Synergy between estrogen receptor alpha activation functions AF1 and AF2 mediated by transcription intermediary factor TIF2

Abstract

The activation function AF2 in the ligand-binding domain of estrogen receptors ER alpha and ER beta signals through the recruitment of nuclear receptor coactivators. Recent evidence indicates that coactivators, such as the transcription intermediary factor TIF2, also bind to and transactivate the N-terminal AF1 function of the two ERs. We have generated TIF2 mutant proteins that are deficient in either AF1 or AF2 interaction and use these mutants to investigate the relative contribution of both AFs to TIF2 recruitment and transactivation. We observe that TIF2 is capable of interacting simultaneously with both the isolated N- and C-terminus of ER alpha in transfected mammalian cells and in vitro, indicating that TIF2 can bridge both receptor domains. The concomitant interaction of TIF2 with both AFs results in synergistic activation of transcription. Thus, synergy between ER alpha AF1 and AF2 is a result of the cooperative recruitment of TIF2 and/or other members of the p160 coactivator family.

Fig. 1. Constructs used in this study. A schematic drawing of the major TIF2 and ERα constructs employed in this study is given. Note that domains are not depicted to scale. On the right a western blot confirming espression of the TIF2 mutant proteins in cDNA transfected cells at levels comparable to wild-type TIF2 is shown. The position of molecular weight markers is indicated on top of the 90° rotated image. Abbreviations used in this study are: hTIF2, human transcription intermediary factor 2 (Voegel et al., 1996); hTIF2m123, a mutant of TIF2 that contains alanine substitutions for critical leucines in all three LxxLL boxes (Voegel et al., 1998); hTIF2ΔQ, a deletion mutant of TIF2 lacking the Q-rich domain (this study); hTIF2m123ΔQ, a double mutant of TIF2 combining the LxxLL to LxxAA mutations with the Q-rich deletion mutant (this study); hERα, human estrogen receptor α; upper case letters denote the different domains of hERα present in the corresponding construct; in parentheses are the names of the different constructs used in previous studies. Note that the expression of hTIF2ΔQ is slightly higher compared with the other constructs (equal amounts of protein have been loaded for western blot analysis).

Fig. 2. TIF2 has two functionally independent ERα interfaces. (A) A GST interaction assay with hTIF2, hTIF2m123, hTIF2ΔQ, hTIF2m123ΔQ and purified GST–hERαAB (lane 3), as well as GST–hERαCDEF (lanes 4 and 5) in the absence and presence of estradiol (E2), is shown. The different cDNAs coding for wild-type and mutant TIF2 proteins were in vitro translated and proteins subsequently analyzed for their interaction pattern with the two ER fusion proteins. Scanned images of autoradiographs from dried gels were used to generate the figure. Note that equal loading of GST fusion proteins was confirmed by Coomassie staining (not shown). (B–D) Transient transfection experiments in COS-1 cells employing as a reporter the 17m-ERE-TATA-CAT construct, the different TIF2 cDNAs, and hERαABC (B), hERαCDEF (C) and the entire hERα (D) to reveal transcription stimulation by the different TIF2 mutants. CAT values were determined by ELISA and standardized with the aid of the activity of co-expressed β-galactosidase. Note that in (C) for the sake of clarity, values for the activity of hERαCDEF in the absence of ligand have been omitted. Hydroxy-tamoxifen (OHT) functions as complete antagonist on hERαCDEF, while being a partial agonist for hERα, which is completely blocked in its activity in the presence of ICI164-384 (ICI).

Fig. 3. TIF2 can bridge both ER AFs. (A) Set-up for the experiment shown in (B). Transient transfections similar to those in Figure 2 were performed. However, the reporter chosen [5× Gal4-binding sites in front of a TATA element directing expression of chloramphenicol acetyltransferase (17m5-TATA-CAT)] is inactive for Gal4-hERαAB. Again, activity of the system was compared in the presence or absence of TIF2 and its mutants, VP16-hERαCDEF and ligands as indicated underneath the columns. (C) Schematic for the set-up of the experiment shown in (D). A GST-based interaction assay similar to Figure 2A is shown. The retention of in vitro translated, ³⁵S-labeled hERαCDEF on GST–hERαAB beads is determined in the absence and presence of TIF2.1 protein (see Figure 1) and ligands. An autoradiograph of a dried gel is shown.

Fig. 4. TIF2 can mediate synergy between AF1 and AF2. (A) Experimental set-up for transient transfections in CEF cells is shown. (B) Actual experiment using indicated reporter and expression vectors, similar to Figure 2B–D.