Ligand-dependent Corepressor LCoR Is an Attenuator of Progesterone-regulated Gene Expression*

Ligand-dependent corepressor LCoR interacts with the progesterone receptor (PR) and estrogen receptor ERα 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 ERα-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 ERα-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 ERα-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.

founding member of a well conserved family of proteins (17)(18)(19). Highly homologous CtBP2 was identified from EST databases (20). Remarkably, CtBPs bind NAD(H) and are related to 2-hydroxy acid dehydrogenases, and CtBP1 has weak dehydrogenase activity. In addition, dinucleotide binding stimulates protein oligomerization activity and corepression.
In addition to its N-terminal CtBP interaction motifs and a central HDAC binding domain (29), LCoR contains a C-terminal helix-turn-helix (HTH) domain that is homologous to motifs encoded by the Eip93F (CG18389 gene product from transcript CG18389-Rb) and MBLK-1 genes of Drosophila and Honeybee (Apis mellifera) (30), respectively. The LCoR HTH domain also bears 35% homology to pipsqueak motifs (PSQ). PSQ motifs are repeated four times in the DNA binding region of the Drosophila pipsqueak transcription factor, which plays a role in gene silencing (31). Multiple repeats of the domain are required for PSQ DNA binding (31), and mutation of one of the two HTH motifs in the MBLK-1 gene strongly reduced sitespecific DNA binding (30). The PSQ domain is homologous to unique motifs found in a number of prokaryotic and eukaryotic proteins that interact with DNA, such as recombinases (31,32), raising the possibility that LCoR itself may interact with DNA. Other studies have shown that HTH domains can function in protein-protein interactions, where the HTH motif, combined with other domains, can induce formation of multisubunit complexes (33). Such proteins with multidomains can act as scaffolds between the basal transcription machinery and transcription factors (33).
The accompanying paper showed that LCoR recruits HDAC6 through a central domain (29). In this study we have analyzed the roles of domains controlling recruitment of CtBPs and the C-terminal HTH motif in corepression by LCoR. We were primarily interested in determining the roles of these domains in corepression of the PR, as our previous work showed that the efficacious corepression of PR-driven gene reporter gene expression by LCoR appeared to be largely insensitive to HDAC inhibition (15). We find that both the PXDLS motifs and the HTH domain are required for corepression of both the PR and ER␣, as disruption of either region markedly attenuated LCoR function. LCoR colocalizes with CtBP1 and CtIP in nuclear foci, and CtBP1 is corecruited with LCoR to PR and ER␣ target genes in a hormone-dependent manner. Ablation of LCoR and/or CtBP1 enhanced progesterone-stimulated gene expression in T47D breast cancer cells. In contrast, loss of LCoR or CtBP1 had gene-specific effects on ER␣-regulated genes and generally led to reduced target gene expression.
Cell Culture and Transfections-All cells were cultured under the recommended conditions. For immunocytochemistry, MCF7 cells grown on collagen IV-treated microscope slides in 6-well plates in DMEM supplemented with 10% FBS. Cells were prepared for immunocytochemistry as described below. For immunoprecipitation of tagged proteins, MCF7 cells in 100-mm dishes were transfected with 10 l of Lipofectamine containing 10 g of pSG5 vectors containing FLAG-LCoR, FLAG-m1, FLAG-m2, or FLAG-m1 ϩ m2. For analysis of the effects of CtBP1 and CtIP on LCoR corepression, MCF7 cells (60 -70% confluent) were grown in DMEM without phenol red supplemented with 10% FBS on 6-well plates. Cells were transfected in medium without serum (Opti-MEM (Invitrogen) with Lipofectamine 2000 (Invitrogen). The amounts of expression vectors were as follows: 100 ng of ER␣ or human PR expression vectors (as indicated), 100 ng of LCoR/pcDNA3.1, 250 ng of ERE3-TATA/pXP2 or pGRE5/pXP2 reporter plasmid, 250 ng of internal control vector pCMV-␤gal. Quantities of expression vectors used are indicated in the legends to Figs. 3 and 5. The medium was replaced 18 h after transfection by a medium containing charcoal-stripped serum and ligand (10 nM) for 30 h as indicated. For the Luciferase reporter assay, cells were harvested in 250 l of reporter lysis buffer (Promega).
