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. Author manuscript; available in PMC: 2009 May 9.
Published in final edited form as: Mol Cell. 2008 May 9;30(3):336–347. doi: 10.1016/j.molcel.2008.03.022

Regulation of Estrogen Receptor Alpha by the SET7 lysine methyltransferase

Krithika Subramanian 1, Da Jia 2, Priya Kapoor-Vazirani 1, Doris R Powell 1, Robert E Collins 2, Dipali Sharma 3,5, Junmin Peng 4, Xiaodong Cheng 2, Paula M Vertino 1,5,*
PMCID: PMC2567917  NIHMSID: NIHMS50808  PMID: 18471979

Summary

Estrogen receptor α (ER) is a ligand-dependent transcription factor. Upon binding estrogen, ER recruits coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes. In addition to histones, coactivators can modify ER itself and other proteins in the transactivation complex. Here, we show that ER is directly methylated at lysine 302 (K302) by the SET7 methyltransferase. SET7-mediated methylation stabilizes ER and is necessary for the efficient recruitment of ER to its target genes, and their transactivation. The SET7-ER complex structure reveals the molecular basis for ER peptide recognition and predicts that modifications or mutations of nearby residues would affect K302 methylation. Indeed, a breast cancer-associated mutation at K303 (K303R) alters methylation at K302 in vitro and in vivo. These findings raise the possibility that generation, recognition, and removal of modifications within the ER hinge region generates “ER modification cassettes” that yield distinct patterns for signaling downstream events.

Introduction

The steroid hormone estrogen plays a critical role in normal mammary gland development. The biological effects of estrogen are mediated by estrogen receptors. Estrogen receptor alpha (ER) is an estrogen-dependent transcription factor that binds to specific DNA sequences called estrogen response elements (ERE) located in ER-target genes. The ER-ERE complex interacts with coactivator proteins, which alter local chromatin structure and facilitate the recruitment of the transcription machinery to activate target gene expression.

ER contains several functional domains that are conserved in the nuclear hormone receptor family of transcription factors (Enmark and Gustafsson, 1999): amino-terminal transcription activation and DNA-binding domains, which are connected to the C-terminal ligand-binding domain via a polypeptide stretch known as the hinge region (Figure 1A). Ligand binding and interaction with DNA induces a conformational change in ER, and synergistic interactions between functional domains generate interaction surfaces that recruit coactivator complexes. The p160 class of coactivators, which include Nuclear receptor Coactivators (NCoA1, NCoA2 and NCoA3), act as bridging complexes that in turn recruit other cofactors (Leo and Chen, 2000) with intrinsic or associated histone acetyltransferase (HAT) and histone methyltransferase (HMTase) activities (Hall and McDonnell, 2005; Hermanson et al., 2002). The HATs recruited to ER-targets include p300/CBP (Hall and McDonnell, 2005). HMTases shown to act as ER coactivators include both lysine-specific (eg. G9a) and arginine-specific HMTases (eg. PRMT1 and CARM1) (Carling et al., 2004; Koh et al., 2001; Lee et al., 2006; Mo et al., 2006; Rayasam et al., 2003). The generally accepted model is that the ordered recruitment of coactivator complexes culminates in alterations in local chromatin structure allowing access of the basal transcription machinery to DNA, and activation of gene expression.

Figure 1. SET7 methylates ER in vitro.

Figure 1

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(A) (Top Panel) Schematic representation of ER indicating domains conserved in the nuclear hormone receptor superfamily- activation function-1 (AF-1), DNA-binding domain (DBD), hinge region (Hinge) and Ligand-binding domain (LBD). (Bottom Panel) The conserved acetylation motif is highlighted in grey. The sequence methylated by SET7 in various known substrates is indicated. The asterisk indicates the target lysine residue.

(B) In vitro methylation assays were carried out using peptide substrates (H3 residues 1-24, ER residues 294-314) and three recombinant purified HMTases, SET7, DIM-5 and G9a (Collins et al., 2005; Zhang et al., 2003). Peptides (50 μM) were incubated with 0.1 μM HMTase at 37° C in the presence of 3H-labeled AdoMet. The reactions were terminated by adding SDS-PAGE loading buffer, resolved on a 20% polyacrylamide gel and analyzed by autoradiography.

(C) In vitro methylation assays were carried out using peptide substrates (ER: SPLMIKRSKKNSLALSLTADQ, GR: MNLEARKTKKKIKGIQQATTG, AR: MTLGARKLKKLGNLKLQEEGE, MR: MNLGARKSKKLGKLKGIHEEQ; ER-K303R: SPLMIKRSKRNSLALSLTADQ; ER-K303Ac: SPLMIKRSK(acetyl-K)NSLALSLTADQ) and recombinant SET7. Reactions contained 50 mM glycine pH 9.8, 1.8 μM [methyl-3H] AdoMet (14.9 Ci/mmol; NEN), 0.25 μM SET7 and 50 μM peptide and were incubated at 37 °C for 20 min. Methylation was analyzed either by precipitation with 20% TCA, collection on Whatman GF/F filters, and liquid scintillation counting (top) or by separation on 17% polyacrylamide-SDS gels and fluorography (bottom). Activity on the various peptides is calculated relative to wild-type ER Data represent the mean ± SD of two independent determinations.

In addition to modifying histones at ER target genes, coactivators also modify components of the transactivation complex, as well as ER itself. For instance, arginine methylation of p300 by CARM1 inhibits the interaction between p300 and NCoA2 (Lee et al., 2005). p300 acetylates several conserved lysines (K266, K268, K299, K302 and K303) within the hinge region of ER (Kim et al., 2006; Wang et al., 2001a). Acetylation of K266/K268 enhances the DNA-binding of ER (Kim et al., 2006). K302 and K303 lie within an acetylation motif ([K/R]XKK) that is conserved in ER across species, in the nuclear hormone receptor family, and in some other transcription factors like p53 (Fu et al., 2003). Acetylation of K303 attenuates ER-driven transcription, in part by antagonizing the phosphorylation of a neighboring serine (S305) (Cui et al., 2004). Moreover, an ER variant (ER-K303R) is susceptible to hyper-phosphorylation at S305 and displays higher transcriptional output at lower estrogen levels (Cui et al., 2004; Wang et al., 2001a). Somatic mutations affecting ER-K303 (K303R) have been identified in primary ductal hyperplasias and invasive breast tumors, suggesting that defects/alterations in post-translational modifications of ER may contribute to breast carcinogenesis (Conway et al., 2005; Fuqua et al., 2000).

SET7 (also known as SET9) is a lysine methyltransferase that methylates histone H3 lysine 4 (H3K4) (Nishioka et al., 2002; Wang et al., 2001b) and non-histone proteins with important roles in transcription, such as p53 and components of the TBP-complex, TAF10 and TAF7 (Chuikov et al., 2004; Couture et al., 2006; Kouskouti et al., 2004). The consensus recognition sequence for SET7-mediated lysine methylation, [R/K][S/T]K (Couture et al., 2006), closely resembles the conserved acetylation motif found in nuclear hormone receptors and some transcription factors (Fu et al., 2003), leading us to propose that this motif in ER is regulated by lysine methylation. Here, we show that K302 in the hinge region of ER is methylated by SET7. Down-regulation of SET7 in breast cancer cells leads to impaired recruitment of ER to its target genes and an attenuated estrogen-driven transcriptional response. SET7-mediated methylation modulates ER function by regulating ER turnover. These results demonstrate an important role for lysine methylation in ER signaling.

Results

SET7 methylates ER peptides in vitro

The conserved lysine K302 and neighboring residues in the hinge region of ER closely resemble the consensus SET7 recognition sequence (Couture et al., 2006) (Figure 1A). Recombinant SET7 methylated an ER peptide encompassing amino acids 294-314 of the hinge region, whereas two other histone H3 lysine 9-specific HMTases, G9a and Dim-5, did not (Figure 1B). In contrast, all three enzymes were able to methylate a histone H3 peptide (amino acids 1-24), suggesting that the methylation of the ER peptide by SET7 is specific.

Three lysines (K299, K302 and K303) are encompassed in the ER peptide. Mass spectrometric analysis of the products of the in vitro methylation reaction showed that the methylated ER peptide differs in mass from the unmodified ER peptides by 14 Da (Figures S1A and S1B), indicating that SET7 mono-methylates ER, akin to its other substrates (Chuikov et al., 2004; Couture et al., 2006; Kouskouti et al., 2004; Zhang et al., 2003). Subsequent MS/MS analysis identified lysine 302 (K302) as the single site of methylation (Figure S1C).

A consensus SET7 motif can be identified in the hinge region of other nuclear hormone receptors (Figure 1A). SET7 also methylated peptides corresponding to the glucocorticoid receptor and the mineralocorticoid receptor, but not that of the androgen receptor, confirming the requirement for a serine or threonine in -1 position relative to the target lysine.

ER is methylated at K302 in vivo

To facilitate our studies of ER methylation, an antibody was raised against an ER peptide mono-methylated at K302 (ER-K302me1). Dot blot analysis indicates that the antibody recognizes the ER peptide mono-methylated at K302 with a 200- to 1000-fold greater efficiency than the unmethylated ER peptide (Figure S2A).

HeLa cells, which do not express ER, were transiently transfected with expression constructs for ER, and K302 methylation was assessed after immunoprecipitation of ER by western blot analysis using the anti ER-K302me1 antibody. Methylated ER was detected in cells expressing wild-type ER, but not the ER-K302R mutant (Figure 2A). Interestingly, mutation of ER-K303 to arginine suppressed methylation at K302 (Figure 2A). Co-expression of exogenous SET7 was able to rescue the methylation defect in the ER-K303R mutant to some extent, but had no impact on methylation of the ER-K302R mutant (Figure 2A). We also evaluated ER methylation in MCF7 cells, which express endogenous ER. Methylated ER was detected in MCF7 cells over-expressing both SET7 and wild-type ER, but not the ER-K302R mutant (Figure 2B, upper left panel). Reciprocal immunoprecipitations performed with the anti ER-K302me1 antibody confirmed the presence of methylated ER in the same MCF7 cell lysates (Figure 2B, upper right panel). These data indicate that ER is methylated at K302 in vivo, and that the neighboring lysine, K303, impacts methylation of K302 by SET7.

Figure 2. ER is methylated in vivo.

Figure 2

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(A) HeLa cells were transfected with expression constructs for Myc-SET7 and wild-type (WT) or mutant (K302R, K303R)) ER, or a vector control (-). (Top) After 48 hr, ER was immunoprecipitated from 1 mg protein lysate using an anti-ER antibody and analyzed by western blotting with anti-ER K302me1 or anti-ER antibodies. (Bottom) One-tenth of input was analyzed by western blotting with the indicated antibodies.

(B) MCF7 cells were transfected with expression constructs for Myc-SET7 and wild-type (WT) or mutant (K302R) ER or vector (-). (Upper left) After 48 hr, total ER was immunoprecipitated from 1 mg protein lysates using an anti-ER antibody and analyzed by western blot with the anti-ER K302me1 or anti-ER antibodies. (Upper right) The same protein lysates (1 mg) used in lanes 5 and 7 were immunoprecipitated with the anti-ER K302me1 antibody and analyzed by western blot with an anti-ER antibody. (Bottom) One-tenth of input was analyzed by western blot with the indicated antibodies.

Despite extensive efforts, we were unable to detect methylation of endogenous ER in MCF7 cells (eg. Figure 2B, lanes 2,3), suggesting that the pools of this form may be very low or transient. ER-K302me1 might be rapidly demethylated by a lysine-specific demethylase, or further modified to the di-or tri-methylated form, as has been shown for SMYD2-mediated methylation of p53 (Huang, et al., 2007). Alternatively, ER-K302me1 may be subject to (or occur primarily in the context of) modification at nearby residues (eg. acetylation at K303), affecting recognition by the K302me1-specific antibody. Indeed, acetylation of K303 reduced the affinity of the antibody for methylated ER peptides by ~ 2-fold (Figure S2B).

The above data suggest that the K303R mutation inhibits methylation at K302 in vivo. Although a reduced affinity of the K302me1-specific antibody for methylated ER resulting from mutation at K303 might contribute to the apparent reduction in methyl ER levels, dot blot analysis of the corresponding ER peptides suggests that this is not the case (Figure S2B). Nevertheless, to directly address the impact of K303R mutation on SET7-mediated methylation at K302, we performed in vitro methylation reactions on ER peptides. Interestingly, in the context of a peptide, the K303R mutant was a better substrate for methylation at K302 than wild-type ER (Figure 1C). These data suggest that in the absence of other factors or modifications, the ER-K303R mutant is an efficient substrate for K302 methylation. However, the situation in cells appears more complex, likely due to the added influence of K303R on nearby modifications (eg. S305 phosphorylation) or protein binding, which may interfere with SET7-mediated methylation.

Regulation of ER protein stability by SET7

To address the role of SET7-mediated methylation in regulation of ER function, we created derivatives of the ER+ breast cancer cell line MCF7 stably knocked-down for SET7 using an shRNA approach (referred to as shSET7 cells, see Experimental Procedures). A correlation between SET7 and ER protein expression was observed: independent clones stably knocked-down for SET7 showed a concomitant decrease in the steady-state levels of ER protein (Figure 3A). Steady-state levels of ER mRNA did not differ significantly between MCF7, control clones and shSET7 clones (Figure S3). These data suggested that knockdown of SET7 might affect post-transcriptional regulation of ER, which is known to occur at the level of proteasome-mediated protein degradation (Nawaz et al., 1999; Wijayaratne and McDonnell, 2001). We tested whether the down-regulation of ER in shSET7 cells was due to alterations in protein stability by pulse-chase analysis. In agreement with previous reports (Nirmala and Thampan, 1995; Pakdel et al., 1993), we found that endogenous ER is a short-lived protein with a half-life of about 3 hr (range 2.3 to 3.4 hr, n=2) (Figure 3B). In contrast, the half-life of ER in shSET7 cells was decreased by about 2-fold (range 1.35-1.4 hr, n=2) (Figure 3B), suggesting that SET7 stabilizes ER. We also tested the impact of mutations to the SET7 methylation site (K302) on ER stability. Flag tagged wild-type ER expressed ectopically in MCF7 cells was more stable than endogenous ER, with a half-life of >8hr (Figure 3C,D). Mutation of K302 to R, or to A, significantly destabilized ER, and reduced the half life by more than 2-fold (range 3-4 hr, n=3). Down-regulation of SET7 resulted in a 2-fold reduction in the half-life of wild-type ER (to ~ 4hrs n=3) but had no effect on the stability of the ER K302 mutants (Figure 3E, F). Taken together, these data indicate that SET7-mediated methylation modulates the stability of ER and this effect is mediated through modification at K302.

Figure 3. SET7 regulates ER protein stability.

Figure 3

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(A) MCF7 and shSET7 cell lysates were analyzed by western blot using antibodies to ER, SET7 and GAPDH (loading control).

