Identification of CtBP1 and CtBP2 as proteins that interact with the vertebrate Pc homologs XPc and HPC2.
To identify genes encoding proteins that interact with HPC2 and XPc, both of which are vertebrate homologs of the Drosophila
PcG protein Pc, we performed two-hybrid screens. The full-length coding regions for XPc (19
) and HPC2 (21
) were cloned into the pAS2 vector (5
). The plasmids pAS2-XPc and pAS2-HPC2 were cotransformed with, respectively, a Xenopus
oocyte and a human fetal brain two-hybrid library. Approximately 106
independent clones were obtained for each screen. One hundred thirty-six growing colonies were obtained from the two-hybrid screen with XPc. Twelve colonies, of which eight colonies contained similar cDNA inserts, remained histidine and β-galactosidase positive after DNA isolation and rescreening. From the two-hybrid screen with HPC2, 100 growing colonies were obtained, of which 3 colonies remained histidine and β-galactosidase positive after DNA isolation and rescreening. A 1,519-bp cDNA clone that we isolated from the two-hybrid screen with HPC2 was identical to CtBP2
). The isolated CtBP2
clone encodes aa 1 to 445 of the 445-aa CtBP2 protein. A 1,414-bp cDNA clone obtained from the XPc screen was homologous to CtBP1
). The predicted 440-aa protein is 85% identical to CtBP1 based on the encoding sequence published by Schaeper et al. (22
) and is 78% identical to CtBP2 (11
) (Fig. ). However, comparison of the open reading frames of XCtBP and CtBP1 revealed potential frameshifts in the reading frame. We therefore searched for different EST clones in the database of CtBP1
and compared these with CtBP1
. Comparison of different EST clones (accession no. H46860
, and AA312167
) indeed revealed that there are several frameshifts in the published sequence of CtBP1
. We confirmed these differences by sequencing the CtBP1
cDNA, which we obtained by PCR. When the corrections are taken into account, the XCtBP protein is 96% identical to CtBP1 instead of 85%. Based on the extensive homology between CtBP1 and XCtBP we therefore named the novel Xenopus
Comparison of the XCtBP1 and the human CtBP1 and CtBP2 proteins. Identical amino acids are indicated as black boxes.
In conclusion, a two-hybrid screen with XPc as a target resulted in the isolation of XCtBP1, a Xenopus homolog of CtBP1. A two-hybrid screen with HPC2 as a target resulted in the isolation of the CtBP2 protein.
A specific 6-amino acid motif in HPC2 is crucial for binding of CtBP.
To define domains that are responsible for the interaction of the Pc proteins and the CtBP proteins, we cloned different parts of HPC2 in frame with the GAL4 DNA binding domain (GAL4 DBD) and tested whether these proteins could still interact with full-length CtBP2 (Fig. ). HPC2 comprises two functional domains. The first domain is the N-terminal chromodomain, which is essential for binding of the Pc protein to chromatin (12
). The other domain is the C-terminal COOH box (aa 540 to 558). This COOH box is necessary for the repression of gene activity (4
) and is also the domain to which the RING1 protein binds (20
). We found that an HPC2 mutant (aa 1 to 540) which lacks the COOH box is still able to interact with CtBP2 (Fig. ). In contrast, a smaller portion of the HPC2 protein (aa 1 to 468) does not interact with CtBP2, whereas a C-terminal fragment (aa 459 to 558) is able to interact with CtBP2. Thus, CtBP2 interacts with a part of the C terminus of HPC2 but not with the extreme C-terminal COOH box (aa 540 to 558), which is involved in gene repression and RING1 binding.
FIG. 2 Mapping of the CtBP2 interaction domain in the HPC2 protein and specificity among vertebrate Pc homologs for binding CtBP. The indicated portions of HPC2 were fused to the GAL4 DBD. The HPC2 regions include the shaded chromodomain (aa 6 to 58), a 6-aa (more ...)
Within the C-terminal fragment to which CtBP2 binds, we observed a 6-aa motif (PIDLRS) (aa 470 to 475) (Fig. ) which is very similar to a 6-aa motif (PLDLSC) present in the extreme C terminus of the Ad2/5 E1A protein. This motif is essential for the interaction between E1A and CtBP1 (22
). Mutations within the first four amino acids of the E1A motif completely abolish the interaction between E1A and CtBP1 (22
). We created a similar mutation within this corresponding 6-aa motif of HPC2 by changing the motif from PIDLRS to PIASRS, using PCR primers which contained the specific mutations. Subsequently, we tested whether the HPC2(DL→AS) mutant protein is still able to interact with CtBP2. We found that the DL-to-AS mutation in the HPC2 protein completely abolishes the interaction with CtBP2 in the two-hybrid system. Importantly, the mutation within the 6-aa motif leaves intact the C-terminal COOH box of the HPC2 protein to which the RING1 protein binds (20
). We therefore tested whether the RING1 protein is still able to interact with the HPC2(DL→AS) mutant protein. We observed no loss of interaction between this mutant HPC2 protein and RING1 (data not shown), underlining the specificity of the interaction between CtBP2 and the conserved 6-aa motif in HPC2.
We have identified the XCtBP1 protein in a two-hybrid screen with the XPc protein as the target. The XPc protein encompasses a specific 6-aa motif, PIDLRC, related to the 6-aa motif in HPC2 which is crucial for binding CtBP (Fig. ). We also tested whether the CtBP protein could interact with another murine homolog, M33, which is more homologous to the human Pc homolog, CBX2/HPC1, than to HPC2 (7
). Surprisingly, we observed no interaction between M33 and CtBP2 in the two-hybrid system (Fig. ) or between M33 and CtBP1 (data not shown). Importantly, M33 does not encompass the conserved 6-amino-acid motif that is present in HPC2 and that is crucial for the interaction with CtBP. It is therefore likely that the lack of this conserved 6-aa motif in M33 is responsible for the lack of interaction between M33 and CtBP. This result is the first indication that, despite the high degree of homology in the chromodomain and the COOH box, there is specificity among different vertebrate Pc proteins, particularly in their ability to interact with other proteins.
In conclusion, the highly homologous proteins CtBP1, CtBP2, and XCtBP1 interact with HPC2 and XPc. Strikingly, no interaction could be observed between CtBP and M33, a murine Pc homolog, indicating specificity among the different vertebrate Pc proteins.
CtBP1 and CtBP2 are able to homo- and heterodimerize, and the interaction domain differs from the domain responsible for interaction with HPC2.
To determine which part of the CtBP proteins is responsible for the interaction with HPC2, we subcloned different protein fragments of CtBP2 in frame with the GAL4 transactivating domain (GAL4 TAD) (Fig. A). The C-terminal region of CtBP2 encompassing aa 361 to 445 is not capable of interaction with HPC2, whereas the N-terminal region containing aa 1 to 362 is still able to interact with HPC2. This region encompasses three domains which have strong homology with various NAD-dependent D
-isomer-specific 2-hydroxy acid dehydrogenases (11
). To analyze whether these dehydrogenase homology domains are responsible for the interaction with HPC2, we made three constructs containing different sets of these dehydrogenase homology domains.
FIG. 3 Mapping of domains of interaction of CtBP2 with HPC2 (A) and CtBP2 (B). (A) The indicated portions of CtBP2 were fused to the GAL4 TAD. These CtBP2 regions include three dehydrogenase homology domains. Plasmids were cotransformed with full-length HPC2 (more ...)
We found that a region of CtBP2 encompassing aa 81 to 362, which contains all three dehydrogenase homology domains, is not able to interact with HPC2. Also, a CtBP2 region (aa 1 to 233) encompassing the N terminus and the first two dehydrogenase homology domains and a CtBP2 region (aa 162 to 337) encompassing the second and the third dehydrogenase homology domains are not able to interact with HPC2. These results indicate that a large region of CtBP2 (aa 1 to 362), which encompasses both the extreme N-terminal part and the dehydrogenase homology domains, is responsible for the interaction with HPC2.
The HPC2 protein (20
) is part of a complex which constitutes the mammalian homologs of the Drosophila
Ph protein, HPH1 and HPH2. These two proteins are able to homo- and heterodimerize with each other (9
). To address the question of whether this is also true for CtBP1 and CtBP2, we cloned the full-length coding region for CtBP2 in frame with the GAL4 DBD and tested whether CtBP2 could interact with itself or CtBP1. Both CtBP1 (data not shown) and CtBP2 (Fig. B) are able to interact with CtBP2 in the two-hybrid system, indicating that these proteins are able to homodimerize and to heterodimerize.
To define the domains that are responsible for the interaction between CtBP2 and CtBP2, we subcloned different parts of CtBP2 in frame with the GAL4 TAD and tested whether these domains are still able to interact with full-length CtBP2. The C-terminal region of CtBP2 encompassing aa 361 to 445 is not able to interact with CtBP2, whereas the N-terminal region containing aa 1 to 362 is still able to interact with CtBP2 (Fig. B). A region containing only the three dehydrogenase homology domains (aa 81 to 361) still interacts with CtBP2. Detailed analysis of this region showed that CtBP2 aa 81 to 233, encompassing the first two dehydrogenase homology domains, exhibits no interaction with CtBP2. In contrast, CtBP2 aa 162 to 337, containing dehydrogenase homology domains two and three, still interacts with CtBP2. Also, CtBP2 aa 225 to 337, containing only the third dehydrogenase homology domain, is still able to interact with CtBP2 (Fig. B). These data indicate that a region in CtBP2 encompassing the third dehydrogenase homology domain is sufficient for the interaction with full-length CtBP2. Interestingly, this relatively small interaction domain, which is necessary to convey homodimerization between CtBP2 and CtBP2, differs from the domain for interaction with HPC2. Above we showed that a much larger region of CtBP2, containing the N terminus as well as all three dehydrogenase domains, is necessary for the interaction with HPC2 (Fig. A).
In summary, the CtBP1 protein and the CtBP2 protein each can interact with itself, and they are also able to interact with each other. The domain responsible for this interaction is a region encompassing the third dehydrogenase homology domain. This interaction domain differs from the domain that is responsible for the interaction with HPC2, which involves the N terminus and all three dehydrogenase homology domains.
The XPc and CtBP2 proteins interact directly in vitro.
To determine whether the interaction between the vertebrate Pc homologs and CtBP is a direct interaction, we employed an in vitro pull-down assay. The previous described (19
) fusion protein of GST and full-length XPc (aa 1 to 521) was expressed in bacteria. The affinity-purified protein was subsequently immobilized on GST-Sepharose and incubated with [35
S]methionine-labelled, in vitro-translated CtBP2. After extensive washing, the [35
S]methionine-labelled proteins bound to GST-XPc were analyzed by SDS-PAGE. The in vitro-translated full-length CtBP2 protein of 48 kDa (Fig. , lane 1) was able to bind to the immobilized GST-XPc (lane 3) but did not bind to the immobilized GST alone (lane 2). We also tested whether CtBP interacted with another GST-XPc (aa 1 to 178) fusion protein (19
). This portion of the XPc protein encompasses the chromodomain of XPc but lacks the entire C-terminal domain that contains the 6-amino-acid motif to which CtBP binds. CtBP does not bind to such a C-terminal deletion HPC2 mutant in the two-hybrid assay (Fig. ). Importantly, we found that the GST-XPc aa 1 to 178 protein does not interact with CtBP2 (Fig. , lane 4). These results confirm the two-hybrid assay data (Fig. ) and underline the specificity of the in vitro pull-down assay.
FIG. 4 XPc and CtBP2 interact directly in vitro. [35S]methionine-labelled CtBP2 protein (lane 1) was incubated with GST-Sepharose alone (lane 2), GST-XPc aa 1 to 521 (lane 3), or GST-XPc aa 1 to 178 (lane 4). The GST-XPc aa 1 to 521 but not the (more ...) Expression of CtBP1 and CtBP2 in human tissues and human cancer cell lines.
