1. Volume 5, Issue 5, Pages 811-820 (May 2000)
Oligomerization of RAR and AML1 Transcription Factors as a Novel Mechanism of Oncogenic Activation 
Saverio Minucci, Marco Maccarana, Mario Cioce, Pasquale De Luca, Vania Gelmetti, Simona Segalla, Luciano Di Croce, Sabrina Giavara, Cristian Matteucci, Alberto Gobbi, Andrea Bianchini, Emanuela Colombo, Ilaria Schiavoni, Gianfranco Badaracco, Xiao Hu, Mitchell A Lazar, Nicoletta Landsberger, Clara Nervi, Pier Giuseppe Pelicci 
Molecular Cell 
Volume 5, Issue 5, Pages (May 2000)
DOI: /S (00)

2. Figure 1
Enhanced Recruitment of N-CoR by PML-RAR Is Due to the CC Region of PML
(A-B) Decreasing amounts of GST-N-CoR (from 10 μg to 150 ng) or GST as a control (10 μg; lane “-”) were incubated with the indicated in vitro translated, 35S-labeled proteins. R, RING finger; and B, B boxes. The input lanes (“I”) are loaded with the same (A) or 50% (B) of the amount used in the assays.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2 Role of the CC in Transcriptional Regulation by PML-RAR
(A) Transcriptional repression. HeLa cells were cotransfected with the RARE-G5-TATA reporter in the absence (C) or presence of increasing amounts (50, 100, 250, and 1000 ng) of the indicated expression vectors (P-R, PML-RAR; ΔCC-PR: ΔCC-PML-RAR; CC-R: CC-RAR; p53-R: p53-RAR). Transfection of the expression vectors yielded comparable levels of protein expression (data not shown).
(B) RA sensitivity. Cells were cotransfected with 500 ng of the indicated expression vectors. RA was added 24 hr after transfection (1, 10, 100 nM, 1, 10 μM).
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 3 PML-RAR Forms Oligomers In Vivo
(A) Role of the CC. Nuclear extracts from U937 clones expressing the indicated proteins, or recombinant PML-RAR from BL21 cells, were fractionated by SEC and analyzed by Western blotting using an anti-RAR antibody. Fraction number is indicated at the top of each lane. Elution fractions of molecular weight markers are indicated by arrows.
(B) Biochemical purification of PML-RAR. Silver stain and Western blot analysis (upper panels) of highly purified PML-RAR after the final DNA-affinity chromatography: asterisks mark nonspecific bands. Western blot analysis of purified PML-RAR after SEC (lower panel).
(C) In vivo cross-linking. Nuclear extracts from in vivo cross-linked, metabolically labeled U937-PML-RAR cells were analyzed by Western blot after SDS PAGE in reducing (R) or nonreducing (NR) conditions: the arrows indicate the cross-linked species. Extracts were subjected to SEC, and then HMW PML-RAR fractions were immunoprecipitated with anti-PML or control (C) antibodies and analyzed (before or after a further SEC step) by SDS PAGE in reducing conditions, followed by autoradiography.
(D) Characterization of the isolated CC domain of PML. (Upper panel) SEC of purified, bacterially expressed (BL21) CC. Fractions were analyzed by SDS-PAGE, followed by Western blotting using an anti-CC antibody. Silver stain (lower left panel) and Western blot analysis (lower right panel) of purified CC cross-linked in vitro with increasing concentrations of BS3. The positions of the mono-, di-, and trimeric CC are indicated by arrowheads.
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 4 PML-RAR Oligomers Associate with N-CoR and DNA
(A) In vivo association of PML-RAR oligomers with N-CoR. Metabolically labeled U937 PML-RAR cells (I, input lane) were immunoprecipitated with anti-N-CoR (αN) or control (PI) antibodies (the PI lane derives from approximately five times more material). The anti-N-CoR immunoprecipitates were incubated in the presence of RA (10 μM) for 2 hr at 4°C (RA lane). Anti-PML immunoprecipitates (αP) are shown in the last lane (upper panel). The RA-eluted material was then analyzed by SEC, followed by SDS PAGE and autoradiography (lower panel).
(B) Recruitment of multiple N-CoR molecules. Anti-N-CoR immunoprecipitates from PML-RAR-expressing cells or control cells were incubated with an in vitro translated, 35S-labeled N-CoR C-terminal fragment (VP16-N-CoR, aa 1944–2453: Hu and Lazar 1999) and then analyzed by SDS-PAGE followed by Western blot with anti-RAR antibodies (left panel) or autoradiography (right panel). I, input; C, control cells; and PR, PML-RAR-expressing cells. Western blot analysis with anti-N-CoR antibodies showed comparable amounts of N-CoR immunoprecipitated from the two cell extracts (data not shown). Note that the anti-N-CoR used in our assays is directed against the N terminus of N-CoR.
(C) HMW PML-RAR complexes bind DNA. Mobility shift assays using the RARE from the RARβ2 promoter as a probe and extracts from Xenopus oocytes programmed with mRNA for RAR, PML-RAR either singly or coinjected with mRNA for RXR. Lanes 3–4, 5–8, and 9–11 correspond to 0.03 (lane 5), 0.05 (lanes 6 and 9), 0.2 (lanes 3, 7, and 10), and 0.5 (lanes 4, 8, and 11) oocyte equivalent extract amounts. The empty arrow indicates the RXR-RAR-DNA complex.
(D) HMW PML-RAR complexes recruit N-CoR on DNA. Mobility shift assays of extracts from Xenopus oocytes coinjected with mRNA for PML-RAR and RXR. Extracts were incubated with the labeled RARE in the presence of recombinant GST-N-CoR (aa 1782–2453) or GST as a control. Where indicated, RA (10 μM) was added during the incubation. Empty arrow, PML-RAR/RXR/DNA complex; filled arrow, PML-RAR/RXR/N-CoR/DNA complex.
Molecular Cell 2000 5, DOI: ( /S (00) )

