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First published online 10 July 2007
doi: 10.1242/jcs.009878

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GNL3L inhibits activity of estrogen-related receptor by competing for coactivator binding

Hiroaki Yasumoto, Lingjun Meng^*, Tao Lin^*, Qubo Zhu and Robert Y. L. Tsai

Center for Cancer and Stem Cell Biology, Alkek Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX 77030, USA

View larger version (63K): [in this window] [in a new window]   Fig. 1. GNL3L interacts with ERR, ERR and ERR. Protein interactions between ERR and nucleostemin family genes (A) and between GNL3L and ERR family genes (B) were examined by in vivo coimmunoprecipitation assays. HEK293 cells were co-transfected with (A) Myc-tagged ERR and HA-tagged nucleostemin, GNL3L or Ngp1 expression plasmids, or (B) HA-tagged GNL3L and Myc-tagged ERR, or expression plasmids. Lysates were immunoprecipitated (IP) with anti-Myc antibody (rows 1 and 2, -Myc) or anti-HA antibody (rows 3 and 4, -HA). The co-purified proteins (rows 1 and 3) and the immunoprecipitates (rows 2 and 4) were immunodetected with the antibodies indicated on the right. Our results show that ERR interacts only with GNL3L, not with nucleostemin or Ngp1, and that GNL3L binds all ERR family proteins. (C) Tissue distribution of GNL3L, ERR and ERR in adult mice is shown by multi-tissue northern blots. GNL3L mRNA is expressed primarily in the neural tissues, including the brain and eye, and at lower levels in the kidney and muscle. The expression levels of GNL3L and ERR match in kidney, muscle and eye, but differ in brain and heart. (D) In U2OS cells, the intensity of GFP-tagged GNL3L (GNL3L-gfp) is higher in the nucleolus than in the nucleoplasm. GFP-tagged ERR (ERR-gfp) is localized exclusively in the nucleoplasm. The nucleolar regions are labeled by anti-fibrillarin (Fib) antibody in the right panels. Dashed lines demarcate nucleo-cytoplasmic boundaries. Bars, 10 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Binding between GNL3L and ERR requires the intermediate (I)-domain of GNL3L and the AF2-domain of ERR. (A) Truncated mutants of GNL3L were used to determine its interacting domain with ERR. B, basic domain; C1 and C2, coiled-coil domain-1 and -2; G, GTP-binding domain; I, intermediate domain. Numbers indicate amino acid positions. (B) GST-ERR fusion proteins fail to bind mutants that lack the I-domain (dI and G3l-G) but can retain the dBC and the non-GTP-binding mutants, N166I and dG. (C) The subcellular distribution of HA-tagged dBC, G3l-G, dG, dI and N166I mutants are shown by confocal analyses double-labeled with anti-HA (left panels) and anti-fibrillarin (Fib, right panels) antibodies. Bar, 10 µm. (D) Truncated mutants of ERR were used to determine the domain interacting with GNL3L. AF1 and AF2, activation function 1 and 2; DBD, DNA-binding domain; LBD, ligand-binding domain. (E) Affinity-binding assays show that GST fusion proteins of the wild-type ERR, the dAF1 mutant and the dLBD mutant can bind GNL3L, but GST fusion proteins of the dAF2, LBD-AF2 and AF1-DBD mutants cannot (top panel). The amount of GST fusion proteins used in each reaction, marked by asterisks, is shown in the bottom panel by Commassie Blue staining. Some degradation occurs at the fusion site of the GST-dLBD protein (arrow).

