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, 19 (1), 777-87

C-Terminal Binding Protein Is a Transcriptional Repressor That Interacts With a Specific Class of Vertebrate Polycomb Proteins

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C-Terminal Binding Protein Is a Transcriptional Repressor That Interacts With a Specific Class of Vertebrate Polycomb Proteins

R G Sewalt et al. Mol Cell Biol.

Abstract

Polycomb (Pc) is part of a Pc group (PcG) protein complex that is involved in repression of gene activity during Drosophila and vertebrate development. To identify proteins that interact with vertebrate Pc homologs, we performed two-hybrid screens with Xenopus Pc (XPc) and human Pc 2 (HPC2). We find that the C-terminal binding protein (CtBP) interacts with XPc and HPC2, that CtBP and HPC2 coimmunoprecipitate, and that CtBP and HPC2 partially colocalize in large PcG domains in interphase nuclei. CtBP is a protein with unknown function that binds to a conserved 6-amino-acid motif in the C terminus of the adenovirus E1A protein. Also, the Drosophila CtBP homolog interacts, through this conserved amino acid motif, with several segmentation proteins that act as repressors. Similarly, we find that CtBP binds with HPC2 and XPc through the conserved 6-amino-acid motif. Importantly, CtBP does not interact with another vertebrate Pc homolog, M33, which lacks this amino acid motif, indicating specificity among vertebrate Pc homologs. Finally, we show that CtBP is a transcriptional repressor. The results are discussed in terms of a model that brings together PcG-mediated repression and repression systems that require corepressors such as CtBP.

Figures

FIG. 1
FIG. 1
Comparison of the XCtBP1 and the human CtBP1 and CtBP2 proteins. Identical amino acids are indicated as black boxes.
FIG. 2
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 motif (PIDLRS) (aa 470 to 475), and the shaded COOH box (aa 540 to 554). The mutation from DL to AS within the 6-aa motif is indicated. The full-length vertebrate Pc proteins M33 and XPc were also fused to the GAL4 DBD. The conserved 6-aa motif (PIDLRC) in the XPc protein is indicated. The three dehydrogenase homology domains within CtBP2 and XCtBP1 are shaded. Constructs that encompass different portions of the HPC2 protein are indicated. The plasmids were cotransformed with full-length CtBP2 (aa 1 to 445) or XCtBP1 (aa 1 to 440), which is fused to the GAL4 TAD. Interactions were positive when cells grew on selective medium lacking histidine and when they were also β-galactosidase positive. When a negative interaction is indicated, no β-galactosidase activity was detected.
FIG. 3
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 which was fused to the GAL4 DBD. (B) Full-length CtBP2 which was fused to the GAL4 DBD was tested for interaction against the indicated portions of CtBP2. When a negative interaction is indicated, no β-galactoctosidase activity was detected.
FIG. 4
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 GST-XPc aa 1 to 178 fusion protein is able to interact with in vitro-translated [35S]methionine-labelled CtBP2 protein. Molecular weights in thousands are indicated on the left.
FIG. 5
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 peripheral blood leukocytes (lane 8). (B) Expression levels in promyelocytic leukemia HL-60 (lane 1), HeLa S3 (lane 2), chronic myelogenous leukemia K-562 (lane 3), lymphoblastic leukemia MOLT-4 (lane 4), Burkitt’s lymphoma Raji (lane 5), colorectal adenocarcinoma SW480 (lane 6), lung carcinoma A549 (lane 7), and melanoma G361 (lane 8) cell lines. Lanes 1 to 8, commercially obtained Northern blot. We also isolated and blotted poly(A)+ RNA from U-2 OS cells (lane 10) and SW480 cells (lane 9), the latter to allow comparison with the commercial multiple-tissue Northern blot. To verify the loading of RNA in each lane, the blots were hybridized with a probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDM).
FIG. 6
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 against T7 (αT7) (lanes 1 and 2) or the polyclonal antibody against CtBP (αCtBP) (lanes 3 and 4). In cell lysates of Xenopus X1 cells (lane 5), colorectal adenocarcinoma SW 480 cells (lane 6), and osteosarcoma U-2 OS cells (lane 7), the polyclonal antibody against CtBP recognizes a doublet of 48 kDa. Molecular weights in thousands are indicated on the left.
FIG. 7
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 immunoprecipitates were Western blotted and analyzed with mouse monoclonal antibodies against T7. The total cell extracts (Input) are shown in lanes 7 to 9. COS-7 cells were transiently transfected with both pcDNA3-T7-HPC2 and pcDNA3-T7-CtBP2 (lanes 1, 4, and 7) or with either pcDNA3-T7-CtBP2 (lanes 2, 5, and 8) or pcDNA3-T7-HPC2 (lanes 3, 6, and 9). Molecular weights in thousands are indicated on the right.
FIG. 8
FIG. 8
HPC2 and CtBP partially colocalize in nuclear domains of U-2 OS cells. Confocal single optical sections are shown. (A to C) Rabbit anti-XCtBP1 and chicken anti-HPC2 double labelling. CtBP (A) colocalizes with HPC2 (B) in large nuclear PcG domains (C; indicated by yellow), but CtBP is also abundantly expressed in a fine granular pattern throughout the nucleus (B and C). (D to F) Rabbit anti-BMI1 (D) and chicken anti-HPC2 (E) double labelling demonstrates colocalization (F) of BMI1 and HPC2 in large nuclear PcG domains. We transiently transfected U-2 OS cells with either T7-tagged CtBP1 (G) or T7-tagged CtBP2 (J). Double labelling was performed with a mouse monoclonal antibody against T7 (G and J) and the chicken anti-HPC2 antibody (H and K). We observed colocalization of HPC2 with either T7-CtBP1 (I) or T7-CtBP2 (L) in large nuclear PcG domains.
FIG. 9
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 indicated plasmids were expressed as percentages of this control value. Bars represent the average degree of repression by LexA, LexA-CtBP1, LexA-HPC2, or LexA-HPC2(DL→AS) in seven independent experiments (means ± standard errors of the means).

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