0888-8809/06/$15.00/0 Molecular Endocrinology 20(7):1479–1493 Printed in U.S.A. Copyright © 2006 by The Endocrine Society doi: 10.1210/me.2005-0531 Acetylation of Estrogen Receptor ␣ by p300 at Lysines 266 and 268 Enhances the Deoxyribonucleic Acid Binding and Transactivation Activities of the Receptor Mi Young Kim, Eileen M. Woo, Yee Ting Esther Chong, Daria R. Homenko, and W. Lee Kraus Department of Molecular Biology and Genetics (M.Y.K., Y.T.E.C., D.R.H., W.L.K.), Cornell University, Ithaca, New York 14853; Laboratory of Chromatin Biology and Epigenetics (E.M.W.), The Rockefeller University, and Department of Pharmacology (W.L.K.), Weill Medical College of Cornell University, New York, New York 10021 Using a variety of biochemical and cell-based ap- tive enzymes (i.e. class III deacetylases, such as proaches, we show that estrogen receptor ␣ (ER␣) sirtuin 1). Acetylation at Lys266 and Lys268, or sub- is acetylated by the p300 acetylase in a ligand- and stitution of the same residues with glutamine (i.e. steroid receptor coactivator-dependent manner. K266/268Q), a residue that mimics acetylated ly- Using mutagenesis and mass spectrometry, we sine, enhances the DNA binding activity of ER␣ in identified two conserved lysine residues in ER␣ EMSAs. Likewise, substitution of Lys266 and (Lys266 and Lys268) that are the primary targets of Lys268 with glutamine enhances the ligand-depen- p300-mediated acetylation. These residues are dent activity of ER␣ in a cell-based reporter gene acetylated in cells, as determined by immunopre- assay. Collectively, our results implicate acetyla- cipitation-Western blotting experiments using an tion as a modulator of the ligand-dependent gene antibody that specifically recognizes ER␣ acety- regulatory activity of ER␣. Such regulation is likely lated at Lys266 and Lys268. The acetylation of ER␣ to play a role in estrogen-dependent signaling out- by p300 is reversed by native cellular deacetylases, comes in a variety of estrogen target tissues in including trichostatin A-sensitive enzymes (i.e. both normal and pathological states. (Molecular class I and II deacetylases) and nicotinamide ade- Endocrinology 20: 1479–1493, 2006) nine dinucleotide-dependent/nicotinamide-sensi- HE GENE-REGULATORY ACTIONS of estrogens tinct gene-regulatory activities under certain promoter Tare mediated through two nuclear estrogen recep- and cell contexts (7–15). tor (ER) proteins, ER␣ and ER␤, which belong to the ER␣ and ER␤ share a conserved structural and nuclear receptor superfamily (1–3). ERs bind estro- functional organization, including the following: 1) an gens with high affinity and function as ligand-regulated amino-terminal A/B region containing a transcriptional transcription factors to control global patterns of gene activation function (AF-1), 2) a DNA-binding domain expression. ER␣ and ER␤ have unique, but overlap- (DBD), and 3) a carboxyl-terminal ligand-binding do- ping, patterns of expression in a variety of estrogen main (LBD) containing a second transcriptional activa- target tissues, including mammary glands, uterus, and tion function (AF-2) (see Fig. 2B) (1, 3). In addition to bone (4–6). In addition, the two ERs may exhibit dis- these canonical nuclear receptor domains, recent studies have also begun to characterize the C-terminal extension (CTE) of the ER␣ and ER␤ DBDs (amino First Published Online February 23, 2006 acids 251–288 and 170–207, respectively), which Abbreviations: acetyl CoA, Acetyl coenzyme A; AR, andro- gen receptor; CBP, cAMP response element binding protein- plays a role in regulating the DNA-binding activities of binding protein; CTE, C-terminal extension; DBD, DNA-bind- the receptors (16, 17). The coordinated actions of the ing domain; E2, 17␤-estradiol; ER, estrogen receptor; GST, aforementioned ER functional domains allow for pre- glutathione S-transferase; HNE, HeLa cell nuclear extract; cisely controlled signal-regulated transcription in re- LBD, ligand-binding domain; MALDI-QqTOF, matrix-assisted sponse to both natural and synthetic ER ligands. laser desorption/ionization-quadrupole-quadrupole-time of ␣ ␤ flight; MS/MS, mass spectrometry/mass spectrometry; The binding of agonistic ligands by ER and ER NADϩ, nicotinamide adenine dinucleotide; PID, p300/CBP promotes a conformational change in the receptor interaction domain; RID, receptor interaction domain; SDS, LBD that allows the receptor to interact directly or sodium dodecyl sulfate; SIRT1, sirtuin 1; SRC, steroid recep- indirectly with a diverse set of coregulatory proteins (1, tor coactivator; TSA, trichostatin A. 3). These include members of the steroid receptor Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the coactivator (SRC) family (i.e. SRC1, -2, and -3), which foremost professional society serving the endocrine function primarily as bridging factors to recruit other community. coregulators (18–20), including a diverse set of pro- 1479 Downloaded from mend.endojournals.org at Rockefeller University Library on December 24, 2007 1480 Mol Endocrinol, July 2006, 20(7):1479–1493 Kim et al. • p300-Mediated Acetylation of ER␣ tein-modifying enzymes (e.g. acetylases, methyltrans- RESULTS ferases, kinases) (21, 22). p300 and its paralog cAMP response element binding protein-binding protein Human ER␣ Is Acetylated by p300 and (CBP) are the two best characterized mammalian Deacetylated by TSA- and acetylases. They function as coregulators for a variety Nicotinamide-Sensitive Deacetylases of transcription factors, including ERs and other nu- clear receptors (23–25). p300 and CBP are recruited to To examine the acetylation of human ER␣ by p300, we 3 ER␣ and ER␤ in a ligand-dependent manner via inter- used an in vitro acetylation assay with [ H]acetyl co- actions with SRC proteins. During an estrogen-depen- enzyme A (acetyl CoA) and the following purified re- ␣ dent transcriptional response, p300 and CBP can combinant proteins: ER , p300, and glutathione acetylate nucleosomal histones to alter chromatin S-transferase (GST)-fused SRC2(RID/PID), which con- structure and function (19, 22), as well as components tains the receptor interaction domain (RID) and p300/ CBP interaction domain (PID) of SRC2 (Fig. 1A). With of the transcription complex to alter transcriptional this assay, we showed previously that SRCs play a key activity (see below). A variety of deacetylases, includ- role in the targeted acetylation of nucleosomal core ing trichostatin A (TSA)-sensitive enzymes (i.e. class I histones by p300, functioning as a bridging factor and II deacetylases, such as histone deacetylase 1) between p300 and 17␤-estradiol (E2)-bound ER␣ (Ref. and nicotinamide adenine dinucleotide (NADϩ)-de- 41 and Fig. 1B, bottom). Interestingly, these same pendent/nicotinamide-sensitive enzymes [i.e. class III interactions also promote the acetylation of ER␣ (Fig. deacetylases, such as sirtuin 1 (SIRT1)], also function 1B, top), indicating that interactions among agonist- as coregulators and can reverse the protein acetyla- bound ER␣, SRC, and p300 are required for efficient tion reactions catalyzed by acetylases (26–29). acetylation of ER␣ by p300 (see also supplemental Fig. A number of recent studies have shown that acet- 1, published as supplemental data on The Endocrine ylation is an important covalent posttranslational mod- Society’s Journals Online web site at http://mend. ification for regulating the activity of transcription- endojournals.org). SRC2(RID/PID), which lacks the pu- related factors, including p53, SRC3 (also known as tative SRC acetylase domain (18, 21), was unable to ACTR), nuclear factor-␬B p65, and poly(ADP-ribose) promote the acetylation of ER␣ in the absence of p300 polymerase-1 (30–39). Interestingly, different acety- (data not shown). lases (e.g. p300/CBP vs. p300/CBP-associated factor) Next, we examined whether the p300-dependent have different substrate preferences as demonstrated acetylation of ER␣ can be reversed by native deacety- by the fact that some factors can be acetylated by one lases present in a HeLa cell nuclear extract (HNE). but not the other (35, 36, 38, 40). With regard to Immobilized [3H]acetylated ER␣ was incubated with modulating the activity of transcription-related factors, HNE, which contains a variety of deacetylases includ- the consequence of acetylation may be either tran- ing TSA-sensitive enzymes (i.e. class I and II deacety- ϩ scriptional activation or inhibition. Acetylation of tran- lases) and NAD -dependent/nicotinamide-sensitive scription-related factors can increase their transcrip- enzymes (i.e. class III deacetylases, such as SIRT1) tional activity by the following: 1) enhancing DNA (26–29). As shown in Fig. 1C, incubation with HNE ␣ binding activity, 2) stimulating interactions with posi- dramatically reduced the acetylation of ER (compare tive transcriptional regulators, such as chromatin re- lanes 1 and 2). The addition of TSA inhibited deacety- ␣ modeling factors or coactivators, 3) inhibiting interac- lation of ER by the HNE (lane 3), indicating that one tions with negative regulators, resulting in a loss of or more class I/II deacetylases present in the HNE can ␣ ϩ transcription repression, 4) increasing the stability of deacetylate ER . The addition of NAD in the pres- ence of TSA (i.e. under conditions in which class I/II the factors, and 5) altering subcellular localization (35, deacetylases were inhibited) also resulted in the 36, 38, 40). Likewise, acetylation may inhibit the ac- deacetylation of ER␣ (compare lanes 3 and lane 4). tivity of transcription-related factors by reducing bind- ϩ The effect of NAD was blocked by the addition of ing to DNA or chromatin, as well as reducing protein- nicotinamide (lane 5), indicating that one or more class protein interactions required for transcriptional III deacetylases present in the HNE, such as SIRT1, activation (35, 36, 38, 40). Thus, the specific biochem- can deacetylate ER␣. To explore this last result further, ical effects of acetylation are varied and differ with we performed a similar set of experiments using puri- each target protein. fied recombinant SIRT1 (Fig. 1A) in place of the HNE.