(B) (Left Panel) MCF7 and shSET7 cells were pulsed with methionine/cysteine-free media containing 100 μCi of L-35S-methionine (Amersham Biosciences) for 1 hr followed by culture in regular growth media. Cells were harvested at various time points during the chase period and immunoprecipitated using an anti-ER antibody. Complexes were resolved by SDS-PAGE and the gel was fixed, dried and exposed to storage phosphor screens. A representative gel image is shown. Arrows indicate labeled ER. (Right Panel) The intensity of the labeled ER band was quantified by phosphorimage analysis using ImageQuant software (Molecular Dynamics) and the percent ER remaining was calculated relative to that at the beginning of the chase period (time zero). The mean values from two independent experiments are shown.

(C-F) MCF7 cells (C, D) or shSET7 cells (E, F) were transfected with expression constructs containing Flag-tagged wildtype (WT) or mutant (K303A, K303R) ER. After 1 day, cells were labeled in L-35S-methionine and subjected to pulse chase analysis as described in A except that immunoprecipitations were carried out using Flag-M2 agarose. (C, E) Representative gel images are shown. (D, F) The intensity of the labeled ER was quantified by phosphorimage analysis using ImageQuant software (Molecular Dynamics) and the percent ER remaining calculated relative to time zero. The mean values from two independent experiments are shown.

Down-regulation of SET7 attenuates estrogen-induced gene activation

We tested the impact of SET7-mediated methylation of ER on estrogen-induced transactivation of ER target genes. MCF7 cells showed a 20-fold increase in ERE-coupled luciferase reporter activity upon stimulation with 17-β estradiol (E2), and this response was stimulated a further 3-fold in the presence of a known ER-coactivator, NCoA2 (Figure 4A). In contrast, E2-driven transactivation was dramatically compromised in two independent SET7 knock-down clones, which showed only 6-fold and 3-fold stimulation in ERE-coupled reporter activity (Figure 4A). The attenuated estrogenic response in shSET7 cells was, however, stimulated by NCoA2 to an extent comparable to that in MCF7 cells (Figure 4A). These data suggest that it is the ability of ER to respond to E2, rather than its interaction with NCoA2, that is impaired in SET7 knock-down cells.

Figure 4. SET7 regulates estrogen-driven transactivation of ER target genes.

Figure 4

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(A) MCF7 and shSET7 cells were grown in EFM for 4 days and transfected with pERE-Luc, pRL-Tk and pNCoA2. One day later, the cells were treated with either ethanol vehicle alone (−) or 1 pM E2 (+) for 24 hr. The fold-increase in firefly luciferase activity compared to that of unstimulated cells in the absence of NCoA2 was determined, after normalization to Renilla luciferase activity. Shown is the mean ± SD of two independent experiments performed in triplicate.

(B-C) MCF7 and its derivatives were cultured in EFM for 4 days, followed by treatment with ethanol vehicle (V) or 1 nM E2 for 1, 2 or 24 hr after which RNA was isolated, reverse-transcribed and analyzed by real-time PCR using primers specific for PS2, PgR, or 18s rRNA. The E2-stimulated fold-increase in PS2 (B) and PgR (C) mRNA levels were determined relative to time zero, after normalization to 18s rRNA. Shown is the mean ± SD of two independent experiments performed in triplicate.

We also evaluated the impact of SET7-mediated methylation on estrogen-induced transactivation of two endogenous ER target genes, PS2 and progesterone receptor (PgR). MCF7 cells and control clones showed a 7-10-fold induction of PS2 mRNA when stimulated with 1 nM E2 for 24 hours (Figure 4B). In contrast, shSET7 cells showed only a 2.5-fold induction of PS2 (Figure 4B). The E2-dependent induction of PgR mRNA was similarly attenuated in shSET7 cells compared to MCF7 cells (Figure 4C). These data suggest that SET7 is required for efficient estrogen-induced transcriptional response.

Estradiol-induced nuclear accumulation and recruitment of ER to target genes

We next determined the impact of ER methylation on nuclear accumulation and recruitment of ER to target genes in response to estradiol. As expected, estradiol induced a rapid influx of ER into the nucleus in MCF7 cells (Figure 5A). In comparison, shSET7 cells showed reduced nuclear accumulation of ER. To determine the impact of SET7-mediated methylation on ER function, we then monitored the estradiol-induced recruitment of ER to the PS2 and PgR genes over time by chromatin immunoprecipitation (ChIP). Estradiol induced a rapid accumulation of ER at the PS2 promoter within 30 min. in MCF7 cells (mean peak induction, ~70-fold, range 28-145-fold, n=3) (Figure 5B, top panel). In contrast, the peak occupancy at PS2 was both delayed (1hr) and less robust in shSET7 cells (mean peak induction 13.7 fold, range 11-15-fold, n=2).

Figure 5. E2-induced nuclear accumulation and recruitment of ER to ER target genes.

Figure 5

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(A) MCF7 and shSET7 cells were cultured in EFM for 3 days and then treated with vehicle alone (-) or 100 nM E2 (+) for 20 min. Samples were split and whole cell extracts (WCE) were prepared by lysis in RIPA buffer. Nuclei were prepared from the remaining cells and extracted with 50mM Tris-HCl, 0.42M KCl, 1mM MgCl2, 10%glycerol, 1mM DTT, 0.1mM PMSF to generate soluble nuclear extracts (NE). Equal cell equivalents were fractionated by SDS-PAGE and subject to western blotting using antibodies to ER, SET7, and DNA polymerase δ (loading control).

(B) Estrogen-induced recruitment of ER to the PS2 and PgR genes was analyzed by ChIP. MCF7 (filled diamonds) and shSET7 cells (open triangles) were cultured in EFM for 3-4 days and then treated with 100 nM E2. At the indicated time points, ChIP was performed with antibodies directed against ER. Immunoprecipitated DNA was analyzed by real-time PCR using primers encompassing the EREs of the PS2 and PgR promoters and a putative PgR enhancer #1 (Carroll, et al., 2006). The E2-stimulated fold increase in recruitment was determined relative to time zero after normalization to input DNA. The mean of three (MCF7) or two (shSET7) independent experiments analyzed in triplicate is shown.

(C-D) MCF7 cells were grown in EFM and transfected with pERE-Luc, pRL-Tk and pMyc-SET7. After 24 hr, the cells were treated with vehicle alone (-) or 1 pM E2 (+). (C) Luciferase activity was determined relative to the unstimulated cells after normalization to the Renilla luciferase internal control. The mean ± SD from two independent experiments performed in triplicate is shown. (D) Protein lysates used in the luciferase assay were immunoblotted with the indicated antibodies. SET7 appears as a doublet; the upper band is Myc-tagged SET7 and the lower band is endogenous SET7.

We also examined ER recruitment to the PgR gene. Recent genome-wide studies mapping estrogen-induced ER occupancy showed that unlike some known estrogen-regulated genes that have defined ER binding sites within their proximal promoter regions (such as PS2), others (such as PgR) lack discernable ER binding at the promoter and may be regulated by distant sites instead (Carroll et al., 2006). Consistent with these studies, we found little impact of estradiol on the recruitment of ER to the PgR proximal promoter, but robust E2-induced recruitment of ER to a putative enhancer 169 Kb upstream of PgR (PgR enhancer #1, Carroll, et al., 2006) (Figure 5B, middle and bottom panel). ER occupancy at the PgR enhancer increased by ~30-fold (range 32-33, n=3) in response to estradiol treatment in the MCF7 cells. In contrast, E2-induced recruitment of ER to the PgR enhancer was attenuated in shSET7 cells (mean peak induction 12.6-fold, range 6-19-fold, n=2) (Figure 5B, middle panel). Estradiol did not affect ER recruitment to a non-estrogen regulated gene (myoglobin) in either cell line (data not shown). These data indicate that the attenuated transcriptional response to E2 in shSET7 cells is accompanied by, and perhaps explained by, a reduced occupancy of ER at ER-target genes.

In addition to modifying ER directly, SET7 may also act as a chromatin-modifying ER-coactivator (Nishioka et al., 2002; Wang et al., 2001b). We tested whether SET7 was recruited to the PS2 promoter along with ER after E2 stimulation in ChIP assays. Under conditions in which both ER and NCoA2 were recruited to the PS2 genes, SET7 was not detected (data not shown). We also tested the ability of SET7 to act as an ER-coactivator in transient transfection assays using an ERE-coupled luciferase reporter assay. Under conditions where MCF7 cells showed a 13-fold enhancement in reporter activity upon stimulation with E2, co-expression of SET7 had no further effect on the estrogenic response (Figure 5C). Similar results were obtained in HeLa and MDA-MB468 cells transfected with ER and SET7 expression constructs (Figure S4). These data suggest that SET7 regulates ER function by altering the stability of transcription-competent ER, rather than acting as a typical ER-coactivator. Taken together, our data suggest that SET7 regulates ER by methylating K302, and that the attenuated transcriptional response to E2 in the absence of SET7 results from destabilization of ER protein and reduced occupancy of ER at its target genes.

Structure of the SET7-ER peptide complex

To analyze the molecular mechanism of ER recognition by SET7, structures of three ternary complexes, in the resolution range of 1.42-1.69 Å, were solved (Table 1). An ER peptide encompassing amino acids 298-307 was used for co-crystallization in the presence of the methyl donor S-adenosyl-L-methionine (AdoMet), termed the AdoMet complex. The reaction occurred during the crystal formation of the complex, with the methyl group transferred to the ε-amino of K302 and AdoMet converted to AdoHcy and retained in the complex (Figure 6A). The enzyme-driven methyl peptide-AdoHcy complex is highly similar to the complex using the peptide pre-methylated at K302 and the reaction product AdoHcy (termed the AdoHcy complex). A third complex was formed using the unmodified substrate peptide and the cofactor analog Sinefungin. The three structures are highly similar to each other with a pair-wise root-mean-square deviation of less than 0.2 Å comparing 240 pairs of Cα atoms. The largest difference lies in the bound ER peptide: while 7 residues (298-304) are ordered in the AdoMet complex, 5 residues (299-303) are observed in the AdoHcy complex with no side chain densities for residues 299 and 303, or 4 residues (299-302) observed in the Sinefungin complex with no side chain density for residue 299. It appears that the enzyme-driven methylated peptide binds most stably in the active site (described below), probably because it went through the catalytic cycle.

Table 1.

Crystallographic data and refinement statistics for SET7

Crystal AdoMet AdoHcy Sinefungin
Wavelength (Å) 1.0 1.0 1.0
Space group P21212
Unit cell dimensions (Å)
 a
 b
 c		 102.3
38.9
66.8		 101.7
38.9
66.3		 102.0
38.8
66.6
Resolution range (Å) 35-1.69 (1.74-1.69) 35-1.65 (1.71-1.65) 25-1.42 (1.47-1.42)
Completeness (%) 93.2 (93.0) 99.9 (100) 98.7 (95.9)
Rmergea 0.068 (0.571) 0.060 (0.450) 0.060 (0.664)
<I/σ> 16.8 (3.4) 16.5 (5.4) 19.5 (3.0)
Observed reflections 218,700 372,337 357,673
Unique reflections 28,681 32,445 50,059
Refinement
Resolution range (Å) 35-1.69 35-1.65 35-1.42
Rfactorb 0.197 0.195 0.239
Rfreec 0.228 0.231 0.243
Rms deviation from ideal
 Bond lengths (Å) 0.005 0.005 0.005
 Bond angels (°) 1.2 1.2 1.3
 Dihedrals (°) 24.8 24.9 24.8
 Improper (°) 0.73 0.72 0.79
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a

Rmerge=Σ|I-<I>|/ΣI, where I is the observed intensity and <I> is the averaged intensity from multiple observations.

b

Rfactor= Σ|Fo-Fc|/Σ|Fo|.

c

Rfree was calculated using a subset (5%) of the reflection not used in the refinement.

Figure 6. Structure of the ER-SET7 complex.

Figure 6

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(A) The reaction occurred during crystallization, the methyl group has transferred to the ε-amino of ER-K302 and AdoMet is converted to AdoHcy, which is still present in the complex (right panel). The AdoMet analog sinefungin (adenosyl ornithine) was used to prepare a ternary complex mimicking the step prior to methyl transfer because it also carries a formal positive charge on the ε amino group (middle panel). The substrate peptide and sinefungin, or the methlyated peptide and the reaction product AdoHcy, are located at the opposite ends of the target lysine-binding channel. The atoms are colored red, nitrogen; blue, oxygen; grey, carbon; and yellow, sulfur.

(B) Three pairs of salt bridges and hydrogen bonds define SET7-ER-peptide interactions (dashed lines). Inset is a surface representation of the peptide-binding groove.

Like all structurally characterized SET domain proteins, the ER peptide binds to a surface groove of the SET domain as an antiparallel β-strand (Figure 6B). The ER peptide specificity is determined primarily through recognition of ordered side chains. The network of interactions involves three pairs of electrostatic salt bridges - SET7-D256 and ER-K299, SET7-E348 and ER-R300, and SET7-D338 and ER-K303 – and three hydrogen bonds - between the side chain hydroxyl oxygen atoms of SET7-S268 and ER-S301, and ER-N304 bridging between the side chain of K317 and the main carbonyl oxygen of Y305 of SET7. Emphasizing the importance of these interactions is the observation that the target lysine ER-K302 is inserted into a narrow channel so that the target nitrogen lies in close proximity to the methyl donor AdoMet at the opposite end, where the methyl group is transferred from the donor to the acceptor during the crystal formation (Figure 6A, right panel). The complex structures also predict that modifications occurring to side chains of ER-K299, R300, S301, or K303 would affect ER-K302 methylation by SET7. To this end, we tested the impact of K303 acetylation and mutation (to R) on methylation at K302 in vitro. In the context of a peptide, acetylation of K303, which would be expected to destroy the interaction with SET7-D388, was a poorer substrate for SET7-mediated methylation at K302 (Figure 1C). In contrast, the breast cancer-associated mutation K303R, which retains its positively-charged side chain, caused increased methylation at K302 in the context of peptide substrate (Figure 1C). Modeling of an arginine side chain at position 303 resulted in a rotamer that maintains the charge-charge interaction with SET7-D338.

Discussion

In this study we show that ER is methylated within the hinge region at K302 in vivo and in vitro. This methylation stabilizes ER and is necessary for the recruitment of ER to estrogen responsive genes and the activation of an estrogen-driven transcriptional response. These studies represent one of the first demonstrations of nuclear hormone receptor regulation by lysine methylation. Tri-methylation of K347 within the ligand binding domain of Retinoic acid receptor-α was recently described, however the identity of the lysine methyltransferase that catalyzes this reaction is currently unknown (Huq et al., 2007). Other SET domain-containing HMTases, including Rb-interacting Zinc-finger protein-1 (RIZ1), G9a, Nuclear receptor-binding SET domain-containing protein-1 (NSD-1) and Mixed lineage leukemia-2 (MLL2) have been shown to act as ER coactivators (Carling et al., 2004; Lee et al., 2006; Mo et al., 2006; Rayasam et al., 2003). The role of SET7 is distinct from these, as it does not appear to be recruited to ER-targets, and its over-expression does not enhance ER-driven transactivation of a reporter construct. Rather, our data are most consistent with the interpretation that SET7 regulates the estrogenic response via direct modification of ER and stabilization of ER protein.

