Guanine nucleotide binding protein-like 3 (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1-null yeasts. To determine the unique function of GNL3L, we identified estrogen-related receptor γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRγ requires the intermediate domain of GNL3L and the AF2-domain of ERRγ. Gain-of- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRγ. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRγ by GNL3L through coactivator competition.
Nucleostemin and its homologues, guanine nucleotide binding protein-like 3 (GNL3L) and Ngp-1 (hereafter referred to as Ngp1), constitute a subfamily of GTP-binding proteins featured by their nucleolar distribution and a unique domain of circularly permuted GTP-binding motifs, where the G4 motif is located N-terminally to the G1, G2, and G3 motifs (Daigle et al., 2002; Leipe et al., 2002). Nucleostemin is enriched in the embryonic, mesenchymal and neural stem cells, in adult testes and several types of human cancers (Baddoo et al., 2003; Kafienah et al., 2006; Tsai and McKay, 2002). It plays a role in maintaining the continuous proliferation of neural stem cells (Tsai and McKay, 2002) and in regulating the protein stability of telomeric-repeat binding factor 1 (TRF1) (Zhu et al., 2006). Targeted deletion of nucleostemin leads to early embryonic lethality in homozygous nucleostemin-null embryos (Beekman et al., 2006; Zhu et al., 2006) and to premature senescence of heterozygous nucleostemin-null mouse embryonic fibroblast cells (Zhu et al., 2006).
Phylogenetically, nucleostemin is most closely related to GNL3L in vertebrates. They share the same yeast orthologue: Grn1p in Schizosaccharomyces pombe and Nug1p in Saccharomyces cerevisiae. Grn1p is involved in the processing of 35S pre-ribosomal RNA (rRNA), the nuclear export of Rpl25, and the maintenance of cell growth (Du et al., 2006). Mutation of Nug1 inhibits the export of 60S subunit from the nucleolus (Du et al., 2006; Kallstrom et al., 2003). Although the yeast orthologue of nucleostemin and GNL3L displays general activities in growth and ribosome biogenesis, rodent nucleostemin and GNL3L are distinctively expressed in different tissues. Furthermore, only human GNL3L, but not nucleostemin, can rescue the Grn1-deficient growth phenotype in fission yeasts (Du et al., 2006). These results suggest that nucleostemin and GNL3L have evolved specific properties in vertebrates, and become functionally diverged from each other and from Grn1p. By comparison, GNL3L retains more characteristics of Grn1p than does nucleostemin.
GNL3L bears 28% identity and 39% similarity to nucleostemin in mice. Very little is known about its function in vertebrates. To delineate the distinct activity of GNL3L, we looked for proteins that interact with GNL3L but not with nucleostemin. We first identified estrogen-related receptor γ (ERRγ) as a GNL3L-binding protein by a yeast two-hybrid screen, and confirmed this interaction by affinity-binding and coimmunoprecipitation assays. ERRγ belongs to a subfamily of the nuclear receptor superfamily. The ERR gene family consists of three members, ERRα, ERRβ, and ERRγ, that most resemble estrogen receptor α (ERα). Like ERα, the ERR proteins contain functionally separable structures that include an AF1 domain (or A/B region), a DNA-binding domain (DBD, or C region) with two zinc fingers, a hinge region (D), and a ligand-binding domain (LBD, or E/F region) with an AF2 domain at the C-terminal end. The LBD is involved in ligand binding, receptor dimerization, and coactivator binding. The AF2 domain is required for the ligand-dependent activation function. ERRα and ERRγ are found in the brain, muscle, heart, kidney and adipose tissues (Bonnelye et al., 1997; Giguere et al., 1988; Hong et al., 1999). ERRβ is expressed in the eye, heart, kidney, cerebellum and testis (Bookout et al., 2006). The functions of the ERR family genes are implicated in many aspects of embryogenesis and tumorigenesis. Mice deficient in ERRα exhibit reduced body weight and peripheral fat deposit, and are resistant to obesity induced by a high-fat diet (Luo et al., 2003). ERRβ-null mice display placentation defects, consistent with its role in the proliferation and differentiation of trophoblastic cells (Luo et al., 1997). In humans, the expression level of ERRα correlates with poor prognosis in ovarian and breast cancers (Ariazi and Jordan, 2006; Suzuki et al., 2004). Conversely, ERRγ is a favorable indicator for human ovarian tumors (Sun et al., 2005). Although the ERR family genes are capable of binding the estrogen response element (ERE), they are different from ERα in that their transcriptional activity and coactivator binding do not require ligand binding (Giguere et al., 1988; Hong et al., 1999; Kallen et al., 2004), which leaves open the question of whether the activities of the ERR family genes are constitutively active or dynamically regulated.
