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. 2000 Apr;20(8):2718-26.
doi: 10.1128/MCB.20.8.2718-2726.2000.

The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes

C Rachez  1 , M Gamble, C P Chang, G B Atkins, M A Lazar, L P Freedman
Affiliations

The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes

C Rachez et al. Mol Cell Biol. 2000 Apr.

Abstract

Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D(3) receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.

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Figures

FIG. 1
FIG. 1
Mapping of regions in DRIP205 required for VDR interaction. (A) Schematic representation of the various DRIP205 fragments fused to GST and used as baits in in vitro pull-down assays. The two potential nuclear receptor interaction motifs (NR1 and NR2) are indicated. (B) GST pull-down assay using GST-DRIP205 deletion mutants and [35S]VDR wild type (top) or [35S]VDR AF-2 deletion mutant (bottom). All incubations were carried out in the presence of 10−6 M 1,25(OH)2D3.
FIG. 2
FIG. 2
DRIP205 ligand-dependent binding with VDR and TR. (A) GST pull-down assay using 5 μg of GST-DRIP205(527-970) together with purified VDR in the presence of 10−6 M 1,25(OH)2D3 (+) or ethanol (−). GST alone and GST-RXR were used as negative and positive controls, respectively. Detection was by immunoblotting with anti-VDR antibody. (B) GST pull-down assay using GST-TR and [35S]DRIP205 in the presence (+) or absence (−) of thyroid hormone (T3). Detection was by autoradiography. (C) Gel mobility shift assay using eluted GST-DRIP205(527-970) or GST alone to supershift a VDR-RXR heterodimer bound to a consensus VDRE in the presence of 10−6 M 1,25(OH)2D3 (+) or ethanol (−). (D) Gel mobility shift assay of GST-DRIP205 binding to a TR-RXR heterodimer bound to a TRE in the presence of 10−6 M T3 (+) or ethanol (−), as described in for panel C.
FIG. 3
FIG. 3
Two functionally distinct complexes bind VDR LBD. (A) Glycerol gradient fractionation of proteins immobilized on the GST-VDR LBD affinity column are shown by silver staining of a SDS–7.5% polyacrylamide gel. In., input; M., myoglobin; Ov., ovalbumin; Gl., gamma globulin; Th., thyroglobulin. (B) SRC-1 and DRIP205 exhibit distinct sedimentation profiles, as determined by Western blot analyses of glycerol gradient fractions in panel A for the presence of DRIP205 (probed with anti-DRIP205 serum) and SRC-1 (probed with MAb GT12 2E9). (C) HAT activity colocalizes with SRC-1 but not with the DRIP complex. Fractions were assayed for HAT activity in the presence of free histones by a filter binding assay as described previously (35). HAT activity was measured as the amount of [3H]acetate transferred from [3H]acetyl coenzyme A to histones.
FIG. 4
FIG. 4
DRIP205-VDR interaction is selectively competed by the NR2 peptide. (A) Sequence of DRIP205 encompassing the two potential nuclear receptor-interacting motifs, NR1 and NR2. Underlined amino acids correspond to the sequences of NR1 and NR2 peptides used in subsequent experiments. (B) GST pull-down assay using 2 μg of GST-DRIP205 fragment (527 to 970) and purified VDR in the presence of 10−6 M 1,25(OH)2D3. NR1 (lanes 2 to 4) and NR2 (lanes 5 to 7) peptides (A) and an unrelated peptide (Flag; lanes 8 to 10) were used as competitors in 2-, 5-, and 20-fold molar excess over GST-DRIP205. The same amounts of NR peptides were coincubated with GST-RXR (lanes 11 to 17), here used as a control bait. (C) NR2 peptide is sufficient to elute the entire DRIP complex from VDR. GST-VDR LBD was used to pull down the DRIP complex from nuclear extracts as described previously (35). The complex immobilized on beads was then incubated with 5 or 30 μM NR1 or NR2 or 30 μM nonspecific (Flag) peptide. Bands matching subunits of the DRIP complex (34) are shown on the right.
FIG. 5
FIG. 5
DRIP205 binds a VDR-RXR heterodimer on DNA through contributions of both NR boxes. (A) Schematic representation of GST-DRIP205(527-970) wild-type protein (WT), or the same fragment containing point mutations in the NR1 or NR2 box that change each LXXLL motif to LXXAA (Mut1 or Mut2). The GST proteins used in the experiments depicted in panels B and C were quantitated by visualization on a Coomassie blue-stained SDS-polyacrylamide gel (right). Black arrowhead, GST-DRIP205(527-970) proteins; white arrowhead, GST alone. (B) GST pull-down assay of in vitro-translated, [35S]methionine-labeled VDR and GST-DRIP205(527-970) protein fragments. The pull-down assays were carried out in the presence (+) or absence (−) of 10−6 M 1,25(OH)2D3, as indicated, and VDR was visualized by autoradiography. (C) Association of DRIP205 to DNA-bound VDR-RXR heterodimers. Gel mobility shift analysis was performed in the presence of purified VDR, RXR, and GST-DRIP205(527-970), together with a VDRE oligonucleotide as a probe. GST-DRIP205 wild-type (wt), Mut1, and Mut2 proteins (A) were used in the presence (+) or absence (−) of 10−6 M of 1,25(OH)2D3 or LG153 ligands.
FIG. 6
FIG. 6
DRIP205 potentiates VDR transactivation, and a 190-amino-acid fragment containing both NR boxes (205-Box) acts as a dominant negative in vivo. (A) Schematic representation of the two NR box motifs (NR1 and NR2) in the full-length DRIP205 (amino acids 1 to 1566) and in 205-Box (527 to 714) used in the transient transfection experiments. (B) U-937 cells were transfected with a luciferase reporter plasmid containing a multimerized VDRE, together with 0.5 μg of VDR expression vector (+) and increasing amounts (in μg) of DRIP205 expression vector, in the absence (−) or presence (+D3) of 10−8 M 1,25(OH)2D3. Luciferase activity (expressed as RLU) was normalized relative to β-Gal activity. (C) Transient transfections were performed as for panel B, with the addition of increasing amounts (in micrograms) of 205-Box expression construct. (D and E) DRIP205 and 205-Box have no effect on transactivation by VP16 and E1A activation domains. Transient transfection assays were performed as for panels B and C except that U-937 cells were transfected with a luciferase reporter plasmid containing a multimerized GAL4 upstream activation sequence enhancer, together with increasing amounts (in micrograms) of DRIP205 and 205-Box expression vectors. Cells were also transfected with expression vectors for Gal4-VP16 (50 ng; D) or Gal4-E1A (100 ng; E).
FIG. 7
FIG. 7
Both NR boxes of DRIP205 are equally required for VDR signaling in vivo. A transient transfection assay was performed as for Fig. 6C, using 205-Box wild-type (WT) and mutant constructs.
FIG. 8
FIG. 8
Model of VDR-coactivator interactions, as described in the text.
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