Interaction between Transcription Factor, Basal Transcription Factor 3, and the NH 2 -Terminal Domain of Human Estrogen Receptor A Chris D. Green, 3 Paul D. Thompson, 2 Patrick G. Johnston, 1 and Mohamed K. El-Tanani 1 1 Queen’s University Belfast, Centre of Cancer Research and Cell Biology, Belfast City Hospital, Belfast, United Kingdom; 2 University of Ulster, Coleraine, Londonderry, United Kingdom; and 3 School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom Abstract The estrogen receptor (ER), like other members of the nuclear receptor superfamily, possesses two separate transcriptional activation functions, AF-1 and AF-2. Although a variety of coactivators and corepressors of AF-2 have been identified, less is known of the mechanism of action of AF-1. We have used the yeast two-hybridsystemtoisolateacDNAcodingforaprotein that binds specifically to the AF-1 region of human ERA. This cDNA codes for the transcription factor basal transcription factor 3 (BTF3). The specificity of the interaction between BTF3 and ERA has been confirmed in vivo and in vitro . Transient transfection experiments reveal that overexpression of BTF3 modulates the transcriptional response of reporter genes to ERA.BTF3 interacts with ERA that has been activated either by 17B-estradiol (ligand-dependent activation) or by epidermal growth factor (ligand-independent activation). The effects of BTF3 on the reporter genes requires the presence of ERA containing an active AF-1 function. BTF3 may be a component of the mechanism by which the AF-1 function of ERA stimulates gene transcription. (Mol Cancer Res 2007;5(11):1191–200) Introduction Steroid hormones, such as 17h-estradiol, produce responses in their target cells by regulating gene expression (1, 2). The receptors for steroid hormones belong to a superfamily of nuclear receptors that, upon binding their individual ligand, are able to bind to specific DNA sequences, hormone response elements, and stimulate the initiation of transcription of nearby hormone-responsive genes (3, 4). Members of the receptor superfamily contain two separate regions that are involved in the activation of transcription (4, 5). These two transactivation functions (AF-1 and AF-2), which are physically distinct from each other, show differences in their mode of action. In particular, the activity of AF-2 is dependent on ligand binding by the receptor, whereas AF-1 activity is ligand-independent (6,7).Morerecently,ithasbeenshownthatnon–ligandsignal molecules such as certain growth factors (8-14) may regulate AF-1 activity. The mechanism by which the receptor, bound to its response element, may influence the assembly/stability of the multiprotein transcription initiation complex, situated at the gene promoter, is unclear. Transcriptional interference (‘‘squelching’’) experiments have provided evidence that both AF-1 and AF-2 require coregulator molecules to achieve stimulation of transcription (15, 16). Subsequently, a variety of molecular candidates for AF-2 coregulators, both coactivators and cosuppressors, have been described (17-21). Such coregu- lator proteins bind in a ligand-dependent manner to regions of the amino acid sequence that are conserved between receptors. Various mechanisms have been suggested by which they may contribute to transcriptional activation (22, 23). The mechanism of action of the AF-1 transactivation function is more obscure, not least because of the lack of sequence conservation at the amino-terminal end of nuclear receptors,whereAF-1islocated.Fewercoregulatorproteinsfor AF-1havebeendescribed(11-14),althoughithasrecentlybeen reported that the steroid receptor RNA activator molecule acts as an AF-1 coactivator (21), and that certain AF-2 coactivators may also bind to the A/B domain of estrogen receptor a (ERa; refs. 24, 25). ER AF-1 binds the COOH terminus of glucocorticoid receptor–interacting protein 1 (NID/AF-1) and other p160s (24). ERa AF-1 transcriptional activity is also enhancedbyp300andDEADboxproteinp68/p72,whichform a protein complex with p160 family proteins and p300/CBP, and directly bind to the A/B domain to potentiate AF-1 activity (11, 26). The phosphorylation of the serine residue at position 118 in the A/B domain stabilizes the complex formation of ER and the coactivator complex containing p68/p72 to potentiate the AF-1 activity (19, 26). Phosphorylation of certain serine residues, within the A/B domain of several nuclear receptors, may play an essential role in the action of AF-1 (10, 26, 27). It has been shown that a point mutation that replaces Ser 118 of human ERa with an unphosphorylatable Ala residue prevents the activation of the receptor by epidermal growth factor (EGF; refs. 10, 26). We have therefore used the yeast two-hybrid system (28) to isolate cDNA coding for a protein that binds specifically to the region of the human ER that contains the AF-1 transactivating Received 3/12/07; revised 6/12/07; accepted 7/5/07. Grant support: NorthWestCancerResearchFund,RRG,NIandActionCancer, Northern Ireland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Mohamed K. El-Tanani, Centre for Cancer Research and Cell Biology, Queen’s University Belfast, University Floor, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, United Kingdom. Phone: 44-28-9026-3486; Fax: 44-9026-3744. E-mail: m.el-tanani@qub.ac.uk Copyright D 2007 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-07-0123 Mol Cancer Res 2007;5(11). November 2007 1191 on May 26, 2016. © 2007 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from