Transrepression by a liganded nuclear receptor via a bHLH activator through co-regulator switching

Abstract

Vitamin D receptor (VDR) is essential for ligand-induced gene repression of 25(OH)D3 1alpha-hydroxylase (1alpha(OH)ase) in mammalian kidney, while this gene expression is activated by protein kinase A (PKA) signaling downstream of the parathyroid hormone action. The mapped negative vitamin D response element (1alphanVDRE) in the human 1alpha(OH)ase gene promoter (around 530 bp) was distinct from those of the reported DR3-like nVDREs, composed of two E-box-like motifs. Unlike the reported nVDREs, no direct binding of VDR/RXR heterodimer to 1alphanVDRE was detected. A bHLH-type factor, designated VDIR, was identified as a direct sequence-specific activator of 1nVDRE. The transactivation function of VDIR was further potentiated by activated-PKA signaling through phosphorylation of serine residues in the transactivation domains, with the recruitment of a p300 histone acetyltransferase co-activator. The ligand-dependent association of VDR/RXR heterodimer with VDIR bound to 1alphanVDRE caused the dissociation of p300 co-activators from VDIR, and the association of HDAC co-repressor complex components resulting in ligand-induced transrepression. Thus, the present study deciphers a novel mechanism of ligand-induced transrepression by nuclear receptor via co-regulator switching.

Figure 1

Identification of 1αnVDRE. (A) CAT assay using a series of human 1α(OH)ase gene promoter deletion mutants in MCT cells. After 3 h, forskolin (1 × 10⁻⁸ M), which activates PKA signaling, and 1α,25(OH)₂D₃ (1 × 10⁻⁸ M) were added, respectively. 1α(OH)ase gene promoter deletion constructs (−889/−30, −537/−30, −514/−30, −889/−537 and −537/−514) as indicated were transfected in MCT cells. Results shown are representative of five independent experiments. (B) Sequence of the 1αnVDRE core element. The 1αnVDRE was composed of two E-box-like motifs in the 1α(OH)ase gene promoter −537 to −514 bp. (C) Absence of direct binding between VDR/RXR and 1αnVDRE. A gel mobility shift assay was performed using bacterially expressed recombinant VDR and RXR proteins or MCT cell nuclear extracts together with a radiolabeled probe (10 ng) comprising 1αnVDRE sequence (lanes 3–7). Unlabeled 1αnVDRE oligonucleotides (100 ng) were used as cold competition (lanes 5–7). Radiolabeled probe DR3 (consensus positive VDRE) (10 ng) was used as positive control for DNA binding of liganded VDR/RXR (lanes 1and 2).

Figure 2

Cloning of the 1αnVDRE-binding factor, VDIR. (A) Sequence of VDIR. VDIR has two transactivation domains (AD1 and AD2), and a bHLH motif. (B) Functional domain sequence homology between VDIR and members of the bHLH-type activator family (rat Pan-1, E47; rat Pan-2, E12; human E47; human E12; mouse E12 ). VDIR exhibits a high homology with rat Pan-1 (E47). (C) Analysis of VDIR mRNA expression in various tissues. Northern blotting analysis was performed using VDIR open reading frame as a probe. GAPDH was used as an internal control. (D) 1α(OH)ase and VDIR gene expression in the kidneys of normal and VDR-deficient mice by Northern blotting. VDR^+/+: wild-type mice; VDR^−/−; VDR-deficient mice.

Figure 3

VDIR as an activator for 1αnVDRE. (A) Plasmid dose dependency of VDIR activation of nVDRE. Luciferase activity under the control of 1αnVDRE after the transfection of VDIR, mTFE3 or hE47 into MCT cells. MCT cells were cotransfected with LUC reporter plasmid (0.3 μg of nVDRE pGL3 TATA-LUC vector), rat VDR, rat RXR expression vector (0.1 μg of pSG5-rat VDR, pSG5-rat RXR), mTFE3(1.0 μg of pcDNA3-mTFE3), hE47 (1.0 μg of pcDNA3-hE47) and increasing amounts of pcDNA3-VDIR (0.01–1.0 μg). Empty vector (pcDNA3) was used to keep the total DNA concentration the same. LUC activity is represented as fold induction. Values are mean±s.d. (B) Gel mobility shift assay using bacterially expressed recombinant VDIR, VDR and RXR proteins together with a radiolabeled probe containing 1αnVDRE. The closed arrow indicates VDIR, and the open arrow indicates supershift of the VDR/RXR-VDIR complex. (C) Luciferase activity under the control of 1αnVDRE in MCT cells. Wild-type and mutated VDR, RXR, VDIR and 1α,25(OH)₂D₃ (1 × 10⁻⁸ M) were added as indicated. DR3-Luc was used as a positive control for VDR/RXR and 1α,25(OH)₂D₃. VDR wt: wild-type VDR; VDR ΔABC and VDRΔAB: VDR mutants with deleted N-terminal A–C and AB domains, respectively.

Figure 4

The DNA-binding domain (C-domain) of VDR leeds to the binding of VDIR. (A) GST pull-down assay using either GST alone, GST wild-type VDR or GST-fused VDRs deletion mutants together with [³⁵S]-labeled VDIR in the presence or absence of 1α,25(OH)₂D₃ (1 × 10⁻⁶ M) (upper panel). GST pull-down assay was observed using either GST alone, GST wild-type VDIR or GST-variant VDIRs together with [³⁵S]-labeled VDR in the presence or absence of 1α,25(OH)₂D₃ (1 × 10⁻⁶ M) (lower panel). Right panel: Schematic diagrams of wild-type and variant VDR or VDIR proteins. The specific residues present in each VDR or VDIR variant are indicated. (B) Schematic diagram of wild-type VDR and the structure of VDR DNA-binding domain. The P-box is located in the bottom of the first Zn finger, and the D-box is located in the second Zn finger. Amino-acid residues indicating shadow replaced into alanine or threonine residues, which inhibit DNA binding (E42A, P61T and F62T). Y236A and E420A mutants lack co-activator-binding activity. I260R (isoleucine → arginine) mutant lacks heterodimerization of VDR and RXR. (C) Transrepression of VDIR via VDR mutants in luc assay. Luciferase activities were tested in either 1αnVDRE or DR3 after co-transfection of either wild-type VDR or point mutant VDRs into MCT cells in the presence or absence of 1α,25(OH)₂D₃ (1 × 10⁻⁸ M). This experiment is representative of five independent experiments performed.

Figure 5

Phosphorylation of VDIR by PKA induced a p300 co-activator recruitment. (A) Association of VDIR and p300 in the mammalian two-hybrid assay. The expression plasmids of fusion proteins with GAL4-DBD (pM) and VP16-AD (pVP) were transiently transfected into MCT cells with a GAL4-DBD-regulated 17mer × 8 TATA luciferase reporter. PKAα or VDR/RXR was co-transfected in the absence or presence of 1α,25(OH)₂D₃ (1 × 10⁻⁸ M) as indicated. (B) Phosphorylation of VDIR by PKAα. Luciferase activity of either wild-type VDIR or its point mutants of potential PKAα phosphorylation residue to alanine was tested on 1αnVDRE with or without PKAα in MCT cells. S56A (M1), T322A (M2) and S528A (M3) were replaced alanine residue, respectively. M1/M2/M3 mutant was indicated to replace alanine residues to all of S56, T322 and S528 amino residues. In the lower panel, the in vitro phosphorylation of the VDIR mutants fused with GST by PKAα is shown by in vitro phosphorylation assay. (C) ChIP assays demonstrate co-localization of VDIR and p300 in MCF7 cells. In the left schematic diagram, the 1αnVDRE-contained region amplified by PCR in ChIP assays is illustrated. Antibodies used in each assay are indicated on the right panel.

Figure 6

Co-regulator switching upon VDIR for the ligand-induced transrepression by VDR. (A) HAT and HDAC activities of the immunoprecipitated VDIR complexes in the MCT cells. Assays were determined in MCT cells after treatment, in the absence or presence of 1α,25(OH)₂D₃ and forskolin. Representative graphs corresponding to means±s.d. for triplicate independent experiments are shown. (B) Forskolin-dependent interaction between p300 and VDIR, and 1α,25(OH)₂D₃-dependent interaction between HDAC complex and VDIR. Western blotting of the immunoprecipitates with α-VDIR, α-VDR, α-NCoR, α-HDAC2 and α-Sin3A antibodies. (C) Effects of HDAC inhibitor TSA on repression by 1α,25(OH)₂D₃. Transfections were performed in the presence of TSA (3 mM) in MCT cells. TSA reduced 1α,25(OH)₂D₃-dependent transrepression. (D) Co-localization of VDIR complex components on 1αnVDRE in ChIP assay. Soluble chromatin was prepared from MCT cells treated with 1α,25(OH)₂D₃ (1 × 10⁻⁸ M) for 45 min and immunoprecipitated with the indicated antibodies.

Figure 7

Schematic illustration of the proposed molecular mechanism of 1α,25(OH)₂D₃-induced transrepression in the 1α-hydroxylase gene promoter. Upon activated-PKA signaling due to PTH, the 1α-hydroxylase gene is transactivated through recruitment of a HAT co-activator complex to VDIR bound to 1αnVDRE, leading to increased serum concentrations of 1α,25(OH)₂D₃. 1α,25(OH)₂D₃ binding to VDR induces association with VDIR, and leads to the dissociation of the HAT co-activator complex, and the recruitment of an HDAC co-repressor complex. This results in ligand-induced transrepression of the 1α(OH)ase gene due to co-regulator switching on VDIR.