The transcriptional activity of estrogen receptor-alpha (ER) is modified by coactivators, corepressors and chromatin remodeling complexes. We have previously shown that the metastasis-associated protein-1 (MTA1), a component of histone deacetylase and nucleosome remodeling complexes, represses ER-driven transcription by recruiting histone deacetylases (HDACs) to the estrogen receptor element (ERE)-containing target gene chromatin in breast cancer cells. Using a yeast two-hybrid screening to clone MTA1-interacting proteins, we identified a previously uncharacterized molecule, which we named as MTA1-interacting coactivator (MICoA). Our findings suggest that estrogen signaling promotes nuclear translocation of MICoA and that MICoA interacts with MTA1 both in vitro and in vivo. MICoA binds to the C-terminal region of MTA1, while MTA1 binds to the N-terminal MICoA containing one nuclear receptor interaction LSRLL motif. We showed that MICoA is an ER coactivator, cooperates with other ER coactivators, stimulates ER-transactivation functions, and associates with the endogenous ER and its target gene promoter-chromatin. MTA1 also repressed MICoA-mediated stimulation of ERE mediated transcription in the presence of ER and ER variants with the naturally occurring mutations such as D351Y and K302R, and that it interfered with the MICoA’s association with the ER-target gene chromatin. Since chromatin is a highly dynamic structure and because MTA1 and MICoA could be detected within the same complex, these findings suggest that MTA1 and MICoA might transmodulate functions of each other and any potential deregulation of MTA1 is likely to contribute to the functional inactivation of ER pathway, presumably by derecruitment of MICoA from ER target promoter chromatin. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Introduction The steroid hormone 17 beta-estradiol (E2) plays an important role in controlling the expression of genes involved in a wide variety of biological processes, including reproduction, development, and breast tumor progression (1-3). The biological effects of estrogen are mediated by its binding to the structurally and functionally distinct estrogen receptors (ERand ER). ERis the major estrogen receptor in the mammary epithelium. Like other steroid nuclear receptors, ERcomprises of an N-terminal transcriptional activation function (AF1) domain, a DNA-binding domain, and a C-terminal ligand-binding domain (LBD) that contains a liganddependent transcriptional activation function 2 (AF2) domain (4). Binding of hormone to ER triggers conformational changes that allow ER to bind the responsive elements in the target gene promoters. The ligand-activated ERthen translocates to the nucleus, binds to the 13-base-pair palindromic estrogen response element (ERE) in the target gene promoters, and stimulates gene transcription, thereby promoting the growth of breast cancer cells. In addition, a series of recent studies also demonstrate other actions of the estrogen receptors, which involve protein-protein interactions (i.e. with AP-1 and SP-1) rather than direct DNA binding. As with hormonal regulation, the transcriptional activity of ER is affected by a number of regulatory cofactors including chromatin-remodeling complexes, coactivators, and corepressors (5 –9). Coactivators generally do not bind to the DNA but are recruited to the target gene promoters through protein-protein interactions with the ER. Examples of ER coactivators include, members of the p160 family, SRC1-3, AIBI, TRAM1, RAC3, CREB binding protein CBP and p300 (10-11). Corepressors preferentially associate with antagonist occupied ER (1214). Among the ER corepressors, NCoR and SMRT are widely characterized molecules, that have been implicated in the transcriptional silencing that happens in the absence of ligands (15). by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from In addition, a few bifunctional coregulators such as PELP1 also exist that can act both as coactivators and corepressors of ER (16) Evidence suggests that multi-protein complexes containing coactivators, ERs and transcriptional regulators assemble in response to hormone binding and that they activate transcription. The molecular mechanisms of ER, the composition of the ER coactivator proteins and the way these hormones illicit tissue or cell-type specific responses are active areas of investigation. A structural analysis of the ER coactivators has identified a five-amino acid NR motif LXXLL (where X is any amino acid) that can mediate coregulator binding to the liganded ERs (17-19). For transcription factors to access DNA, the repressive chromatin structure must be remodeled. Dynamic alterations in the chromatin structure resulting from the acetylation of histones can facilitate or suppress the access of the transcription factors to nucleosomal DNA, leading to transcriptional regulation (20-22). Hyperacetylated chromatin is generally associated with