SET domain-containing protein with histone methyltransferase activity on histone H3-K4. Recent studies showed that SMYD3 is frequently overexpressed in different types of cancer cells, but how SMYD3 regulates the development and progression of these malignancies remains unknown. Here, we report the previously unrecognized role of SMYD3 in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity. We demonstrate that SMYD3 functions as a coactivator of ERalpha and potentiates ERalpha activity in response to ligand. SMYD3 directly interacts with the ligand binding domain of ER and is recruited to the proximal promoter regions of ER target genes upon gene induction. Importantly, our chromatin immunoprecipitation analyses provide compelling evidence that SMYD3 is responsible for the accumulation of di- and trimethylation of H3-K4 at the induced ER target genes. Furthermore, RNA interference-directed down-regulation of SMYD3 reveals that SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathway. Thus, our results identify SMYD3 as a new coactivator for ER-mediated transcription, providing a possible link between SMYD3 overexpression and breast cancer.

FIGURE 1.

Direct interaction between SMYD3 and ER. A, schematic diagrams of the full-length ERα and SMYD3. B, in vitro interaction of SMYD3 with ERα. Recombinant ER and SMYD3 and in vitro-translated LBD+DBD and LBD were immunoprecipitated with anti-FLAG M2-agarose beads and analyzed by Western blot analysis with indicated antibodies. Asterisks indicate the purified protein. C, specific interaction of SMYD3 with ER LBD. In vitro translated SMYD3 was incubated with GST, GST-NTD+DBD, or GST-LBD immobilized on glutathione beads, and SMYD3 interaction was analyzed by Western blot analysis using anti-SMYD3 antibody. D, cellular interaction of SMYD3 with ER. FLAG-tagged SMYD3 and ER were transiently expressed in 293T cells, and cell extracts were immunoprecipitated with anti-FLAG antibody. Coimmunoprecipitation of ER was analyzed by Western blot using ER antibody. E, interaction between endogenous SMYD3 and ER. Cell lysates were prepared from MCF-7 cells and subjected to immunoprecipitation using anti- antibody. Coimmunoprecipitation of endogenous SMYD3 was determined by Western blot using anti-SMYD3 antibody. Linker histone H1 is also included as a negative control.

FIGURE 2.

Requirement of NHSC and EEL motifs for SMYD3 HMT activity. A, conserved amino acid sequences within SET domains. B, H3-targeted activity of SMYD3. HMT assays were performed with full-length SMYD3 or NHSC/EEL-deleted SMYD3 proteins expressed in 293T cells using [³H]AdoMet and recombinant histone octamers. Recombinant SET7/9 was included as a control. C, di-/trimethylation of H3–K4 by SMYD3. HMT assays were performed as in Fig. 2B, but using cold AdoMet. H3–K4 methylation was determined by Western blot with antibodies recognizing mono-/di-/trimethylation of H3–K4.

FIGURE 3.

Coactivator function of SMYD3 in ER transcription. A and C, effect of SMYD3 on ER-mediated transcription in vivo. MCF-7 (A) and 293T (C) cells in 12-well plates were transiently transfected with MMTV-ERE reporter gene together with ER expression vector and various amounts of SMYD3 expression vectors in uninduced and estrogen-induced conditions as indicated. The representative data from three independent experiments are shown, and the error bars indicate as the means ±S.E. B and D, requirement of HMT activity for SMYD function. Reporter gene assays were as in Fig. 3A and 3C, but with mutant SMYD3 expression vectors. E, in vitro interaction of ER with SMYD3 lacking NHSC/EEL motifs. ER NTD+DBD and LBD fused to GST were incubated with SMYD3 lacking NHSC/EEL motifs, and SMYD3 binding was analyzed by immunoblot with anti-SMYD3 antibody.

FIGURE 4.

Estrogen-induced accumulation of SMYD3 and H3–K4 methylation at ER target genes. MCF-7 cells were cultured under estrogen-deprived conditions for 3 days and subjected to ChIP analysis after treating with estrogen for 0, 30, 60, 90, and 120 min. ChIP assays were performed using antibodies specifically recognizing ER, SMYD3, and H3–K4 mono-/di-/trimethylation. Input DNA and immunoprecipitated DNA were quantified by real-time PCR using specific primer sets as indicated. The results are shown as percentage of input. The error bar indicates the means ± S.E.

FIGURE 4.

Estrogen-induced accumulation of SMYD3 and H3–K4 methylation at ER target genes. MCF-7 cells were cultured under estrogen-deprived conditions for 3 days and subjected to ChIP analysis after treating with estrogen for 0, 30, 60, 90, and 120 min. ChIP assays were performed using antibodies specifically recognizing ER, SMYD3, and H3–K4 mono-/di-/trimethylation. Input DNA and immunoprecipitated DNA were quantified by real-time PCR using specific primer sets as indicated. The results are shown as percentage of input. The error bar indicates the means ± S.E.

FIGURE 5.

Requirement of SMYD3 for ER transcription. A, validation of SMYD3 knockdown. MCF-7 cells were transfected with SMYD3 shRNA or control shRNA, and expression of SMYD3 protein was checked by Western blotting of whole cell lysates using anti-SMYD3 antibody. Actin was used as an internal control (lower panel). B, repression of ER transcription by SMYD3 knockdown. Cells were transfected with SMYD3 shRNA, and treated with 100 nm estrogen 24 h post-transfection. mRNA levels were analyzed by real-time PCR. Error bars indicates as the means ± S.E. from the experiments performed in duplicate, and the experiments were repeated three times.

FIGURE 6.

Reduction of H3–K4 methylation upon SMYD3 depletion. A, Western blot analysis of SMYD3 knockdown. MCF7 cells were transfected with SMYD3 shRNA for 24 h and subjected to Western blot analysis after E2 treatment for 0, 30, 60, 90, and 120 min. Actin was used as a loading control (lower panel). B–F, inhibition of H3–K4 methylation at pS2 gene upon SMYD3 knockdown. ChIP assays were performed using antibodies against ER, SMYD3, and mono-/di-/trimethylated H3–K4 as in Fig. 4, but after SMYD3 knockdown.