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Differential interaction of nuclear receptors with the putative human transcriptional coactivator hTIF1
S Thénot1, C Henriquet, H Rochefort
1University 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 alpha-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 vitro hormone-dependent binding is only observed with some members of the nuclear receptor superfamily.
Insights
Researchers identified a human protein, hTIF1, that interacts with estrogen receptors (ER). This interaction is crucial for hormone-dependent gene regulation, highlighting a key intermediary factor in nuclear receptor signaling pathways.
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- Nuclear receptors are key regulators of gene activity, functioning as ligand-activated transcription factors.
- Transcriptional stimulation by nuclear receptors requires intermediary factors that interact with their activation functions.
Purpose of the Study:
- To identify and characterize novel proteins that interact with transcriptionally active estrogen receptors (ER).
- To elucidate the role of identified proteins in mediating nuclear receptor-mediated gene regulation.
Main Methods:
- Screening of a human cDNA expression library to identify ER-interacting proteins.
- In vitro interaction assays, including precipitation assays, to confirm and characterize binding.
- Sequence analysis and point mutagenesis to define binding domains and requirements.
Main Results:
- Isolation and identification of human TIF1 (hTIF1), a homolog of mouse TIF1, which interacts with active ER.
- Characterization of a 26-residue region on hTIF1 sufficient for hormone-dependent binding to ER.
- Demonstration that ER's AF2 domain is necessary but not sufficient for hTIF1 binding, and that estradiol is required for hTIF1-ER association.
- Selective binding of hTIF1 to specific members of the nuclear receptor superfamily.
Conclusions:
- hTIF1 is a crucial intermediary factor that binds to estrogen receptors in a hormone-dependent manner.
- The interaction between hTIF1 and ER is specific and requires the ligand estradiol, playing a significant role in transcriptional regulation.
- hTIF1 represents a key component in the signaling pathway of nuclear receptors, influencing gene activity through direct interaction.