1. Volume 18, Issue 6, Pages 623-635 (June 2005)
Binding of pRB to the PHD Protein RBP2 Promotes Cellular Differentiation 
Elizaveta V. Benevolenskaya, Heather L. Murray, Philip Branton, Richard A. Young, William G. Kaelin 
Molecular Cell 
Volume 18, Issue 6, Pages (June 2005)
DOI: /j.molcel
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2. Figure 1
Interaction of the Nucleolar Protein RBP2 with pRB in Yeast Two-Hybrid Assays
(A and D) Chromogenic GusA assays of yeast coproducing the cI DNA binding domain fused to the indicated large pocket (LP) pRB (A) and full-length pRB (D) variants (listed on top), and indicated prey cDNAs fused to B42 transactivation domain (listed on side). Control preys indicated in italics. In (D), clones were replica plated and assayed after growth at 23°C, 30°C, or 37°C. (B and C) Immunoblot assays showing bait proteins in (A) and (D). (E) Schematic representation of RBP2. Two pRB binding domains are shown in red. (F) Confocal immunofluorescence images of WI 38 fibroblasts grown in the presence of 0.1% or 10% fetal bovine serum (FBS). All cells grown at 0.1% had exited the cell cycle as shown by failure to incorporate BrdU (data not shown). ANA positive was used as a positive control for nucleolar staining.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Salt Extraction of RBP2 and pRB from Nuclei (A) Schema.
(B–D) SAOS-2 (RB−/−) cells were transiently transfected with plasmids encoding WT, Δ663, or Δex22 pRB. Aliquots of the cells were lysed directly (B) or processed as in (A). (B and C) Immunoblot assays with the indicated antibodies. Triangle in (C) represents 0.15, 0.25, and 0.50 M NaCl fractions. “R” indicates remaining fraction resistant to high salt extraction. (D) 0.25 M NaCl nuclear extracts (indicted by asterisks in [C]) or pooled cytosolic, nucleosolic, and salt-extractable proteins were immunoprecipitated with anti-RBP2 or control antibodies and immunoblotted with anti-pRB antibody. RBP2 is not seen in input due to dilution (data not shown).
(E) 0.15, 0.25, and 0.50 M NaCl fractions, as indicated by the triangle, of differentiated U937 RB+/+ leukemic cells were immunoprecipitated with anti-RBP2 antibody and immunoblotted with anti-pRB antibody. Cytosolic (C) and nucleosolic (N) fractions were loaded on the same gel for comparison to the salt-extractable material.
(F) U937 cells grown in the presence or absence of the differentiation agent TPA and SAOS-2 RB−/− were analyzed as in (D).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
RBP2 and pRB Interact and Redistribute in Chromatin Fractions during Differentiation
(A) Schema.
(B) Ethidium bromide-stained agarose gel after electrophoresis of chromatin isolated as in (A). Nuclease digestion was omitted where indicated in (B) and (C). Note that under limiting conditions micrococcal nuclease digests chromatin in hypersensitive regions, which have an open configuration. S1 is composed of mononucleosomal-sized DNA and S2 is di-, tri- and higher oligonucleosomes.
(C) Immunoblot analysis of S1, S2, and P chromatin fractions from U937 cells induced to differentiate with TPA for the indicated periods of time. As cells differentiate, there is a shift of RBP2 from S2 to S1 fraction coincident with peak appearance of pRB in S1.
(D) S1 and S2 fractions from the indicated time points in hours (C) were immunoprecipitated with anti-RBP2 or control antibodies and immunoblotted with anti-pRB antibody.
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5. Figure 4
Knockdown of RBP2 with siRNA Potentiates GRα and CBFA1 Activity
(A) Immunoblot analysis of SAOS-2 cells transfected with the indicated siRNAs. (B) Phase-contrast microscopy of SAOS-2 cells 5 days after transfection with siRNA against luciferase (GL3), RBP2 (siRNAs 1 and 4), or scrambled RBP2 siRNA (4sc) showing induction of “flat” or “senescent” phenotype. (C) Normalized firefly luciferase values for SAOS-2 cells transfected with a GRα expression plasmid (50 ng), a GRE-driven firefly luciferase reporter plasmid (100 ng), and a renilla luciferase expression plasmid (5 ng). Where indicated, transfection mix also contained 20 ng of a pRB expression plasmid (wild-type or 567L) and/or 2 nM RBP2 siRNA (“2” or scrambled “2sc”). 24 hr after transfection, cells were split 1 to 2 and grown in the presence of dexamethasone. 48 hr later the cells were lysed. Normalized firefly luciferase values are expressed as percentage of the values obtained in the absence of pRB or siRNA (“none”). Error bars indicate mean ± SEM. (D) Immunoblot analysis of SAOS-2 cells transfected as in (C). ([E], [F,] [G], and [I]) Normalized firefly luciferase values for SAOS-2 cells transfected with a CBFA1 expression plasmid (50 ng), an OSE-driven firefly luciferase reporter plasmid (100 ng), and a renilla luciferase expression plasmid (5 ng). Where indicated, transfection mix also contained an expression plasmid for pRB (wild-type or mutant; 50 ng except where indicated by triangle representing 10 and 200 ng plasmid DNA) and/or RBP2 siRNA (“1,” “2,” or scrambled versions thereof; 2 nM except where indicated by triangle representing 0.2, 1, and 4 nM). In panel (I), an RBP2 expression plasmid containing an RBP2 cDNA with silent mutations in the RBP2 oligo 1 site was included as indicated by the triangle (8 and 80 ng). Normalized firefly luciferase values are expressed as percentage of the values obtained in the absence of pRB, RBP2, or siRNA (“none”) ([E], [F], and [G]) or with scrambled siRNA (I). Error bars indicate mean ± SEM.