View larger version (117K): [in this window] [in a new window]   Fig. 3. Overexpression of GNL3L brings ERR and ERR into the nucleolus. (A) To generate a nucleolar form of GNL3L (NoG3l), we replaced the N-terminal nucleolus-targeting domain of GNL3L with the corresponding region of nucleostemin (grey bar), which has a stronger nucleolus-targeting activity than GNL3L but lacks the ability to bind ERR. To create a nucleoplasmic form of GNL3L (nls-I), we fused the I-domain of GNL3L to an SV40 nuclear localization signal (oval). (B) Affinity-binding assays show that both nls-I and NoG3l maintain the ability to bind ERR. To measure the effect of GNL3L overexpression on the distribution of ERR, U2OS cells were transfected with Myc-tagged ERR alone (C1), Myc-tagged ERR and HA-tagged wild-type GNL3 (C2) or mutant GNL3L (C3,C4) or HA-tagged GNL3L constructs alone (C5 and C6). Double-transfected cells were labeled with anti-Myc (red) and anti-HA (green) antibodies, and visualized by confocal analyses. Single-transfected cells were immunostained with anti-fibrillarin antibody (Fib) and anti-Myc or anti-HA antibody. The ERR (red) fluorescence intensities are measured quantitatively along the lines indicated by arrows, shown in the right panels of (C1'-C3'), and the nucleolar regions (No) are indicated by the increase of green fluorescence. Compared to cells transfected with only ERR, the fluorescence intensity of ERR in the nucleolus is increased in cells cotransfected with NoG3l or the wild-type GNL3L. By contrast, the nls-I mutant does not change the distribution of ERR (C4). Neither does ERR alter the distribution of GNL3L (C5) or NoG3l (C6). The same analyses were performed using ERR (D1-D3) and ERR (E1-E3). Our results showed that, when coexpressed with wild-type GNL3L (D2) or NoG3l (D3), ERR begins to accumulate in the nucleolus. GNL3L overexpression has little or no effect on the distribution of ERR (E2,E3). Bars, 10 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Overexpression of GNL3L inhibits transcriptional activity of ERR proteins independently of nucleolar distribution. (A1) Estrogen response element (ERE)-specific transcriptional activities were measured in CV-1 cells by the ratio between the ERE-driven firefly luciferase activity and the Renilla-null luciferase activity. ERR elicits a six-fold increase in the ERE-specific transcriptional activity. Coexpression of wild-type GNL3L (WT) leads to a 50% reduction in the ERR-mediated transcriptional activity. This decrease is reversed by a deletion of the ERR-binding I-domain of GNL3L (dI). Coexpression of either the nucleolar form (NoG3l) or the nucleoplasmic form (dBC) of GNL3L suppresses the ERR transcriptional activity more than or to the same extent as the wild-type GNL3L protein. (B1,C1) Using the same approach, we show that this inhibitory activity of GNL3L can also work on (B1) ERR and (C1) ERR with the exception that the dBC mutant has little effect on the ERR-mediated transactivation. Error bars represent the standard error of the mean (± s.e.m.). ***P<0.0001. (A2,B2,C2) Expression levels of wild-type and mutant GNL3L proteins and ERR proteins in the experimental samples are compared in western blots side-by-side using anti-HA and anti-Myc antibodies, respectively; -tubulin (-Tub) was used as a loading control. (D) GNL3L fails to suppress the estradiol (E2)-induced transcriptional activity of ER on the ERE-driven promoter in the same cell-based reporter system.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Endogenous GNL3L suppresses transcriptional activity of ERR family genes. (A) To confirm the GNL3L-mediated negative regulation of ERR activities from a loss-of-function angle, a small interfering RNA (siRNA) approach was used to knock down the endogenous expression of GNL3L. Compared to the control knockdown sample (siNEG), the protein knockdown efficiencies of GNL3L-specific siRNA duplexes (siGNL3L-1 and siGNL3L-2) in HEK293 cells stably expressing HA-tagged GNL3L are estimated to be 83% and 84%, respectively. (B) Consistent with our overexpression data, the transcriptional activity of ERR is increased 2.5 times by siGNL3L-1 and siGNL3L-2 treatment as compared with the siNEG-treated sample. (C,D) GNL3L knockdown has the same effect on the ERR- and ERR-mediated transactivation, although their increase is less dramatic than the increase in the ERR-mediated transactivation. Error bars represent the standard error of the mean (± s.e.m.). **P<0.001; ***P<0.0001.

View larger version (32K): [in this window] [in a new window]   Fig. 6. GNL3L competes with SRC1 and SRC2 for ERR binding. Agarose-bound GST fusion proteins of ERR (1 µg) were used to pull down whole-cell lysates containing a fixed amount of SRC1 (A) or SRC2 (B), mixed with increasing amounts of the wild-type GNL3L (A1,B1) or the dI mutant lacking the ERR-interacting domain (A2,B2). Whole-cell proteins in each sample were adjusted to the same amount. In the agarose-retained portions (R), the interaction between GNL3L and ERR can reduce the amount of SRC1 and SRC2 bound by ERR in a dose-dependent manner, but the dI mutant fails to do so. Conversely, when GST-ERR fusion proteins were used to pull down the same amount of GNL3L in the presence of increasing amounts of SRC1 (C) or SRC2 (D), SRC1 and SRC2 were able to reduce the amount of GNL3L bound by ERR in a dose-dependent way as well. Proteins in the agarose-bound fraction and in the supernatant are indicated by (R) and (S), respectively.

View larger version (87K): [in this window] [in a new window]   Fig. 7. Coexpression of GNL3L increases the electrophoretic mobility of the DNA-bound ERR protein complex and reduces its binding with SRC1 and SRC2. (A) The GNL3L effect on the binding of ERR to DNA was examined by EMSA using ERE-containing probes and whole-cell lysates expressing the indicated recombinant proteins. Compared with the probe alone (lane 1) and the vector-transfected control sample (lane 2), ERR-specific DNA-protein complex was identified in lane 3 (arrow b), competed by excess non-labeled probes (lane 4), and supershifted by anti-Myc antibody (lane 5, arrow a). Coexpression of GNL3L produces fast-moving complexes (lane 6, arrows d and e) that can be supershifted by anti-Myc antibody (lane 7, arrow c) but not by anti-HA antibody (lane 8). GNL3L itself cannot bind the ERE probe (lane 9). The intensity of the fast-moving complex d is reduced by a deletion of the ERR-binding I-domain of GNL3L (lanes 10-12). (B) The fast-moving complex d and the slow-moving complex b were retrieved from the EMSA gel, fractionated in SDS-denaturing PAGE and analyzed for their ERR (-Myc), GNL3L (-HA), SRC1, and SRC2 protein components by western blotting. Our results indicate that the increase in the electrophoretic mobility of the ERR-DNA complex by GNL3L coexpression is due to a loss of SRC1 binding (arrow) and diminished SRC2 binding, rather than by protein cleavage of ERR.

View larger version (18K): [in this window] [in a new window]   Fig. 8. GNL3L suppresses SRC-mediated transcriptional coactivation of ERR. (A) Using the same cell-based reporter system as described in Fig. 4, we show that the ERE-specific transcriptional activity in cells coexpressing ERR and SRC1 (8.0±0.3) is 1.7 times higher than that of the ERR-expressing sample (4.8±0.3). When coexpressed with the wild-type GNL3L (WT), this ERR and SRC1-mediated ERE-specific transcriptional activity is reduced by 55% and 70% compared with the sample expressing both ERR and SRC1 in a dose-dependent manner. This inhibitory effect of GNL3L on the SRC1-mediated coactivation of ERR requires the I-domain of GNL3L because deletion of this domain (dI) fails to suppress the transcriptional activity of ERR and SRC1 (P=0.17). (B) Using the same approach, we show that GNL3L can also suppress the coactivator function of SRC2 on the ERR-dependent transcriptional activity in a dose-dependent (54% reduction for 100 ng of GNL3L and 71% reduction for 200 ng of GNL3L) and I-domain-dependent (P=0.58) manner. Error bars represent the stand error of mean (± s.e.m.). ***P<0.0001.

View larger version (28K): [in this window] [in a new window]   Fig. 9. GNL3L inhibits the transcriptional activities of ERR family genes by coactivator competition. Our data reveal a novel mechanism that regulates the activity of ERR family genes by the nucleolar GTP-binding protein GNL3L. GNL3L decreases the transcriptional activity of ERR proteins. This event takes place in the nucleoplasm and does not require the nucleolar localization of GNL3L. The interaction between GNL3L and ERR displaces coactivators such as SRC1 and SRC2 from the ERR complex. The SRC-depleted ERR protein binds DNA without GNL3L, resulting in transcriptional inhibition. In this model, the nucleolar accumulation of GNL3L does not appear to affect its ability to suppress the transcriptional function of ERR proteins (grey arrows). Protein domains of GNL3L and ERR are: B, basic domain; C1 and C2, coiled-coil domain-1 and -2; G, GTP-binding domain; I, intermediate domain; AF1 and AF2, activation function 1 and 2; DBD, DNA-binding domain; LBD, ligand-binding domain.