JBC Papers in Press. Published on March 28, 2011 as Manuscript M110.206029 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M110.206029 ARGLU1 interacts with MED1 and is required for estrogen receptor-mediated gene transcription and breast cancer cell growth Dingxiao Zhang§, Pingping Jiang§‡, Qinqin Xu§‡, and Xiaoting Zhang §* §Department of Cancer and Cell Biology, College of Medicine, University of Cincinnati, OH 45267; and ‡College of Life Sciences, Zhejiang University, Hangzhou 310058, China Running title: ARGLU1 in ER-Mediated Transcription 1Address correspondence to: Xiaoting Zhang Ph.D., Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267. Tel.: 513-558-3017, Fax: 513-558-4454, E-mail: [email protected] we found that depeltion of ARGLU1 The estrogen receptor is a nuclear receptor significantly impairs the growth, as well as superfamily member of transcriptional anchorage-dependent and -independent colony activators that regulate gene expression by formation of breast cancer cells. Taken together, recruiting diverese transcriptional coregulators. these results establish ARGLU1 as a new Downloaded from The Mediator complex is a central MED1-interacting protein required for transcriptional coactivator complex that acts as estrogen-dependent gene transcription and a bridge between transcriptional activators and breast cancer cell growth. RNA polymerase II. MED1 (Mediator subunit 1) www.jbc.org is the key Mediator subunit that directly INTRODUCTION interacts with estrogen receptor to mediate its The estrogen receptor belongs to nuclear receptor functions both in vitro and in vivo. Interestingly, superfamily of transcriptional activators that, in a by guest, on September 12, 2011 our previous biochemical analyses indicated ligand-dependent manner, regulates the expression that MED1 exists only in a subpopulation of of specific genes controlling the development, Mediator complex that is enriched with a reproduction, homeostasis, and metabolism of an number of distinct Mediator subunits and RNA organism (1,2). Like other nuclear receptors, polymerase II. Here, we report ARGLU1 as a estrogen receptor shares a common organization of MED1/Mediator-associated protein. We found functional domains: a highly variable N-terminal ARGLU1 (arginine and glutamate rich 1) not activation domain 1 (AF-1) that mediates only colocalizes with MED1 in the nucleus, but ligand-independent transcription, a highly also directly interacts with a far C-terminal conserved central DNA-binding domain (DBD) region of MED1. Reporter assays indicate that that specifically binds to hormone-response ARGLU1 is able to cooperate with MED1 to elements (HRE) in cognate promoters of target regulate estrogen receptor-mediated gene genes, and a moderately conserved C-terminal transcription. Importantly, ARGLU1 is ligand-binding domain (LBD) containing recruited, in a ligand-dependent manner, to activation domain 2 (AF-2), which mediates endogenous estrogen receptor target genes ligand-dependent transcription (1,2). Upon ligand promoters and is required for their expression. binding, the ultimate action of these receptors on Furthermore, by ChIP-reChIP assay, we regulating target gene expression is to enhance the confirm that ARGLU1 and MED1 colocalize on recruitment and/or function of the general the same estrogen receptor target gene transcriptional machinery, including RNA promoter upon estrogen induction. Moreover, polymerase II and general transcription factors 1 Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc. (3-6). In the past decades, increasingly diverse Mediator subunits (20,24). Interestingly, MED1 groups of transcriptional coregulators have been displays a relative functional specificity to its found to play key roles in this process (3,4). corresponding transcription factors. Thus, Among them, Mediator has emerged as a key knockdown of MED1 expression abolishes the transcriptional coregulator complex that is expression of estrogen receptor-dependent target responsible for communicating the signals from genes but not that of genes controlled by other transcription activators to RNA polymerase II and activators such as p53, which interacts with the general transcription machinery (7-9). another subunit, MED17 (20). Importantly, our ChIP analysis revealed that the MED1/Mediator Mediator is a large protein complex composed of subpopulation is specifically recruited to estrogen about 30 distinct subunits and is conserved from receptor target gene promoters upon estrogen yeast to humans (9-13). Recent studies have stimulation but not to the p53 target gene promoter established the direct interactions between by UV stimulation where a general population of estrogen receptor and