1. Acquisition of Oncogenic Potential by RAR Chimeras in Acute Promyelocytic Leukemia through Formation of Homodimers 
Richard J Lin, Ronald M Evans 
Molecular Cell 
Volume 5, Issue 5, Pages (May 2000)
DOI: /S (00)

2. Figure 1
PML-RARα Dimerization Is Necessary for Its Differentiation Blocking Effect
(A) Association of in vitro translated PML-RARα and its dimerization-deficient mutant with bacterially expressed GST-SMRT ID in a GST pulldown assay. C-C: coiled-coil domain of PML-RARα, which is required for dimerization.
(B) Dimerization-deficient PML-RARα mutant fails to inhibit RA-induced transactivation of a TK-RARE-LUC reporter in CV1 cells.
(C) Expression of dominant-negative RXR AF2 deletion mutant (RXR443) blocks RA-mediated granulocytic differentiation of PML-RARα-negative U937 cells, but not PML-RARα-positive NB4 cells. Granulocytic differentiation of U937 and NB4 was measured by the percentage of NBT positive cells after 6 days of RA (1 μM) treatment.
(D) Retroviral transduction of RXR443 in U937 and NB4 cells. Western blotting of whole-cell extracts was performed using anti-RXRα antibody. Asterisks and arrows indicate the position of wild-type RXRα and exogenous RXR443, respectively.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2 Biochemical Properties of RAR Homodimeric Mutants
(A) Schematic representation of x-RARα fusion proteins and RAR homodimeric mutants used in this study.
(B) DNA binding by X-RARα fusion proteins and RAR homodimeric mutants. In vitro translated receptors were mixed with 32P-labeled DR5 oligos in a EMSA. The inclusion of RXR is indicated. Asterisks indicate positions of corresponding dimers and multimers. Arrows indicate nonspecific bands.
(C) Interaction of bacterially expressed GST-SMRT ID with RAR homodimeric mutants. In vitro translated receptors were mixed with GST-SMRT ID and 32P-labeled DR5 oligos in a EMSA. The amount of ATRA is indicated.
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 3 Effects of RAR Homodimeric Mutants on RA Signaling
(A) CV1 cells were transient transfected with expression vectors encoding X-RARα fusion proteins and RAR homodimeric mutants along with TK-RARE-Luc reporter. Relative luciferase activity in the absence (hatched bar) or presence (black bar) of 100 nM RA was presented.
(B) Expression of X-RARα fusion proteins and RAR homodimeric mutants in U937 cells. Western blotting was performed using whole-cell extracts of retroviral infected U937 cells. The positions of X-RARα fusion proteins and RAR homodimeric mutants were indicated by asterisks, and the position of wild-type RARα was indicated by the arrow.
(C) Expression of X-RARα fusion proteins and RAR homodimeric mutants in U937 cells blocks granulocytic differentiation under low concentrations of RA (10 nM). The percentage of NBT positive cells was measured after 5 days of RA treatment.
(D) Expression of X-RARα fusion proteins and RAR homodimeric mutants in U937 cells has no effect on granulocytic differentiation induced by high concentrations of RA (1 μM). The percentage of NBT positive cells was measured after 5 days of RA treatment.
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 4
Effects of X-RARα Fusion Proteins and RAR Homodimeric Mutants on Other Nuclear Receptor Signaling Pathways
(A) CV1 cells were transient transfected with expression vectors encoding X-RARα fusion proteins and RAR homodimeric mutants along with VDR expression plasmid and TK-SPP1-Luc reporter. Relative luciferase activity in the absence (hatched bar) or presence (black bar) of 100 nM VD3 was presented.
(B) Expression of X-RARα fusion proteins and RAR homodimeric mutants in U937 cells reduces monocytic differentiation induced by 100 nM VD3. The percentage of monocytic marker CD14 positive cells was measured by FACS after 5 days of VD3 treatment.
(C) CV1 cells were transient transfected with expression vectors encoding X-RARα fusion proteins and RAR homodimeric mutants along with PPAR expression plasmid and TK-PPRE-Luc reporter. Relative luciferase activity in the absence (hatched bar) or presence (black bar) of 5 μM Pd-J2 was presented.
(D) Expression of X-RARα fusion proteins and RAR homodimeric mutants in U937 cells reduces monocytic differentiation induced by a combination of Pd-J2 (3 μM) and LG268 (100 nM). The percentage of CD14 positive cells was measured by FACS after 5 days of ligand treatment.
Molecular Cell 2000 5, DOI: ( /S (00) )

6. Figure 5
Biochemical and Biological Properties of an AF2 Deletion Mutant, RXRα443
(A) Reduced RA sensitivity of SMRT association with RARα/RXRα443 heterodimers. The RA sensitivity of interactions of between in vitro translated receptors and bacterially expressed GST-SMRT ID proteins is measured in an EMSA.
(B) CV1 cells were transiently transfected with expression vectors encoding RXRα443 along with the TK-RARE-Luc reporter. Relative luciferase activity in the absence (hatched bar) or presence (black bar) of 100 nM ATRA was presented. When indicated, cells were treated with TSA for 24 hr.
(C) Expression of RXRα443 in U937 cells blocks granulocytic differentiation induced by 1 μM ATRA, and this inhibition can be reversed by TSA (100 nM). The percentage of NBT positive cells was measured after 5 days of RA and/or TSA treatment.
Molecular Cell 2000 5, DOI: ( /S (00) )

7. Figure 6 Stoichiometry of PML-RARα/SMRT Interaction
(A and B) Utilization of SMRT core IDs by RARα/RXRα heterodimers versus PML-RARα homodimers. Mammalian two-hybrid assay was performed to assess the interaction between receptors and SMRT core ID domains. GAL4-DBD fusion of CoR box1 (ID1) or CoR box2 (ID2) was cotransfected into CV1 cells with indicated VP16 fusion of RARα, RXRα, PML-RARα, and reporter TK-MH100-Luc. Relative luciferase activity in the absence (black bar) or presence (hatched bar) of 100 nM TTNPB was presented.
(C) A PML-RARα homodimer can associate with two molecules of SMRT. A modified GST pulldown assay is performed using GST-SMRT ID proteins and unlabeled receptor dimers and 35S-labeled C-SMRT protein. This is a type of coincidence assay since labeled C-SMRT can only be retained on beads when two halves of a PML-RARα homodimer bind to both GST-SMRT ID and 35S-C-SMRT (left panel). The arrow indicates the position of 35S-labeled C-SMRT.
Molecular Cell 2000 5, DOI: ( /S (00) )

8. Figure 7
Model for Molecular Mechanism of Repression-Mediated Leukemogenesis by PML-RARα and Possibly RXRα443
RAR interacts strongly with SMRT ID1, while RXR interacts weakly with SMRT ID2. In a RAR/RXR heterodimer, combination of RAR and RXR results in cooperative binding to a single SMRT molecule. In contrast, both halves of a PML-RAR homodimer can only bind SMRT ID1 and recruit independently two molecules of SMRT. This leads to increased local concentrations of corepressors and stronger repression on promoters in the presence of physiological concentrations of ligand and results in differentiation block and leukemia. In a RARα/RXRα443 heterodimer, increased affinity of RXRα443 to SMRT ID2 (denoted by three small bands) leads to stronger repression of RA target genes and possible leukemia.
Molecular Cell 2000 5, DOI: ( /S (00) )