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. 2000 Apr;20(7):2411-22.
doi: 10.1128/MCB.20.7.2411-2422.2000.

A tissue-specific coactivator of steroid receptors, identified in a functional genetic screen

D Knutti  1 , A Kaul, A Kralli
Affiliations

A tissue-specific coactivator of steroid receptors, identified in a functional genetic screen

D Knutti et al. Mol Cell Biol. 2000 Apr.

Abstract

Steroid receptors mediate responses to lipophilic hormones in a tissue- and ligand-specific manner. To identify nonreceptor proteins that confer specificity or regulate steroid signaling, we screened a human cDNA library in a steroid-responsive yeast strain. One of the identified cDNAs, isolated in the screen as ligand effect modulator 6, showed no homology to yeast or Caenorhabditis elegans proteins but high similarity to the recently described mouse coactivator PGC-1 and was accordingly termed hPGC-1. The hPGC-1 DNA encodes a nuclear protein that is expressed in a tissue-specific manner and carries novel motifs for transcriptional regulators. The expression of hPGC-1 in mammalian cells enhanced potently the transcriptional response to several steroids in a receptor-specific manner. hPGC-1-mediated enhancement required the receptor hormone-binding domain and was dependent on agonist ligands. Functional analysis of hPGC-1 revealed two domains that interact with steroid receptors in a hormone-dependent manner, a potent transcriptional activation function, and a putative dimerization domain. Our findings suggest a regulatory function for hPGC-1 as a tissue-specific coactivator for a subset of nuclear receptors.

