Corepressor/coactivator paradox: potential constitutive coactivation by corepressor splice variants

Abstract

The functional consequences of the interaction of transcriptional coregulators with the human thyroid hormone receptor (TR) in mammalian cells are complex. We have used the yeast, Saccharomyces cerevisiae, which lack endogenous nuclear receptors (NRs) and NR coregulators, as a model to decipher mechanisms regulating transcriptional activation by TR. In effect, this system allows the reconstitution of TR mediated transcription complexes by the expression of specific combinations of mammalian proteins in yeast. In this yeast system, human adenovirus 5 early region 1A (E1A), a natural N-CoR splice variant (N-CoR(I)) or an artificial N-CoR truncation (N-CoR(C)) coactivate unliganded TRs and these effects are inhibited by thyroid hormone (TH). E1A contains a short peptide sequence that resembles known corepressor-NR interaction motifs (CoRNR box motif, CBM), and this motif is required for TR binding and coactivation. N-CoR(I) and N-CoR(C) contain three CBMs, but only the C-terminal CBM1 is critical for coactivation. These observations in a yeast model system suggest that E1A and N-CoR(I) are naturally occurring TR coactivators that bind in the typical corepressor mode. These findings also raise the possibility that alternative splicing events which form corepressor proteins containing only C-terminal CBM motifs could represent a novel mechanism in mammalian cells for regulating constitutive transcriptional activation by TRs.

Figure 1. Analysis of the importance of the various CBMs for N-CoR_C coactivation in hTR dependent gene activation in yeast.

Left Side: A schematic depiction of the portions of N-CoR_C used in this study, which were fused to the GAL4-DNA binding domain. The amino acid residues present in each fragment are as indicated, as are the positions of the CBMs. Right Side: β-gal assays were performed with a yeast strain containing a β-galactosidase reporter gene regulated by single copy of the chicken lysozyme (F2) TRE and expressing hTRβ1, the indicated portions of N-CoR_C or empty vector. Cultures were treated with either vehicle (grey bars) or 10^-6 M Triac (black bars). β-gal activity is expressed as Miller units per mg of protein. Data shown were pooled from three independent experiments and calculated as mean ± SE. Assay conditions as described previously [Meng et al., 2005; Walfish et al., 1997].

Figure 2. Schematic model in yeast of transcriptional regulation by corepressor or coactivator regulators.

Left Side: The transcriptional response of FL-N-CoR and a p160 coactivator in the absence and presence of TH. Addition of TH leads to hormone dependent coactivation (DC). Right Side: The constitutive coactivation (CC) of a natural splice variant N-CoR_I in the absence and presence of TH.