There are several mechanisms by which SET7-mediated methylation could stabilize ER (Figure 7). Estrogen-stimulated ubiquitylation and subsequent degradation by the proteasome plays an important role in ER transcriptional activity (Alarid et al., 1999; Nawaz et al., 1999; Reid et al., 2003). Ubiquitin-dependent degradation of ligand-bound receptor molecules is thought to be critical for promoter clearance and additional rounds of transcriptional response to estrogen (Reid et al., 2003). Methylation at K302 could directly compete with ubiquitylation of the same lysine residue (Figure 7, Upper right). ER has been shown to be a target for ubiquitylation by several E3 ubiquitin ligases, such as MDM2, CHIP, E6-AP and EFP in vivo (Duong et al., 2007; Eakin et al., 2007; Li et al., 2006; Nakajima et al., 2007; Tateishi et al., 2004), although the lysine residues targeted by these ligases is currently unknown. In this regard, recent work showed that the BRCA1/BARD1 complex can ubiquitylate ER at K302, at least in vitro (Eakin et al., 2007).

Figure 7. Models for the regulation of ER turnover by SET7-mediated lysine methylation.

Figure 7

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(Upper) Methylation of ER at K302 by SET7 could prevent the ubiquitylation of K302 by an E3 ubiquitin ligase, such as the BRCA1/BARD1 complex. Loss of SET7-mediated methylation at K302 would allow ubiquitylation of K302, and premature proteasome-mediated degradation of ER. (Lower) Methylated ER may recruit specific proteins, such as CaM or a methyl lysine binding protein. These methyl-K302-specific binding proteins would then directly or indirectly block the action of E3 ligases and ER degradation. Subsequently, these binding proteins may be released to allow the ubiquitylation and degradation of methylated ER after it completes the transactivation cycle.

Alternatively, the methylation of ER by SET7 may serve as a platform for the recruitment of proteins that affect ER turnover (Figure 7, Bottom right). K302 falls within a region of ER (amino acids 293-309) that constitutes a calmodulin (CaM)-binding motif. Interaction with CaM has been shown to prevent ubiquitylation by E6-AP and subsequent degradation of ER (Li et al., 2006). Although the effect of post-translational modifications on the ER-CaM association is currently unknown, mutation of K302 and K303 to glycines has been shown to reduce binding of ER to CaM (Garcia Pedrero et al., 2002). Therefore, methylation of K302 by SET7 could limit ER ubiquitylation and degradation by promoting the formation of ER-CaM complexes. K302 methylation could also recruit a methyllysine binding protein that may in turn prevent ER degradation by interfering with the action of E3 ligases or the proteasome. Protein domains that recognize methylated lysines in histones and p53 have been described (Huang et al., 2007, Ruthenburg et al., 2007; Zhang, 2006). It remains to be seen whether the same is true for methylated ER.

The hinge region of ER is a target of extensive post-translational modifications. In particular, the residues from K299 through S305 have been shown to undergo acetylation (K299, K302 and K303), ubiquitylation (K302), and phosphorylation (S305) (Eakin et al., 2007; Kim et al., 2006; Wang et al., 2001a). There might be direct competition at K302 between methylation by SET7, acetylation by p300, and ubiquitylation, as well as cross-regulation by modifications at nearby residues (K299, K303 and S305), resembling the cross talk amongst post-translational modifications in histones (Rea et al., 2000), and p53 (Huang et al., 2006). Acetylation of K303 and phosphorylation of S305 are mutually antagonistic and regulate hormone sensitivity (Cui et al., 2004). Acetylation of K303 attenuates ER-driven transcription, which has been attributed in part to the suppression of S305 phosphorylation (Cui et al., 2004), but could also be due to the inhibition of K302 methylation and subsequent destabilization of ER. Indeed, we find that an ER peptide acetylated at K303 is a poorer substrate for SET7-mediated methylation at K302. These findings raise the possibility that generation, recognition, and removal of modifications within the hinge region of ER may generate distinct “ER modification cassettes” that modulate specific steps in the transcription life cycle or the transcriptional output. LSD1, a lysine-specific demethylase that demethylates di- or mono-methylated H3K4 (a mark generated by SET7), was unable to demethylate ER-K302me1 peptides in vitro (data not shown). It remains to be seen whether, like the involvement of histone demethylases in nuclear hormone receptor function, JmjC-domain-containing demethylases can act on methyl-ER (Garcia-Bassets et al., 2007; Metzger et al., 2005; Shi and Whetstine, 2007).

The expression of ER is of critical importance in the clinical management of breast cancers. Approximately two-thirds of breast cancers express ER at diagnosis. Such ER positive (ER+) breast cancers generally have a better prognosis and respond to treatment with anti-estrogens. However, ER+ breast cancers can become resistant to anti-estrogens during treatment, though they retain ER expression (Clarke et al., 2001; Clarke et al., 1993; Taylor et al., 1982). Development of anti-estrogen resistance has been linked to defects in the ER-signaling pathway, altered assembly of ER-coactivator complexes, or cross-talk between ER and growth factor signaling pathways (Clarke et al., 2001). Post-translational modifications are also implicated in the aberrant activation of ER in some anti-estrogen-resistant breast cancers (Clarke et al., 2001). In particular, hyper-phosphorylation of S305 results in constitutive activation of ER, which renders ER-driven transcription ligand-independent, estrogen-hypersensitive and anti-estrogen-insensitive (Michalides et al., 2004; Rayala et al., 2006). An acquired missense mutation in the ER gene (A908G), resulting in a lysine to arginine substitution at K303 (K303R), has been observed in primary ductal hyperplasias and invasive breast carcinomas (Conway et al., 2005; Cui et al., 2004; Fuqua et al., 2000). This mutation has been linked to more aggressive clinical features and a poorer prognosis, and may be associated with increased use of oral contraceptives (Conway et al., 2007; Herynk et al., 2007). The ER-K303R mutant was shown to be hypersensitive to low doses of estrogen, a phenotype attributed to hyper-phosphorylation at S305 (Cui et al., 2004; Fuqua et al., 2000). Our studies suggest that there is a complex interplay between the K303R mutation and methylation at K302. Whereas ER peptides bearing the K303R mutation are better substrates for K302 methylation in vitro, the mutation appears to suppress K302 methylation in vivo, potentially due to the added effects of K303R on other ER modifications or coactivator binding (Fuqua et al., 2000). Given the prevalence of the ER-K303R mutation in breast cancers and its effect on SET7-mediated K302 methylation, it will be of great interest to determine the impact of K302 methylation on other modifications, estrogen-mediated transcription and breast cancer cell growth.

Experimental Procedures

Cell culture

MCF7 and HeLa cells were maintained in DMEM plus 10% FBS and 2 mM L-glutamine. For estrogen stimulation experiments, cells were cultured in estrogen-free medium (EFM) (phenol-red-free DMEM supplemented with 10% charcoal-stripped FBS and 2 mM L-glutamine) for 3-4 days prior to transfection or stimulation with estrogen. 17β-estradiol (E2) (Sigma) was dissolved in ethanol. MCF7 derivatives stably knocked-down for SET7 were created using the GeneEraser system (Stratagene). A SET7-specific small-hairpin RNA (shRNA) against the target sequence 5’-GTAGACGGAGAGCTGAACG-3’ was constructed in pGE1. pGE-shSET7, vector alone or vector expressing a non-targeting shRNA, were transfected into MCF7 cells. Stable clones were selected with 1 mg/ml G418 and tested for knock-down of SET7. The experiments described here were carried out with two independent shSET7 clones.