Here, we uncover a GNL3L-mediated pathway that regulates the transcriptional activity of the ERR family genes. We show that only GNL3L, but not nucleostemin or Ngp1, can interact with ERR family genes. Coexpression of GNL3L inhibits the transcriptional activities of the ERR family genes. Conversely, knocking down the endogenous expression of GNL3L increases the ERR-mediated transactivation. Furthermore, GNL3L can compete with steroid receptor coactivators (SRCs) for their ERRγ binding and block the SRC-mediated coactivation of ERRγ. Our study reveals a GNL3L-mediated mechanism that modulates the transcriptional activities of ERR proteins.
GNL3L interacts with ERR family genes
To determine the unique function of GNL3L in vertebrates, we searched for proteins that interact with GNL3L but not with nucleostemin. A yeast two-hybrid approach was employed, where full-length GNL3L was fused to a GAL4 DNA binding-domain, and used to screen a mouse E17.5 brain cDNA library. From a total of 5 million clones screened, two positive clones were identified. They encoded the same in-frame partial sequence of ERRγ (Clone 43, residues 27-458). The interaction between GNL3L and clone 43 or full-length ERRγ was confirmed by affinity-binding assays in which agarose-bound GST fusion proteins of clone 43 and full-length ERRγ, but not the GST backbone protein, were able to retain hemagglutinin (HA)-tagged GNL3L specifically (Fig. S1A in supplementary material). We use coimmunoprecipitation assays to verify the interaction between GNL3L and ERRγ and to determine whether nucleostemin or Ngp1 can bind ERRγ. HEK293 cells were co-transfected with Myc-tagged ERRγ and one of the nucleostemin family genes tagged with an HA-epitope. Protein complexes were precipitated with anti-Myc or anti-HA antibodies, and immunodetected for the HA-tagged or Myc-tagged proteins (Fig. 1A). We found that only GNL3L, but not nucleostemin or Ngp1, was co-purified with ERRγ by anti-Myc immunoprecipitation (Fig. 1A, row 1). Consistently, ERRγ was detected only in the GNL3L protein complex, but not in the nucleostemin or Ngp1 protein complexes, precipitated by anti-HA antibody (Fig. 1A, row 3). Like GNL3L, ERRγ belongs to a family of three genes. To determine whether GNL3L interacts with only ERRγ or with multiple members of the ERR gene family, HEK293 cells were transfected with HA-tagged GNL3L and Myc-tagged ERRα, ERRβ, or ERRγ expression plasmids. Our results show that GNL3L and all members of the ERR gene family can be co-purified in the same protein complexes precipitated by either anti-Myc or anti-HA antibodies (Fig. 1B, rows 1 and 3). By contrast, no physical interaction between ERRα (or ERRβ) and nucleostemin (or Ngp1) was detected by affinity-binding assays (Fig. S1B in supplementary material). Based on these results, we conclude that only GNL3L of the nucleostemin family can form stable protein complexes with the ERR family genes.
ERRγ colocalizes with GNL3L in the nucleoplasm
To determine whether the interaction between GNL3L and ERRγ is physiologically possible, we examined the expression patterns of GNL3L, ERRβ and ERRγ by multi-tissue northern blot analyses. GNL3L was expressed most abundantly in the neural tissues, including the brain and eye, and was also detected in the muscle and kidney at low levels (Fig. 1C). Parallel blots showed that ERRβ was expressed most abundantly in the kidney, followed by the eye, testis, heart and muscle. High-level expression of ERRγ was seen in the heart and eye, followed by the brain, kidney and muscle. To decide where within the cell this interaction might occur, green fluorescent protein (GFP)-tagged GNL3L or ERRγ were expressed in U2OS cells, which express both GNL3L and ERRγ endogenously. We found that GNL3L was distributed both in the nucleolus and in the nucleoplasm, whereas ERRγ was localized exclusively in the nucleoplasm (Fig. 1D). These data show that the tissue expression pattern of GNL3L correlates better with that of ERRγ than that of ERRβ. Within the cell, ERRγ and GNL3L colocalize in the nucleoplasm. In the nucleolus, only GNL3L is found.
