Ligand-dependent corepressor LCoR is an attenuator of progesterone-regulated gene expression - PubMed (original) (raw)
Ligand-dependent corepressor LCoR is an attenuator of progesterone-regulated gene expression
Ana Palijan et al. J Biol Chem. 2009.
Abstract
Ligand-dependent corepressor LCoR interacts with the progesterone receptor (PR) and estrogen receptor ERalpha in the presence of hormone. LCoR contains tandem N-terminal PXDLS motifs that recruit C-terminal-binding protein (CtBP) corepressors as well as a C-terminal helix-turn-helix (HTH) domain. Here, we analyzed the function of these domains in coregulation of PR- and ERalpha-regulated gene expression. LCoR and CtBP1 colocalize in nuclear bodies that also contain CtBP-interacting protein CtIP and polycomb group repressor complex marker BMI1. Coexpression of CtBP1 in MCF7 or T47D breast cancer cells augmented corepression by LCoR, whereas coexpression of CtIP did not, consistent with direct interaction of LCoR with CtBP1, but not CtIP. The N-terminal region containing the PXDLS motifs is necessary and sufficient for CTBP1 recruitment and essential for full corepression. However, LCoR function was also strongly dependent on the helix-turn-helix domain, as its deletion completely abolished corepression. LCoR, CtBP, and CtIP were recruited to endogenous PR- and ERalpha-stimulated genes in a hormone-dependent manner. Similarly, LCoR was recruited to estrogen-repressed genes, whereas hormone treatment reduced CtBP1 binding. Small interfering RNA-mediated knockdown of LCoR or CtBP1 augmented expression of progesterone- and estrogen-stimulated reporter genes as well as endogenous progesterone-stimulated target genes. In contrast, their ablation had gene-specific effects on ERalpha-regulated transcription that generally led to reduced gene expression. Taken together, these results show that multiple domains contribute to LCoR function. They also reveal a role for LCoR and CtBP1 as attenuators of progesterone-regulated transcription but suggest that LCoR and CtBP1 can act to enhance transcription of some genes.
Figures
FIGURE 1.
The association of endogenous LCoR with endogenous CtBP1 and CtIP (A and B) is shown. Confocal microscopic analysis of the subcellular colocalization of LCoR with CtBP1 (A) and CtIP (B) by immunocytochemistry (see “Experimental Procedures” for details) is shown. C, shown is the analysis of the association of LCoR with CtBP1 and CtIP by coimmunoprecipitation (Co-IP). Extracts of MCF7 cells were immunoprecipitated (IP) with antibodies against CtBP1 or CtIP as indicated and probed by Western blotting for enrichment of target proteins in immunoprecipitates. Immunoprecipitates were also probed for the coimmunoprecipitation of LCoR.
FIGURE 2.
Direct association of LCoR with CtBP1 but not CtIP. A, shown are a schematic representation of the primary structure of LCoR along with LCoR mutated in the P_X_DLS motifs that bind CtBPs (m1m2) and C-terminal deletion mutants of LCoR. aa, amino acids. B, shown is GST pulldown analysis of the interaction of CtBP1 present in MCF7 cell extracts (MCF7 extr.) with GST fusions of wild-type LCoR or mutants described in A. CtBP1 bound to fusion proteins was detected by Western (W) blotting. C, mutation of both CtBP binding sites of LCoR disrupts its interaction with CtBPs in MCF7 cell extracts. MCF7 cells were transfected with FLAG-tagged wild-type LCoR or tagged LCoR mutated in one (m1 or m2) or both (m1m2) CtBP binding sites, as indicated. Top panel, extracts and immunoprecipitations (IP) with the anti-FLAG antibody of transfected MCF7 cells show that tagged proteins are expressed at similar levels in all cases. Middle panel, control immunoprecipitation with anti-CtBP1 antibody and Western blots shows that CtBP1 is expressed at similar levels in all cases. Bottom panel, shown is coimmunoprecipitation of tagged LCoR derivatives from extracts of transfected MCF7 cells. D, shown are Western blots of MCF7 extracts expressing FLAG-LCoR or FLAG-m1m2 blotted for FLAG (first row) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; second row), used as the loading control. E, GST pulldown assays show no direct binding between LCoR and CtIP. Binding of CtBP1 to GST-LCoR fusions was used as a positive control.
