Differential Interaction of Nuclear Receptors with the Putative Human Transcriptional Coactivator hTIF1*
- From the University of Montpellier and INSERM, Hormones and Cancer (U148), 60 rue de Navacelles, 34090 Montpellier, France
Abstract
Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. Intermediary factors interacting with their activation functions are required to mediate transcriptional stimulation. In search of such receptor interacting proteins, we have screened a human cDNA expression library and isolated a human protein that interacts in vitro with transcriptionally active estrogen receptors (ER). Sequence analysis reveals that this protein is the human homolog of mouse TIF1 (transcription intermediary factor 1) shown to enhance nuclear receptor ligand-dependent activation function 2 (AF2) in yeast. We have characterized the nuclear receptor binding site on hTIF1 and shown that a region of 26 residues is sufficient for hormone-dependent binding to the estrogen receptor. As shown by point mutagenesis, the AF2 activation domain of ER is required for the binding of hTIF1 but not sufficient, since a short region encompassing the conserved amphipathic α-helix corresponding to this domain fails to precipitate hTIF1. We also demonstrate that hTIF1 association with DNA-bound ER requires the presence of estradiol. Finally, we show that the interaction of hTIF1 with receptors is selective since strong in vitrohormone-dependent binding is only observed with some members of the nuclear receptor superfamily.
Footnotes
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↵* This work was supported by INSERM, the University of Montpellier I, the Ligue Nationale contre le Cancer, and the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Tel.: 33-4-67-04-37-60; Fax: 33-4-67-54-05-98; E-mail:cavaille{at}u148.montp.inserm.fr.
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↵1 The abbreviations used are: ER, estrogen receptor; GST, glutathione S-transferase; TIF, transcription intermediary factor; RIP, receptor interacting protein; AF, activating function; AD, activation domain; NRBS, nuclear receptor binding site; PCR, polymerase chain reaction; ERE, estrogen-responsive element; PAGE, polyacrylamide gel electrophoresis; PR, progesterone receptor; MR, mineralocorticoid receptor; TR, thyroid hormone receptor; RAR, retinoic acid receptor; RXR, 9-cis-retinoic acid receptor; GR, glucocorticoid receptor; VDR, vitamin D3 receptor; AR, androgen receptor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; HNF4, hepatocyte nuclear factor 4; mut, mutant; RA, retinoic acid; TBP, TATA box-binding protein; TF, transcription factor; HBD, hormone binding domain.
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↵2 S. Dauvois, unpublished observation.
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- Received July 15, 1996.
- Revision received December 27, 1996.