(H) Immunoblot analysis of SAOS-2 cells transfected as in (E) and (F).
(J) Immunoblot analysis of SAOS-2 cells transfected as in (I).
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6. Figure 5 pRB Displaces RBP2 from the OC Promoter
(A) Decreased binding of pRB to RBP2 mutants. Anti-RBP2 (top) and anti-Flag (bottom) immunoblot assays of SAOS-2 cells transfected to produce Flag-pRB (where indicated by “+”) and the indicated RBP2 variants. C, cytosolic fraction; N, nucleosolic fraction; NaCl fr, 0.25 M NaCl fraction (lanes 6–10) or pooled NaCl-extractable material (lanes 11–30); IP, anti-RBP2, anti-Flag, or control IgG immunoprecipitates.
(B) Cells were transfected with expression plasmids that encode the indicated RBP2 variants (that contain the silent oligo 1 site mutations) and RBP2 siRNA (1 or 1sc). Normalized firefly luciferase values are expressed as percentage of the values obtained in the absence RBP2 or siRNA (“none”). Error bars indicate mean ± SEM.
(C and D) Autoradiograms of radiolabeled PCR products using primers for the indicated promoter regions and chromatin immunoprecipitated with RBP2 antibodies, anti-Flag, or control IgG from SAOS-2 cells transfected to produce CBFA1 and, where indicated, Flag-tagged wild-type or mutant pRB.
(E) Model for transcriptional repression by RBP2 and antagonism by pRB. For simplicity, RBP2 is shown binding directly to DNA.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Knockdown of RBP2 Cooperates with MyoD to Promote Differentiation
(A) Representative photomicrographs of RB−/− mouse embryo fibroblasts (MEFs) transfected with a plasmid encoding MyoD and, where indicated, a plasmid encoding pRB (WT, Δ663, or Δ22) or RBP2 siRNA (“2” or scrambled “2sc”). “Mock” indicates MyoD plasmid alone. After 3 days of growth in differentiation media, the cells were fixed and stained with anti-MHC (myosin heavy chain) antibody (red), which is a marker of myogenic differentiation, and counterstained with the nuclear stain DAPI (blue).
(B) Approximately 200 cells from each transfection (2 slides per transfection) were scored for the presence of multiple nuclei. Error bars indicate mean ± SEM.
(C) Representative photomicrographs of RB−/− mouse embryo fibroblasts (MEFs) transfected with a plasmid encoding p21 or dominant-negative cdk2 and processed as in (A).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Binding of RBP2 and pRB to BRD2 and BRD8 Promoters Coincides with Increased BRD2 and BRD8 Expression
(A) Schematics of BRD2 and BRD8 promoter regions. Thick lines show PCR fragments generated in ChIP experiments (B) relative to BRD2 and BRD8 transcription start sites (arrows). Thin lines (tick marks = 0.25 kb) show genomic DNA fragments subcloned upstream of firefly luciferase cDNA to make BRD2 and BRD8 reporter plasmids (used in Figure 8).
(B) Autoradiograms of radiolabeled PCR products using primers for the indicated promoter regions, including control Hoxa5 promoter, and chromatin immunoprecipitated with the indicated antibodies from U937 cells treated with TPA for the indicated time periods. Also shown are PCR products using input DNA (no ChIP). Results shown are representative of four independent experiments and did not use the enriched DNA that was subjected to LM-PCR and promoter microarray analysis.
(C and D) Ethidium stained gel showing RT-PCR products obtained with primers for the indicated genes from U937 cells induced to differentiate with TPA for the indicated amount of time (C) or SAOS-2 cells (D). SAOS-2 cells were transfected to produce pRB where indicated.
(E) Immunoblot analysis of SAOS-2 cells transfected with the indicated RBP2 siRNAs.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 8 pRB Cooperates with RBP2 to Activate BRD2 Promoter.
(A–E) Normalized firefly luciferase values of SAOS-2 cells transfected with the BRD2 reporter plasmid (50 ng) together with a renilla luciferase expression plasmid ([A], left panel, [B], [C], and [D]) or a LacZ expression plasmid ([A], right panel, and [E]). Where indicated, transfection mix also contained an expression plasmid for pRB (wild-type or mutant; 50 ng), RBP2 (wild-type or mutant; 500 ng), E7 (wild-type or mutant; 5 or 50 ng as indicated by the triangle), p21 (wild-type or mutant; 20 ng), and/or RBP2 siRNA (“2” or scrambled “2sc;” 1 nM). Error bars indicate mean ± SEM.
(F) Model for cooperation between pRB and RBP2. For simplicity, RBP2 is shown binding directly to DNA.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions