P/CAF associates with cyclin D1 and potentiates its activation of the estrogen receptor

Abstract

Cyclin D1 is overexpressed in a significant percentage of human breast cancers, particularly in those that also express the estrogen receptor (ER). We and others have demonstrated previously that experimentally overexpressed cyclin D1 can associate with the ER and stimulate its transcriptional functions in the absence of estrogen. This effect is separable from the established function of cyclin D1 as a regulator of cyclin-dependent kinases. Here, we demonstrate that cyclin D1 can also interact with the histone acetyltransferase, p300/CREB-binding protein-associated protein (P/CAF), thereby facilitating an association between P/CAF and the ER. Ectopic expression of P/CAF potentiates cyclin D1-stimulated ER activity in a dose-dependent manner. This effect is largely dependent on the acetyltransferase activity of P/CAF. These results suggest that cyclin D1 may trigger the activation of the ER through the recruitment of P/CAF, by providing histone acetyltransferase activity and, potentially, links to additional P/CAF-associated transcriptional coactivators.

Figure 1

The association between P/CAF and cyclin D1 in vivo. (A) COS cells were transfected with the indicated combinations of pCX-FLAG-P/CAF (F-P/CAF) and pRcCMV-cyclin D1-HA, pCMX-Gal4-cyclin D1, and pCMX-Gal4. Immunoprecipitations were carried out with antibodies against the FLAG epitope or the Gal4 DNA-binding domain. Precipitated proteins were separated by SDS/PAGE on a 10% gel, transferred to a polyvinylidene difluoride membrane, and analyzed by Western blotting with antibodies against cyclin D1 or the FLAG epitope. (Lower) Whole-cell lysate equivalent to one-tenth of the input for each immunoprecipitation. (B) SAOS-2 cells were transfected with the indicated combinations of FLAG-P/CAF, FLAG-ER, and cyclin D1 expression plasmids. They then were metabolically labeled with [³⁵S]methionine for 4 hr, and immunoprecipitations were carried out with the M2 antibody. Precipitated proteins were resolved on a 10% gel and detected by autoradiography. (C) COS cells were transfected with pRcCMV-cyclin D1-HA, pRcCMV-cyclin D2-HA, or pRcCMV-cyclin D3-HA, plus either pCX-FLAG-P/CAF or the empty vector. Immunoprecipitations were carried out with anti-FLAG antibodies. Proteins were detected by Western blotting with an antibody against the HA-epitope.

Figure 2

Cyclin D1-dependent recruitment of the ER into a complex with P/CAF. MCF-7 cells were transfected with the indicated combinations of pCX-FLAG-P/CAF (2 μg), pcDNA3.1-hER (1 μg), pRcCMV-cyclin D1 (2 μg), plus the empty pRcCMV vector to make up a total of 6 μg DNA. Immunoprecipitations were carried out with the anti-FLAG antibody. Precipitated proteins were analyzed by SDS/PAGE followed by Western blotting of the indicated antibodies.

Figure 3

Potentiation of cyclin D1-stimulated ER activity by wild-type P/CAF, but not by a mutant derivative that lacks HAT activity. (A) SAOS-2 cells were transfected with the reporter plasmids p(ERE)₂-TATA-luc (0.25 μg), pCMV-β-galactosidase (0.5 μg), and expression plasmids encoding the ER (50 ng), cyclin D1 (0 or 1.0 μg), plus FLAG-P/CAF or HAT(−) (0, 0.5, 1.0, 1.5, or 2.0 μg). To assess the effects of cyclin D1, P/CAF, and HAT(−) on the reporter in the absence of the ER, 2 μg of each coactivator was used. The respective empty vectors were used to make up a total of 3 μg DNA in each transfection. Where indicated, the cells were treated with estradiol (10 nM) for 24 hr. The fold activation is calculated relative to that of the ER alone, which is set to 1. The graph shows the averages + SEs from three independent experiments, each of which was done in duplicate. (B) Acetyl-transferase activity of P/CAF and the HAT(−) mutant. Immunoprecipitations using an anti-FLAG antibody were performed on lysates from COS cells transfected with plasmids encoding FLAG-tagged P/CAF or the HAT(−) mutant derivative. The precipitated proteins then were incubated with histones in the presence of ¹⁴C-acetyl-CoA. Proteins were resolved by SDS/PAGE in a composite gel cast, with 15% acrylamide in the lower half and 10% acrylamide in the upper half, and subsequently transferred to a polyvinylidene difluoride membrane and exposed to film for 48 hr. (C) The association between cyclin D1 and P/CAF or the HAT(−) derivative. Immunoprecipitations were carried out with anti-FLAG antibodies on lysates from COS cells transfected with the indicated plasmids. Precipitates were analyzed by Western blotting for their cyclin D1 and FLAG-P/CAF content.