Plasmid constructs

The pERE-Luc reporter and pCMV5-hER2 human ER expression construct were gifts from Dr. Michael Wang (Xu et al., 2003). Flag-tagged human ER in pcDNA3.1 was provided by Dr. Jin-Tang Dong (Emory University). Site-directed mutants of ER (pCMV5-hER2-K302R, pCMV5-hER2-K303R, pFlag-ERK303A, pFlag-ER-K303R) were generated using the QuickChange site-directed mutagenesis kit (Stratagene). The Myc-tagged human SET7 (pMyc-SET7) expression construct was a gift from Dr. Iannis Talianidis (Kouskouti et al., 2004). Expression constructs for human NCoA1, NCoA2 and NCoA3 were gifts from Dr. Myles Brown.

Pulse-chase analysis

MCF7 and shSET7 cells were plated in 10 cm dishes. The next day, cells were incubated in DMEM lacking methionine and cysteine (Cellgro) containing 100 μCi of L-35S-methionine (10 mCi/ml, Amersham Biosciences) for 1hr. The media was replaced with estrogen-replete growth medium (DMEM plus 10 % FBS and 2 mM glutamine). At timed intervals during the chase period, cells lysates were prepared in IP buffer and immunoprecipitated using an anti-ER antibody. For analysis of ER mutants, MCF7 or shSET7 cells were transfected with Flag-tagged expression constructs for either wild-type or mutant ER using Fugene 6.0 (Roche Biochemicals). One day later the cells were subjected to pulse chase-analysis as described above except that immunoprecipitation was carried out using anti Flag-M2-agarose (Sigma).

Immunoprecipitation

Cell lysates prepared in IP buffer (50 mM Tris at pH 8, 150 mM NaCl, 0.5 % NP-40, 5 mM EDTA plus protease inhibitor cocktail (Roche Applied Biosciences)) were pre-cleared with Protein A-agarose beads for 30 min. and incubated overnight with an anti-ER antibody (D-12, Santa Cruz Biotechnology) or mouse IgG (Santa Cruz Biotechnology) at 4°C. Immunocomplexes were captured on Protein A-agarose beads, washed with IP buffer and boiled in 2X Laemmli sample buffer. For Flag immunoprecipitations, lysates were incubated with anti Flag M2-agarose for 2 hr at 4°C. Immunocomplexes were resolved by SDS-PAGE and analyzed by fixation, drying, and phosphorimage analysis (for pulse-chase studies) or by western blot analysis.

Immunoblotting and antibodies

Cell lysates were prepared in RIPA buffer (50 mM Tris.HCl at pH 8, 150 mM NaCl, 0.5 % sodium deoxycholate, 0.1 % SDS and 1 % NP-40) containing protease inhibitors (Roche Applied Sciences). Clarified lysates were resolved by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with the following antibodies: SET7 (07-134, Upstate Biotechnology), GAPDH (6C5, Abcam), ER (H-184 and G-20, Santa Cruz Biotechnology), and β-tubulin (Clone TUB 2.1, Sigma). An affinity purified rabbit antisera against methyl-ER (ER-K302me1) was generated by New England Peptide, LLC using a peptide corresponding to amino acids 297-308 of ER mono-methylated at K302 (NH2-MIKRS(meK)KNSLALC-COOH) as the immunogen.

Reporter assays

MCF7 cells and shSET7 cells were transfected with pERE-Luc and pNCoA2 or pMyc-SET7 using Fugene (Roche Applied Science). pRl-Tk (Promega) Renilla luciferase construct was included as an internal control. The total mass of DNA was equalized (200ng) by adding vector DNA. HeLa cell transfections additionally contained 100 ng of pCMV5-hER2 (WT/ K302R). One day after transfection, cells were treated with either vehicle alone or E2 for 24 hr. Luciferase activities were measured using the Dual Luciferase assay (Promega).

Quantitative reverse-transcriptase PCR

Total RNA was prepared using the RNeasy kit (Qiagen) and reverse-transcribed using MMLV-RT (Invitrogen) as described (Conway et al., 2000). The cDNA was amplified using primers specific for PS2, PgR or 18s rRNA, as an internal control (Supplemental Table 1). PCR reactions were monitored in real-time using SyBr Green dye detection and relative starting quantities calculated by comparison to a common standard curve generated with MCF7 cDNA.

Chromatin immunoprecipitation

Cells were cross-linked with 1% formaldehyde for 10 min and quenched with 0.1 M glycine. Chromatin was isolated, sheared by sonication, pre-cleared with Protein A-agarose blocked with sheared salmon sperm DNA and BSA (Upstate Biotechnology) and incubated overnight with antibodies against ER (H-184, Santa Cruz Biotechnology). Complexes were collected on Protein A-agarose, washed, and eluted with 0.1 M NaHCO3, 1% SDS. Crosslinks were reversed and the DNA recovered after Proteinase K digestion, phenol-chloroform extraction and ethanol precipitation. DNA was resuspended in H2O and used as a template for PCR using primers encompassing the EREs of PS2 promoter, the PgR promoter, and PgR Enhancer#1 (Carroll et al., 2006) (Supplemental Table 1). PCR reactions were monitored in real-time using SyBr Green dye detection. Relative starting quantities were calculated by comparison to a common standard curve of MCF7 genomic DNA.

Crystallography

The ternary structures of SET7-ER peptide-AdoMet analog were solved by molecular replacement. Purification of recombinant human SET7 and crystallography methods can be found in the Supplemental Methods.

Supplementary Material

01

Acknowledgments

We thank Dr. Paul Wade for materials and advice, Dr. Xing Zhang for technical and conceptual advice and members of the Vertino and Cheng laboratories for helpful discussions and support. We also thank Drs. Myles Brown, Michael Wang, Xue-Yuan Dong, Jin-Tang Dong and Iannis Talianidis for plasmid constructs. This work was supported in part by National Institutes of Health grants 2RO1 CA077337 (PMV), 2RO1 GM068680 (XC) and P50 AG025688 (JP), an American Cancer Society Research Scholar grant RSG-02-144-01 (PMV), and funds from the Burris Foundation (KS) and the Georgia Research Alliance (XC).

Footnotes

Accession The coordinates of Set7/9-ER complex structures have been submitted to PDB as 3CBM (AdoMet), 3CBO (AdoHcy), and 3CBP (Sinefungin), respectively.