To investigate the expression patterns of CtBP1 and CtBP2, we needed unique cDNA fragments in order to avoid cross-hybridization between CtBP1 and CtBP2 mRNA species. Since there is no homology between the UTRs of CtBP1 and CtBP2, we used a 560-bp fragment of the 3′ UTR of CtBP1 and a 470-bp fragment of the 3′ UTR of CtBP2 as probes. These probes were hybridized to Northern blots containing poly(A)+ mRNAs from different human cancer cell lines or human tissues (Clontech). We detected single transcripts of approximately 2.4 kb for CtBP1 and approximately 3.0 kb for CtBP2. In all human tissues present on the commercial Northern blot (Fig. A), CtBP1 was expressed at approximately the same level as CtBP2, with the exception of the thymus and peripheral blood leukocytes. In these two tissues, the CtBP2 transcript was hardly detectable (Fig. A, lanes 2 and 8).
FIG. 5 Expression patterns of CtBP1 and CtBP2 in human tissues (A) and in human cancer cell lines (B). (A) Expression levels in spleen (lane 1), thymus (lane 2), prostate (lane 3), testis (lane 4), ovary (lane 5), small intestine (lane 6), colon (lane 7), and (more ...)
In human cancer cell lines, differences in expression of either CtBP1 or CtBP2 were more pronounced than in normal tissues. In the case of CtBP1, high expression of the commercial blot was detected in HL-60 cells (Fig. B, lane 1) and in the adenocarcinoma SW480 cell line (lane 6). Expression of CtBP1 was still well pronounced in HeLa S3 cells (Fig. B, lane 2), K-562 cells (lane 3), MOLT-4 cells (lane 4), and U-2 OS cells (lane 10). Low expression of CtBP1 was detected in Raji cells (lane 5) and G361 cells (lane 8), whereas almost no CtBP1 expression was found in A549 cells. In the case of CtBP2, high expression was detected in HeLa S3 cells (Fig. B, lane 2) and SW480 cells (lane 6), whereas significantly lower expression was detected in HL-60 (lane 1), G361 (lane 8), and U-2 OS (lane 10) cells. A very low level of CtBP2 expression was found in A549 cells (lane 7), but no detectable CtBP2 transcript could be observed in K-562 (lane 3), MOLT-4 (lane 4), and Raji (lane 5) cells. Interestingly, CtBP2 was highly expressed in the spleen (Fig. A, lane 1), whereas no expression could be observed in a B-cell-derived cell line, Raji (Fig. B, lane 5). Strikingly, in all tissues or cell lines either one or two CtBP transcripts could be detected, with the exception of lung carcinoma cells (lane 7), in which both CtBP transcripts were hardly detectable.
A polyclonal antibody raised against XCtBP1 recognizes both CtBP1 and CtBP2.
To determine the distribution of the CtBP proteins in the cell nucleus and to be able to detect CtBP proteins in immunoprecipitates, we raised a polyclonal antibody against full-length XCtBP1. To test whether the polyclonal antibody also recognizes both CtBP1 and CtBP2, we created constructs containing the full-length coding region for either CtBP1 or CtBP2, with a T7 tag at the N terminus. Fusion proteins were produced in E. coli BL21(DE), and the bacterial cell lysates were subsequently separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed with either a mouse monoclonal antibody against T7 (Fig. , lanes 1 and 2) or our rabbit polyclonal antibody against XCtBP1 (lanes 3 to 7). The T7 antibody recognizes both the 48-kDa T7-tagged CtBP1 (lane 1) and T7-tagged CtBP2 (lane 2) proteins. Also, the anti-XCtBP1 polyclonal antibody recognizes the 48-kDa T7-tagged CtBP1 (lane 3) and T7-tagged CtBP2 (lane 4) proteins, indicating that both CtBP1 and CtBP2 are recognized by the polyclonal antibody raised against XCtBP1. We further analyzed cell extracts of Xenopus X1 cells (Fig. , lane 5), SW480 cells (lane 6), and U-2 OS cells (lane 7). In all three cell extracts a doublet protein band of approximately 48 kDa was observed. We conclude that the antibody against XCtBP1 recognizes both the CtBP1 and CtBP2 proteins.
FIG. 6 A rabbit polyclonal antibody recognizes XCtBP1, CtBP1, and CtBP2. T7-tagged CtBP1 (lanes 1 and 3) and T7-tagged CtBP2 (lanes 2 and 4) were expressed in E. coli. Cell lysates were analyzed by Western blotting and probed with either a mouse monoclonal antibody (more ...) An in vivo interaction between CtBP2 and HPC2.
To determine whether the interaction between CtBP proteins and HPC2 also exists in vivo, we performed coimmunoprecipitation experiments. We transiently transfected COS-7 cells with T7-tagged HPC2 and T7-tagged CtBP2. We used polyclonal rabbit antibodies directed against XCtBP1 and HPC2 for the immunoprecipitations and a mouse monoclonal antibody against T7 to detect either the 82-kDa T7-HPC2 (21
) or the 48-kDa T7-CtBP2 protein.
We found that CtBP2 and HPC2 coimmunoprecipitate with each other (Fig. ). The anti-HPC2 antibody coimmunoprecipitated both T7-CtBP2 and T7-HPC2 (Fig. , lane 1) from cells expressing both T7-HPC2 and T7-CtBP2 (lane 7), as was detected with the anti-T7 monoclonal antibody. No T7-CtBP2 could be detected in the anti-HPC2-immunoprecipitated material (lane 2) when T7-CtBP2 but not T7-HPC2 was expressed (lane 8). Also, no T7-CtBP2 could be detected in the anti-HPC2 immunoprecipitated material (lane 3) when T7-HPC2 but not T7-CtBP2 was expressed (lane 9).
FIG. 7 In vivo interaction between HPC2 and CtBP2. Immunoprecipitation (IP) was performed with polyclonal rabbit antibodies against HPC2 (αHPC2) (lanes 1 to 3) or polyclonal rabbit antibodies against XCtBP1 (αCtBP) (lanes 4 to 6). The resulting (more ...)
Similarly, the anti-CtBP antibody immunoprecipitated both T7-HPC2 and T7-CtBP2 (Fig. , lane 4) from cells expressing both T7-HPC2 and T7-CtBP2 (lane 7). No T7-HPC2 could be detected in the anti-CtBP-immunoprecipitated material (lane 5) when T7-CtBP2 but not T7-HPC2 was expressed (lane 8). Finally, no T7-HPC2 could be detected in the anti-CtBP-immunoprecipitated material (lane 6) when T7-HPC2 but not T7-CtBP2 was expressed (lane 9).
Also, in extracts of SW480 cells, in which the PcG proteins are highly expressed (9
) and in which the CtBP proteins are expressed, we observed coimmunoprecipitation of either HPC2 and CtBP or BMI1 and CtBP (data not shown). However, in both cases the recovery of the proteins in the immunoprecipitations was approximately 20% of the input. This result further strengthens the notion that an interaction between CtBP and HPC2 exists in vivo. The low recovery might indicate that the interaction between CtBP and the PcG complex is of a transient nature.
In conclusion, we show that CtBP2 and HPC2 coimmunoprecipitated with each other from extracts of COS-7 cells in which we overexpressed CtBP2 and HPC2. These findings indicate that CtBP2 and HPC2 interact with each other in vivo.
CtBP1 and CtBP2 partially colocalize with HPC2 in nuclei of U-2 OS cells.
To determine the subcellular distribution of the CtBP1 protein and the CtBP2 protein in relation to the HPC2 protein, we performed immunofluorescence labelling experiments. Previously we have shown that the HPC2 protein colocalizes in large nuclear domains, termed PcG domains, with BMI1, HPH1, HPH2, and RING1 (9
). To compare the distributions of the CtBP proteins relative to the distribution of HPC2, we performed double-labelling experiments with the rabbit anti-XCtBP1 antibody, which recognizes both CtBP1 and CtBP2 (Fig. ), and a chicken anti-HPC2 antibody (20
). We found that the CtBP proteins are abundantly present in nuclei of U-2 OS cells in a fine granular pattern but also in larger nuclear domains (Fig. A). Within these larger nuclear domains, the CtBP proteins colocalize with HPC2 (Fig. B and C). However, the colocalization within these domains differs slightly from the colocalization of the BMI1 protein (Fig. D) with the HPC2 protein (Fig. E and F). The BMI1 and HPC2 proteins completely colocalize in bright, sharply edged PcG domains (Fig. F). This specific labelling pattern has also been observed with antibodies against the human PcG homologs HPH1 and HPH2 (9
) and the RING1 protein (20
). The nuclear domains that are detected by the anti-XCtBP1 antibody and that colocalize with the more sharply edged PcG domains have a more diffuse shape (Fig. A, B, and C). Another difference between the CtBP and PcG labelling patterns is that most of the BMI1 and HPC2 proteins appear to be concentrated within the large PcG domains (Fig. D, E, and F). In contrast, most of the CtBP labelling is detected in the smaller domains throughout the nucleoplasm and not in the larger domains that colocalize with the PcG domains. This fine granular pattern is too complex to allow analysis of any systematic colocalization.
Since the rabbit anti-CtBP antibody recognizes both the CtBP1 protein and the CtBP2 protein, it is not possible to directly test for differences in nuclear localization of the CtBP1 protein and the CtBP2 protein. In order to distinguish between the distributions of the CtBP1 protein and the CtBP2 protein, we transiently transfected U-2 OS cells with either the T7-tagged CtBP1 protein (Fig. G) or the T7-tagged CtBP2 protein (Fig. J). Double labelling was performed with a mouse monoclonal antibody against T7 and the affinity-purified chicken antibody against HPC2. We found that T7-tagged CtBP1 (Fig. G) colocalizes with HPC2 (Fig. H) in the large PcG domains (Fig. I). Also, T7-tagged CtBP2 (Fig. J) colocalizes with HPC2 (Fig. K) within these large PcG domains (Fig. L). These results indicate that there are no major detectable differences in the localizations of CtBP1 and CtBP2 and that both proteins are present in the same PcG domains.
CtBP acts as a transcriptional repressor when targeted to a reporter gene.
The PcG proteins are involved in the repression of gene expression, but the identified PcG proteins do not bind directly to DNA. Nevertheless, the ability of the PcG proteins to repress gene activity can be tested by targeting LexA fusion proteins to a reporter gene (20
). Previously, we have shown that LexA-HPC2 was able to repress gene activity (20
). We asked whether this is also true for the CtBP proteins. We therefore tested whether LexA-CtBP1 was able to repress gene expression when targeted to a reporter gene. U-2 OS human osteosarcoma cells were transfected with a construct containing a tandem of four LexA operators, binding sites for the HSF transcriptional activator, and the hsp70
TATA promoter region, immediately upstream of the LUC reporter gene. The endogenous HSF was used as transcriptional activator. In absence of this activator, no LUC expression could be measured (data not shown). In the presence of the HSF, expression was maximal and was set at 100%. Cotransfection with LexA alone had no significant effect on LUC expression (Fig. ) (97% ± 6% [n
= 7]). We found that LexA-CtBP1 was able to repress LUC expression significantly (16% ± 4% [n
= 7]). This degree of LUC repression was also found for LexA-CtBP2 (data not shown). In the same experiment we found that LexA-HPC2 could repress LUC activity most efficiently (9% ± 3% [n
= 7]). Previously, we have shown that a LexA-HPC2 mutant which lacks the C-terminal domain, to which the RING1 protein binds, was no longer able to repress LUC expression (21
). We tested whether the HPC2(DL→AS) mutant also has lost the ability to repress LUC expression. In this HPC2 mutant the specific 6-aa motif is mutated, which leads to abolishment of the interaction with CtBP (Fig. ). We observed a slight but significant decrease in the ability of the HPC2 protein to repress gene activity when the DL→AS mutation is introduced. However, the LexA-HPC2(DL→AS) mutant still represses LUC activity significantly (20% ± 5% [n
FIG. 9 Repression of HSF-induced LUC gene activity by CtBP. Activation of LUC expression is maximally induced by endogenous HSF in the absence of any LexA fusion protein. This LUC activity was set at 100%. LUC activities in cells cotransfected with the (more ...)
We conclude that CtBP is able to repress gene activity when targeted to a reporter gene, almost as efficiently as HPC2. Furthermore, mutating the specific 6-aa motif within HPC2 which is crucial for CtBP binding has a significant but small effect on the ability of HPC2 to repress gene activity.