The intermediate domain of GNL3L interacts with the AF2 domain of ERRγ
To gain insight into the functional importance of the GNL3L-ERRγ interaction, we first identified the interacting domains of these two proteins using a panel of truncated GNL3L and ERRγ mutants (Fig. 2A,D). To define the ERRγ-binding domain in GNL3L, agarose-bound GST-ERRγ fusion protein was used to pull down the wild-type and mutant GNL3L proteins (Fig. 2B). Our results show that ERRγ can bind GNL3L mutants that lack the BC-domain (dBC) or the G-domain (dG), as well as the N166I mutant that contains an Asn166 to Isl mutation in the G4 domain, which abolishes the GTP-binding capability of GNL3L (our unpublished data). Notably, the GST-ERRγ fusion protein failed to retain mutants without the intermediate (I)-domain (dI and G3l-G, Fig. 2B), indicating that the I-domain is necessary for the ERRγ binding of GNL3L. Different GNL3L mutants displayed distinctive subcellular distribution patterns not related to their ERRγ-binding abilities. The dBC and G3l-G mutants were localized in the nucleoplasm and cytoplasm (Fig. 2C1,C2). The dG and dI mutants were localized more in the nucleolus than in the nucleoplasm (Fig. 2C3,C4), whereas the N166I mutant was diffusely distributed in the nucleus (Fig. 2C5). The ERRγ protein structure consists of the AF1, DBD, LBD and AF2 domains (Fig. 2D). GST fusion proteins of the full-length ERRγ, the AF1-domain deletion mutant (dAF1) and the LBD deletion mutant with an intact AF2-domain (dLBD) were able to bind wild-type GNL3L. By contrast, GST fusion proteins of the AF2-domain deletion mutant (dAF2), the last 245 residues containing the LBD and AF2 domains (LBD-AF2) and a mutant deleted of the LBD and AF2 domain (AF1-DBD) were unable to retain GNL3L, demonstrating that the AF2 domain is necessary but not sufficient for the binding of ERRγ to GNL3L (Fig. 2E). Based on these results, we conclude that the interaction between GNL3L and ERRγ requires the I-domain of GNL3L and the AF2 domain of ERRγ, and is independent of the GTP binding and nucleolar localization of GNL3L.
Overexpression of a nucleolar form of GNL3L brings ERRβ and ERRγ into the nucleolus
Given that GNL3L, but not ERRγ, is localized in the nucleolus, we tested the idea whether coexpression of GNL3L can bring ERRγ into the nucleolus. Using confocal analysis, we determined the distribution of ERRγ when coexpressed with wild-type GNL3L (WT), a nucleolar form of GNL3L (NoG3l), or an I-domain mutant of GNL3L fused to an SV40 nuclear localization sequence (NLS) (nls-I). NoG3l was created by replacing the BC-domain of GNL3L with the BC-domain of nucleostemin (indicated by the grey bar in Fig. 3A) because nucleostemin has a stronger nucleolar localization capability than GNL3L and does not bind ERRγ. The I-domain mutant was fused with an SV40 NLS because it lacks endogenous NLS of its own. Affinity-binding assays confirmed that both NoG3l and nls-I mutants were capable of binding ERRγ (Fig. 3B). Whereas ERRγ by itself was distributed outside of the nucleolus (Fig. 3C1,C1′), overexpression of wild-type GNL3L (Fig. 3C2,C2′) and NoG3l (Fig. 3C3,C3′) increased the ERRγ fluorescence signal in the nucleolar region compared with cells expressing ERRγ by itself. Notably, in cells overexpressing the NoG3l mutant, the ERRγ signal accumulated in the nucleolar region, particularly in the periphery of the nucleolus. By contrast, the nls-I mutant failed to alter the ERRγ distribution (Fig. 3C4), and distributions of wild-type GNL3L and NoG3l were unaltered by coexpression of ERRγ (Fig. 3C2 vs C5, and C3 vs C6). Overexpression of GNL3L and its mutants exerts the same effects on the distribution of ERRβ. Whereas ERRβ by itself displayed a nucleoplasmic distribution (Fig. 3D1,D1′), both the wild-type GNL3L protein (Fig. 3D2,D2′) and the NoG3l mutant (Fig. 3D3,D3′) were able to increase the nucleolar intensity of ERRβ. By comparison, NoG3L had a stronger effect on bringing ERRβ into the nucleolus than wild-type GNL3L. By contrast, overexpression of wild-type GNL3L or NoG3l had little or no effect on changing the distribution of ERRα (Fig. 3E). These results demonstrate that overexpression of a nucleolar form of GNL3L can change the distribution of ERRβ and ERRγ in living cells, suggesting the possibility that nucleolar sequestration underlies the regulation of ERRβ and ERRγ by GNL3L.