6. Figure 5
A Heterologous Oligomerization Domain Activates the Transforming Potential of RAR
(A) p53-RAR form oligomers. (Upper panel) SEC analysis of RA binding capacity of nuclear extracts from COS-1 cells transfected with p53-RAR or RAR expression vectors, incubated with tritiated RA (10 nM), in the absence (black squares and circles) or in the presence (white squares) of a 100-fold excess of cold RA. (Lower panel) SEC fractions of in vitro translated, 35S-labeled p53-RAR were analyzed by SDS-PAGE, followed by autoradiography. Two products are generated in the reaction: full-length p53-RAR oligomers and monomeric RAR, starting from its internal ATG (maintained in the construct).
(B) Differentiation of murine hematopoietic progenitors. Lin− cells were transduced with the indicated retroviral vectors (C: control; P-R: PML-RAR; ΔCC-P-R: ΔCC-PML-RAR; CC-R: CC-RAR; GPR: GFP-PML-RAR; GΔCC: GFP-ΔCC-PML-RAR; and Gp53R: GFP-p53-RAR), and GFP+ cells were sorted by FACS. ΔCC-PML-RAR, GFP+ cells were sorted in GFPhigh or GFPlow expressors. After sorting, cells were either plated in differentiation medium in the absence or in the presence of RA (3 nM or 1 μM) (upper panel) or analyzed by Western blot (lower panels). RA delays myeloid differentiation of control cells, and this effect is counteracted by expression of PML-RAR, CC-RAR, and by high levels of ΔCC-PML-RAR. As described for wild-type RAR, expression of high levels of ΔCC-PML-RAR in the presence of physiological concentrations of RA relieves the differentiation block observed in the absence of ligand (Du et al. 1999). Note that p53-RAR was expressed as a GFP-fusion protein: GFP-PML-RAR and GFP-ΔCC-PML-RAR were also tested in some experiments, with identical results to PML-RAR and ΔCC-PML-RAR, respectively (data not shown).
Molecular Cell 2000 5, DOI: ( /S (00) )

7. Figure 6
APL Fusion Proteins Form HMW Complexes Due to the Partners of RAR in the Chromosomal Translocations
(A) SEC analysis of nuclear extracts from COS-1 cells (NPM) or COS-1 cells transfected with PML or PLZF expression vectors, followed by SDS-PAGE and Western blotting.
(B) Nuclear extracts from COS-1 cells transfected with the indicated expression vectors were fractionated and analyzed by Western blotting using anti-RAR antibodies.
Molecular Cell 2000 5, DOI: ( /S (00) )

8. Figure 7 Oligomerization of AML1-ETO
(A) AML1-ETO forms HMW complexes. A scheme of AML1-ETO and the deletions used (upper panel); Z, zinc fingers (N-CoR interaction domain). AML1-ETO (A-E) and ΔP-AML1-ETO were in vitro translated, fractionated by SEC, and analyzed by SDS PAGE followed by autoradiography (lower panel).
(B–C) Interaction of AML1-ETO with N-CoR and DNA. (B) Pull-down assays of in vitro translated AML1-ETO and ΔP-AML1-ETO incubated with GST-N-CoR (RDIII), or GST beads (C) as control. Input lanes (I) represent 100% of the total. (C) Nuclear extracts from U937 AML1-ETO cells were incubated with biotinylated oligos containing a specific AML1 binding site or an unrelated sequence (C) and then pulled-down with streptavidine-agarose beads (left panel). SEC analysis of high-salt eluted material (right panel).
(D) Transcriptional repression by AML1-ETO. C33A cells were transiently transfected with the MDR1-luc reporter and the indicated expression vectors.
(E) Analysis of myeloid differentiation. Lin− cells transduced with the indicated retroviral vectors were sorted and then treated as described in the legend to Figure 5.
Molecular Cell 2000 5, DOI: ( /S (00) )