Western Blotting-A Western blot was performed as previously described (34) using MCF7 cells extracts. Cells were grown in 10-cm dishes (70% confluent) and transiently transfected with 500 ng of FLAG-tagged full-length of mutated LCoR. Cells were harvested 30 h later.
Chromatin Immunoprecipitation (ChIP) Assays and reChIP Assays-ChIP and reChIP assays were performed as previously described (35) in MCF7 and T47D cells. Cells were grown in 10-cm dishes (70% confluent) and transiently transfected with 500 ng of FLAG-tagged LCoR (or FLAG-tagged LCoR⌬HTH as indicated). After transfection, cells were starved for 2 days in DMEM-phenol free and FBS-free media and treated with 2.5 M ␣-amanitin (Sigma, A2263) for 2 h before hormone treatment to properly synchronize cells. Cells were collected, and cofactor recruitment was evaluated on promoter regions containing either estrogen or progesterone response element (as indicated) of target genes. For ChIP primers sequences, please refer to supplemental Table 1.
siRNA Knockdowns-siRNAs were purchased from Thermo Scientific Dharmacon (Lafayette, CO). The following ON-TARGETplus SMART pool siRNAs were used: LCoR (L-026303-00), CtBP1 (L-008609-00), CyPB (D-001820-10), and nontargeting (D-001818-10). siRNAs were resuspended per the manufacturer's instructions. Transfections were done in 6-well plates as described previously. Lipofectamine 2000 (10 l) was used as the transfection reagent. DMEM phenol-free with 10% stripped FBS was added 12 h after transfection. For Western blot analysis, cells were collected 48 h after transfection. Luciferase reporter assays after siRNA knockdowns were performed as follows. 100 ng of ER␣ expression vector and 250 ng of ERE3-TATA-pXP2 vector were transfected with the corresponding siRNA. DMEM phenol-free with 10% stripped FBS was added 12 h after transfection. Ligand was added 36 h after transfection, and cells were collected 24 h later. Luciferase activity was measured as previously described.
RNA Isolation, cDNA Synthesis, and Quantitative Real-time (qRT)-PCR-Cells were grown in 100-mm dishes. Media were replaced with charcoal-strippedmedium-containing ligand. Total RNA was extracted with TRIZOL reagent. cDNA synthesis was performed with iScript cDNA synthesis kit (Bio-Rad) according to the instructions of the manufacturer. The MiniOpticon real-time PCR system with the iQ SYBR Green Supermix (Bio-Rad) was used for qRT-PCR expression analysis of target genes. The program used was as follows: 1) incubation at 94°C for 60 s, 2) incubation at 95°C for 20 s, 3) incubation at 60°C for 30 s (decreasing temperature by 1°per cycle), 4) incubation at 72°C for 30 s, 5) plate reading, 6) repetition from step 2 five more times, 7) incubation at 95°C for 20 s, 8) incubation at 57.5°C for 30 s, 9) incubation at 72°C for 30 s, 10) plate reading, 11) repetition from step 7 thirty-five more times, 12) performance of melting curve and end. Results were normalized to ␤-actin mRNA expression. For qRT-PCR primers sequences, please refer to supplemental Table 2.

Association of LCoR with CtBP1 and CtIP-Colocalization
of LCoR with CtBP1 in MCF7 cells was confirmed by immu-nocytochemical analyses (Fig. 1A). Both proteins were broadly distributed in the nucleus and were also concentrated in discrete nuclear bodies. A similar colocalization of CtBP2 and LCoR was also observed (data not shown). Given the extensive overlap of CtBPs and LCoR, we determined whether LCoR colocalized with CtIP, which was identified as a CtBP-interacting protein containing an extended PXDLS motif (22). Similar to results obtained with CtBP, CtIP and LCoR showed strongly overlapping patterns of expression concentrated in discrete nuclear bodies (Fig. 1B). In addition, endogenous LCoR coimmunoprecipitates with antibodies directed against either CtBPs or CtIP (Fig. 1C).