GNL3L suppresses the transcriptional activities of ERR family genes
To investigate whether GNL3L can modulate the transcriptional activity of the ERR genes, an in vivo cell-based luciferase assay system was set up where CV-1 cells were co-transfected with a firefly luciferase reporter construct driven by three repeats of a consensus palindromic estrogen response element (ERE, see Materials and Methods), an ERRγ expression plasmid, and a pRL-null reporter construct. The ERE-specific transcriptional activity was determined by the ratio between the Firefly and Renilla luciferase activities in the same sample, which represent ERE-driven and basal activities, respectively. This dual luciferase assay system was used to eliminate variations caused by transfection and non-specific effects on the common transcription-translational machinery. The Firefly-to-Renilla luciferase activity ratio for each experiment was expressed as the fold increase over the negative sample not transfected with ERRγ. Our results show that ERRγ can increase the ERE-specific transcriptional activity six times higher than that of the control sample (6.0±0.5, mean±s.e.m.), and coexpression of GNL3L attenuates ERRγ-dependent increase by 50% (2.9±0.2) (Fig. 4A1, WT). This ERRγ inhibitory effect of GNL3L requires its ERRγ-interacting domain, because samples that coexpress the dI mutant fail to show such a repressive activity. To determine whether this inhibition is caused by nucleolar sequestration of ERRγ by GNL3L, a nucleolar form of GNL3L (NoG3l) or a nucleoplasmic mutant of GNL3L (dBC) was coexpressed with ERRγ. Our results demonstrate that both NoG3l and dBC can inhibit ERRγ-mediated transcriptional activity more than or as much as the wild-type GNL3L. To test whether this transcriptional repressive effect of GNL3L can act on other members of the ERR family, we set up the same transactivational assay for ERRβ and ERRα. Our data show that GNL3L reduces the transcriptional activity of ERRβ from 4.7 times (±0.3) to 2.1 times (±0.1) over the control sample in an I-domain-dependent manner, and both NoG3l and dBC inhibit ERRβ as much as the wild-type protein (Fig. 4B1). Compared with ERRβ and ERRγ, the ERRα-dependent increase of ERE-driven transactivation is less (2.7±0.1). Although the wild-type GNL3L and the NoG31 mutant can reduce the ERRα-mediated transcriptional activity, the dBC mutant fails to do so (Fig. 4C1). The GNL3L effect on the transcriptional activities of the ERR family genes is specific, because GNL3L does not suppress estradiol (E2)-induced ERα-mediated transactivation when using the same reporter assay system (P=0.27, Fig. 4D). Finally, we confirm that these different effects of wild-type and mutant GNL3L on the transcriptional activities of ERR genes are not caused by different expression levels of the GNL3L or ERR proteins (Fig. 4A2,B2,C2).
To confirm the inhibitory effect of GNL3L from a loss-of-function angle, a small interfering RNA (siRNA) approach was used to knock down the expression of endogenous GNL3L in HEK293 cells. The knockdown efficiency of the GNL3L-specific siRNA duplex-1 (siGNL3L-1) and duplex-2 (siGNL3L-2) was determined at the protein level in an HEK293 cell line that stably expresses HA-tagged GNL3L, and was estimated to be 83% and 84%, respectively, compared with the control siRNA knockdown sample (siNEG) (Fig. 5A). In siNEG-treated HEK293 cells, ERRγ yielded an 11-fold induction on the ERE-driven transcription. In siGNL3L-1 and siGNL3L-2-treated cells, the ERRγ-mediated transcriptional activities were significantly increased compared with the 11-fold induction seen in the siNEG-treated sample (25.7±1.3 for siGNL3L-1 and 25.3±1.0 for siGNL3L-2; P<0.0001, n=9) (Fig. 5B). Reducing the amount of GNL3L also increased the ERRβ- and ERRα-mediated transactivation on the ERE-driven promoter (Fig. 5C,D). By comparison, GNL3L-specific siRNA treatment had a lesser effect on ERRβ- and ERRα-mediated transcription than on ERRγ-dependent transactivation. The ERRα (or ERRβ)-dependent transcriptional activities were 1.7±0.1 (or 6.8±0.2), 2.4±0.1 (or 10.7±0.3), and 2.6±0.1 (or 11.7±0.4) in the siNEG, siGNL3L-1, and siGNL3L-2-treated samples, respectively. Together, our data demonstrate that GNL3L inhibits the transcriptional functions of the ERR genes without entering the nucleolus.
GNL3L competes with SRC1 and SRC2 (GRIP1) for ERRγ binding
To look for a mechanism other than nucleolar sequestration in order to explain the GNL3L-mediated inhibition of ERR transactivation, we examined the possibility that GNL3L binding of ERRγ prevents ERRγ from accessing coactivators, such as SRC1 and SRC2 (GRIP1), which have been shown to also bind the AF2 domain of ERRγ (Hong et al., 1999). To test this idea, GST fusion proteins of ERRγ were used to pull down whole-cell lysates that contained a fixed amount of SRC1 or SRC2, mixed with increasing amounts of GNL3L (Fig. 6A1,B1). In each sample, whole-cell proteins were adjusted to the same concentration. Western blot analyses of the agarose-bound protein fractions showed that less SRC1 or SRC2 proteins were retained by GST-ERRγ as more GNL3L protein was bound by ERRγ [Fig. 6A1,B1, (R)]. The ability of GNL3L to compete with SRC1 and SRC2 for ERRγ binding is abolished by a deletion of its ERRγ-interacting I-domain (Fig. 6A2,B2). Conversely, to determine whether SRC1 or SRC2 can displace GNL3L from the ERRγ protein complex, GST-ERRγ fusion proteins were used to pull down a fixed amount of GNL3L in the presence of increasing amounts of SRC1 or SRC2. Our results show that both SRC1 (Fig. 6C) and SRC2 (Fig. 6D) can reduce GNL3L bound by GST-ERRγ in a dose-dependent manner. These data demonstrate that binding between ERRγ and GNL3, and between ERRγ and SRC1 or SRC2 are mutually exclusive, and suggest that blocking ERRγ from accessing SRC1 and SRC2 may be responsible for the inhibitory transcriptional activity of GNL3L.
Coexpression of GNL3L increases the mobility and decreases the SRC1 component of the ERRγ DNA-protein complex
To determine whether GNL3L forms a high-order DNA-protein complex with ERRγ, electrophoretic mobility shift assays (EMSAs) were conducted using a radiolabeled probe containing a canonical ERE sequence (TCAGGTCACTGTGACCTGA) and cell extracts expressing the indicated proteins (Fig. 7A). Compared with the probe alone (lane 1) and vector-transfected cell lysate samples (lane 2), the Myc-tagged ERRγ-transfected sample (lane 3) yields an ERE-ERRγ-specific DNA-protein complex (Fig. 7A, arrow b). The signal of this complex can be competed by excess unlabeled probes (lane 4), and supershifted by anti-Myc antibody (lane 5, arrow a). When ERRγ was coexpressed with GNL3L, two fast-moving DNA-protein complexes were identified (arrows d and e, lane 6), and no additional slow-moving complexes were seen. The mobility of the fast-moving complexes can be retarded by anti-Myc antibody (lane 7, arrow c), but not by anti-HA antibody (lane 8), suggesting that they contain ERRγ but not GNL3L. GNL3L alone fails to interact with the ERE probe (lane 9). The intensity of the fast-moving complex, complex d, was reduced when ERRγ was coexpressed with a GNL3L mutant lacking its ERRγ-binding I-domain (dI), indicating that the appearance of this fast-moving complex d depends on the interaction between GNL3L and ERRγ.
The increased mobility of the ERRγ DNA-protein complex by GNL3L may be caused by cleavage of the ERRγ protein or by changes in the protein conformation or components of the ERRγ DNA-protein complex; failure of this fast-moving complex d to be supershifted by anti-HA antibody indicates that it does not contain GNL3L or the HA-epitope of GNL3L is masked in this particular protein conformation. To address these different possibilities, we retrieved the fast-moving and slow-moving protein complexes (complex d and complex b, respectively) from the EMSA gel and analyzed the protein amount and size of ERRγ, GNL3L, SRC1, and SRC2 in these two complexes by western blottings (Fig. 7B). Anti-Myc western analysis shows that the size of the ERRγ protein remains the same in both complexes, excluding the possibility that the increased mobility is a result of ERRγ protein cleavage. Anti-HA western blotting detects no GNL3L protein in the retrieved protein complexes, consistent with the idea that GNL3L does not bind the DNA-bound ERRγ. Notably, we are able to detect SRC1 in the slow-moving complex b but not in the fast-moving complex d, demonstrating that coexpression of GNL3L reduces SRC1 binding to the ERRγ DNA-protein complex. Although the SRC2 protein is present in both complexes, the amount of SRC2 relative to ERRγ is reduced in the fast-moving complex d compared with the slow-moving complex b, which suggests that SRC2 binding to ERRγ is also diminished by GNL3L coexpression.
GNL3L suppresses the SRC-mediated transcriptional coactivation on ERRγ
Next, we addressed the issue whether GNL3L interferes with the function of SRC proteins as coactivators for ERRγ. In a cell-based reporter system similar to that described in Fig. 4, coexpression of ERRγ and SRC1 is able to produce an eight-fold increase (8.0±0.3) in the ERE-specific transcriptional activity compared with the control sample – which is 1.7 times higher than the sample expressing only ERRγ (4.8±0.3) (Fig. 8A). When coexpressed with GNL3L, the luciferase activity is reduced to 3.6 times (± 0.2, for 100 ng of GNL3L) and 2.5 times (± 0.2, for 200 ng of GNL3L) that of the control sample, representing respectively a 55% and 70% reduction compared with the sample expressing both ERRγ and SRC1. Again, a deletion of the ERRγ-interacting domain (dI) abolishes the suppressive activity of GNL3L (P=0.17). The ability of GNL3L to inhibit the transcriptional coactivator activity can also work on SRC2 (Fig. 8B). Here, we show that GNL3L, but not the dI mutant, can reduce the ERRγ-SRC2-mediated 7.4-fold increase (7.4±0.2) in the luciferase activity down to 3.4-fold (± 0.2, for 100 ng of GNL3L) and 2.1-fold (± 0.2, for 200 ng of GNL3L) compared with the control sample. The dI mutant fails to exhibit this inhibitory activity (P=0.58). These data demonstrate that GNL3L can suppress the SRC1-mediated and SRC2-mediated coactivation of ERRγ in an I-domain-dependent manner.
In this manuscript, we identify a GNL3L-mediated mechanism that suppresses the transcriptional activity of ERR family genes by coactivator competition (Fig. 9). We show that ERR binding is specific to GNL3L, but not to nucleostemin or Ngp1. GNL3L and ERRγ colocalize in the nucleoplasm, and their interaction requires the I-domain of GNL3L and the AF2-domain of ERRγ. Gain-of-function and loss-of-function studies reveal that GNL3L has the ability to suppress the transcriptional activity of ERR genes, and does not require the nucleolar localization of GNL3L to do so. We also demonstrate that GNL3L can compete with SRC1 and SRC2 for their binding to ERRγ, resulting in an increased electrophoretic mobility of the DNA-bound ERRγ complex and the inhibition of the SRC1 and SRC2 coactivator function on ERRγ. The AF2-domain binding, SRC competition and transcriptional inhibition activities suggest that GNL3L represents a new class of transcriptional co-repressor for nuclear receptors. However, GNL3L fails to form a stable complex with the DNA-bound ERRγ, and the I-domain of GNL3L – which is necessary and sufficient for ERRγ binding – lacks the LxxLL motif found in most transcriptional coactivators and co-repressors that interact with the AF2-domain (Hentschke et al., 2002; Huss et al., 2002; Lee et al., 1998; Rosenfeld and Glass, 2001; Webb et al., 2000; Zhang et al., 2000). These data support the role of GNL3L as a novel regulator for the ERR gene family and argue against its role as a classical transcriptional co-repressor. It is worth noting that the absence of interaction between GNL3L and the DNA-bound ERRγ in vitro does not exclude the possibility that these two proteins still coexist in the same DNA-bound complex in the native chromatinized context, because the binding of transcription factors to the core response element in vivo is aided by a number of cofactors as well as by histone proteins involved in chromatin remodeling. Furthermore, the ability of GNL3L to compete for SRC binding and to inhibit the transcriptional function of ERRγ might depend on specific chromatin structures or involve cofactors other than SRC proteins.
GNL3L and ERR proteins are colocalized in the nucleoplasm, but only GNL3L is also found in the nucleolus. The nucleolar localization of GNL3L might have created two potential mechanisms that affect its activity in the nucleoplasm. First, GNL3L can enter or exit the nucleolus by itself. In this case, signals that promote the nucleolar accumulation of GNL3L may cause a disinhibition of the ERR activities, and signals that release the nucleolus-bound GNL3L into the nucleoplasm may allow more GNL3L to bind ERR proteins. Alternatively, GNL3L may carry some ERR proteins with it when entering the nucleolus, in which case nucleolar sequestration of ERR proteins may account for some of the inhibitory activity of GNL3L. Although overexpression of a nucleolar form of GNL3L and, to a less extent, the wild-type GNL3L increase the nucleolar intensity of ERRβ and ERRγ, the non-nucleolar dBC mutant is able to suppress ERRβ and ERRγ activities as much as wild-type GNL3L. These results demonstrate that the suppressive effect of GNL3L on the ERR activity is not mediated by a nucleolar sequestration mechanism and that, under physiological conditions, the ERR-binding and transcriptional inhibition events of GNL3L take place in the nucleoplasm. Nevertheless, the fact that overexpression of NoG3l can bring ERRβ and ERRγ into the nucleolus supports the notion that GNL3L can interact with these two proteins in vivo.