FIGURE 3.
Roles of CtBP1 and CtIP in LCoR-dependent corepression in MCF7 cells. Cells were transiently transfected with expression vectors for either PR (100 ng) or ERα (100 ng) and their corresponding reporter plasmids (250 ng) for 18 h. Media was then changed, and cells were treated with vehicle or hormone for 30 h. A and B, shown are dose-response curves analyzing the effects of CtBP1 on reporter gene expression in cells treated with P4 (10 n
m
; A) or E2 (10 n
m
; B). Increasing amounts of CtBP1 were transfected (200, 400, and 600 ng). C and D, shown are dose-response curves analyzing the effects of CtIP on reporter gene expression in cells treated with P4 (C) or E2 (D). Increasing amounts of CtIP1 were transfected (200, 400, and 600 ng). E and F, shown is analysis of the effects of coexpression of LCoR and CtBP1 on hormone-dependent gene expression. Cells were transiently transfected with either vector alone, LCoR alone (100 ng), CtBP1 alone (200 ng), or with both LCoR and CtBP1 and treated with P4 (E) or E2 (F). *, p < 0.05 for results of LCoR and CtBP1 coexpression versus LCoR expression alone. G and H, shown is analysis of the effects of coexpression of LCoR and CtIP on hormone-dependent gene expression. Cells were transiently transfected with either vector alone, LCoR alone, CtIP1 alone, or with both LCoR and CtIP1 and treated with P4 (G) or E2 (H). I and J, shown are dose-response curves of either LCoR or m1m2 in cells treated with P4 (I) or E2 (J). Increasing amounts of wild-type or mutant LCoR were transfected (200, 400, and 600 ng). *, p < 0.05 for results of corresponding wild-type LCoR versus mutant form m1m2. RLU, relative luciferase units.
FIGURE 4.
ChIP and reChIP assays of protein association with progesterone and estrogen target genes. MCF7 and T47D cells were transiently transfected with FLAG-tagged LCoR, synchronized for 2 h with α-amanitin (2.5 μ
m
), and treated with P4 (10 n
m
) for 45 min or E2 (10 n
m
) for 30 min, as indicated. Cell extracts were collected and immunoprecipitated with IgG or antibodies against PR, ERα, FLAG, CtBP1, or CtIP, as indicated. A, shown is a schematic representation of the progesterone-sensitive IGFBP1 promoter (left-hand side) and estrogen-stimulated pS2 promoter (right-hand side). The hormone response element (HRE), transcription start site, and both PCR-amplified sequences (target and non-target control regions) are depicted. B, shown is a ChIP assay of factor binding to the IGFBP1 promoter in MCF7 cells (left-hand side) and T47D cells (right-hand side). Results of semiquantitative and quantitative PCR analyses are presented. Note that no signal was detected by quantitative PCR in the non-target controls. C, shown are reChIP assay in the extracts of T47D cells treated with P4 and immunoprecipitated with FLAG. A second round of immunoprecipitations with IgG, PR, or CtBP1 was performed, as indicated. D, ChIP assays of factor binding to the pS2 promoter in MCF7 cells treated with E2 are shown. E, a reChIP assay in extracts of MCF7 cells treated with E2 and immunoprecipitated with FLAG is shown. A second round of immunoprecipitations with IgG, ERα, or CtBP1 was performed, as indicated.
FIGURE 5.
The HTH domain of LCoR is essential for corepression. A, shown are schematic representations of full-length LCoR (upper panel) and a deletion mutant lacking the HTH domain (lower panel; LCoR_Δ_HTH) in which amino acids 350–395 were deleted, leaving the C-terminal portion of the protein (395–433 amino acids) intact. B, LCoR and LCoRΔHTH are expressed equally in T47D cells. Shown is a Western blot of T47D cell extracts expressing FLAG-LCoR or FLAG-LCoRΔHTH blotted for FLAG (first row) or loading control glyceraldehyde-3-phosphate dehydrogenase (GAPDH, second row). C, LCoRΔHTH was recruited to the progesterone target gene encoding IGFBP1. ChIP assays in T47D cells treated with P4 (10 n
m
) for 45 min and immunoprecipitated with FLAG are shown. The upper panel shows a schematic view of IGFBP1 promoter. PRE, progesterone response element. D and E, deletion of HTH domain of LCoR abolishes corepressor function is shown. Dose-response curves were performed analyzing corepression in the presence of increasing amounts of LCoR or LCoRΔHTH expression vectors (0, 100, 400, and 600 ng, as indicated). T47D cells were treated with P4 (10 n
m
; panel D) and MCF7 cells were treated with E2 (10 n
m
; panel E). Cells were transiently transfected with expression vectors of either PR (100 ng) or ERα (100 ng) and their corresponding reporter plasmid (250 ng) for 18 h. Media was then changed, and cells were treated for 30 h. RLU, relative luciferase units.
