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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nuclear receptor corepressor (NCoR) mediates repression (silencing) of basal gene transcription by nuclear receptors for thyroid hormone and retinoic acid. The goal of this study was to create novel estrogen receptor (ER) mutants by fusing transferable repressor domains from the N-terminal region of NCoR to a functional ER fragment. Three chimeric NCoR-ER proteins were created and shown to lack transcriptional activity. These fusion proteins silenced basal transcription of the ERE2-tk-Luc reporter gene and inhibited the activity of co-transfected wild-type ER (wtER), indicating that they possess dominant negative activity. One of the fusion proteins (CDE-RD1), containing the ER DNA-binding and ligand-binding domains linked to the NCoR repressor domain (RD1), was selected for detailed examination. Its hormone affinity, intracellular localization, and level of expression in transfected cells were similar to wtER, and it bound to the estrogen response element (ERE) DNA in gel shift assays. Glutathione-S-transferase pull-down assays showed that CDE-RD1 retains the ability to bind to steroid receptor coactivator-1. Introduction of a DNA-binding domain mutation into the CDE-RD1 fusion protein eliminated silencing and dominant negative activity. Thus, the RD1 repressor domain prevents transcriptional activation despite the apparent ability of CDE-RD1 to bind DNA, ligand, and coactivators. Transcriptional silencing was incompletely reversed by trichostatin A, suggesting a histone deacetylase-independent mechanism for repression. CDE-RD1 inhibited ER-mediated transcription in T47D and MDA-MB-231 breast cancer cells and repressed the growth of T47D cells when delivered to the cells by a retroviral vector. These ER-NCoR fusion proteins provide a novel means for inhibiting ER-mediated cellular responses, and analogous strategies could be used to create dominant negative mutants of other transcription factors.
Mol Endocrinol 1999 Dec
PMID:A fusion protein of the estrogen receptor (ER) and nuclear receptor corepressor (NCoR) strongly inhibits estrogen-dependent responses in breast cancer cells. 1059 86

In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TRE(pal)) but not the glucocorticoid response element (GRE). This intrinsically activated state of hERR1 and hERR2 resides in the ligand-binding domains of the two genes and is transferable to a heterologous receptor. In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner. Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors.
Mol Endocrinol 1999 Dec
PMID:Constitutive activation of transcription and binding of coactivator by estrogen-related receptors 1 and 2. 1059 88

In order to approach the molecular basis of the tissue-specific agonistic activity of antioestrogens, we have compared, at the mRNA level, the expression of various transcriptional cofactors (activators or repressors) of estrogen receptors in different breast (MCF7, ZR75-1, T47D, MDA-MB231) and endometrial (Ishikawa, RL-95-2 and HEC1A) human cancer cell lines. We showed that for SRC-1, CBP, TIF1alpha, RIP140, N-CoR, and SMRT, no significant differences in the expression levels were observed between breast and endometrial cells. For TIF1alpha mRNA, both isoforms were also detected at similar levels in all the cells tested. By contrast, over-expression of AIB1 mRNA was observed in MCF7 cells, but not in other breast or endometrial cells, irrespective of their ER-status. We then used protein-protein interaction assay (far-Western blot) to confirm the increased expression of at least one of the p160 proteins in MCF7 cells. Finally, we demonstrated that RIP140 mRNA is directly induced by estrogens in ER-positive MCF7 breast cancer cell lines but not in Ishikawa endometrial cells. Together these results indicate that some differences exist between breast and endometrial cancer cell lines at the level of estrogen receptor transcription cofactor expression.
Mol Cell Endocrinol 1999 Oct 25
PMID:Estrogen receptor cofactors expression in breast and endometrial human cancer cells. 1061 26

The glucocorticoid receptor (GR) regulates target gene expression in response to corticosteroid hormones. We have investigated the mechanism of transcriptional activation by the GR by studying the role of the receptor interacting protein RIP140. Both in vivo and in vitro protein-protein interaction assays revealed a ligand-dependent interaction between the GR and RIP140. The ligand binding domain of the GR was sufficient for this interaction, while both the N- and C-terminal regions of RIP140 bound to the receptor. In a yeast transactivation assay RIP140 and SRC-1, a member of the steroid receptor coactivator family of proteins, both enhanced the transactivation activity of a GR protein (GRA-1) in which the potent N-terminal tau1 transactivation domain has been deleted. In contrast, in COS-7 cells increasing amounts of RIP140 significantly inhibited GRdeltatau1 function. In cotransfection studies in COS-7 cells, RIP140 also inhibited receptor activity in presence of both SRC-1 and the coactivator protein CBP together. Thus, in yeast cells a stimulation of receptor activity was observed, while in mammalian cells RIP140 repressed GR function. Taken together, these data suggest that, (1) RIP140 is a target protein for the GR and (2) RIP140 can modulate the transactivation activity of the receptor.
J Steroid Biochem Mol Biol 1999 Dec 15
PMID:The nuclear-receptor interacting protein (RIP) 140 binds to the human glucocorticoid receptor and modulates hormone-dependent transactivation. 1065 97

We have characterized the mechanism by which coactivator p300 facilitates transcriptional activation mediated by the heterodimer of thyroid hormone (T3) receptor and 9-cis retinoid acid receptor (TR-RXR) in the context of chromatin. We demonstrate that, while p300 can enhance the transcriptional activation mediated by both liganded TR-RXR and GAL4-VP16, its histone acetyltransferase activity (HAT) is required for its ability to facilitate liganded TR-RXR- but not GAL4-VP16-mediated transcriptional activation. To understand how p300 is recruited by liganded TR-RXR, we have analyzed the interactions between TR-RXR and p300 as well as SRC-1 family coactivators. We show that, in contrast to a strong hormone-dependent interaction between TR-RXR and SRC-1 family coactivators, p300 displays minimal, if any, T3-dependent interaction with TR-RXR. However, p300 can be recruited by liganded TR-RXR through its interaction with SRC-1 family coactivators. Consistent with the protein-protein interaction profile described above, we demonstrate that the SRC-1 interaction domain of p300 is important for its ability to facilitate transcriptional activation mediated by TR-RXR, whereas its nuclear receptor interaction domain is dispensable. Collectively, these results reveal the functional significance of the HAT activity of p300 and define an indirect mode for the action of p300 in TR-RXR activation.
Mol Cell Biol 2000 Mar
PMID:p300 requires its histone acetyltransferase activity and SRC-1 interaction domain to facilitate thyroid hormone receptor activation in chromatin. 1068 50

The steroid hormone progesterone acts via high-affinity nuclear receptors that interact with specific DNA sequences located near the promoter of the hormone-responsive gene. Recent studies suggested that the hormone-occupied progesterone receptor (PR) mediates gene activation by recruiting a cellular coregulatory factor, termed coactivator, to the target promoter. The identity and mechanism of action of the coactivator(s) that regulates transcriptional activity of PR are currently under investigation. Here we provide evidence that the hormone-occupied PR forms a multisubunit receptor-coactivator complex containing two previously described coactivators, CREB-binding protein (CBP) and steroid receptor coactivator 1 (SRC-1, a member of the p160 family of coactivators), in nuclear extracts of human breast tumor T47D cells. The association of CBP and SRC-1/p160 with the receptor complex is entirely hormone dependent. Both CBP and SRC-1/p160 possess intrinsic histone acetyltransferase (HAT) activity, and it has been recently proposed that these coactivators function by modulating chromatin structure at the promoter of the target gene. Interestingly, addition of purified CBP to the nuclear extracts of T47D cells markedly stimulated progesterone- and PR-dependent transcription from a nucleosome-free, progesterone response element (PRE)-linked reporter DNA template. Furthermore, depletion of SRC-1/p160 by immunoprecipitation from these transcriptional extracts also significantly impaired PR-mediated RNA synthesis from a naked PRE-linked DNA template. These results strongly implied that CBP and SRC-1/p160 facilitate receptor-mediated transcription in these cell extracts through mechanisms other than chromatin remodeling. We also observed that the adenoviral oncoprotein E1A, which interacts directly with CBP, repressed PR-mediated transactivation when added to the nuclear extracts of T47D cells. Supplementation with purified CBP overcame this inhibition, indicating that the inhibitory effect of E1A is indeed due to a blockade of CBP function. Most importantly, we noted that binding of E1A to CBP prevented the assembly of a coactivation complex containing PR, CBP, and SRC-1/p160, presumably by disrupting the interaction between CBP and SRC-1/p160. These results strongly suggested that E1A repressed receptor-mediated transcription by blocking the formation or recruitment of coactivation complexes. Collectively, our results support the hypothesis that the assembly of a multisubunit coactivation complex containing PR, CBP, and SRC-1/p160 is a critical regulatory step during hormone-dependent gene activation by PR and that the fully assembled complex has the ability to control transcription through mechanisms that are independent of the histone-modifying activities of its component coactivators.
Mol Cell Biol 2000 Mar
PMID:E1A-mediated repression of progesterone receptor-dependent transactivation involves inhibition of the assembly of a multisubunit coactivation complex. 1068 60

The human progesterone receptor (PR) exists as two functionally distinct isoforms, hPRA and hPRB. hPRB functions as a transcriptional activator in most cell and promoter contexts, while hPRA is transcriptionally inactive and functions as a strong ligand-dependent transdominant repressor of steroid hormone receptor transcriptional activity. Although the precise mechanism of hPRA-mediated transrepression is not fully understood, an inhibitory domain (ID) within human PR, which is necessary for transrepression by hPRA, has been identified. Interestingly, although ID is present within both hPR isoforms, it is functionally active only in the context of hPRA, suggesting that the two receptors adopt distinct conformations within the cell which allow hPRA to interact with a set of cofactors that are different from those recognized by hPRB. In support of this hypothesis, we identified, using phage display technology, hPRA-selective peptides which differentially modulate hPRA and hPRB transcriptional activity. Furthermore, using a combination of in vitro and in vivo methodologies, we demonstrate that the two receptors exhibit different cofactor interactions. Specifically, it was determined that hPRA has a higher affinity for the corepressor SMRT than hPRB and that this interaction is facilitated by ID. Interestingly, inhibition of SMRT activity, by either a dominant negative mutant (C'SMRT) or histone deacetylase inhibitors, reverses hPRA-mediated transrepression but does not convert hPRA to a transcriptional activator. Together, these data indicate that the ability of hPRA to transrepress steroid hormone receptor transcriptional activity and its inability to activate progesterone-responsive promoters occur by distinct mechanisms. To this effect, we observed that hPRA, unlike hPRB, was unable to efficiently recruit the transcriptional coactivators GRIP1 and SRC-1 upon agonist binding. Thus, although both receptors contain sequences within their ligand-binding domains known to be required for coactivator binding, the ability of PR to interact with cofactors in a productive manner is regulated by sequences contained within the amino terminus of the receptors. We propose, therefore, that hPRA is transcriptionally inactive due to its inability to efficiently recruit coactivators. Furthermore, our experiments indicate that hPRA interacts efficiently with the corepressor SMRT and that this activity permits it to function as a transdominant repressor.
Mol Cell Biol 2000 May
PMID:The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding. 1075 95

We have analyzed ligand-dependent, subnuclear movements of the estrogen receptor-alpha (ERalpha) in terms of both spatial distribution and solubility partitioning. Using a transcriptionally active green fluorescent protein-ERalpha chimera (GFP-ERalpha), we find that 17beta-estradiol (E2) changes the normally diffuse nucleoplasmic pattern of GFP-ERalpha to a hyperspeckled distribution within 10-20 min. A similar reorganization occurs with the partial antagonist 4-hydroxytamoxifen; only a subtle effect was observed with the pure antagonist ICI 182,780. To examine the influence of ligand upon ERalpha association with nuclear structure, MCF-7 cells were extracted to reveal the nuclear matrix (NM). Addition of E2, 4-hydroxytamoxifen, or ICI 182,780 causes ERalpha to partition with the NM-bound fraction on a similar time course (10-20 min) as the spatial reorganization suggesting that the two events are related. To determine the effects of E2 on the redistribution and solubility of GFP-ERalpha, individual cells were directly examined during both hormone addition and NM extraction and showed that GFP-ERalpha movement and NM association were coincident. Colocalization experiments were performed with antibodies to identify sites of transcription (RNA pol Ilo) and splicing domains (SRm160). Using E2 treated MCF-7 cells, minor overlap was observed with transcription sites and a small amount of the total ERalpha pool. Experiments performed with bioluminescent derivatives of ERalpha and steroid receptor coactivator-1 (SRC-1) demonstrated both proteins colocalize to the same NM-bound foci in response to E2 but not the antagonists tested. Deletion mutagenesis and in situ analyses indicate intranuclear colocalization requires a central SRC-1 domain containing LXXLL motifs. Collectively, our data suggest that ERalpha transcription function is dependent upon dynamic early events including intranuclear rearrangement, NM association, and SRC-1 interactions.
Mol Endocrinol 2000 Apr
PMID:Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1. 1077 Apr 89

Mutants isolated from the Y1 mouse adrenocortical tumor cell line (clones 10r-9 and 10r-6) are resistant to ACTH because they fail to express the melanocortin-2 receptor (MC2R). In this study, we show that a luciferase reporter plasmid driven by 1,800 bp of the proximal promoter region of the MC2R was expressed poorly in the mutant cells compared with parent Y1 cells. The differential expression of the MC2R in parent and mutant cells resulted from impaired activity of the orphan nuclear receptor NR5A1 (SF1) on the promoter as determined by 5'-deletion analysis. Furthermore, the activity of an SF1 expression plasmid on an SF1-dependent reporter plasmid was compromised in mutant clones. The site-specific DNA binding properties of SF1 from parent and mutant cells did not differ as determined in electrophoretic mobility shift assays, and the addition of the activation domain of VP16 to the amino terminus of SF1 restored the transcriptional activity of the protein. In addition, the levels of SF1 and other cofactors including WT1, CBP/p300, and steroid receptor coactivator 1 did not differ appreciably between parent and mutant cells. Taken together, these results suggest that ACTH resistance in the mutant clones resulted from a defect that affected the activation properties of SF1 rather than its DNA binding activity. Consistent with the observed impairment in SF1 function, other SF1-dependent genes, including Cyp11b1 and steroidogenic acute regulatory protein (StAR), were poorly expressed and global steroidogenesis, as evidenced by the metabolism of 22(R)-hydroxycholesterol to steroid products, was impaired. Interestingly, MC2R, Cyp11a, Cyp11b1, and StAR transcripts were not affected to the same degree, suggesting that each of these genes may have a different absolute requirement for SF1. These mutants thus provide an experimental paradigm to identify factors that influence SF1 function and to evaluate the relative importance of SF1 in the expression of genes essential for adrenal steroidogenesis.
Mol Endocrinol 2000 Apr
PMID:Impaired steroidogenic factor 1 (NR5A1) activity in mutant Y1 mouse adrenocortical tumor cells. 1077 Apr 90

Steroid and thyroid hormone receptors are members of the superfamily of nuclear receptors (NR) that participate in developmental and homeostatic mechanisms by changes in the transcription of specific genes. These activities are governed by the receptors' cognate ligands and through interaction with the components of the transcriptional machinery. A number of coactivator molecules of the steroid receptor coactivator (SRC)/nuclear receptor coactivator (NCoA) family interact with activation functions within NRs through a conserved region containing helical domains of a core LXXLL sequence and, thereby, participate in transcriptional regulation. Using a mammalian-two-hybrid assay, we show that the thyroid hormone receptor beta (TRbeta) and estrogen receptor beta (ERbeta) have different LXXLL motif preferences for interactions with SRC-1. Using large random and focused (centered on the LXXLL motif) recombinant peptide diversity libraries, we have obtained novel peptide sequences that interact specifically with ERbeta or with TRbeta in a ligand-dependent manner. Random sequence libraries yielded LXXLL-containing peptides, and sequence analysis of selected clones revealed that the preferred residues within and around the LXXLL motif vary significantly between these two receptors. We compared the receptor binding of library-selected peptides to that of peptides derived from natural coactivators. The affinities of selected peptides for the ligand binding domains of ERbeta and TRbeta were similar to the best natural LXXLL motifs tested, but showed a higher degree of receptor selectivity. These selected peptides also display receptor-selective dominant inhibitory activities when introduced into mammalian cells. Finally, by directed mutations in specific residues, we were able to alter the receptor binding preference of these peptides.
Mol Endocrinol 2000 May
PMID:Selection of estrogen receptor beta- and thyroid hormone receptor beta-specific coactivator-mimetic peptides using recombinant peptide libraries. 1080 26


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