Unlike ERα whose activity is controlled by hormone binding, no ligand has so far been identified for ERR family genes and their transactivation works in a ligand-independent manner. This GNL3L-mediated inhibition on the activities of ERRs provides one mechanism to regulate their functions in a cell-context-dependent and dynamic way. At the transcriptional level, although the relative abundance of GNL3L matches that of ERRγ in most tissues we examined, they are distinctively different in the brain and heart. GNL3L is expressed highly in the brain but little in heart, whereas ERRγ is found at high levels in heart but not in brain. The differences between the expression levels of GNL3L and ERRγ in those organs indicate that tissues that express the same level of ERRγ may exhibit differential ERR activities depending on their GNL3L expression. In the adult brain where little ERRγ is found, GNL3L might have other regulatory targets. At the post-translational level, GNL3L is partitioned between the nucleolus and the nucleoplasm by a dynamic process (L.M. and R.Y.L.T., unpublished data). Like nucleostemin (Meng et al., 2006; Tsai and McKay, 2005), the nucleolar accumulation of GNL3L is controlled by its GTP binding and a N-terminal basic domain (Rao et al., 2006) (L.M. and R.Y.L.T., unpublished data). Notably, the nucleolar residence of GNL3L is significantly shorter than that of nucleostemin (L.M. and R.Y.L.T., unpublished data). The transient residence of GNL3L in the nucleolus might explain why nucleolar compartmentalization of GNL3L does not seem to play a role in its ability to suppress the ERR transcriptional function.
In conclusion, it is known that the ERR family genes are transcriptionally active without the ligand, but it is unclear if and how their activities can be controlled in a dynamic manner. Our work unravels a GNL3L-mediated mechanism that modulates the transcriptional activity of ERRγ by coactivator competition. Given the important role of the ERR genes in embryogenesis and tumorigenesis, the differential regulation of their activities by GNL3L can provide us with new insight into these two processes in a cell-type-specific manner.
Materials and Methods
Recombinant plasmids and mutation analyses
Full-length ERR family genes were cloned from mouse brain cDNAs by reverse transcription (RT)-PCR. Deletions and point mutations of GNL3L and ERRγ were introduced by the stitching PCR method as described previously (Tsai and McKay, 2002; Tsai and McKay, 2005). The final PCR products were subcloned into pCIS expression vectors and confirmed by sequencing.
Cell culture, transfection, siRNA knockdown and immunostaining
We used HEK293 cells for biochemical studies because of their high transfection efficiency and protein production, and U2OS cells for distribution analyses. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS, Hyclone), penicillin (50 IU/ml), streptomycin (50 μg/ml), and glutamine (1%). Plasmid transfections were performed using a standard Ca2+ phosphate method for HEK293 cells or the Lipofectamine-Plus reagent (Invitrogen) for U2OS cells. Immunofluorescence studies were performed 1 day after transfection as described previously (Tsai and McKay, 2005). For siRNA knockdown experiments, cells were transfected with siRNA duplex (100 nM) for 12-24 hours using Oligofectamine reagent (Invitrogen) and analyzed 2 days later. Targeted sequences for siRNA duplexes were as followed: siGNL3L-1, 5′-AAAAACGCAGGACCAUUGAGA-3′; siGNL3L-2, 5′-AACUAUUGCCGCCUUGGUGAA-3′; siNEG, 5′-AAUGACGAUCAGAAUGCGACU-3′.
Primary antibodies were monoclonal anti-HA antibody (×2000; HA.11, Covance), monoclonal anti-Myc antibody (×1000, Covance) and monoclonal anti-fibrillarin antibody (×1000, EnCor). Secondary antibodies were conjugated with Rhodamine-X or FITC.
Yeast two-hybrid screen
Full-length mouse GNL3L was subcloned in the pAS2-1 vector and used as a bait to screen an E17.5 mouse brain cDNA library in the pACT2 vector (Clontech). The bait and library plasmids were co-transformed into the Saccharomyces cerevisiae strain Y190, and selected for both histidine+ and β-galactosidase+ phenotypes. cDNA plasmids were re-transformed into Escherichia coli HB101 by electroporation and expanded for further analysis.
Cells were harvested in NTEN buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.1 mM DTT, supplemented with 1 mM PMSF, 1 μg/ml leupeptin, 0.5 μg/ml aprotinin, 0.7 μg/ml pepstatin A, and 1 μM E64). Lysates were incubated with monoclonal anti-HA (HA.11, Covance), monoclonal anti-Myc (9E10, Covance), or mouse IgG for 1 hour, followed by incubation with protein G sepharose beads (Pharmacia) for an additional 4 hours at 4°C. Immunoprecipitates were washed 5 times with RIPA buffer (1× PBS, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, 1 mM PMSF, 1 μg/ml leupeptin, 0.5 μg/ml aprotinin, 0.7 μg/ml pepstatin A, and 1 μM E64), fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to Hybond-P membranes (Amersham). Specific signals were detected by western blotting with polyclonal anti-HA or anti-Myc antibodies and horseradish-peroxidase-conjugated secondary antibody.
