Inhibition of nuclear receptor signalling by poly(ADP-ribose) polymerase - PubMed (original) (raw)

Inhibition of nuclear receptor signalling by poly(ADP-ribose) polymerase

T Miyamoto et al. Mol Cell Biol. 1999 Apr.

Abstract

Mammalian poly(ADP-ribose) polymerase (PARP) is a nuclear chromatin-associated protein with a molecular mass of 114 kDa that catalyzes the transfer of ADP-ribose units from NAD+ to nuclear proteins that are located within chromatin. We report here the identification of a novel property of PARP as a modulator of nuclear receptor signalling. PARP bound directly to retinoid X receptors (RXR) and repressed ligand-dependent transcriptional activities mediated by heterodimers of RXR and thyroid hormone receptor (TR). The interacting surface is located in the DNA binding domain of RXRalpha. Gel shift assays demonstrated that PARP bound to TR-RXR heterodimers on the response element. Overexpression of wild-type PARP selectively blocked nuclear receptor function in transient transfection experiments, while enzyme-defective mutant PARP did not show significant inhibition, suggesting that the essential role of poly(ADP-ribosyl) enzymatic activity is in gene regulation by nuclear receptors. Furthermore, PARP fused to the Gal4 DNA binding domain suppressed the transcriptional activity of the promoter harboring the Gal4 binding site. Thus, PARP has transcriptional repressor activity when recruited to the promoter. These results indicates that poly(ADP-ribosyl)ation is a negative cofactor in gene transcription, regulating a member of the nuclear receptor superfamily.

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Figures

FIG. 1

FIG. 1

PARP interacts with nuclear receptors in vitro. (a) Schematic diagram of the domain structure of full-length rat PARP. The isolated interacting fragment (amino acids 82 to 220) is indicated. (b) PARP interacts with RXR. 35S-labelled PARP(82-220) or luciferase was synthesized by in vitro translation and incubated separately with GST (lanes 2 and 5) or GST-RXR (lanes 3 and 6) bound to glutathione-Sepharose beads. Ten percent of input 35S-labelled proteins are indicated (lanes 1 and 4). (c) Endogenous PARP interacts with hormone receptors. Crude COS1 cell extracts were allowed to interact with immobilized RXR. COS1 cell extracts were incubated with matrix-bound GST-RXR or GST. After a wash, PARP activity which associated with beads was measured in the absence or presence of 3-aminobenzamide (3AB). The initial velocity of [32P]NAD incorporation into acid-insoluble acceptors was measured at 25°C for 1 min. The results represent averages and standard deviations from three independent experiments.

FIG. 2

FIG. 2

Domains within nuclear receptors required for PARP interactions. (a) Series of N- and C-terminal deletions of RXR used in pull-down experiments. (b) In vitro interaction of PARP(82-220) with RXR. Bacterially produced GST-RXR deletions or GST alone was bound to glutathione-Sepharose beads and incubated with equivalent amounts of 35S-labelled PARP(82-220) produced by in vitro translation. Associated proteins were analyzed by SDS–15% PAGE and visualized by BAS 1500 (Fuji, Tokyo, Japan). (c) 35S-labelled PARP(82-220) was incubated with matrix-bound GST-RXRα in the absence or presence of 1 U of DNase I, and associated proteins were analyzed by SDS–15% PAGE. (d) Differential binding of PARP to nuclear receptors. 35S-labelled PARP(82-220) or 35S-labelled RXRα was incubated with GST alone (lane 2), GST-RXRα (lane 3), GST-TRα (lane 4), or GST-VDR (lane 5). Associated proteins were analyzed by SDS–15% PAGE and visualized.

FIG. 2

FIG. 2

Domains within nuclear receptors required for PARP interactions. (a) Series of N- and C-terminal deletions of RXR used in pull-down experiments. (b) In vitro interaction of PARP(82-220) with RXR. Bacterially produced GST-RXR deletions or GST alone was bound to glutathione-Sepharose beads and incubated with equivalent amounts of 35S-labelled PARP(82-220) produced by in vitro translation. Associated proteins were analyzed by SDS–15% PAGE and visualized by BAS 1500 (Fuji, Tokyo, Japan). (c) 35S-labelled PARP(82-220) was incubated with matrix-bound GST-RXRα in the absence or presence of 1 U of DNase I, and associated proteins were analyzed by SDS–15% PAGE. (d) Differential binding of PARP to nuclear receptors. 35S-labelled PARP(82-220) or 35S-labelled RXRα was incubated with GST alone (lane 2), GST-RXRα (lane 3), GST-TRα (lane 4), or GST-VDR (lane 5). Associated proteins were analyzed by SDS–15% PAGE and visualized.