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References

  1. Alarid ET, Bakopoulos N, Solodin N. Proteasome-mediated proteolysis of estrogen receptor: a novel component in autologous down-regulation. Mol Endocrinol. 1999;13:1522–1534. doi: 10.1210/mend.13.9.0337. [DOI] [PubMed] [Google Scholar]
  2. Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J, Brodsky AS, Krasnickas Keeton E, Fertuck KC, Hall GF, et al. Genome-wide analysis of estrogen receptor binding sites. Nat Genet. 2006;38:1289–1297. doi: 10.1038/ng1901. [DOI] [PubMed] [Google Scholar]
  3. Carling T, Kim KC, Yang XH, Gu J, Zhang XK, Huang S. A histone methyltransferase is required for maximal response to female sex hormones. Mol Cell Biol. 2004;24:7032–7042. doi: 10.1128/MCB.24.16.7032-7042.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chuikov S, Kurash JK, Wilson JR, Xiao B, Justin N, Ivanov GS, McKinney K, Tempst P, Prives C, Gamblin SJ, et al. Regulation of p53 activity through lysine methylation. Nature. 2004;432:353–360. doi: 10.1038/nature03117. [DOI] [PubMed] [Google Scholar]
  5. Clarke R, Leonessa F, Welch JN, Skaar TC. Cellular and molecular pharmacology of antiestrogen action and resistance. Pharmacol Rev. 2001;53:25–71. [PubMed] [Google Scholar]
  6. Clarke RB, Laidlaw IJ, Jones LJ, Howell A, Anderson E. Effect of tamoxifen on Ki67 labelling index in human breast tumours and its relationship to oestrogen and progesterone receptor status. Br J Cancer. 1993;67:606–611. doi: 10.1038/bjc.1993.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collins RE, Tachibana M, Tamaru H, Smith KM, Jia D, Zhang X, Selker EU, Shinkai Y, Cheng X. In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases. J Biol Chem. 2005;280:5563–5570. doi: 10.1074/jbc.M410483200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conway K, Parrish E, Edmiston SN, Tolbert D, Tse CK, Geradts J, Livasy CA, Singh H, Newman B, Millikan RC. The estrogen receptor-alpha A908G (K303R) mutation occurs at a low frequency in invasive breast tumors: results from a population-based study. Breast Cancer Res. 2005;7:R871–880. doi: 10.1186/bcr1315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Conway K, Parrish E, Edmiston SN, Tolbert D, Tse CK, Moorman P, Newman B, Millikan RC. Risk factors for breast cancer characterized by the estrogen receptor alpha A908G (K303R) mutation. Breast Cancer Res. 2007;9:R36. doi: 10.1186/bcr1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conway KE, McConnell BB, Bowring CE, Donald CD, Warren ST, Vertino PM. TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers. Cancer Res. 2000;60:6236–6242. [PubMed] [Google Scholar]
  11. Couture JF, Collazo E, Hauk G, Trievel RC. Structural basis for the methylation site specificity of SET7/9. Nat Struct Mol Biol. 2006;13:140–146. doi: 10.1038/nsmb1045. [DOI] [PubMed] [Google Scholar]
  12. Cui Y, Zhang M, Pestell R, Curran EM, Welshons WV, Fuqua SA. Phosphorylation of estrogen receptor alpha blocks its acetylation and regulates estrogen sensitivity. Cancer Res. 2004;64:9199–9208. doi: 10.1158/0008-5472.CAN-04-2126. [DOI] [PubMed] [Google Scholar]
  13. Duong V, Boulle N, Daujat S, Chauvet J, Bonnet S, Neel H, Cavailles V. Differential regulation of estrogen receptor alpha turnover and transactivation by Mdm2 and stress-inducing agents. Cancer Res. 2007;67:5513–5521. doi: 10.1158/0008-5472.CAN-07-0967. [DOI] [PubMed] [Google Scholar]
  14. Eakin CM, Maccoss MJ, Finney GL, Klevit RE. Estrogen receptor alpha is a putative substrate for the BRCA1 ubiquitin ligase. Proc Natl Acad Sci U S A. 2007;104:5794–5799. doi: 10.1073/pnas.0610887104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Enmark E, Gustafsson JA. Oestrogen receptors - an overview. J Intern Med. 1999;246:133–138. doi: 10.1046/j.1365-2796.1999.00545.x. [DOI] [PubMed] [Google Scholar]
  16. Fu M, Wang C, Zhang X, Pestell R. Nuclear receptor modifications and endocrine cell proliferation. J Steroid Biochem Mol Biol. 2003;85:133–138. doi: 10.1016/s0960-0760(03)00223-1. [DOI] [PubMed] [Google Scholar]
  17. Fuqua SA, Wiltschke C, Zhang QX, Borg A, Castles CG, Friedrichs WE, Hopp T, Hilsenbeck S, Mohsin S, O’Connell P, Allred DC. A hypersensitive estrogen receptor-alpha mutation in premalignant breast lesions. Cancer Res. 2000;60:4026–4029. [PubMed] [Google Scholar]
  18. Garcia-Bassets I, Kwon YS, Telese F, Prefontaine GG, Hutt KR, Cheng CS, Ju BG, Ohgi KA, Wang J, Escoubet-Lozach L, et al. Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors. Cell. 2007;128:505–518. doi: 10.1016/j.cell.2006.12.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Garcia Pedrero JM, Del Rio B, Martinez-Campa C, Muramatsu M, Lazo PS, Ramos S. Calmodulin is a selective modulator of estrogen receptors. Mol Endocrinol. 2002;16:947–960. doi: 10.1210/mend.16.5.0830. [DOI] [PubMed] [Google Scholar]
  20. Hall JM, McDonnell DP. Coregulators in nuclear estrogen receptor action: from concept to therapeutic targeting. Mol Interv. 2005;5:343–357. doi: 10.1124/mi.5.6.7. [DOI] [PubMed] [Google Scholar]
  21. Hermanson O, Glass CK, Rosenfeld MG. Nuclear receptor coregulators: multiple modes of modification. Trends Endocrinol Metab. 2002;13:55–60. doi: 10.1016/s1043-2760(01)00527-6. [DOI] [PubMed] [Google Scholar]
  22. Herynk MH, Parra I, Cui Y, Beyer A, Wu MF, Hilsenbeck SG, Fuqua SA. Association between the estrogen receptor alpha A908G mutation and outcomes in invasive breast cancer. Clin Cancer Res. 2007;13:3235–3243. doi: 10.1158/1078-0432.CCR-06-2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Huang J, Perez-Burgos L, Placek BJ, Sengupta R, Richter M, Dorsey JA, Kubicek S, Opravil S, Jenuwein T, Berger SL. Repression of p53 activity by Smyd2-mediated methylation. Nature. 2006;444:629–632. doi: 10.1038/nature05287. [DOI] [PubMed] [Google Scholar]
  24. Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, Richter M, Opravil S, Shiekhattar R, Bedford MT, Jenuwein T, Berger SL. p53 is regulated by the lysine demethylase LSD1. Nature. 2007;449:105–108. doi: 10.1038/nature06092. [DOI] [PubMed] [Google Scholar]
  25. Huq MD, Tsai NP, Khan SA, Wei LN. Lysine Trimethylation of Retinoic Acid Receptor-{alpha}: A Novel Means To Regulate Receptor Function. Mol Cell Proteomics. 2007;6:677–688. doi: 10.1074/mcp.M600223-MCP200. [DOI] [PubMed] [Google Scholar]
  26. Kim MY, Woo EM, Chong YT, Homenko DR, Kraus WL. Acetylation of estrogen receptor alpha by p300 at lysines 266 and 268 enhances the deoxyribonucleic acid binding and transactivation activities of the receptor. Mol Endocrinol. 2006;20:1479–1493. doi: 10.1210/me.2005-0531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koh SS, Chen D, Lee YH, Stallcup MR. Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J Biol Chem. 2001;276:1089–1098. doi: 10.1074/jbc.M004228200. [DOI] [PubMed] [Google Scholar]