Affinity binding and competition assays
Full-length cDNAs of GNL3L and ERRγ were subcloned into the pGEX4T-2 vector. GST fusion proteins were expressed in BL21/DE3 as described previously (Tsai and McKay, 2002; Tsai and Reed, 1997). Epitope-tagged proteins were expressed in HEK293 cells and extracted in phosphate-buffered saline (PBS)-Triton X-100 (1%) buffer, supplemented with protease inhibitors. Sepharose-bound GST fusion proteins (2-5 μg) were incubated with whole-cell lysates for 2 hours at 4°C, washed five times with extraction buffer, including twice with high-salt buffer (extraction buffer plus 500 mM of NaCl), fractionated on 10% SDS-PAGE, and detected by western blot analyses.
Northern blot analyses
Ten micrograms of total RNA were isolated from CD-1 mice using Trizol solutions (Invitrogen), fractionated on 1% formamide denaturing agarose gels, and transferred onto Hybond XL membrane (Amersham). Filters were then hybridized with 32P-labeled probes at 65°C overnight and washed under high stringency conditions. Plaque date was counted as embryonic day 0.5 (E0.5).
Confocal images were captured on a Zeiss LSM510 confocal microscope using a 63× plan-apochromat oil objective. Images were scanned using the multi-track program, a 512×512 frame size, 3× zoom, and <1.4 μm optical thickness. Detector gain and amplifier offset were adjusted to ensure that all signals were appropriately displayed within the linear range. Fluorescence intensities were digitally quantified in Fig. 3C′,D′,E′ using the profile display mode along the path indicated by arrow.
Dual luciferase assays
For gain-of-function experiments, CV1 cells were grown in DMEM supplemented with 5% charcoal/dextran-treated FBS. Transient transfection was performed in 24-well plates using the Lipofectamine-Plus reagent. Total DNA amount in each well was adjusted to 2 μg using the empty expression vector. Cell extracts were prepared 30 hours after transfection. HEK293 cells were used for siRNA knockdown experiments because of their high GNL3L expression levels. HEK293 cells were split and grown in DMEM supplemented with 5% chacoal/dextran-treated FBS. On the next day, transfections of siRNA duplexes were performed in 24-well plates using the Oligofectamine reagent (Invitrogen). Firefly (100 ng) and Renilla (10 ng) reporter plasmids, ERR (50 ng), ERα (100 ng), GNL3L-related genes (200 ng or as specified), and/or SRC (50 ng) expression vectors were transfected on the fourth day, and cell extracts were prepared 1 day later. For E2 stimulation (Fig. 4D), cells were treated with 100 nM of E2. Firefly and Renilla luciferase activities were measured by the Dual-Luciferase Reporter Assay System (Promega). The expression of the firefly luciferase reporter gene was driven by three repeats of a synthetic consensus palindromic estrogen response element (ERE, GGTCACTGTGACC).
EMSA and post-EMSA western blot
Electrophoretic mobility shift assays (EMSAs) were carried out as described previously (Tsai and Reed, 1997; Tsai and Reed, 1998) with the following modifications. Recombinant proteins were expressed in HEK293 cells. Whole-cell lysates were extracted in buffer containing 40 mM HEPES-KOH (pH 7.9), 0.4 M KCl, 1 mM DTT, 10% glycerol, 0.1 mM PMSF and complete protease inhibitor cocktail (Roche) mixed with specified amounts of probes in 20 μl binding reactions, and incubated on ice for 20 minutes. The binding-reaction mixture contained 10 mM HEPES (pH 7.9), 70 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 4% glycerol, 20 μg/ml salmon sperm DNA and 200 μg/ml poly-deoxyinosinic-deoxycytidylic acid. The reaction products were subjected to electrophoresis on a 4% polyacrylamide gel (29:1) in 0.5× Tris-borate-EDTA (TBE) buffer at 4°C, and detected by autoradiography. To generate EMSA probes, RT1006 primer was radiolabeled with [γ-P32]ATP in a T4 kinase reaction, annealed with excess amounts of RT1007 primer, and purified using a QIAquick nucleotide removal kit (Qiagen); RT1006, 5′-GATCTCTTTGATCAGGTCACTGTGACCTGACTTTG-3′; RT1007: 5′-GATCCAAAGTCAGGTCACAGTGACCTGATCAAAGA-3. To determine the protein components in the shifted complexes, fast (d) and slow (b) mobility complexes were identified by autoradiography, retrieved, and subjected to SDS-PAGE. Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, ×500), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, ×250) antibodies.
We thank Seokwoon Kim for his technical support in the beginning of this work. SRC1 and SRC2 expression plasmids were kindly provided by Jiemin Wong, and the ERE reporter construct was kindly provided by Stephen Safe. This work was supported by a grant from the National Cancer Institute (R01 CA113750-01) to R.Y.L.T.