Other studies have shown that CtIP interacts directly with the retinoblastoma gene product (22) and link CtBP1 and Rb to polycomb group repressor complexes (26,36). PRC1 complexes form large foci containing numerous factors, including BMI1 polycomb ring finger oncogene (BMI1), visible as discrete nuclear structures (37). Indeed, we found that BMI1 and LCoR coimmunoprecipitated and colocalized in nuclear bodies (supplemental Fig. 1). Taken together, these studies show that LCoR extensively colocalizes with CtBP1 and CtIP in the nucleus, including in PRC1 complexes.
The specificity of the interactions of LCoR with CtBP1 were further analyzed in vitro using glutathione S-transferase (GST) fused to a series of C-terminal deletion mutants of LCoR or LCoR mutant m1m2, which lacks the tandem PXDLS motifs ( Fig. 2A). CtBP1 bound to full-length LCoR (Fig. 2B, top) and all C-terminal deletion mutants (Fig. 2B, bottom) but not to LCoR m1m2 ( Fig. 2A). Moreover, tagged LCoR mutated in either one of the two PXDLS motifs coimmunoprecipitated with endogenous CtBPs from extracts of MCF7 cells (m1, amino acids 64 -70 deleted; m2, amino acids 82-88 deleted; Fig. 2C, bottom panel). In contrast, no coimmunoprecipitation was observed in cells express-  OCTOBER 30, 2009 • VOLUME 284 • NUMBER 44

JOURNAL OF BIOLOGICAL CHEMISTRY 30279
ing the m1m2 derivative lacking both sites (Fig. 2C, bottom), although the wild-type and m1m2 proteins were expressed at similar levels (Fig. 2D). Taken together, these results show that the integrity of at least one of the two PXDLS motifs of LCoR is required for binding to CtBP1.
We also analyzed the association of LCoR with CtIP by GST pull down assay. The coimmunoprecipitation of CtIP and LCoR is remarkable given that CtIP and LCoR interact with CtBPs through common motifs. However, no evidence was found for LCoR binding directly to CtIP in vitro (Fig. 2E), suggesting that their association in cells is indirect.
CtBP1 but Not CtIP Augments LCoR-dependent Corepression-The function of CtBP1 and CtIP as corepressors of progesterone-or estrogen-regulated gene expression in the absence or presence of LCoR was analyzed in MCF7 (Fig. 3) and T47D (supplemental Fig. 2) breast cancer cells. Neither protein repressed (hormone-dependent) gene expression in MCF7 cells in the absence of LCoR when coexpressed in dose-response experiments with either the PR or ER␣ (Fig. 3, A-D). On the other hand, cotransfection of a limiting amount (200 ng) of a CtBP1 expression vector (Fig. 3, E and F) significantly augmented LCoR-dependent corepression, whereas coexpression of CtIP had no effect on LCoR function (Fig. 3, G and H). This is consistent with the direct interaction of CtBP1, but not CtIP, with LCoR, observed above. In agreement with a role of CtBP recruitment in LCoR function, corepression of progesteroneor estrogen-induced gene expression by the m1m2 mutant of LCoR lacking the CtBP binding motifs was markedly attenuated (Fig. 3, I and J). Similar results were obtained in T47D cells (supplemental Fig. 2).
CtBP1 Is Corecruited with LCoR to PR-and ER␣-stimulated Target Genes in the Presence of Hormone-The binding of LCoR and CtBP1 to hormone-responsive promoters was also analyzed by ChIP assay. As we do not have an antibody that immunoprecipitates endogenous LCoR efficiently, LCoR was expressed as a FLAG-tagged protein in these experiments. Treatment of T47D or MCF7 cells with hormone induced PR binding to the progesterone response element of the insulinlike growth factor binding protein 1 (IGFBP1) promoter (Fig. 4, A-C, left-hand side). Similarly, LCoR and CtBP1 as well as CtIP were recruited to the promoter under the same conditions in both cell lines. Non-target controls showed that the binding of cofactors and nuclear receptors was specific for their corresponding hormone response elements (Fig. 4, A and B). In re-ChIP experiments, proteins from progesterone-treated T47D cells were immunoprecipitated with an anti-FLAG antibody to concentrate LCoR and were re-immunoprecipitated with antibodies against either the PR or CtBP1 (Fig. 4C), which confirmed that LCoR is recruited to the same promoters in vivo as the PR and CtBP1. Similar results were obtained when recruitment of ER␣, LCoR, CtBP1, and CtIP to the estrogen-responsive trefoil factor 1 (pS2) promoter was analyzed in MCF7 cells (Fig. 4, A, right-hand side, D, and E). Note that essentially identical results were obtained in multiple biological replicates with both cell lines.
The recruitment of CtIP is intriguing, given its indirect association with LCoR and the lack of effect of CtIP on hormonedependent transactivation either in the presence or absence of LCoR (Fig. 3). The data raise the possibility that at least a portion of CtIP may be recruited to hormone-responsive promoters through its colocalization with LCoR in PRC1 complexes. Consistent with this hypothesis, we found that PRC1 marker BMI1 was also recruited to the pS2 promoter in the presence of estradiol (supplemental Fig. 1).
Deletion of the HTH Domain of LCoR Abolishes Corepressor Function-Function of the C-terminal HTH motif of LCoR in corepression of progesterone-and estrogen-regulated gene expression was analyzed by deletion of the domain (LCoR⌬HTH; Fig. 5A) and comparison of the function of the resulting mutant to full-length LCoR. Western blotting and ChIP assays showed that the ⌬HTH mutant was expressed at similar levels in T47D cells as full-length LCoR and that it was recruited to the IGFBP1 promoter in the presence of progesterone (Fig. 5, B and C). Remarkably, however, the LCoR⌬HTH protein was essentially devoid of corepressor activity on either progesterone-or estrogen-responsive promoters in transient expression experiments (Fig. 5, D and E). In similar studies (not shown) corepressor activity of the LCoR⌬HTH mutant was even more attenuated than that of LCoR derivative m1m2 lacking PXDLS motifs, identifying the HTH region as being critical to LCoR corepressor function.
Function of LCoR and CtBP1 as Attenuators of Progesteroneregulated Gene Expression-To further address the function of LCoR and CtBP1 in regulating hormone-dependent gene transcription, we knocked down expression of both proteins using siRNAs in T47D and MCF7 cells. Knockdown of cyclophilin B was used as the control for off-target effects of siRNA in these studies (Fig. 6, A-C). Loss of either LCoR or CtBP1 augmented progesterone-induced reporter gene expression in T47D cells (Fig. 6D) and estrogen-stimulated reporter expression in MCF7 cells (Fig. 6E), consistent with a role in corepression of hormone-inducible gene transcription.
We extended this analysis to the regulation in T47D cells of progesterone target genes encoding IGFBP1, mucin 1 (MUC1) and FK506-binding protein 5 (FKBP51) (4,38,39) (Fig. 7). Knockdown of LCoR markedly enhanced progesterone-stimulated expression of the IGFBP1 gene (Fig. 7A). Unexpectedly, ablation of CtBP1 expression alone or in combination with 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 ␣-amanitin (2.5 M), and treated with P4 (10 nM) for 45 min or E2 (10 nM) for 30 min, as indicated. Cell extracts were collected and immunoprecipitated with IgG or antibodies against PR, ER␣, 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, ER␣, or CtBP1 was performed, as indicated. OCTOBER 30, 2009 • VOLUME 284 • NUMBER 44

LCoR Is a Corepressor of Progesterone-regulated Gene Expression
LCoR slightly attenuated basal and hormone-induced expression of the gene. In contrast, loss of LCoR or CtBP1 individually or in combination substantially augmented progesterone-stimulated expression of the mucin 1 (MUC1) and FK506binding protein 5 (FKBP51) genes (Fig. 7, B and C). Taken together, the data in Figs. 6 and 7 provide evidence for roles of LCoR and CtBP1 as hormone-recruited attenuators of progesterone-regulated gene transcription. However, they also suggest that CtBP1 function may enhance the expression of some genes, similar to what was observed below in an analysis of estrogen-regulated gene expression.