  28. Kouskouti A, Scheer E, Staub A, Tora L, Talianidis I. Gene-specific modulation of TAF10 function by SET9-mediated methylation. Mol Cell. 2004;14:175–182. doi: 10.1016/s1097-2765(04)00182-0. [DOI] [PubMed] [Google Scholar]
  29. Lee DY, Northrop JP, Kuo MH, Stallcup MR. Histone H3 lysine 9 methyltransferase G9a is a transcriptional coactivator for nuclear receptors. J Biol Chem. 2006;281:8476–8485. doi: 10.1074/jbc.M511093200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lee YH, Coonrod SA, Kraus WL, Jelinek MA, Stallcup MR. Regulation of coactivator complex assembly and function by protein arginine methylation and demethylimination. Proc Natl Acad Sci U S A. 2005;102:3611–3616. doi: 10.1073/pnas.0407159102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Leo C, Chen JD. The SRC family of nuclear receptor coactivators. Gene. 2000;245:1–11. doi: 10.1016/s0378-1119(00)00024-x. [DOI] [PubMed] [Google Scholar]
  32. Li L, Li Z, Howley PM, Sacks DB. E6AP and calmodulin reciprocally regulate estrogen receptor stability. J Biol Chem. 2006;281:1978–1985. doi: 10.1074/jbc.M508545200. [DOI] [PubMed] [Google Scholar]
  33. Metzger E, Wissmann M, Yin N, Muller JM, Schneider R, Peters AH, Gunther T, Buettner R, Schule R. LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature. 2005;437:436–439. doi: 10.1038/nature04020. [DOI] [PubMed] [Google Scholar]
  34. Michalides R, Griekspoor A, Balkenende A, Verwoerd D, Janssen L, Jalink K, Floore A, Velds A, van’t Veer L, Neefjes J. Tamoxifen resistance by a conformational arrest of the estrogen receptor alpha after PKA activation in breast cancer. Cancer Cell. 2004;5:597–605. doi: 10.1016/j.ccr.2004.05.016. [DOI] [PubMed] [Google Scholar]
  35. Mo R, Rao SM, Zhu YJ. Identification of the MLL2 complex as a coactivator for estrogen receptor alpha. J Biol Chem. 2006;281:15714–15720. doi: 10.1074/jbc.M513245200. [DOI] [PubMed] [Google Scholar]
  36. Nakajima A, Maruyama S, Bohgaki M, Miyajima N, Tsukiyama T, Sakuragi N, Hatakeyama S. Ligand-dependent transcription of estrogen receptor alpha is mediated by the ubiquitin ligase EFP. Biochem Biophys Res Commun. 2007;357:245–251. doi: 10.1016/j.bbrc.2007.03.134. [DOI] [PubMed] [Google Scholar]
  37. Nawaz Z, Lonard DM, Dennis AP, Smith CL, O’Malley BW. Proteasome-dependent degradation of the human estrogen receptor. Proc Natl Acad Sci U S A. 1999;96:1858–1862. doi: 10.1073/pnas.96.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nirmala PB, Thampan RV. Ubiquitination of the rat uterine estrogen receptor: dependence on estradiol. Biochem Biophys Res Commun. 1995;213:24–31. doi: 10.1006/bbrc.1995.2093. [DOI] [PubMed] [Google Scholar]
  39. Nishioka K, Chuikov S, Sarma K, Erdjument-Bromage H, Allis CD, Tempst P, Reinberg D. Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev. 2002;16:479–489. doi: 10.1101/gad.967202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pakdel F, Le Goff P, Katzenellenbogen BS. An assessment of the role of domain F and PEST sequences in estrogen receptor half-life and bioactivity. J Steroid Biochem Mol Biol. 1993;46:663–672. doi: 10.1016/0960-0760(93)90307-i. [DOI] [PubMed] [Google Scholar]
  41. Rayala SK, Talukder AH, Balasenthil S, Tharakan R, Barnes CJ, Wang RA, Aldaz M, Khan S, Kumar R. P21-activated kinase 1 regulation of estrogen receptor-alpha activation involves serine 305 activation linked with serine 118 phosphorylation. Cancer Res. 2006;66:1694–1701. doi: 10.1158/0008-5472.CAN-05-2922. [DOI] [PubMed] [Google Scholar]
  42. Rayasam GV, Wendling O, Angrand PO, Mark M, Niederreither K, Song L, Lerouge T, Hager GL, Chambon P, Losson R. NSD1 is essential for early post-implantation development and has a catalytically active SET domain. EMBO J. 2003;22:3153–3163. doi: 10.1093/emboj/cdg288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun ZW, Schmid M, Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature. 2000;406:593–599. doi: 10.1038/35020506. [DOI] [PubMed] [Google Scholar]
  44. Reid G, Hubner MR, Metivier R, Brand H, Denger S, Manu D, Beaudouin J, Ellenberg J, Gannon F. Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling. Mol Cell. 2003;11:695–707. doi: 10.1016/s1097-2765(03)00090-x. [DOI] [PubMed] [Google Scholar]
  45. Ruthenburg AJ, Allis CD, Wysocka J. Methylation of lysine 4 on histone H3: intricacy of writing and reading a single epigenetic mark. Mol Cell. 2007;25:15–30. doi: 10.1016/j.molcel.2006.12.014. [DOI] [PubMed] [Google Scholar]
  46. Shi Y, Whetstine JR. Dynamic regulation of histone lysine methylation by demethylases. Mol Cell. 2007;25:1–14. doi: 10.1016/j.molcel.2006.12.010. [DOI] [PubMed] [Google Scholar]
  47. Tateishi Y, Kawabe Y, Chiba T, Murata S, Ichikawa K, Murayama A, Tanaka K, Baba T, Kato S, Yanagisawa J. Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor. EMBO J. 2004;23:4813–4823. doi: 10.1038/sj.emboj.7600472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Taylor RE, Powles TJ, Humphreys J, Bettelheim R, Dowsett M, Casey AJ, Neville AM, Coombes RC. Effects of endocrine therapy on steroid-receptor content of breast cancer. Br J Cancer. 1982;45:80–85. doi: 10.1038/bjc.1982.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang C, Fu M, Angeletti RH, Siconolfi-Baez L, Reutens AT, Albanese C, Lisanti MP, Katzenellenbogen BS, Kato S, Hopp T, et al. Direct acetylation of the estrogen receptor alpha hinge region by p300 regulates transactivation and hormone sensitivity. J Biol Chem. 2001a;276:18375–18383. doi: 10.1074/jbc.M100800200. [DOI] [PubMed] [Google Scholar]
  50. Wang H, Cao R, Xia L, Erdjument-Bromage H, Borchers C, Tempst P, Zhang Y. Purification and functional characterization of a histone H3-lysine 4-specific methyltransferase. Mol Cell. 2001b;8:1207–1217. doi: 10.1016/s1097-2765(01)00405-1. [DOI] [PubMed] [Google Scholar]
  51. Wijayaratne AL, McDonnell DP. The human estrogen receptor-alpha is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators. J Biol Chem. 2001;276:35684–35692. doi: 10.1074/jbc.M101097200. [DOI] [PubMed] [Google Scholar]
  52. Xu Y, Traystman RJ, Hurn PD, Wang MM. Neurite-localized estrogen receptor-alpha mediates rapid signaling by estrogen. J Neurosci Res. 2003;74:1–11. doi: 10.1002/jnr.10725. [DOI] [PubMed] [Google Scholar]
  53. Zhang X, Yang Z, Khan SI, Horton JR, Tamaru H, Selker EU, Cheng X. Structural basis for the product specificity of histone lysine methyltransferases. Mol Cell. 2003;12:177–185. doi: 10.1016/s1097-2765(03)00224-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhang Y. It takes a PHD to interpret histone methylation. Nat Struct Mol Biol. 2006;13:572–574. doi: 10.1038/nsmb0706-572. [DOI] [PubMed] [Google Scholar]

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