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. 2008 Apr;28(8):2549-58.
doi: 10.1128/MCB.01199-07. Epub 2008 Feb 4.

Acinus-S' represses retinoic acid receptor (RAR)-regulated gene expression through interaction with the B domains of RARs

Zivjena Vucetic  1 , Zhenping Zhang, Jianhua Zhao, Fang Wang, Kenneth J Soprano, Dianne Robert Soprano
Affiliations

Acinus-S' represses retinoic acid receptor (RAR)-regulated gene expression through interaction with the B domains of RARs

Zivjena Vucetic et al. Mol Cell Biol. 2008 Apr.

Abstract

The diverse biological actions of retinoic acid (RA) are mediated by RA receptors (RARs) and retinoid X receptors (RXRs). Modulation of transcription by RARs/RXRs is achieved through two activation functions, ligand-independent AF-1 and ligand-dependent AF-2, located in the A/B and E domains, respectively. While the coregulatory proteins that interact with the E domain are well studied, the A/B domain-interacting partners and their influence(s) on the function of RARs are poorly understood. Acinus-S' is an ubiquitous nuclear protein that has been implicated in inducing apoptotic chromatin condensation and regulating mRNA processing. Our data demonstrate that Acinus-S' can specifically repress ligand-independent and ligand-dependent expression of a DR5 RA response element(RARE)-dependent reporter gene and several endogenous RAR-regulated genes in a dose-dependent and gene-specific manner. Chromatin immunoprecipitation assays show that Acinus-S' associates with RAREs within the promoters of endogenous genes independent of RA treatment. Furthermore, the C-terminal end of Acinus-S' and the B domain of RARbeta interact independently of ligand, and the C-terminal end of Acinus-S' is sufficient for the repression of RAR-regulated gene expression. Finally, histone deacetylase activity only partially accounts for the repressive effect of Acinus-S' on RAR-dependent gene expression. These findings identify Acinus-S' as a novel RAR-interacting protein that regulates the expression of a subset of RAR-regulated genes through direct binding to the N-terminal B domains of RARs.

