Regulation of estrogen receptor α by histone methyltransferase SMYD2-mediated protein methylation

Abstract

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor. Upon estrogen stimulation, ERα recruits a number of coregulators, including both coactivators and corepressors, to the estrogen response elements, modulating gene activation or repression. Most coregulator complexes contain histone-modifying enzymes to control ERα target gene expression in an epigenetic manner. In addition to histones, these epigenetic modifiers can modify nonhistone proteins including ERα, thereby constituting another layer of transcriptional regulation. Here we show that SET and MYND domain containing 2 (SMYD2), a histone H3K4 and H3K36 methyltransferase, directly methylates ERα protein at lysine 266 (K266) both in vitro and in cells. In breast cancer MCF7 cells, SMYD2 attenuates the chromatin recruitment of ERα to prevent ERα target gene activation under an estrogen-depleted condition. Importantly, the SMYD2-mediated repression of ERα target gene expression is mediated by the methylation of ERα at K266 in the nucleus, but not the methylation of histone H3K4. Upon estrogen stimulation, ERα-K266 methylation is diminished, thereby enabling p300/cAMP response element-binding protein-binding protein to acetylate ERα at K266, which is known to promote ERα transactivation activity. Our study identifies a previously undescribed inhibitory methylation event on ERα. Our data suggest that the dynamic cross-talk between SMYD2-mediated ERα protein methylation and p300/cAMP response element-binding protein-binding protein-dependent ERα acetylation plays an important role in fine-tuning the functions of ERα at chromatin and the estrogen-induced gene expression profiles.

Keywords: ERα hinge region; LSD1; lysine methylation.

Fig. 1.

SMYD2 methylates ERα in vitro. (A) Schematic representation of ERα protein domains. (B and C) ERα is methylated at K266 by SMYD2. Autoradiograms of SMYD2-dependent in vitro methylation of recombinant ERα fragments (B) and point mutant proteins (C) are shown. GelCode Blue staining shows equal amount of ERα and SMYD2 proteins used in the assays. (D) ERα is monomethylated and dimethylated by SMYD2. MS analysis of ERα peptide (amino acids 258–276) with or without SMYD2 incubation is shown. (E) ERα–K266 is evolutionally conserved. Alignment of amino acid sequences surrounding K266 in human ERα and ERα proteins from several other species is shown.

Fig. 2.

ERα is methylated by SMYD2 in cells. (A) SMYD2 methylates ectopic ERα at K266 in cells. Western blot analysis of ERα–K266me1 and total ERα levels in WCE and Flag IP of 293T cells cotransfected with Flag-WT ERα or K266R mutants and Myc-SMYD2 are shown. (B) The enzymatic activity of SMYD2 is required for ERα–K266 methylation in cells. Western blot analysis of ERα–K266me1 and total ERα levels in WCE and Flag IP of 293T cells cotransfected with Flag-ERα and MYC-WT SMYD2 or the Y240F catalytic mutant are shown. (C) SMYD2 is required for endogenous ERα–K266 methylation in MCF7 cells. Western blot analysis of ERα–K266me1 and total ERα levels in WCE and ERα IP in control (shCtrl) and SMYD2-knockdown (shSMYD2) MCF7 cells are shown. IgG IP is shown as a negative control. Tubulin is shown as a loading control. (D) K266-methylated ERα mainly resides in the nucleus. Western blot analysis of ERα, SMYD2, and ERα–K266me1 levels in the cytoplasmic and nuclear fractions of MCF7 cells is shown. Tubulin and histone H3 are shown as markers of the cytoplasm and nucleus, respectively. (E) ERα–K266 methylation levels decrease upon E2 treatment. Western blot analysis of ERα–K266me1 and total ERα levels in WCE and ERα IP of MCF7 cells with (+) or without (−) E2 treatment are shown.

Fig. 3.

Depletion of SMYD2 enhances the expression of ERα target genes. (A) Western blot analysis of ERα and SMYD2 protein levels in control and SMYD2-knockdown MCF7 cells. (B and C) Knockdown of SMYD2 enhances the expression of ERα target genes in MCF7 cells. Quantitative reverse transcription PCR (qPCR) analysis of gene expression in cells as in A at 3 and 6 h after E2 treatment. (D) Western blot analysis of ERα proteins in MDA-MB 231 cells stably expressing the WT ERα or ERα–K266R mutant. (E and F) ERα–K266R mutant exhibits increased transactivation activity compared with the WT ERα. qPCR analysis of gene expression in cells as in D at 3 and 6 h after E2 treatment is shown. Error bars represent SEM of three experiments.

Fig. 4.

SMYD2 attenuates ERα chromatin recruitment. (A and B) Depletion of SMYD2 enhances ERα chromatin recruitment. qPCR analysis of ERα ChIP in control and SMYD2-knockdown MCF7 cells 15 and 45 min after E2 treatment is shown. ERα occupancies on the ERE sites of target genes are shown in Upper. (C and D) Depletion of SMYD2 does not affect H3K4me2 levels on the enhancers of ERα target genes. qPCR analysis of H3K4me2 ChIP in control and SMYD2-knockdown MCF7 cells as in A is shown. (E and F) Depletion of SMYD2 does not decrease H3K4me3 levels on the promoters of ERα target genes. qPCR analysis of H3K4me3 ChIP in cells as in A is shown. All error bars indicate SEM of two or three independent experiments.

Fig. 5.

SMYD2-mediated methylation inhibits ERα–K266/268 acetylation. (A) Overexpression of SMYD2 increases ERα–K266 methylation and decreases K266/268 acetylation. Western blot analysis of ERα–K266me1 and ERα–K266/268ac and total ERα levels in WCE and Flag-ERα IP of 293T cells transfected with Flag-ERα with or without SMYD2 are shown. (B) Depletion of SMYD2 decreases K266 methylation and increases K266/268 acetylation on endogenous ERα protein. Western blot analysis of ERα–K266me1 and ERα–K266/268ac and total ERα levels in WCE and ERα IP in control and SMYD2-knockdown MCF7 cells are shown. (C) Depletion of LSD1 increases K266 methylation and decreases K266/268 acetylation on endogenous ERα protein. Western blot analysis of ERα–K266me1 and ERα–K266/268ac and total ERα levels in WCE and ERα IP in control and LSD1-knockdown MCF7 cells are shown. (D) Model of ERα–K266 methylation dynamically regulated by SMYD2 and LSD1 and its cross-talk with p300/CBP-dependent ERα–K266/268 acetylation in regulating ERα target gene expression.