Alternate surfaces of transcriptional coregulator GRIP1 function in different glucocorticoid receptor activation and repression contexts

Abstract

Members of the mammalian p160 family, such as GRIP1, are known as glucocorticoid receptor (GR) coactivators; at certain glucocorticoid response elements (GREs), however, GRIP1 acts as a GR corepressor. We characterized functional interactions of GR and GRIP1 in a repression complex where GR tethers to DNA-bound activator protein-1 (AP-1), as at the human collagenase-3 gene, and tested whether the identified interactions were similar or different at other response elements. At the AP-1 tethering GRE, we mapped the GRIP1 corepressor activity to a domain distinct from the two known GRIP1 activation domains; it exhibited intrinsic GR-independent repression potential when recruited to DNA via Gal4 DNA-binding domain. Interestingly, neither the domain nor the activity was detected in the other two p160 family members, SRC1 and RAC3. The same GRIP1 corepression domain was required for GR-mediated repression at the nuclear factor-kappaB (NF-kappaB) tethering GRE of the human IL-8 gene. In contrast, at the osteocalcin gene GRE, where GR represses transcription by binding to a DNA site overlapping the TATA box, both GRIP1 and SRC1 corepressed, and the GRIP1-specific repression domain was dispensable. Thus, in a single cell type, GR and GRIP1 conferred one mode of activation and two modes of repression by selectively engaging distinct surfaces of GRIP1 in a response element-specific manner.

Fig 1.

Mapping a GRIP1 corepression domain. (A) The domain structure of WT and mutant GRIP1 derivatives. The basic helix–loop–helix (bHLH)/Per-ARNT-Sim (PAS) domain, NR boxes (NR box3 is shown in black), AD1, and AD2 are diagrammed. GRIP1 derivatives act as corepressors (CoR) or dominant-negatives (DN), as indicated, with respect to GR-mediated repression of AP-1. Residues 765–1,007 (shaded) are required for corepression. U2OS.G cells were transfected with indicated amounts of WT (wt) GRIP1 or ΔAD1/ΔAD2 (B), N1007 and N765 (C), or GRIP1_648–1007 (D) along with 40 ng per well each of the AP-1-Luc reporter and β-actin-LacZ plasmid. Total amounts of transfected DNA were equalized with pCDNA3. Cells were treated overnight with 25 ng/ml PMA in the absence (gray) or presence (black) of 100 nM dexamethasone, and reporter activity was measured, normalized to β-galactosidase activity, and expressed as relative luminescence units (RLU). The y axis is broken to better visualize reporter activity in the absence and presence of Dex. Fold repression in each case is shown.

Fig 2.

GRIP1 corepressor activity in heterologous context. (A) GRIP1 repression domain is active in the absence of GR. Parental U2OS cells were transfected with increasing amounts of pSG424 plasmid expressing Gal4DBD (Gal), Gal4DBD fused to GRIP1_631–1007 (Gal:GRIP1_631–1007) or to MAD repression domain (Gal:MAD), along with the 2xGal4/2xAP-1-Luc reporter and β-actin-LacZ. Total amount of DNA was equalized with empty pSG424. Reporter activity was measured in the presence of PMA, normalized to β-galactosidase activity, and expressed as the percentage of activity observed for empty pSG424. (B) Full-length GRIP1 fused to Gal4DBD is a transcriptional activator. U2OS cells were transfected with increasing amounts of Gal4DBD (Gal) or Gal:GRIP1, and the activity of 2xGal4/2xAP-1-Luc reporter was assayed with/without PMA and normalized as described in A. (C) Liganded GR blocks GRIP1-mediated transcriptional activation. U2OS.G cells were transfected with Gal:GRIP1 or empty pSG424, 2xGal4-Luc reporter, and β-actin-LacZ. Reporter activity in the presence of PMA−/+Dex, as indicated, was assayed as described in A.

Fig 3.

GRIP1 corepressor activity at the AP-1 tethering GRE is not conserved across the p160 family. (A) GRIP1 corepression domain is unique in the p160 family. Diagrammed are the p160 proteins: SRC1, GRIP1, and RAC3. Numbers indicate the amino acid similarity of SRC1 and RAC3 to GRIP1 throughout their basic helix–loop–helix (bHLH)/Per-ARNT-Sim (PAS), NID, RD, AD1, and AD2 domains, as assessed by using blast software (National Center for Biotechnology Information). (B and C) The effects of SRC1 and RAC3 on GR repression (B) and activation (C). Indicated amounts of SRC1 and RAC3 were transfected into U2OS.G cells and AP-1-Luc- (B) or XG₄₆TL- (C) reporter activity was measured as described in Fig. 1.

Fig 4.

GRIP1 potentiates GR-mediated repression of NF-κB. (A) IL-8 gene is active in U2OS.G cells. U2OS.G cells were treated for 2 h with PMA−/+Dex, as indicated, and the IL-8 mRNA level was assessed by real-time PCR, as described in Materials and Methods, and expressed relative to untreated control cells. (B–D) GRIP1 residues 648–1,007 mediate corepression at the NF-κB tethering GRE. Indicated amounts of wt GRIP1, GRIP1 NID (B and C) or GRIP1_648–1007 (D) (equalized with pCDNA3) were transfected into U2OS.G cells, along with the IL-8-Luc (B) or IL-8(κB)-Luc (C and D) reporter and β-actin-LacZ. Cell treatments and reporter activities were measured as described in Fig. 1.

Fig 5.

p160 corepression at the octeocalcin GRE. (A) GRIP1 and SRC1 potentiate GR-mediated repression of osteocalcin. U2OS.G cells were transfected with a pOS-344-Luc reporter, β-actin-LacZ, and indicated amounts of GRIP1 or SRC1 (equalized with pCDNA3). Reporter activity was assessed in the absence (gray) or presence (black) of Dex as described in Fig. 1. (B) GRIP1 repression domain is dispensable for corepression at the osteocalcin GRE. U2OS.G cells were transfected with indicated amounts of WT (wt) GRIP1 or GRIP1 ΔRD (RD−), and the activities of the pOS-344-Luc (OC) and AP-1-Luc (AP1) reporters were assessed in the absence or presence of Dex and plotted as “fold repression” at each amount of transfected GRIP1 derivative.