Linking the Rb and Polycomb Pathways Anjali Dahiya, Sharon Wong, Susana Gonzalo, Mark Gavin, Douglas C Dean  Molecular Cell  Volume 8, Issue 3, Pages 557-568 (September 2001) DOI: 10.1016/S1097-2765(01)00346-X

Figure 1 Rb and CtBP Do Not Repress the SV40 Promoter/Enhancer in HPC2(−) Cells (A) Rb and CtBP are unable to repress the SV40 enhancer in HPC2(−) CV-1 cells. The Rb large pocket (amino acids 379–928) fused to the DNA binding domain of Gal4 (G-Rb) (Weintraub et al., 1995) was coexpressed in CV-1 cells along with G5MLPCAT, which contains Gal4 DNA binding sites upstream of the MLP driving the CAT gene, or pSVEC-G reporter, containing Gal4 binding sites upstream of the SV40 enhancer (Chow and Dean, 1996; Chow et al., 1996). Where indicated, the HDAC inhibitor trichostatin A (TSA) was added to transfected cells as described previously (Luo et al., 1998). An expression vector for HPC2 or DN-HPC2 (Satijn et al., 1997b) was cotransfected where indicated. CAT activity from the reporter was assayed as described (Weintraub et al., 1995). (B) Rb and CtBP repress the SV40 enhancer in HPC2(+) cells. Transfections and assays were carried out as in (A), in HPC2(+) C33a cells (similar results were seen in other HPC2[+] cells; data not shown). (C) CV-1 cells do not express HPC2. Lysates from C33a, CV-1, U2OS, and SAOS-2 cells were prepared and Western blotted for endogenous HPC2 as described (Dahiya et al., 2000) Molecular Cell 2001 8, 557-568DOI: (10.1016/S1097-2765(01)00346-X)

Figure 2 Coimmunoprecipitation Assays to Detect Rb-Interacting Proteins (A–D) The indicated expression vectors were coexpressed in the Rb(−) C33A cells. Protein-protein interactions were detected by coimmunoprecipitation assays as indicated at the top of each panel (see Experimental Procedures). In each case, the top panel is coimmunoprecipitated protein (Co-I.P.), and the middle panel shows control immunoprecipitated protein. The bottom panel is a Western blot of input protein, representing 10% of the cell lysate. (E–G) U2OS cells stably expressing p16 under control of an IPTG-inducible promoter were treated for 2 days with IPTG to induce p16 (Zhang et al., 1999), and coimmunoprecipitation assays were performed as indicated Molecular Cell 2001 8, 557-568DOI: (10.1016/S1097-2765(01)00346-X)

Figure 3 Rb and HPC2 Cooperate to Growth Arrest HPC2(−) Cells in G2/M and to Repress Expression of Cyclin A and Cdc2 (A) Rb and HPC2 cooperate to arrest growth. HPC2(−) CV-1 cells were cotransfected with an expression vector for neomycin resistance and for Rb, HPC2, or BRG1 as indicated. Cells were selected in G418 for 2 weeks, and colonies were stained with crystal violet and counted. (B) Rb-HPC2-mediated growth arrest of CV-1 cells is reversed by E1a. CV-1 cells were transfected with the indicated expression vectors, along with a GFP expression vector. GFP(+) cells were counted at 12 hr (open bars) and at 48 hr (filled bars). E1a(2–36) has the p300/CBP binding domain deleted. (C) HPC2 causes a G2/M arrest in CV-1 cells. Along with an expression vector for GFP, an expression vector for Rb or HPC2 was transfected into CV-1 cells. Cells were harvested 72 hr later, followed by analysis of GFP(+) cells for DNA content by flow cytometry. Where indicated, nocodazole (Noc) was added for the final 24 hr. (D) Expression of HPC2 in HPC2(−) cells represses expression of cyclin A and cdc2 but not cyclin E. Expression vectors for HPC2 or Rb, along with a puromycin resistance plasmid, were transfected into HPC2(−) CV-1 cells, followed by a 72 hr puromycin selection. Selected cells were lysed and immunoblotted for endogenous cyclin A, cdc2, and cyclin E. The lower panel shows the level of tubulin in each of the lysates as a control Molecular Cell 2001 8, 557-568DOI: (10.1016/S1097-2765(01)00346-X)

