Ligand-dependent activation of transcription in vitro by retinoic acid receptor alpha/retinoid X receptor alpha heterodimers that mimics transactivation by retinoids in vivo

Abstract

All-trans and 9-cis retinoic acids (RA) signals are transduced by retinoic acid receptor/retinoid X receptor (RAR/RXR) heterodimers that act as functional units controlling the transcription of RA-responsive genes. With the aim of elucidating the underlying molecular mechanisms, we have developed an in vitro transcription system using a chromatin template made up of a minimal promoter and a direct repeat with 5-spacing-based RA response element. RARalpha and RXRalpha were expressed in and purified from baculovirus-infected Sf9 cells, and transcription was carried out by using naked DNA or chromatin templates. Transcription from naked templates was not affected by the presence of RA and/or RAR/RXR heterodimers. In contrast, very little transcription occurred from chromatin templates in the absence of RA or RAR/RXR heterodimers whereas their addition resulted in a dosage-dependent stimulation of transcription that never exceeded that occurring on naked DNA templates. Most importantly, the addition of synthetic agonistic or antagonistic retinoids to the chromatin transcription system mimicked their stimulatory or inhibitory action in vivo, and activation by a RXR-specific retinoid was subordinated to the binding of an agonist ligand to the RAR partner. Moreover, the addition of the p300 coactivator generated a synergistic enhancement of transcription. Thus, the dissection of this transcription system ultimately should lead to the elucidation of the molecular mechanisms by which RAR/RXR heterodimers control transcription in a ligand-dependent manner.

Figure 1

DNA templates and S1 nuclease probe. The structures of the (DR5)5β2G, (17 m)5β2G, and internal control pG1 reporter templates are schematically represented with the positioning of the response elements relative to the transcription start site.

Figure 2

Analysis of RARα/RXRα heterodimers and chromatin structure. (A) Purification of RARα/RXRα heterodimers. FhRARα and HmRXRα were coexpressed in Sf9 cells and were affinity-purified by using a Ni²⁺ column followed by anti-Flag agarose column that bind the HmRXR moiety and the FhRAR moiety of the heterodimer, respectively. Purified heterodimers (100 ng of protein) were separated on a 10% SDSPAGE gel before staining with Coomassie blue (lane 1) or Western blot analysis using mAbs recognizing either human RARα (lane 2) or mouse RXRα (lane 3). (B) Overall chromatin structure was not affected by RARα/RXRα heterodimers. Chromatin or naked (DR5)5β2G templates (200 pM) incubated in the presence or absence of FhRARα/HmRXRα (1 nM) and tRA (10⁻⁶ M) were digested with varying concentrations of micrococcal nuclease in a final volume of 80 μl, were separated on a 1.5% agarose gel, and were Southern blotted by using a [³²P] probe corresponding to the −40 to +5 region of the (DR5)5β2G promoter. DNA supercoiling was estimated as described (35) on DNA (200 ng) treated (or not treated) by topoisomerase I (10 units; final volume of 45 μl). DNA was separated on a 1% agarose gel in the presence or absence of 1.2 μM chloroquine. Migration of relaxed and supercoiled template DNA is indicated.

Figure 3

RARα/RXRα heterodimers activate transcription from chromatin templates in a ligand- and template-specific manner. (A) tRA-induced derepression of transcription from chromatin templates by RARα/RXRα. In vitro transcription was performed on chromatin or naked (DR5)5β2G templates (200 pM) by using a HeLa cell nuclear extract (100 μg) for 45 min in the presence or absence of FhRARα/HmRXRα (1 nM) and tRA (1 μM) in a final reaction volume of 50 μl as indicated. S1 nuclease analysis was carried out after deproteinization. (B) Template specificity of activated transcription. Activation of transcription on chromatin (DR5)5β2G or (17M)5β2G templates was determined in the presence of 1 nM of activator [either Gal4(1–147), Gal4-VP16, or FhRARα/HmRXRα] with or without tRA (1 μM) as above. S1 nuclease digestion of RNA transcripts originating from β2G and pG1 templates generated 179- and 60-nt fragments, respectively (see Fig. 1).

Figure 4

RARα/RXRα heterodimers bind all five RAREs in the promoter region of the (DR5)5β2G chromatin template, irrespective of the presence of tRA. Chromatin or naked (DR5)5β2G templates (250 ng) were incubated in the presence or absence of FhRARα/HmRXRα and tRA (10⁻⁶ M) (under the conditions described above for transcription reactions) for 30 min, were subjected to DNase I digestion (5 units; final volume of 50 μl), then were analyzed by primer extension footprinting (see Materials and Methods). Sites of increased (closed triangle) or decreased (open triangle) sensitivity to DNase I are shown.

Figure 5

Dose-dependent synergistic effects of specific retinoids on activation of transcription by RARα/RXRα heterodimers. (A) Dose-dependent activation by tRA and 9cRA. Transcription reactions were performed as described in Fig. 3 on a (DR5)5β2G template by using FhRARα/HmRXRα in the presence of varying concentrations (5 × 10⁻¹⁰ to 10⁻⁶ M) of tRA (open circles) or 9cRA (closed squares). (B) Receptor-selective and synergistic activation of transcription. Transcription reactions were performed as described above by using synthetic retinoid agonists and antagonists at the concentrations indicated. The receptor specificity of retinoids used are as follows: tRA (panRAR-specific ligand), 9cRA (panRAR- and panRXR-ligand), BMS 753 (RARα-specific agonist), BMS961 (RARγ-specific agonist), BMS649 (panRXR-specific agonist), and BMS614 (RARα-specific antagonist). Transactivation by FhRARα/HmRXRα is expressed relative to that observed from the internal control template (pG1). Induction by tRA (10⁻⁶ M) was arbitrarily set to 100%. All points are the average of at least two independent experiments run in duplicate.

Figure 6

p300 enhances transactivation by RARα/RXRα heterodimers in vitro. (A) Addition of exogenous acetyl CoA (AcCoA) does not effect ligand-dependent transactivation by RARα/RXRα. Transcription reactions were performed in the presence or absence acetyl CoA (1 μM) on naked or chromatin (17M)5β2G or (DR5)5β2G templates plus or minus 1 nM activator [either Gal4(1–147), Gal4-VP16, or FhRARα/HmRXRα] and/or tRA (1 μM), as described in Fig. 3. (B) Addition of acetyl CoA does not further enhance p300-activated transcription. Transcription was performed on (DR5)5β2G templates in the presence or absence of FhRARα/HmRXRα and/or tRA (5 × 10⁻⁸ M). Where indicated, the coactivator p300 (0.5 nM) and acetyl CoA (1 μM) were added.