E1A and a nuclear receptor corepressor splice variant (N-CoRI) are thyroid hormone receptor coactivators that bind in the corepressor mode

Abstract

Unliganded thyroid hormone (TH) receptors (TRs) and other nuclear receptors (NRs) repress transcription of hormone-activated genes by recruiting corepressors (CoRs), such as NR CoR (N-CoR) and SMRT. Unliganded TRs also activate transcription of TH-repressed genes. Some evidence suggests that these effects also involve TR/CoR contacts; however, the precise reasons that CoRs activate transcription in these contexts are obscure. Unraveling these mechanisms is complicated by the fact that it is difficult to decipher direct vs. indirect effects of TR-coregulator contacts in mammalian cells. In this study, we used yeast, Saccharomyces cerevisiae, which lack endogenous NRs and NR coregulators, to determine how unliganded TRs can activate transcription. We previously showed that adenovirus 5 early-region 1A coactivates unliganded TRs in yeast, and that these effects are blocked by TH. We show here that human adenovirus type 5 early region 1A (E1A) contains a short peptide (LDQLIEEVL amino acids 20-28) that resembles CoR-NR interaction motifs (CoRNR boxes), and that this motif is required for TR binding and coactivation. Although full-length N-CoR does not coactivate TR in yeast, a naturally occurring N-CoR variant (N-CoR(I)) and an artificial N-CoR truncation (N-CoR(C)) that retain CoRNR boxes but lack N-terminal repressor domains behave as potent and direct TH-repressed coactivators for unliganded TRs. We conclude that E1A and N-CoR(I) are naturally occurring TR coactivators that bind in the typical CoR mode and suggest that similar factors could mediate transcriptional activation by unliganded TRs in mammals.

Fig. 1.

Structure of N-CoR and adenovirus 5 E1A. (Upper) A schematic depiction of full-length N-CoR (amino acids 1-2453); N-CoR truncations; and the longer E1A splice variant, 289R. RDs in the N-CoR N terminus are shown with darker shading, and NR IDs (1-3) in the C terminus, each containing CBMs (black bars), are shown in light shading. Also shown are schematic representations of N-CoR_I (amino acids 1539-2453) and N-CoR_c (amino acids 1944-2453). The position of the E1A CBM and conserved regions (CR)1-3, which are important for E1A activity but not TR binding, are marked. (Lower) A comparison of the CoRNR box consensus [(I/L)XX(I/H/L)IXXX(I/L)] with similar motifs from the corepressors N-CoR, SMRT, and E1A.

Fig. 2.

The E1A CBM is required for TR coactivation. (A) E1A amino acids 4-29 are required for TR coactivation. Shown are the results of β-gal assays performed with a yeast strain containing a single copy of the chicken lysozyme (F2) TRE and expressing hTRβ1, wild-type E1A_1-82, or E1A_1-82 internal deletion mutants or empty vector. Cultures were treated with either vehicle (hatched bars) or 10^-7 M Triac (solid bars). β-Gal activities were expressed as Miller units per mg of protein. Data shown were pooled from three independent experiments and calculated as mean ± SE. (B) Point mutations in the E1A CBM abrogate TR coactivation as for A, except that E1A_1-82 point mutants were used. A2, Ala substitution for L23 and I24; A4, Ala substitution at L20, L23, L28, and I24. (C) TR interactions with E1A require the CBM. Shown is an exposure of a 10% SDS-polyacrylamide gel loaded with input labeled TRβ or TRβ retained on GST beads in the presence of GST-E1A proteins ± T₃ (10^-6 M). (D) CBM peptide blocks TR binding to E1A and N-CoR. The sequence of the N-CoR CBM1 peptide is shown (Upper) with key hydrophobic residues that are NR contact points boxed in black. (Lower) An exposure of a 10% SDS-polyacrylamide gel loaded with TRs retained on GST-E1A or GST-N-CoR columns plus increasing amounts of CBM peptide (0.3-10 μg). Experiments were performed in the absence of TH except where indicated.

Fig. 3.

N-CoR_I is a coactivator for unliganded TRs. (A) Triac blocks actions of unliganded activators. Shown are the results of β-gal assays performed as in Fig. 2 A, with yeast strains expressing E1A_1-82, N-CoR_FL, N-CoR_I, and N-CoR_C as galactosidase fusions in the presence of increasing concentrations of Triac (10^-9-10^-5 M). (B) N-CoR CBM mutations block TR coactivation as in A, with wild-type or mutated N-CoR_C. Point mutations were all Ala substitutions. Leu-2277, Ile-2281 (M1), and Leu-2285 are in CBM1; Ile-2073 is in CBM2 (M2); and Ile-1953 is in CBM3 (M3). A201 combines mutations at Ile-2281 and Ile-2073 to disrupt CBM1 and -2 (M₁₊₂), A202 combines mutations at Ile-2281 and Ile-1953 to disrupt CBM1 and -3 (M₁₊₃), A203 combines mutations at Ile-2073 and Ile-1953 to disrupt CBM2 and the remaining CBM3 core (M₂₊₃), and A204 combines mutations at all three residues to disrupt all CBMs (M₁₊₂₊₃).

Fig. 4.

E1A and N-CoR coactivation requires the TR CoR-binding surface. (A) Mutations in the TR CoR-binding surface disrupt coactivation by E1A and N-CoR truncations. Shown are results of β-gal assays performed as in Fig. 2 A, with yeast strains expressing E1A_1-82 or N-CoR_C as galactosidase fusions along with wild-type TRs or TR mutants. (B) Interaction of E1A1-82 with hTR ligand-binding domain is impaired by the TR I280K mutant. Shown are results of a yeast two-hybrid experiment performed with a LexA-DNA-binding domain to either wild-type hTRβ or the I280K mutant as bait and E1A_1-82-AD as prey.