Progress 01/01/03 to 12/31/06
Outputs
Estrogen receptors (ERs) are ligand-regulated, DNA-binding transcription factors that mediate the physiological effects of estrogens. Using a variety of biochemical and cell-based approaches, we have shown that estrogen receptor alpha (ERa), but not ER beta (ERb), is a target for acetylation by the p300 acetylase and deacetylation by cellular deacetylases (e.g., members of both the HDAC and Sirtuin deacetylase families). Using mutagenesis and mass spectrometry, we identified two conserved lysine residues in ERa (Lys266 and Lys268) that are the primary targets of p300-mediated acetylation. These residues are acetylated in cells, as determined by immunoprecipitation-Western blotting experiments using an antibody that specifically recognizes ERa acetylated at Lys266 and Lys268. Functionally, acetylation by p300 at Lys266 and Lys268 enhances the DNA binding and transactivation activities of ERa, suggesting a role for ERa in determining outcomes in estrogen signaling
pathways. Collectively, our results implicate acetylation as modulator of the ligand-dependent gene regulatory activity of ERa. Such regulation is likely to play a role in estrogen-dependent signaling outcomes in a variety of estrogen target tissues in both normal and pathological states.
Impacts
These studies have lead to new insights about the molecular mechanisms of estrogen signaling and transcriptional regulation. The information gained from these studies can be applied to the development of new, more effective drugs that could be marketed to treat human diseases, such as cancers. In addition, these studies have produced an antibody reagent that can specifically detect acetylated human estrogen receptor alpha, which could have utility as a diagnostic tool for breast cancers.
Publications
- Kim M.Y., Woo E., Homenko D.R., Chong Y.T.E., Kraus W.L. 2006. 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. 20:1479-1493. (Featured on the cover).
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Progress 01/01/05 to 12/31/05
Outputs
In these studies, we show that estrogen receptor alpha (ERa) is acetylated by the p300 acetylase in a ligand- and steroid receptor coactivator (SRC)-dependent manner. Using mutagenesis and mass spectrometry, we show that two conserved lysine residues in ERa (Lys266 and Lys268) are the primary targets of p300-mediated acetylation. These residues are acetylated in cells, as determined by immunoprecipitation-Western blotting experiments using an antibody that specifically recognizes ERa acetylated at Lys266 and Lys268. The acetylation of ERa by p300 is reversed by native cellular deacetylases, including TSA-sensitive enzymes (i.e., Class I and II deacetylases) and NAD+-dependent/nicotinamide-sensitive enzymes (i.e., Class III deacetylases, such as SIRT1). Acetylation at Lys266 and Lys268, or substitution of the same residues with glutamine (i.e., K266/268Q), a residue that mimics acetylated lysine, enhances the DNA binding activity of ERa in gel electrophoretic mobility
shift assays. Likewise, substitution of Lys266 and Lys268 with glutamine enhances the ligand-dependent activity of ERa in a cell-based reporter gene assay. Collectively, our results implicate acetylation as modulator of the ligand-dependent gene regulatory activity of ERa. Such regulation is likely to play a role in estrogen-dependent signaling outcomes in a variety of estrogen target tissues in both normal and pathological states.
Impacts
These studies have lead to new insights about the molecular mechanisms of estrogen signaling and transcriptional regulation. The information gained from these studies can be applied to the development of new, more effective drugs that could be marketed to treat human diseases, such as cancers. In addition, these studies have produced an antibody reagent that can specifically detect acetylated human estrogen receptor alpha, which could have utility as a diagnostic tool for breast cancers.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The post-translational modification of transcription-related factors by phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation has emerged as a critical regulatory mechanism in activator-dependent transcription. Interestingly, we have found that ER alpha is acetylated in a ligand-dependent manner by a complex of the same cofactors that it uses to stimulate transcription by RNA Pol II, namely p300 and SRC. Using mass spectrometry and mutagenesis, we have identified the lysine residues in ER alpha that are the primary and secondary targets for acetylation by the acetyltransferase p300. Acetylation of ER alpha enhances ER alpha DNA binding activity and transcriptional activity without effecting ligand binding. Together, our studies suggest that acetylation of ER alpha by p300 plays an important role in regulating the receptor's activity and may provide another mechanism for modulating estrogen responsiveness in vivo. Through these studies, we hope to
gain a greater understanding of the role of covalent factor modification in signal-regulated transcription. Furthermore, since the acetylation state of ER alpha is indicative of its transcriptional activity, we hope to determine if it can be used as a prognostic indicator in ER alpha-positive, estrogen-dependent breast cancers.
Impacts
These studies have lead to new insights about the molecular mechanisms of estrogen signaling and transcriptional regulation. The information gained from these studies can be applied to the development of new, more effective drugs that could be marketed to treat human diseases, such as cancers.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The post-translational modification of transcription-related factors by phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation has emerged as a critical regulatory mechanism in activator-dependent transcription. Interestingly, we have found that ER alpha is acetylated in a ligand-dependent manner by a complex of the same cofactors that it uses to stimulate transcription by RNA Pol II, namely p300 and SRC (Kim and Kraus). Acetylation increases the DNA binding activity of ER alpha and correlates with a transcriptionally active state. We have identified at least one lysine residue in ER alpha that is a target for acetylation by p300 and plays a critical role in the enhancement of ER alpha DNA binding activity upon acetylation. Together, our studies suggest that acetylation of ER alpha by p300 plays an important role in regulating the receptor's activity and may provide another mechanism for modulating estrogen responsiveness in vivo. Through these
studies, we hope to gain a greater understanding of the role of covalent factor modification in signal-regulated transcription. Furthermore, since the acetylation state of ER alpha is indicative of its transcriptional activity, we hope to determine if it can be used as a prognostic indicator in ER alpha-positive, estrogen-dependent breast cancers.
Impacts
These studies have lead to new insights about the molecular mechanisms of estrogen signaling and transcriptional regulation. The information gained from these studies can be applied to the development of new, more effective drugs that could be marketed to treat human diseases, such as cancers.
Publications
- Kim M. Y., Kraus W. L. 2003. Analysis of the Mechanisms of p300-mediated acetylation of estrogen receptor a (ERa). Abstract, Keystone Meeting on the Enzymology of Chromatin and Transcription, Santa Fe, NM. March, 2003.
- Malherbe T. F., Kim, M. Y., Homenko D. R., Kraus W. L. 2003. Analysis of the mechanisms and consequences of p300-mediated acetylation of estrogen receptor a (ERa). Abstract, Penn State Summer Symposium on Chromatin Structure and Function, University Park, PA. July, 2003.
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