Ablation of LCoR or CtBP1 Diminishes Expression of Estrogen Target Genes in a Gene-specific
Manner-The effects of LCoR and/ or CtBP1 knockdown on expression of a series of ER␣ target genes (40) were examined in MCF7 cells. Unlike the general stimulatory effect on progesterone-induced gene expression, knockdown of LCoR elicited distinct and gene-specific effects on estrogen target gene regulation. Notably, loss of CtBP1 but not LCoR attenuated estrogen-induced pS2 transcription (Fig. 8A). This effect of CtBP1 ablation is in agreement with previous studies showing that CtBP1 or CtBP2 knockdown attenuated estrogen-stimulated pS2 expression in MCF7 cells (41) and is consistent with the reduced expression of the IGFBP1 gene seen above in CtBP1-deficient cells. Although loss of CtBP1 had no effect on regulation of the GREB1 gene (the gene regulated by estrogen in breast cancer; Fig. 8B), its knockdown attenuated both basal and estrogen-regulated expression of the serum/ glucocorticoids-regulated kinase 3 (SGK3) and cytochrome P450, family 26, subfamily b, polypeptide 1 (CYP26B1) genes (Fig. 8, C  and D). Knockdown of LCoR had no effect on basal or estrogen-induced expression of GREB1 or SGK3, whereas estrogen-induced expression of CYP26B1 was eliminated (Fig. 8D), mimicking the effect of CtBP1 ablation. These results are remarkable given the augmented progesterone-stimulated gene transcription seen in LCoR-deficient cells.
Given the unexpected effects of knockdowns on estrogeninduced expression, we also analyzed the potential roles of LCoR and CtBP1 on genes whose transcription is repressed by OCTOBER 30, 2009 • VOLUME 284 • NUMBER 44 estrogen (40). Loss of CtBP1, but not LCoR, attenuated basal expression of the bone morphogenetic protein 7 (BMP7) gene (Fig. 8E), whereas loss of either protein attenuated basal expression of the keratin 4 (KRT4) gene (Fig. 8F). Effects of ablation on estrogen-repressed transcription were variable, with no change in estrogen-repressed BMP7 transcription and a slight increase in expression of the keratin 4 gene. In general, these effects are not consistent with CtBP1 or LCoR functioning as corepressors on these genes.

LCoR Is a Corepressor of Progesterone-regulated Gene Expression
We further analyzed the molecular basis of these results by performing ChIP assays on the BMP7 promoter, where the ER␣ binding region has been identified (42). Remarkably, we found that whereas LCoR was strongly recruited to the promoter after 30 min of estrogen treatment, CtBP1 partially dissociated from the promoter, indicating that the two factors function independently (Fig. 8G). ReChIP analysis found evidence for corecruitment of LCoR with ER␣, but not CtBP1, on the BMP7 promoter after 30 min of estrogen treatment (not shown). These results are consistent with an effect of CtBP1 ablation on basal but not estrogen-regulated expression of the BMP7 gene.

DISCUSSION
We have analyzed the roles of LCoR and its cofactor CtBP1 in coregulation of progesterone and estrogen receptor-regulated gene expression. Our previous findings showed that LCoR was a particularly efficacious inhibitor of progesterone-regulated reporter gene expression and that corepression was largely resistant to HDAC inhibitor trichostatin A (15). This study showed that CtBP1 did not repress hormone-regulated gene expression on its own in transient expression studies but did contribute to corepression by LCoR. GST pulldown experiments and coimmunoprecipitations with a series of LCoR mutants suggested that the N-terminal region of LCoR containing the tandem PXDLS motifs was necessary and sufficient for interactions of CtBP1 with LCoR. Other experiments showed that deletion of the C-terminal HTH domain severely abrogated corepression, although this domain had no apparent role in CtBP1 recruitment. The HTH domain is distinct from the central region of LCoR identified in the accompanying manuscript (29) as being required for interaction with HDAC6. Together, these findings emphasize that LCoR is a multidomain protein that exerts its coregulator function through diverse mechanisms. siRNA-mediated knockdown of LCoR expression established its role in attenuation of progesterone-regulated gene transcription, as hormone-stimulated expression was enhanced on all three PR target genes tested in cells lacking LCoR.