FIG. 2

FIG. 2

Domains within nuclear receptors required for PARP interactions. (a) Series of N- and C-terminal deletions of RXR used in pull-down experiments. (b) In vitro interaction of PARP(82-220) with RXR. Bacterially produced GST-RXR deletions or GST alone was bound to glutathione-Sepharose beads and incubated with equivalent amounts of 35S-labelled PARP(82-220) produced by in vitro translation. Associated proteins were analyzed by SDS–15% PAGE and visualized by BAS 1500 (Fuji, Tokyo, Japan). (c) 35S-labelled PARP(82-220) was incubated with matrix-bound GST-RXRα in the absence or presence of 1 U of DNase I, and associated proteins were analyzed by SDS–15% PAGE. (d) Differential binding of PARP to nuclear receptors. 35S-labelled PARP(82-220) or 35S-labelled RXRα was incubated with GST alone (lane 2), GST-RXRα (lane 3), GST-TRα (lane 4), or GST-VDR (lane 5). Associated proteins were analyzed by SDS–15% PAGE and visualized.

FIG. 2

FIG. 2

Domains within nuclear receptors required for PARP interactions. (a) Series of N- and C-terminal deletions of RXR used in pull-down experiments. (b) In vitro interaction of PARP(82-220) with RXR. Bacterially produced GST-RXR deletions or GST alone was bound to glutathione-Sepharose beads and incubated with equivalent amounts of 35S-labelled PARP(82-220) produced by in vitro translation. Associated proteins were analyzed by SDS–15% PAGE and visualized by BAS 1500 (Fuji, Tokyo, Japan). (c) 35S-labelled PARP(82-220) was incubated with matrix-bound GST-RXRα in the absence or presence of 1 U of DNase I, and associated proteins were analyzed by SDS–15% PAGE. (d) Differential binding of PARP to nuclear receptors. 35S-labelled PARP(82-220) or 35S-labelled RXRα was incubated with GST alone (lane 2), GST-RXRα (lane 3), GST-TRα (lane 4), or GST-VDR (lane 5). Associated proteins were analyzed by SDS–15% PAGE and visualized.

FIG. 3

FIG. 3

PARP associates with receptors on hormone response elements. Shown is the interaction of PARP(82-220) with an RXR-TR heterodimer on a DR4 element comprising an AGGTCA direct repeat spaced by four nucleotides in a gel retardation assay. Bacterially expressed and purified RXR and TR were incubated with radiolabelled DR4 probe in the presence or absence of GST-PARP(82-220) or GST alone. The RXR-TR heterocomplex and PARP-RXR-TR ternary complex are indicated by arrows.

FIG. 4

FIG. 4

Overexpression of PARP inhibits ligand-dependent transactivation by nuclear receptors. Luciferase reporter gene activities under control of the DR4-TK or SREX2-TK promoter were measured from extracts of COS1 cells after the cells were transiently transfected by the corresponding reporter and expression plasmids. Relative luciferase activities in the presence (solid bars) or absence (hatched bars) of cognate ligands after being normalized by the internal control for β-galactosidase activities are presented. The results represent averages and standard deviations from at least three independent experiments.

FIG. 5

FIG. 5

PARP has transcriptional repression activity. The pM-PARP eukaryotic expression construct encoding a Gal4 DNA binding domain-PARP fusion protein was cotransfected into COS1 cells with a luciferase reporter plasmid containing five Gal4 binding sites. Compared with cells cotransfected with the reporter construct and Gal4 DNA binding domain, luciferase activity was lower in cells cotransfected with pM-PARP. The C908R mutation, which results in loss of poly(ADP-ribosyl) enzyme activity, eliminated the repressor activity of the protein. All luciferase activity was corrected for transfection efficiency by measuring β-galactosidase activity of cells transfected together. The results represent averages and standard deviations from three independent experiments.

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