FIGURE 6.
siRNA knockdown of LCoR and CtBP1 expression in MCF7 and T47D cells. A, B, and C, Western blots of MCF7 and T47D extracts are shown. Cells were transfected for 48 h with pools of scrambled siRNAs (Scr.) or siRNAs targeting LCoR (A) or CtBP1 (B) as well as siRNAs targeting CyPB (C) to control for off-target effects. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a control. Densities of bands on Western blots, scanned with Bio-Rad Gel Doc XR system and analyzed with Quantity One, are presented below the images of the blots. D and E, luciferase reporter assays in siRNA-transfected cells. A PR expression vector was transfected along with scrambled, LCoR, CtBP1, or CyPB siRNAs (D), or an ERα expression vector was transfected along with scrambled, LCoR, CtBP1, or CyPB siRNAs (E). After 24 h of treatment with P4 (10 n
m
; D) or E2 (10 n
m
; E), cells were harvested, and luciferase activity was measured. Data are shown as relative luciferase units (RLU). Data are the averages of three or more independent experiments, and error bars represent the S.E.; *, p < 0.05 for results of specific knockdown (LCoR, CtBP1, or CyPB) versus results with scrambled siRNA.
FIGURE 7.
Effects of LCoR and CtBP1 ablation in T47D cells on regulation of endogenous PR target genes. Cells were transfected with the corresponding siRNAs (scrambled, CyPB, LCoR, or CtBP1 or LCoR and CtBP1) for 36 h, then treated with vehicle (EtOH) or P4 (10 n
m
) for 24 h. qRT-PCR was performed to analyze regulated expression of IGFBP1 (A), mucin 1 (MUC1; B), or FK506-binding protein 5 (FKBP51; C), and β-actin expression was used as an internal control. Results are shown as -fold induction. Data are the averages of three or more independent experiments. Error bars represent the S.E.; *, p < 0.05 for results of specific knockdown (LCoR, CtBP1, or LCoR and CtBP1) versus scrambled results with scrambled siRNA.
FIGURE 8.
Effects of LCoR and/or CtBP1 ablation in MCF7 cells on regulation of endogenous estrogen target genes. Cells were transfected with corresponding siRNAs (scrambled, CyPB, LCoR, or CtBP1 or both LCoR and CtBP1) for 36 h, then treated with vehicle (DMSO) or E2 (10 n
m
) for 24 h. qRT-PCR was performed to analyze regulated expression of pS2 (A), GREB1 (B), SGK3 (C), CYP26B1 (D), BMP7 (E), or keratin 4 KRT4; F), and β-actin was used as an internal control. Results are shown as -fold induction. Data are the averages of three or more independent experiments. Error bars represent the S.E.; *, p < 0.05 for results of specific knockdown (LCoR, CtBP1, or both LCoR and CtBP1) versus scrambled results. G, shown are ChIP assays analyzing factor binding to the BMP7 promoter (upper panel) in MCF7 cells treated with E2 (10 n
m
). Cell extracts were collected and immunoprecipitated with IgG or antibodies against ERα, FLAG, or CtBP1, as indicated. Results of semiquantitative and quantitative PCR analyses are presented. Note that no signal was detected by quantitative PCR in the non-target controls.
References
- Edwards D. P. (2005) Annu. Rev. Physiol. 67, 335–376 - PubMed
- Hall J. M., McDonnell D. P. (2005) Mol. Interv. 5, 343–357 - PubMed
- Boonyaratanakornkit V., McGowan E., Sherman L., Mancini M. A., Cheskis B. J., Edwards D. P. (2007) Mol. Endocrinol. 21, 359–375 - PubMed
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