The function of LCoR in corepression of PR-stimulated gene expression may be of considerable physiological importance. Progesterone signaling is essential for normal development and homeostasis of a number of physiological processes including female sexual behavior, ovulation, protection against seizures, maintenance of quiescence of the uterus during pregnancy, induction of germ cell maturation, and oocyte maturation (1,5,6). Progestins and antiprogestins are used clinically in contraception, hormone replacement therapy, induction of labor, treatment of endometriosis, and endometrial cancer (43). Moreover, expression of the PR along with ER␣ and human epidermal growth factor receptor 2 (HER2) are used as predictive markers for breast cancer therapy (44).  There is considerable overlap in the expression patterns of LCoR and the PR. LCoR is widely expressed throughout development and in the adult (15). In tissue blots we observed the highest expression of LCoR in the placenta along with robust expression in several fetal tissues. Notably, the placenta is a site of progesterone biosynthesis, and in situ hybridization analysis

LCoR Is a Corepressor of Progesterone-regulated Gene Expression
of near-term placenta (15) revealed that LCoR mRNAs were predominantly expressed in the syncytiotrophoblast layer of terminally differentiated cells, a site of PR expression and signaling (45,46). The syncytiotrophoblast layer acts as a barrier between maternal circulation and the fetus, and its function is critical for controlling maternal signals that modulate fetal metabolism and development (47).
Compared with the substantial increases observed in progesterone-stimulated gene expression, the effects of LCoR or CtBP1 ablation on estrogen target genes were distinct and gene-specific. We observed no effect of LCoR knockdown on expression of three of four estrogen-stimulated genes studied. This may reflect a redundancy in corepressor function on the genes tested. For example, knockdown in MCF7 cells of NRIP1, another corepressor recruited in the presence of hormone, had gene-specific effects on estrogen-dependent transactivation (48). LCoR ablation did augment both basal and hormonestimulated expression of an estrogen-sensitive reporter gene, pointing to a potential role as an attenuator of ER␣ signaling. Remarkably, however, its knockdown blocked estrogen-stimulated expression of CYP26B1. This may reflect its function as a cofactor of CtBP1 on the CYP26B1 promoter, as ablation of CtBP1 also abolished estrogen-induced transcription. Notably, we found in the accompanying manuscript (29) that knockdown of HDAC6 had no effect on CYP26B1 regulation. A diminution in estrogen-stimulated gene expression in the absence of CtBP1 was also observed on two of three other estrogenstimulated genes tested. Although these results were unexpected, they are consistent with other findings that knockdown of CtBP1 led to reduced expression of the ATP binding cassette, subfamily B (MDR/TAP) member 1 (MDR1) gene (49).
We also found that LCoR and CtBP1 are corecruited to the progesterone-stimulated IGFBP1 gene and the ER␣ target gene pS2. This behavior is in apparent contrast to the results of Stossi et al. (41), who found estrogen-induced dissociation of CtBP1 from the pS2 promoter. They found that CtBP1 controlled estrogen-mediated gene repression and that CtBP1 was recruited to repressed genes in the presence of estradiol. On the other hand, our observations are entirely consistent with other findings (50) in which peak recruitment of CtBP1 to the pS2 promoter was observed after 30 min of estradiol treatment.
CtBP1 and LCoR were extensively colocalized in the nucleus, including in pronounced foci. This pattern was similar to the colocalization of LCoR with CtIP and PRC1 marker BMI1 and strongly suggests that a substantial portion of CtBP1-bound LCoR is associated with PRC1 complexes. We found no evidence for direct interaction of LCoR with CtIP (Fig. 2) or BMI1 7 by GST pulldown assay. However, their indirect association is supported by coimmunoprecipitation of LCoR with both proteins and their hormone-dependent recruitment to estrogen or progesterone-regulated genes. Polycomb group proteins and complexes, including PRC1, form nuclear foci visualized as PcG (polycomb group proteins) bodies (51). Immunocytochemical studies have estimated these bodies to measure between 0.2 and 1.5 m and to vary greatly in composition, suggesting that they represent foci with numerous independently functioning transcriptional regulatory complexes (25,52).
More than 30 transcription factors have been shown to interact with CtBP1 (53). Recent studies characterized a CtBP-corepressor complex that contained a great number of proteins with opposing enzymatic activities. This included histone modifying enzymes (53) and other coregulatory proteins that can either activate or repress transcription depending on the context. Additionally, LCoR was identified as one of the components of a CtBP-corepressor complex purified from the nuclear extracts of HeLa cells (54). Taken together, these studies suggest that LCoR is associated with several distinct multisubunit transcriptional regulatory complexes; hence, implying the importance of LCoR in transcriptional control.