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Figures

FIG. 1.
FIG. 1.
Effect of Acinus-S′ on reporter gene activity. The bar graph indicates relative reporter gene activity levels from transfected CV-1 cells treated with ethanol (E), 1 μM RA, or 1 μM ciglitazone (Cig) for 24 h. The change in normalized CAT or Luc activity was calculated relative to cells that were transfected with empty vector DNA (no Acinus-S′) and treated with ethanol (set as 1). Error bars indicate standard deviations.
FIG. 2.
FIG. 2.
Effect of Acinus-S′ on the expression of early RAR-regulated genes in P19 cells in the absence (A) and in the presence (B) of RA. P19 cells were plated on 100-mm tissue culture dishes and transfected with 2, 5, or 10 μg of pEGFP-Acinus-S′ DNA or mock transfected with 10 μg of empty pEGFP vector. At 24 h after transfection, the cells were treated with ethanol (A) or 1 μM RA (B) for 4 h. The expression levels of a panel of early RAR-responsive genes (HNF3a, Stra4, CYP26A1, RARβ2, Hoxa1, Meis2, and Hoxb1 genes) and control genes (36B4 and β-actin genes) were measured by real-time RT-PCR. The expression level of each gene examined was normalized to the endogenous GAPDH levels and expressed relative to the cells transfected with empty vector and treated with ethanol (set to 1). Error bars indicate standard deviations.
FIG. 3.
FIG. 3.
(A) Interaction of Acinus-S′ with RARs and RXR. GST pull-down assays were performed with purified GST-Acinus-S′ and in vitro-transcribed and -translated [35S]methionine-labeled RARs (RARα1, RARβ1, RARβ2, RARβ3, RARβ4, and RARγ2), RXRα, and luciferase. (B) Interaction of Acinus-S′ with members of steroid/thyroid hormone receptor superfamily. The interaction was tested in GST pull-down assays with GST-Acinus-S′ and in vitro-transcribed and -translated labeled ER, TR, and PPARγ. (C) In vivo interaction of Acinus-S′ with RARβ. P19 cells were plated on 100-mm tissue culture dishes and transfected with 10 μg of pEGFP-Acinus-S′ DNA and 10 μg of pOPRSVICAT-RARβ3 DNA. Twenty-four hours later, the cells were treated with ethanol (E) or 1 μM RA for 4 h. Protein complexes were immunoprecipitated from whole-cell lysates using anti-pan-RAR, anti-GFP or mouse IgG. Western blot analysis was performed with either GFP antibody or pan-RAR antibody using the One-Step Complete IP-Western kit (Genscript).
FIG. 4.
FIG. 4.
Interaction of Acinus-S′ on the promoters of the RAR-regulated RARβ2 and CYP26 genes. P19 cells were transfected with pEGFP-Acinus-S′ DNA (+Acinus), and untransfected P19 cells were used as a control (WT). At 24 h after transfection, the cells were treated with ethanol or 1 μM RA for 4 h. Chromatin was immunoprecipitated from cells in each experimental condition using antibodies to GFP, pan-RAR, RNA polymerase (RNAP), and acetylated histone H3. Mouse IgG was used as a negative control. The precipitated DNA was amplified by PCR using specific primers flanking the RAREs of RARβ2 and CYP26A1 and a region located ∼1 kb upstream of the RARβ2 and CYP26A1 RAREs. PCR products were separated by 10% polyacrylamide gel electrophoresis and visualized using ethidium bromide staining.
FIG. 5.
FIG. 5.
(A and B) Schematic representation of Acinus-S′ (A) and RARβ (B) deletion mutants used in GST pull-down assays to map the interaction domains. (C) Purification of GST-Acinus-S′ deletion mutants. GST fusion Acinus-S′ plasmid DNAs representing the full-length GST-Acinus-S′, GST-Acinus-S′ N terminus, GST-Acinus-S′ C terminus, and GST-Acinus-S′ RRM were expressed in E. coli BL21, purified using a glutathione-agarose affinity purification protocol, resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized using Coomassie blue staining. Equal amounts of purified proteins were used for GST-pull down assays. (D) The C terminus of Acinus-S′ binds to the N terminus of RARβ. GST-Acinus-S′, GST-Acinus-S′ N terminus, GST-Acinus-S′ RRM, and GST-Acinus-S′ C terminus were incubated with in vitro-transcribed and -translated full-length [35S]methionine-labeled RARβ3, RARβ4, RARβ(CDEF), and RARβ(DEF). (E) Interaction of the C-terminal region of Acinus-S′ with RARs and RXRα. GST pull-down assays were performed using GST-Acinus-S′ C terminus and in vitro-transcribed and -translated [35S]methionine-labeled RARs (RARα1, RARβ1, RARβ2, RARβ3, RARγ2), RXRα, and luciferase. (F) The C terminus of Acinus-S′ binds to the A/B domain of RARβ. Full-length GST-Acinus-S′ and GST-Acinus S′ C terminus were incubated with in vitro-transcribed and -translated [35S]methionine-labeled A/B domains of RARβ isoforms [(RARβ1(AB), RARβ2(AB), and RARβ3(AB)]. (G) The C terminus of Acinus-S′ interacts with the B domain of RARβ. Interaction assays were performed with full-length GST-Acinus-S′ and in vitro-transcribed and -translated [35S]methionine-labeled RARβ4 and RARβ4 deletion mutants lacking the EF domain [RARβ4(BCD)] or lacking the DEF and partial C domains [RARβ4(BC)].
FIG. 6.
FIG. 6.
(A) Schematic representation of Acinus-S′ constructs used to determine the functional importance of the Acinus-S′ C-terminal domain. (B) Expression of Acinus-S′ C-terminal deletion mutants in P19 cells. P19 cells were plated on 100-mm tissue culture dishes and transfected with 10 μg of one of the following DNAs: His-Acinus-S′, His-Acinus-S′ C terminus, GFP-Acinus-S′, or GFP-Acinus-S′ ΔC. At 24 h after transfection, the cells were treated with ethanol (E) or 1 μM RA for 4 h and nuclear extracts were prepared. Western blot analysis was performed using primary anti-His or anti-GFP antibody followed by a secondary horseradish peroxidase-anti-rabbit antibody. Anti-lamin B primary antibody was used as a loading control. (C and D) Effect of the C-terminal end of Acinus-S′ on the expression of RAR-regulated genes in the absence (C) and presence (D) of RA. P19 cells were transfected and treated as described above. The expression levels of RAR-dependent genes (RARβ2 and CYP26A1) and control genes (36B4 and β-actin) were measured by real-time RT-PCR. The expression level of each gene examined was normalized to the endogenous GAPDH levels and expressed relative to the ethanol-treated P19 cells. Error bars indicate standard deviations.
FIG. 7.
FIG. 7.
Role of histone deacetylation on repression of RARE reporter activity by Acinus-S′. The bar graph indicates relative RARE reporter gene activity levels from transfected Cos-7 cells treated with ethanol (EtOH), 1 μM RA, or 100 nM TSA for 24 h. The change in normalized Luc activity was calculated relative to cells that were transfected with empty vector DNA (no Acinus-S′) and treated with ethanol and vehicle (set as 1). Error bars indicate standard deviations.
FIG. 8.
FIG. 8.
Alignment of the B domains of RARs and RXRα. Amino acids conserved in all three RARs and RXRα are indicated in bold.
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