The adenoviral E1A protein displaces corepressors and relieves gene repression by unliganded thyroid hormone receptors in vivo

Abstract

The human adenovirus type 5 early region 1A (E1A) is one of two oncogenes present in the adenovirus genome and functions by interfering with the activities of cellular regulatory proteins. The E1A gene is alternatively spliced to yield five products. Earlier studies have revealed that E1A can regulate the function of thyroid hormone (T3) receptors (TRs). However, analysis in yeast compared with transfection studies in mammalian cell cultures yields surprisingly different effects. Here, we have examined the effect of E1A on TR function by using the frog oocyte in vivo system, where the effects of E1A can be studied in the context of chromatin. We demonstrate that different isoforms of E1A have distinct effects on TR function. The two longest forms inhibit both the repression by unliganded TR and activation by T3-bound TR. We further show that E1A binds to unliganded TR to displace the endogenous corepressor nuclear receptor corepressor, thus relieving the repression by unliganded TR. On the other hand, in the presence of T3, E1A inhibits gene activation by T3-bound TR indirectly, through a mechanism that requires its binding domain for the general coactivator p300. Taken together, our results thus indicate that E1A affects TR function through distinct mechanisms that are dependent upon the presence or absence of T3.

Figure 1. Schematic representation of E1A variants used in this study

The E1A gene is alternatively spiced to yield 5 mRNA products ranging in size from 13S to 9S. These encode proteins ranging in size from 289 residues (R) to 55 R. The positions for the ends of the regions encoded by alternatively spliced exons are indicated on the top. Note that splicing preserves reading frame except where indicated by hatched box in E1A9S. E1A9S was not used in this study because it lacks most of the highly conserved functional domains.

Figure 2. E1A13S protein relieves unliganded-TR induced gene repression and inhibits liganded TR-induced gene activation in the reconstituted frog oocyte system

The mRNAs for FLAG-TRa/RXR (5.75 ng/oocyte each) with or without increasing amounts of myc-tagged E1A13S mRNA (0.92 ng/oocyte or 4.6 ng/oocyte) were injected into the cytoplasm of the frog oocytes. The firefly luciferase reporter vector (TRE-Luc) together with the control Renilla luciferase plasmid (tk-Luc) was then injected into the nucleus. After overnight incubation with or without 100 nM of T3, the oocytes were lysed and assayed for luciferase activities (top panel). As a measure of the reporter gene transcription level, the ratio of firefly luciferase activity to Renilla luciferase activity was determined and was normalized with the basal level in the absence of T3 and TR as 1. The result from each group was expressed as a percentage of the basal transcription level that was obtained from the oocytes without TRα/RXR mRNA injection. This experiment was repeated 3 times. The same oocyte samples used in luciferase assay were subjected to Western blotting with anti-myc and anti-FLAG antibodies to detect the E1A and TR expression, respectively and representative results were shown in the lower panels, confirming the protein expression of E1A13S and FLAG-TRα.

Figure 3. Differential effects of E1A variants on TR-regulated gene transcription

The mRNAs for FLAG-TRα/RXR (5.75 ng/oocyte each) with or without increasing amounts of myc-tagged E1A13S, 12S, 11S, or 10S mRNA (0.92 ng/oocyte or 4.6 ng/oocyte) were injected into the cytoplasm of the frog oocytes as indicated. After overnight incubation with or without 100 nM of T3, the oocytes were lysed for luciferase assays. The ratio of firefly luciferase activity to Renilla luciferase activity was determined as a measure of the reporter gene transcription level with the basal level in the absence TR set to 1 (top panels). The same oocyte samples were subjected to Western blotting with anti-myc and anti-FLAG antibodies to confirm the protein expression (lower panels). This experiment was repeated twice with similar results. As observed with E1A13S, E1A12S derepressed unliganded TR-induced gene repression and inhibits liganded TR-induced gene activation. In contrast, the shorter forms of E1A, E1A11S and E1A10S, enhanced liganded TR-induced gene activation. E1A11S and E1A10S had little effect on unliganded TR-induced gene repression. Note that the + and −T3 samples were plotted on different scales to highlight the effects of E1A.

Figure 4. E1A 12S competes against corepressor binding to unliganded TR but not coactivator binding to T3-bound TR

The mRNAs for FLAG-TRα/RXR (23 ng/oocyte each) with or without myc-E1A 12S mRNA (4.6 ng/oocyte) were injected into the cytoplasm of oocytes as indicated. After overnight incubation with or without 100 nM of T3, the oocytes were lysed and subjected to IP with anti-FLAG antibody against TRα. Pre-IP lysates and IP samples were immunoblotted with anti-FLAG, anti-myc, anti-N-CoR, and anti-SRC3 antibodies. Myc-E1A 12S was co-immunoprecipitated with FLAG-TR in the sample without T3 treatment (lane 4). The amount of co-immunoprecipitated myc-E1A12S was markedly reduced in T3 treated sample (lane 6). Overexpression of myc-E1A12S dissociated the endogenous corepressor N-CoR from FLAG-TR, resembling T3 treatment (lanes 3-5). Unlike T3 treatment, however, the coactivator SRC3 binding to FLAG-TR was not affected by myc-E1A12S in the presence or absence of T3 (lanes 3-6). Note that the N-CoR signal in the pre-IP samples was expected to be the same in all lanes as it was from endogenous N-CoR in the oocyte. However, it appeared to be stronger in the center lanes but weaker in the flanking ones, especially lane 6. This was likely due to difficulty to transfer the large protein, resulting in some variation with the center lanes transferred better than the flanking ones. However, this does not affect the conclusion about the competition by myc-E1A12S against endogenous N-CoR for binding to TR as shown by lanes 3 and 4 in the center.

Figure 5. Effects of mutant E1A12S on TR-regulated reporter gene transcription

(A). Schematic diagrams of E1A12S and its mutants. The regions in wild-type E1A12S protein that are required for interaction with TR, p300, and pCAF are shown. Three deletion mutant constructs, Δ30–49, Δ48–60, and Δ61–69 were generated in the context of the E1A 12S cDNA by PCR based mutagenesis. All of these mutants retained the TR binding site (amino acids 4-29 [52]) and the ability to bind pCAF (amino acids 1-25 [57, 58]). Mutants Δ30–49, Δ48–60 and Δ61–69 are unable to bind p300 [56]). (B). The mRNAs for FLAG-TRα/RXR (5.75 ng/oocyte each) with or without the mRNA for myc-tagged E1A12S, Δ30–49, Δ48–60, or Δ61–69 (4.6 ng/oocyte) were injected into the cytoplasm of the frog oocytes as indicated. The reporter DNA was injected next. After overnight incubation with (lanes 7-12) or without (lanes 1-6) 100 nM of T3, the oocytes were lysed for luciferase assays. The ratio of firefly luciferase activity to Renilla luciferase activity was determined as a measure of the reporter gene transcription level with the basal level in the absence TR set to 1 (top panels). The same oocyte samples were subjected to Western blotting with anti-myc antibody to show similar levels of the expression of different E1A mutants (bottom panels). As shown in left panel, all of these mutants de-repressed unliganded TR-induced gene repression just like E1A12S. In contrast to E1A12S, which inhibited liganded TR-induced gene activation, all these mutants had minimal effect on liganded TR-induced gene activation. This experiment was repeated twice with similar results.

Figure 5. Effects of mutant E1A12S on TR-regulated reporter gene transcription

(A). Schematic diagrams of E1A12S and its mutants. The regions in wild-type E1A12S protein that are required for interaction with TR, p300, and pCAF are shown. Three deletion mutant constructs, Δ30–49, Δ48–60, and Δ61–69 were generated in the context of the E1A 12S cDNA by PCR based mutagenesis. All of these mutants retained the TR binding site (amino acids 4-29 [52]) and the ability to bind pCAF (amino acids 1-25 [57, 58]). Mutants Δ30–49, Δ48–60 and Δ61–69 are unable to bind p300 [56]). (B). The mRNAs for FLAG-TRα/RXR (5.75 ng/oocyte each) with or without the mRNA for myc-tagged E1A12S, Δ30–49, Δ48–60, or Δ61–69 (4.6 ng/oocyte) were injected into the cytoplasm of the frog oocytes as indicated. The reporter DNA was injected next. After overnight incubation with (lanes 7-12) or without (lanes 1-6) 100 nM of T3, the oocytes were lysed for luciferase assays. The ratio of firefly luciferase activity to Renilla luciferase activity was determined as a measure of the reporter gene transcription level with the basal level in the absence TR set to 1 (top panels). The same oocyte samples were subjected to Western blotting with anti-myc antibody to show similar levels of the expression of different E1A mutants (bottom panels). As shown in left panel, all of these mutants de-repressed unliganded TR-induced gene repression just like E1A12S. In contrast to E1A12S, which inhibited liganded TR-induced gene activation, all these mutants had minimal effect on liganded TR-induced gene activation. This experiment was repeated twice with similar results.