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Figures

FIG. 1
FIG. 1
A yeast screen can identify mammalian proteins that enhance steroid hormone signaling. (A) Schematic illustration of the yeast strain and strategy. The yeast strain YNK441 expresses GR constitutively and has two reporter genes, encoding His3 and β-gal, under the control of GREs. The expression of mammalian proteins that increase the response to hormone enables the activation of the pathway in the presence of low hormone concentrations, thereby allowing for growth and β-gal expression in selective media lacking histidine (−his) and containing the His3 inhibitor 3-aminotriazole (+3AT). The star-shaped forms indicate some of the possible interaction points for the mammalian modulators. (B) Mammalian p23, LEM5, and LEM6 (hPGC-1) enhance the response to corticosterone. Yeast strain YNK441 carrying a vector alone (control) or expression plasmids for the indicated cDNAs was incubated overnight with 0, 1, or 10 μM corticosterone and assayed for β-gal activity. Data represent the mean ± standard deviation of results from six independent yeast transformants. LEMGH, HepG2 library isolate; LEM6/hPGC-1, full-length cDNA.
FIG. 2
FIG. 2
(A) Schematic representation of the hPGC-1 protein. The N terminus is rich in charged residues (A, acidic; B, basic). The putative receptor interaction motif, LXXLL, is indicated (aa 144 to 148). The C terminus harbors two SR-rich stretches (aa 566 to 598 and aa 621 to 632) and a putative RNA-binding domain (RNP; aa 678 to 750). NLS, nuclear localization signals. (B) hPGC-1 is a nuclear protein. COS7 cells were transfected with a GFP–hPGC-1 expression vector, fixed, and analyzed for the localization of the fusion protein. (Left) Differential interference contrast (DIC) image. (Right) Fluorescence acquisition of the same field. (C) hPGC-1 mRNA is expressed in a tissue-specific manner. A human multiple-tissue northern blot (Clontech) was hybridized with an hPGC-1 probe (upper panel) or a β-actin-specific probe (lower panel). The positions of RNA markers (kilobases) are shown at the right of each panel. PBL, peripheral blood leukocytes.
FIG. 3
FIG. 3
hPGC-1 is a potent activator of hormone-dependent, receptor-mediated transcription in mammalian cells. COS7 cells were transfected with the receptor expression plasmid p6RGR, the indicated amounts of either the hPGC-1 expression vector pcDNA3/HA-hPGC-1 (black bars) or the empty vector pcDNA3 (hatched bars), and the GR-responsive luciferase reporter pTAT3Luc. The cells were treated for 24 h with either control vehicle (−) or 50 nM indicated ligand and assayed for luciferase activity. Luciferase activity in the presence of corticosterone (cort) and the absence of hPGC-1 (42,947 ± 17,676 luciferase units) was set equal to 100 within each experiment and used to normalize values from different experiments. Data represent the mean ± standard deviation of 8 to 10 transfections from four independent experiments. RLU, relative luciferase units; dex, dexamethasone.
FIG. 4
FIG. 4
(A) hPGC-1 enhances responses to steroids in a receptor-selective manner. Expression plasmids for ER, AR, MR, or GR were cotransfected into COS7 cells with control (hatched bars), hPGC-1 (black bars), or SRC-1e (gray bars) expression vectors and the following luciferase reporters: pERE-TK-luc for ER, pMMTVDLO (MMTV) for AR and GR, and pTAT3Luc (TAT3) for MR and GR. Cells were treated with 50 nM estradiol (ER), aldosterone (MR), or corticosterone (GR) or 100 nM dihydroxytestosterone (AR) for 24 h and assayed for luciferase activity. Results are expressed as fold enhancement by the coactivator in the presence of hormone; i.e., activity in the presence of hormone and absence of coactivator was set equal to 1 for all receptors. (B) hPGC-1 is not a general activator of transcription. COS7 cells were transfected with expression and reporter plasmids for either the two NF-κB subunits p50 and p60 (left panel) or the chimeric activator Gal4-VP16 (right panel) and assayed for luciferase activity. LU, luciferase units. Error bars show standard deviations.
FIG. 5
FIG. 5
hPGC-1 interacts functionally with the LBD of the receptor. COS7 cells were transfected with expression plasmids for either full-length GR (N795) (aa 1 to 795), the truncated GR variant N525 (aa 1 to 525) or 407C (aa 407 to 795), or the chimeric activator Gal4-LBD (aa 525 to 795 of GR); the GR- or Gal4-responsive reporter pTAT3Luc or pGK-1, respectively; and either vector alone (hatched bars) or the hPGC-1 expression vector (black bars). Cells were treated with 50 nM corticosterone (+) or carrier ethanol (−) for 24 h and assayed for luciferase activity. Data are the mean ± standard deviation luciferase units of eight transfections from four independent experiments.
FIG. 6
FIG. 6
hPGC-1 carries a transcriptional AD. (A) Gal4–hPGC-1 activates transcription in mammalian cells. COS7 cells transfected with the luciferase reporter plasmid pGK-1 and an expression vector for either the Gal4 DBD (aa 1 to 147 of Gal4) or the Gal4–hPGC-1 chimera were assayed for luciferase activity. Data are expressed relative to the activity seen with the Gal4 DBD alone (Gal4), which was set equal to 1, and are the mean ± standard deviation of six transfections from three independent experiments. (B) The hPGC-1 AD is in the N terminus. Yeast carrying a Gal4-responsive β-gal reporter and expression vectors for fusions of Gal4 (aa 1 to 147) to full-length or truncated hPGC-1 was assayed for β-gal activity. hPGC-1, aa 1 to 798; N408, aa 1 to 408; N293, aa 1 to 293; 91C, aa 91 to 798; 189C, aa 189 to 798. Data are the mean ± standard deviation β-gal activity of six or more independent yeast transformants.
FIG. 7
FIG. 7
Two domains in hPGC-1 mediate a hormone-dependent interaction with the LBD of GR. hPGC-1 variants fused to the Gal4 AD were assayed for their ability to interact with a Gal4-LBD fusion (aa 525 to 795 of GR) in yeast carrying a Gal4-responsive β-gal reporter. Cells were treated with no hormone, 25 μM corticosterone (cort), or 25 μM RU486 for 20 h and assayed for β-gal activity. Values in the absence of hormone were <1 β-gal unit. Values shown are the mean ± standard deviation β-gal units of at least four independent yeast transformants. AD−, AD alone; nd, not determined. hPGC-1 variants are named as in Fig. 6; hPGC-1 Δ1, Δ2, and Δ3 carry deletions of aa 91 to 186, 189 to 293, and 91 to 293, respectively. 294C, aa 294 to 798; 91/186, aa 91 to 186; 189/293, aa 189 to 293.
FIG. 8
FIG. 8
Functional analysis of hPGC-1 domains. (A) The AD and NIDs are essential for hPGC-1 coactivation of GR. (B) The C-terminal domain contributes to hPGC-1 function. (A and B) COS7 cells were transfected with the GR expression vector p6RGR, the GR-responsive luciferase reporter plasmid pTAT3Luc, and either the control vector pcDNA3 or hPGC-1 variants as indicated, treated with 50 nM corticosterone for 24 h, and assayed for luciferase activity. Results are expressed as fold enhancement by hPGC-1, with activity in the presence of hormone and just the control vector pcDNA3 set equal to 1. Data are the mean ± standard deviation of 6 to 12 values from at least three independent experiments. hPGC-1 variants are named as in Fig. 6 and 7; hPGC-1 Δ2L carries a deletion of aa 189 to 482. N88, aa 1 to 88; N186, aa 1 to 186. (C) hPGC-1 variants are expressed and present in the nucleus. COS7 cells transfected with the indicated hPGC-1 constructs were analyzed for the expression and localization of the HA epitope-tagged protein by fluorescence microscopy. Note that cells transfected with hPGC-1 variants that lack the putative nuclear localization signals (N293 and N293Δ1) show some cytoplasmic staining but still have comparable levels of hPGC-1 in the nucleus.
FIG. 9
FIG. 9
The C terminus of hPGC-1 interacts with itself. Yeast carrying a Gal4-responsive β-gal reporter and expressing Gal4-294C was transformed with vectors for the Gal4 AD, either alone (AD) or fused to full-length and truncated hPGC-1 variants as indicated, and assayed for β-gal activity. hPGC-1 variants are named as in Fig. 6 and 7. Data are the mean ± standard deviation β-gal activity of six or more independent yeast transformants.
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