The amino-terminus of Pc2 is required for enhanced sumoylation. (A) COS-1 cells were cotransfected with T7-CtBP, H6-SUMO1 and the indicated Flag-tagged Pc2 expression constructs. Cells were lysed directly by boiling in SDS–PAGE loading buffer. Proteins were separated by SDS–PAGE and analyzed by Western blot with a T7 or Flag antibody. Sumoylated and unsumoylated CtBP are shown. (B) COS-1 cells were transfected with T7-CtBP alone or with the indicated Flag-tagged Pc2 expression constructs. Following lysis, proteins were precipitated with anti-Flag-agarose, separated by SDS–PAGE and analyzed by Western blot with a T7 antibody. Expression of T7 and Flag constructs is shown below. The bar indicates the immunoglobulin heavy chain. The positions of molecular weight markers (kDa) are indicated.

The amino-terminus of Pc2 is required for E3 activity. COS-1 cells were transfected with the indicated expression constructs and analyzed as in Figure 1. (A, B) T7-CtBP and H6-SUMO1 were coexpressed alone or with the indicated amino- or carboxyl-terminal Fl-Pc2 fragments. Lysates were blotted for CtBP and CtBP-SUMO with a T7 antibody, and expression of Fl-Pc2 constructs is shown below. (C) The Flag-tagged Pc2 expression constructs used here, and in other figures, are shown schematically: CD: chromodomain; His: polyhistidine stretch; PIDLR: CtBP interaction motif; VKPE: sumoylation consensus. The hatched region at the extreme carboxyl-terminus is the region required for correct localization (see Figure 4) and interaction with RING1. (D) HA-Smad4 was expressed with H6-SUMO1, Fl-PIASxα and Fl-Pc2(2–288) as indicated. Sumoylated and unmodified Smad4 were detected by HA Western blot. (E) T7-CtBP and H6-SUMO1 were coexpressed in the presence of the indicated combinations of Fl-Pc2, Fl-Pc2(401–558), Fl-Pc2(2–375) and Fl-PIASxα. CtBP and sumoylated CtBP detected by T7 Western blot are shown. The positions of molecular weight markers (kDa) are indicated.

Pc2(2–288) enhances sumoylation of recruited CtBP. (A) COS-1 cells were cotransfected with T7-CtBP, H6-SUMO1 and Fl-Ubc9 either alone or with the indicated Fl-Pc2 or YFP constructs. Following lysis, proteins were separated by SDS–PAGE and analyzed with a T7 antibody to determine relative levels of CtBP sumoylation. Expression of the Flag and YFP constructs is shown below. (B) COS-1 cells were transfected and analyzed as in panel A, except that Fl-Ubc9 was not present. (C, D) COS-1 cells were transfected with the indicated CtBP, Pc2 and YFP expression constructs. Cell lysates were precipitated with anti-Flag-agarose and analyzed for co-precipitating YFP-PLDLS (C) or T7-CtBP (D). Expression of YFP, Flag and T7 constructs is shown below. The bar denotes the immunoglobulin heavy chain. (E) The expression constructs used in (A–D) are shown schematically (labeling as in Figure 3). (F) T7-CtBP and H6-SUMO1 were coexpressed alone or with Fl-Pc2, Fl-Pc2(401–558), Fl-Pc2(2–288) or a mutant in which cysteine 183 has been altered to alanine. Cell lysates were analyzed by Western blot with a T7 antibody to detect SUMO modified and unmodified CtBP. The positions of molecular weight markers (kDa) are indicated.

Analysis of the carboxyl-terminal region of Pc2. (A) COS-1 cells were transfected with Fl-Pc2 or a mutant form in which lysine 492 is altered to arginine, either with or without H6-SUMO1. Lysates were analyzed by Flag Western blot and the unmodified and sumoylated Pc2 are indicated. (B) COS-1 cells were transfected with T7-CtBP, H6-SUMO1 and the indicated Flag-Pc2 constructs. Cell lysates were analyzed by Western blot with a T7 antibody to detect SUMO modified and unmodified CtBP. Expression of Flag constructs is shown below. (C) A series of carboxyl-terminal deletions of Pc2 was tested for their ability to enhance SUMO modification of CtBP in COS-1 cells. (D) COS-1 cells were transfected with the indicated Fl-Pc2 expression constructs and T7-Ubc9. Protein complexes were precipitated with anti-Flag-agarose and analyzed by Western blot for T7-Ubc9. Expression of Ubc9 and the Fl-Pc2 constructs in the lysate was analyzed by direct Western blot. The positions of molecular weight markers (kDa) are indicated. (E) The Pc2 deletion constructs tested in panels C and D are shown schematically (labeling as in Figure 3).

Pc2(401–558) colocalizes Ubc9 and CtBP. COS-1 cells were transfected with the indicated expression constructs and visualized by live cell fluorescence microscopy 24 h later. (A) YFP, YFP-Pc2(401–558) or YFP-Pc2 was expressed and cells were stained with Hoechst before visualization. YFP and Hoechst images are shown. (B, C) CFP-CtBP or YFP-Ubc9 was expressed alone (upper panel) or with the indicated Fl-Pc2 constructs (lower panels). CFP and YFP images are shown. (D) CFP-CtBP and YFP-Ubc9 were coexpressed in the presence or absence of Fl-Pc2(401–558). Individual CFP and YFP images along with the merged (overlay) are shown. Scale bar=10 μm.

Pc2 localization to polycomb bodies is not required for E3 activity. (A) T7-CtBP and H6-SUMO1 were coexpressed alone or with the indicated Fl-Pc2 constructs. Cell lysates were analyzed by Western blot with a T7 antibody to detect SUMO modified and unmodified CtBP. Expression of Flag constructs is shown below. (B) YFP-Pc2(1–529), YFP-Pc2(401–529) or YFP-Pc2 was expressed in COS-1 cells and visualized by live cell fluorescence microscopy 24 h later. YFP and Hoechst images are shown. (C) T7-CtBP and H6-SUMO1 were expressed without or with Fl-Pc2, Fl-Pc2ΔPIDLR, Fl-Pc2(2–529) or Fl-Pc2(2–529)ΔPIDLR. Cell lysates were analyzed by Western blot with a T7 antibody to detect sumoylated and unsumoylated CtBP, and with a Flag antibody for expression controls. (D) COS-1 cells were transfected with the indicated Fl-Pc2 expression constructs and T7-CtBP. Protein complexes were precipitated with anti-Flag-agarose and analyzed by Western blot for T7-CtBP. The bar denotes the immunoglobulin heavy chain. (E) COS-1 cells were transfected with T7-Pc2(2–288) and the indicated Flag-tagged Pc2 constructs. Proteins were collected on anti-Flag-agarose and analyzed for co-precipitating T7-Pc2(2–288). Expression of Flag and T7 constructs in the lysates is shown below. The positions of molecular weight markers (kDa) are indicated.

Pc2(2–288) stimulates CtBP sumoylation in vitro. Recombinant T7-CtBP, E1, SUMO1 and Ubc9 (as indicated) were incubated with increasing amounts (1, 5, 10 or 25 ng) of GST-Pc2(2–288)-PIDLRS (A) or GST-Pc2(2–288) (B). In (C), ‘+' indicates 25 ng GST-Pc2(2–288)-PIDLRS or GST-Pc2(2–288). Reactions were terminated by the addition of SDS loading buffer, and SUMO modified CtBP and unmodified CtBP were detected by T7 Western blot. (D) Either GST or GST-Pc2(2–288) bound to glutathione Sepharose was incubated with recombinant Ubc9. Following extensive washing, bound Ubc9 was analyzed by Western blot with a Ubc9 antibody. The input lane is 100 ng Ubc9. (E) Ubc9, E1 and SUMO1 were incubated in vitro for the indicated times with or without GST-Pc2(2–288), and analyzed by nonreducing SDS–PAGE and Western blot with a Ubc9 antibody. The positions of Ubc9 and Ubc9 with SUMO1 attached via a thioester linkage are indicated. The right-hand lanes are reducing SDS–PAGE to disrupt the thioester. (F) SUMO1-loaded E1 and T7-CtBP were incubated with apyrase and the indicated amounts of Pc2(2–288). CtBP and sumoylated CtBP were detected by Western blot with a T7 antibody. The positions of molecular weight markers (kDa) are indicated.

A Ubc9 binding domain required for enhancement of CtBP sumoylation. (A) Recombinant T7-CtBP, E1, SUMO1 and Ubc9 (as indicated) were incubated with increasing amounts (1–500 ng) of GST-Pc2(401–558). (B) SUMO1-loaded E1 and T7-CtBP were incubated with apyrase and the indicated amounts of GST-Pc2(401–558) or GST. Reactions were terminated by the addition of SDS loading buffer and SUMO modified and unmodified CtBP were detected by T7 Western blot. (C) COS-1 cells were transfected with T7-CtBP, H6-SUMO1 and the indicated Flag-Pc2 constructs. Cell lysates were analyzed by Western blot with a T7 antibody to detect SUMO modified and unmodified CtBP. (D) The Fl-Pc2 constructs used in (B) were coexpressed with T7-CtBP in COS-1 cells. Lysates were precipitated with anti-Flag-agarose and analyzed for co-precipitating T7-CtBP. The positions of molecular weight markers (kDa) are indicated. (E) The constructs used in panels C and D are shown schematically.