transcriptional activation, whereas hypoacetylated chromatin is associated with transcriptional repression (23-28). Transcriptional outcome is regulated by a dynamic interaction of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Coactivators like SRC1-3, CBP/p300 have been shown to possess intrinsic histone acetyltransferase activity (HAT) (4,29-32) while corepressors such as NCoR and metastasis associated 1 (MTA1) protein are associated with HDACs (33-35). The MTA1 gene was originally identified by differential expression in rat mammary adenocarcinoma metastatic cells and is now known to correlate well with the metastatic potential of several human cell lines and tissues (14,35-37). MTA1 has also been shown to physically interact with HDAC and repress the estrogen receptor alpha-driven transcription by recruiting HDAC to the ERE-containing target gene chromatin in breast cancer by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from cells (35) Although MTA1 is known to be a part of the HDAC complex, the nature of its target or targets remains unidentified. To better understand the cellular functions of MTA1 in breast cancer cells, we performed a yeast two-hybrid screen to clone MTA1-interacting proteins, and identified a previously uncharacterized protein (Genebank accession number S 82447), which we named as MTA1interacting coactivator (MICoA). Here, we show that MICoA is a bona-fide coactivator of ER transactivation functions and that its interaction with MTA1 controls the dynamics of ER-driven transactivation by influencing its association with ER-target gene promoter chromatin. Materials and Methods Plasmid Construction and Two-hybrid Library Screening. The full length MTA1 (1 – 715 aa) was digested at BamHI and XbaI (blunt end) and ligated to the pGBKT7 vector that expresses proteins fused to amino acids 1-147 of the GAL4-DNA binding domain (DNA-BD) at BamHI and PstI (blunt end) (Clontech). MTA1 baits were constructed by deleting 1-254 amino acids from the N-terminal of MTA1 by cutting and self-ligating with Nco1 that cleave first 254 amino acids. The remaining 255-715 amino acids of the C-terminal MTA1 (CT-MTA1) were used as bait. This bait was used to screen a mammary gland cDNA library fused to Gal4 activation domain (Clontech) was screened according to manufacturer's instructions. Positive clones were also verified by one-on-one transformations and selection on agar plates lacking leucine and tryptophan (LT) or adenine, histidine, leucine and tryptophan (AHLT) and also processed for by -galactosidase ( -gal) assay. Full length MICoA was either cloned into pCDNA 3.1A or pGEX 5X-1 vectors at Eco R1 and Xho 1 sites. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Cell Cultures and Reagents. Human breast cancer cells were cultured in the DMEM/F12 medium supplemented with 10% fetal bovine serum. For estrogen treatment experiments, regular medium was replaced by medium containing 3% DCC (charcoal-stripped serum). Antibodies against c-myc tag were from MBL International, Watertown, MA. Anti-ER was from Upstate Biotechnology, USA, whereas anti-mouseand anti-rabbit-horseradish peroxidase-conjugate were from Amersham, Piscataway, NJ. In-situ Hybridization. For in-situ hybridization, mouse mammary gland tissues or 13.5 day old embryos were cut out and fixed with 4% paraformaldehyde and frozen sections were cut (35). In situ hybridization was done by using the digoxigenin (Roche) labeled riboprobe. A 375 bp of mouse MICoA cDNA was amplified by RT-PCR, subcloned into TOPO II vector (Promega) and used for riboprobe synthesis under the control of T7 promoter. Primers used are, FCCAGCCCGGAATTCCCATGC-TGTCCCGCCTC; RGGAGGGAACTCGAGCTAGGAAGGGGCAGAC; RNA probes were labeled with digoxigenin and hybridized for 16-20 h in buffer containing 1 g/ml riboprobes, 50% formamide, 300 mM NaCl, 10 mM Tris (pH 7.4), 10 mM NaH2PO4 (pH 6.8), 5 mM EDTA (pH 8.0), 0.2% Ficoll 400, 0.2% polyvinyl pyrolidone, 10% dextran sulfate, 200 ug/ml yeast total RNA, and 50 mM dithiothreitol. Alkaline phosphatase labeled sheep anti-digoxigenin antibody was applied and signals were visualized by NBT-BCIP. Hybridization with sense-probe was used as background control. Chromatin Immunoprecipitation (ChIP) Assay. Approximately 10 cells were treated with 1% formaldehyde (final concentration, v/v) for 10 min at 37 C to cross-link histones to DNA. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from ChIP assay was performed as described (35). The sequence of the forward and reverse primers for pS2 used in this study is GAATTAGCTTAGGCCTAGACGGAATG and AGGATTTGCTGATAGACAGAGACGAC respectively. Histone Acetyl-transferase (HAT) Assay. Cells were either treated with/ without estrogen (10M). Then cells were lysed and immunoprecipitated with anti T7 antibody. Immunoprecipitate was taken for histone acetyl transferase assay by HAT-Check (Histone acetyl transferase) activity assay kit (Pierce, IL). HAT assay with positive c