the MED1 (also known as Mediator is recruited by activated p53 (20). These Downloaded from TRAP220/PBP/DRIP205) subunit of the Mediator results, together with our finding that MED1 is complex (14-17). Ectopic MED1 expression has selectively expressed and required for luminal been shown to markedly enhance estrogen mammary epithelial cell differentiation, revealed receptor-dependent transcription, both in vitro and additional functional specificity for this complex www.jbc.org in cellular systems (15,16,18). Further studies (17). indicated that MED1 is not only required for estrogen receptor-dependent reporter and In this study, we report the novel finding that by guest, on September 12, 2011 endogenous gene expression, but is also required ARGLU1 (arginine and glutamate rich 1) is a for estrogen-dependent breast cancer cell growth MED1/Mediator- interacting protein by mass (15,18-20). The estrogen receptor interacts in a spectrometry and protein-protein interaction ligand-dependent manner with two LxxLL motifs studies. Interestingly, we found that ARGLU1 not of MED1 through its AF-2 domain (16,19,21-23). only colocalizes with MED1, but that it is also More recently, we have generated MED1 LxxLL able to directly interact with MED1. Further motif-mutant knockin mice and found that MED1 deletion mapping experiments were carried out to is required for estrogen receptor functions in dissect the region required for these interaction pubertal mammary gland development and and establish ARGLU1 as the first protein reported luminal-cell differentiation (17). Surprisingly, to interact with a far C-terminal region of MED1. these MED1 LxxLL motifs were apparently Moreover, the role of ARGLU1 in dispensible for mammary gland development estrogen-mediated gene transcription and the during pregnancy and the development of other requirment of ARGLU1 for estrogen-depedent cell estrogen-responsive tissues such as uterus (17). growth and anchorage-dependent and independent colony formation of breast cancer cells was Our biochemical analyses indicated that MED1 investigated. exists only in a subpopulation of Mediator complexes (20). Further characterization revealed EXPERIMENTAL PROCEDURES that MED1/Mediator is enriched with RNA Plasmids and Constructs: pERE-TK-Luc, polymerase II and a number of additional pRL-CMV, and the MED1 expression construct 2 pCDNA3.1-TRAP220 have been described immunoblotting. previously (20). MED1 deletions and other Immunofluorescent staining: Immunofluorescent Mediator subunits used for TNT assays were staining was carried out by first seeding MCF-7 constructed by inserting the appropriate cells on glass coverslips in 6-well plates. Cells PCR-amplified cDNA coding regions into the were then grown to about 75% confluency and BamHI/EcoRI sites of the pIRES-neo vector fixed for 15 min at room temperature (RT) in 2% (20,23,25). The ARGLU1 expression plasmid (w/v) formaldehyde. After washing, cells were pCDNA3.1-ARGLU1 was constructed by incubated overnight at 4°C with control IgG, or inserting the coding region of ARGLU1 into the primary anti-MED1 (20) and anti-ARGLU1 BamHI/EcoRI sites of pCDNA3.1 after (H00055082-B01, Novus Biologicals) rabbit amplication using primers polyclonal antibodies, followed by Alexa 555- or 5’-atggatccaccatgGGCCGGTCTCGGAGC-3’ and Alexa 488-conjugated secondary goat anti-rabbit 5’- acgaattcTTAATCCTGGGTTTTTAATG-3’. antibodies (Invitrogen). DAPI staining was then The GST (glutathione S-transferase)-fusion carried out for 15 min at RT before mounting of Downloaded from proteins of ARGLU1 were produced by the coverslips. The images were visualized and subcloning the cDNA fragments encoding captured using a LSM510 NLO two-photon full-length human ARGLU1 (primers described confocal microscope (Zeiss). above), N-terminal (amino acids 1-89, www.jbc.org 5’-atggatccaccatgGGCCGGTCTCGGAGC-3’ and Luciferase Reporter Assay: Cells were first grown 5’-acgaattcCACCGTGCGCCCGAAGATGT-3’) in DMEM medium containing 10% and C-terminal (amino acids 83-273, charcoal-dextran-stripped fetal bovine serum for at by guest, on September 12, 2011 5’-atggatccaccatgGACATCTTCGGGCGCACGG least 3 days, and then seeded in 24-well plates at a T-3’ and density of 105 cells/well. The estrogen-responsive 5’-acgaattcTTAATCCTGGGTTTTTAATG-3’) reporter plasmid pERE-TK-Luc was cotransfected into the pGEX-4T1 vector. with control plasmids or plasmids expressing ARGLU1 or MED1 by Oligofectamine Immunoprecipitation (IP) and GST Pull-down: (Invitrogen) according to the manufacturer's Hela nuclear extract was first cleared with protein instructions. The pRL-CMV plasmids were also A/G agarose beads (Santa Cruz Biotechnology) for cotransfected to serve as an internal control for