Figure 4 Rb Colocalizes with HPC2 to Form Nuclear PcG Protein Complexes, and HPC2 Represses Expression of Cyclin A and Cdc2 in an Rb-Dependent Fashion (A) p16(−) U2OS cells containing an IPTG-inducible stably integrated p16 expression vector were treated with IPTG for 2 days to induce p16 as in Figures 2E–2G. Cells were immunostained with anti-Rb and anti-HPC2 antibodies as indicated. Purple color indicates nuclear staining with DAPI. Results are typical of two independent experiments. (B) DN-E2F displaces Rb-E2F complexes from E2F sites in the promoters of cell cycle genes (Zhang et al., 2000). (C) DN-E2F blocks HPC2-mediated inhibition of cdc2 expression. U2OS cells were transfected with expression vectors for HPC2 and puromycin resistance. Cells were selected for 72 hr in puromycin, followed by lysis and Western blotting for endogenous cdc2 (a similar result was seen with cyclin A; data not shown). The lower panel shows control tubulin levels for each lysate. (D) DN-E2F reverses the accumulation of cells in G2/M, triggered by HPC2. U2OS cells transfected with the indicated expression vectors were analyzed by flow cytometry as in Figure 3 Molecular Cell 2001 8, 557-568DOI: (10.1016/S1097-2765(01)00346-X)

Figure 5 A Dominant-Negative HPC2 Blocks Rb-Dependent Repression of Cyclin A and Cdc2 but Not Cyclin E, and it Reverses the Rb-Dependent Growth Arrest by BRG1 (A) Ring1 interacts with Rb. Expression vectors for Ring1 and Rb were cotransfected as indicated. Ring1 was immunoprecipitated from lysates, followed by immunoblotting for associated Rb (see Experimental Procedures). Coimmunoprecipitated proteins are shown in the top lane. The middle lane shows control immunoprecipitated proteins, and the bottom lane shows the input protein (as in Figure 2). (B) Ring 1, Rb, and E2F can form a complex. Ring1, Rb, and E2F expression vectors were transfected as indicated, and Ring1 was immunoprecipitated from cell lysates. The coimmunoprecipitated E2F was detected by Western blotting for E2F1. (C) DN-HPC2, with a Ring1 binding site mutation, still interacts with CtBP and Rb. Coimmunoprecipitation assays were done as in Figure 2. The black box indicates the Ring1 binding site deleted in DN-HPC2 (Sewalt et al., 1999). (D) DN-HPC2 reverses Rb-mediated repression of cyclin A and cdc2 expression. Expression vectors for DN-HPC2 or vector alone were cotransfected along with a puromycin resistance plasmid into U2OS cells containing an IPTG-inducible p16 expression vector (McConnell et al., 1999). Cells were selected in puromycin for 4 days and IPTG was added for the final 65 hr to induce p16 expression. Lysates were prepared and endogenous cyclin E, cyclin A, and cdc2 were detected by Western blotting. Tubulin levels are shown in the bottom panel. (E) DN-HPC2 reverses the Rb-BRG1-dependent growth arrest of BRG1/Brm(−) SW13 cells. SW13 cells were cotransfected with a neomycin resistance expression vector along with the indicated expression vectors. Cells were selected in G418 for 2 weeks, followed by crystal violet staining and counting of colonies Molecular Cell 2001 8, 557-568DOI: (10.1016/S1097-2765(01)00346-X)

Figure 6 Chromatin Immunoprecipitation Assays of the Cyclin E and Cyclin A Gene Promoters p16 was induced in U2OS cells by treatment with IPTG for 2 days, and extracts from the cells were used for chromatin immunoprecipitation assays with the indicated antibodies. Immunoprecipitated promoter fragments were then analyzed by PCR. “No crosslinking” indicates control assays in the absence of crosslinking Molecular Cell 2001 8, 557-568DOI: (10.1016/S1097-2765(01)00346-X)

Figure 7 Proposed Model for the Role of Polycomb Proteins in the Rb Pathway See text for discussion of the model Molecular Cell 2001 8, 557-568DOI: (10.1016/S1097-2765(01)00346-X)