Amino-terminal domain of TIF2 is involved in competing for corepressor binding to glucocorticoid and progesterone receptors.
Both agonist- and antagonist-bound glucocorticoid receptors (GRs) and progesterone receptors (PRs) regulate gene transcription with the assistance of corepressors (NCoR and SMRT) and coactivators (TIF2/GRIP1, SRC1, and AIB1). Receptor binding of these cofactors is competitive and is considered to involve interactions between the C-terminal ligand binding domain of receptors and receptor interaction domains (RIDs) in the middle and C-terminus of coactivators and corepressors, respectively. Therefore, our recent finding that an amino terminal fragment of TIF2 (TIF2.0 = amino acids 1-627) competed for GR and PR interactions with corepressors in mammalian two-hybrid assays was unexpected. Here, we use biochemical approaches (mammalian two-hybrid, pull-down, and coimmunoprecipitation assays) to locate an N-terminal GR region that is sufficient to bind TIF2.0. In contrast, an N-terminal sequence of PR-B that is largely missing in the shorter PR-A is necessary but not sufficient for TIF2.0 binding. Mutagenesis studies of NCoR establish that the more amino-terminal RID#1, but not RID#2, is necessary for binding to both GR and PR agonist and antagonist complexes. ChIP assays indicate that PR and NCoR each selectively localize to the enhancer element (PRE) of a transiently transfected PREtkLUC reporter in the presence of antagonist steroid, whereas exogenous TIF2.0 reduces the amount of PRE-associated NCoR. Importantly, exogenous TIF2.0 also inhibits the biological responses to added NCoR under the same conditions as those used in the ChIP assays. These results suggest that both N-terminal and middle sequences of TIF2 participate in competing with corepressor for regulating the gene transcriptional responses of GRs and PRs.
Wang
Dongqing
D
Steroid Hormones Section, NIDDK/CEB, National Institutes of Health, Bethesda, Maryland 20892-1772, USA.
Wang
Qi
Q
Awasthi
Smita
S
Simons
S Stoney
SS
D001483:Base Sequence; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D051958:Nuclear Receptor Coactivator 2 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone