www.sciencemag.org/cgi/content/full/328/5975/240/DC1

Supporting Online Material for

Arsenic Trioxide Controls the Fate of the PML-RARα Oncoprotein by Directly Binding PML

Xiao-Wei Zhang, Xiao-Jing Yan, Zi-Ren Zhou, Fei-Fei Yang, Zi-Yu Wu, Hong-Bin Sun, Wen-Xue Liang, Ai-Xin Song, Valérie Lallemand-Breitenbach, Marion Jeanne, Qun-Ye Zhang, Huai-Yu Yang, Qiu-Hua Huang, Guang-Biao Zhou, Jian-Hua Tong, Yan Zhang,

Ji-Hui Wu, Hong-Yu Hu, Hugues de Thé, Sai-Juan Chen,* Zhu Chen*

*To whom correspondence should be addressed. E-mail: E-mail: zchen@stn.sh.cn (Z.C.); sjchen@stn.sh.cn (S-J.C.)

Published 9 April 2010, Science 328, 240 (2009)

DOI: 10.1126/science.1183424

This PDF file includes

Materials and Methods Figs. S1 to S8 Tables S1 and S2 References

Supporting Online Material

Arsenic Trioxide Controls the Fate of the PML-RAR Oncoprotein by

Directly Binding PML

MATERIALS AND METHODS

Reagents and cell culture. The antibodies used for the experiments were as follows:

anti-RAR (Santa Cruz), anti-PML (PG-M3, Santa Cruz), anti-FLAG (M2, Sigma),

anti-ubiquitin (Biomol), anti-SUMO-1 (Santa Cruz), anti-GFP (Santa Cruz),

anti-SUMO-2 (Zymed) and anti-His (Santa Cruz). As2O3 and p-arsanilic acid were

purchased from Sigma, ReAsH-EDT2 (ReAsH) from Invitrogen, PFP-Biotin and

streptavidin agarose from Pierce. p-Aminophenylarsine oxide (PAPAO) was synthesized

from p-arsanilic acid as described previously (1). EGFP-TRIM30 was a gift from Prof.

Bing Sun (2). NB4 cells were grown in RPMI-1640 with 10% FBS. HEK 293T cells and

HeLa cells were grown in DMEM with 10% FBS.

Plasmid constructs. cDNA PML (IV) (3), PML-RAR(L isoform) PML 1-394, PML

RBCC (261-633), RAR and SP100 were amplified by PCR and cloned into

pFLAG-CMV-4 (Sigma) or pEGFP-C1 (Clontech) vectors. The deletion and point

mutants of PML were constructed by QuikChange® Site-Directed Mutagenesis Kit

(Stratagene). The primers designed by QuikChange® Primer Design Program

(Stratagene online) were as follows: PML ΔR (Forward 5’-GAGCCCCCGCTTCG

GATAACGTCTTTTTC; Reverse 5’-GAAAAAGACGTTATCCGAAGCGGGGGC

TC); PML ΔB1 (Forward 5’-TACCGGCAGATTGTGGCAGAGCTGCGCAAC;

Reverse 5’-GTTGCGCAGCTCTGCCACAATCTGCCGGTA); PML ΔB2 (Forward

5’-GACGGCACCCGCCAGGAGGAGCTG; Reverse 5’-CAGCTCCTCCTGGCGG

GTGCCGTC). cDNAs encoding residue 49-104 of PML were amplified by PCR, and

inserted into pET32M using restriction sites BamH I and Xho I to construct pET32M

PML-R.

Measurement of arsenic contents. HEK 293T cells transfected with mock or

FLAG-PML plasmid were treated with 2 M As2O3 for 2 hr, and lysed in RIPA buffer.

Pellets were collected by centrifugation, and then dissolved in 2% SDS to facilitate

protein quantification. Arsenic contents in pellets of cells were quantified by atomic

fluorescence spectrometry and standardized by the arsenic content per microgram of

protein.

Streptavidin agarose affinity assay. Biotin-As synthesis was performed by conjugation

of PAPAO to activated PFP-Biotin according to the published procedures (4). For

streptavidin agarose affinity, HEK 293T cells transfected with FLAG-PML,

FLAG-PML-RAR or FLAG-RAR were treated with 10 μM Biotin-As for 2 hr and

then lysed in 8 M urea buffer. The cell lysates were incubated with streptavidin agarose

overnight at 4 0C. After washing with urea buffer, streptavidin agarose beads were

resuspended in SDS-PAGE loading buffer. For PAPAO or As2O3 blocking, cells were

pretreated with 10 μM As2O3 or 10 μM PAPAO for 1 hr, followed by 10 μM Biotin-As

treatment for 2 hr in fresh cell media.

Fluorescence microscopy. Time-lapse confocal microscopy and immunofluorescence

confocal microscopy analysis were performed on Leica TCS SP5. HeLa cells stably

expressing EGFP-PML (EGFP-PML HeLa) were cultured at 37 0C and treated with 2

μM As2O3. Time-lapse images of the same cells under confocal microscope were

captured for the indicated time points. For immunofluorecence assay, NB4 cells were

fixed in 4% paraformaldehyde buffer, and endogenous PML/PML-RAR were revealed

by anti-PML antibody.

For arsenic-induced detergent resistance of PML NBs, EGFP-PML HeLa cells were

treated with or without 2 μM As2O3 for 30 min, and the cells (5×105) were subsequently

lysed in 100 μL of 1% Triton X-100 buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1%

Triton X-100), RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100,

0.1% SDS, 0.5% sodium deoxycholate) or 3 M urea buffer (50 mM Tris-HCl, pH 8.0,

150 mM NaCl, 3 M urea). Aliquotes of cell lyates (3 L) were smeared onto glass slides,

and directly observed under fluorescence microscope.

ReAsH labeling in cells. NB4 cells were labeled with 5 M ReAsH for 3 hr at 37 0C in

serum-free Opti-MEM (GIBCO). For As2O3 interference assay, NB4 cells were

pretreated with 10 μM As2O3 for 30 min in RPMI-1640 medium containing 10% FBS,

and then changed to Opti-MEM with 5 M ReAsH for 3 hr. For zinc interference assay,

NB4 cells were pretreated with 10 μM ZnSO4 for 30 min, and then changed to

Opti-MEM with 5 M ReAsH and 10 μM ZnSO4 for 3 hr. HeLa cells expressing

EGFP-fused constructs were grown on cover glasses and were labeled with 5 to 10 M

ReAsH for 1 hr at 37 0C in serum-free Opti-MEM. After thoroughly washing with 250

M BAL buffer, cells were fixed with 4% paraformaldehyde and imaged by confocal

microscope (Leica TCS SP5).

Preparation of recombinant PML-R. Prokaryotic pET32M PML-R was transformed

into E.coli BL21 (DE3) cells. Cells were grown in M9 minimal media and induced by

100 M IPTG and 20 M ZnCl2. For NMR study, 15

N-labelled PML-R was prepared by

using M9 minimal media containing 15

NH4Cl as the nitrogen resource. The Trx tag of

Trx-PML-R was removed by thrombin cleavage. PML-R was further purified by

Superdex-75 column chromatography. For arsenic binding assays, apo-PML-R was

prepared by dialysis against 50 mM NaAc buffer (pH 5.0) containing 50 mM NaCl, 10

mM EDTA and 5 mM DTT to remove zinc ions as described previously (5). The

As-PML-R was initially obtained by using the HiTrap Desalting column (Amersham

Biosciences) against a buffer containing As2O3, and used directly for the assays. For

convenience, we prepared lyophilized apo-PML-R by using a desalting column against

Millipore water for titration with ZnCl2 or As2O3. The consistent results between

re-suspended apo-PML-R titrated with ZnCl2 and Zn-PML-R directly purified from

E.coli suggested that the preparation was valid. The preparation of apo-PML-R for

arsenic or zinc binding assays were performed in anaerobic condition by using nitrogen

gas-inflated buffers and assay tubes.

MALDI-TOF mass spectrometry. Arsenic binding to apo-PML-R was analyzed by

MALDI-TOF-MS in a linear model on an AutoFlex MALDI-TOF-TOF-MS (Bruker). 10

M protein in 10 mM Tris-HCl (pH 7.4) buffer was incubated with varying

concentrations of As2O3 at room temperature for 1 hr, and then aliquots (1 L) of

samples were added to an equal volume of a standard cyano-4 hydroxy-cinnamic acid

matrix solution for spotting on a sample plate.

XAS analysis. The K-edge XAS spectra (EXAFS and XANES) were collected in the

fluorescence yield mode using a Si (111) double crystal monochromator at the XAFS

station of the 4W1B beam line of the Beijing Synchrotron Radiation Facility (BSRF).

The storage ring was working at 2.5 GeV with an electron current decreasing from 240

to 160 mA within approximately 8 hours. To avoid the effect of the high harmonic, a

filter foil of Cu or Ge both with a thickness of 3 μm were used at the Zn and As K-edge,

respectively. Solutions of samples were kept in a cell sealed by Kapton films. In order to

increase the S/N ratio, each sample was scanned twice. The analyzed data are the

average of the raw experimental data.

The WinXAS3.1 package (6) was used to fit the EXAFS oscillation with the

theoretical models generated by FEFF8.0 (7). S02 value of each element was extracted

from the standard sample and was maintained constant during the fit. Based on

information available from the previous analysis (8), we set the coordination number of

the Zn-N·His at 0.5 and the Zn-S·Cys at 3.5 for the fit of the Zn-PML-R. For the

As-PML-R, the coordination number of the As-S·Cys was set free and that of As-N·His

was limited to the range of 0 to 0.5. It turned out that As-N·His bond length was longer

than 3.0 Å, so the N·His was too far to be considered as a first shell atom and was

removed from the first coordinated shell for the final fit. As shown in fig. S5, a

single-shell model was used for the best fit of the raw data, both in the momentum space

(k space) and in the real space (R space). Results are summarized in Table S1 and clearly

show a coordination number of 3 for the As3+

and the metal-S bond length is slightly

shorter than that in Zn-PML.

To extract “stereo” structural information around the metal ion site, we applied the

software code MXAN (Minuit XANes) (9) to fit the XANES spectra. This code is

capable of performing a quantitative analysis of a XAS spectrum from the absorption

edge up to 200 eV via a comparison of the experimental data with theoretical calculations

generated by changing relevant geometrical parameters of the atomic sites. Two initial

input clusters including 50 atoms were considered from the available PDB structure

(code 1BOR). The quality of the two fits is shown in Fig. 3D and both show a very good

agreement with the experimental data. It indicates that the final geometrical structure

around metal ion sites with a large accuracy approaches the “real” structure. Detailed

result is shown in Table S2, which is consistent with that from the EXAFS analysis.

Spectroscopic studies. For Near-UV and circular dichroism (CD) studies, lyophilized

apo-PML-R was dissolved in a buffer containing 20 mM Tris-HCl (pH 7.4) and 100 mM

NaCl. 100 M apo-PML-R was titrated stepwise with increasing amount of As2O3. The

Near-UV absorbance spectra were recorded on an HP 8542 diode spectrophotometer

using a 1 cm path length in the wavelength region of 200-400 nm. CD spectra were

recorded on a Jasco J-600 spectropolarimeter. The spectra were scanned between 190 nm

and 260 nm. NMR experiments were performed at 298K on a Bruker DMX600

spectrometer with self-shielded z-axis gradients. 15

N-labelled apo-PML-R was dissolved

to the concentration of 250 M in 50 mM NaAc (pH 5.6) buffer containing 50 mM NaCl.

As2O3 or ZnCl2 was added stepwise with each step monitored by acquiring 2D [1H,

15N]

HSQC.

Gel filtration. apo-PML-R (100 M) was incubated with ZnCl2 and As2O3 at the molar

ratio of 1:2 and 1:1 respectively at room temperature in 20 mM Tris-HCl buffer (pH 7.4)

containing 100 mM NaCl. Gel filtration was performed with Tris-HCl buffer or Tris-HCl

buffer containing 6 M urea through SuperdexTM

75 10/30 GL column (Amersham

Biosciences). BSA (67 KD) and Trx (13 KD) were used as protein markers to calculate

the relative molecular weights of the PML-R oligomers.

In vitro SUMOylation assay. FLAG-PML was immunoprecipitated by anti-FLAG M2

beads from transfected HEK 293T cells and used as substrates for in vitro SUMOylation

assay. 10 µL of M2 beads with FLAG-PML were incubated in 20 μL of reaction buffer

(50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 2 mM ATP and 2 mM MgCl2), in

the presence of SAE1/SAE2 (100 nM, BostonBiochem), UBC9 (2 μM) and SUMO-2 (80

μM). After incubation at 37 0C for 90 min, the reactionwas terminated by addition of

SDS-PAGE loading buffer. To analyze arsenic effects on conjugation, M2 beads with

FLAG-PML was incubated with 1 mM As2O3, and then stringently washed to remove

free arsenic.

Pull down assay. FLAG-PML or FLAG-PML ΔR was immunoprecipitated (IP) from

cells by anti-FLAG M2 beads, and then incubated with 2 M His-UBC9 for 2 hr. The

M2 beads were washed to remove unbound protein and were eluted with SDS-PAGE

loading buffer. For arsenic binding, the PML-M2 beads were incubated with 1 mM

As2O3 and then washed to remove free arsenic before the pull down assay. The PML

input was detected with anti-FLAG antibody, and the UBC9 input and pull down

products with anti-His antibody.

Mammalian two-hybrid assay. The mammalian two-hybrid assay was performed by

using the CheckMateTM

Mammalian Two-Hybrid System (Promega) in HeLa cells. The

cDNA of PML and UBC9 were inserted into pBIND and pACT vectors, respectively.

HeLa cells were transfected with pG5/luc, pBLIND-PML and pACT-UBC9 at a ratio of

1:1:1 by using the calcium phosphate precipitation method. Twenty-four hours after

transfection, cells were treated with 1 M As2O3 for another 24 hr. The luciferase activity

was determined with the Dual-Luciferase Reporter Assay System (Promega).

Construction of a predicted structural model of UBC9/PML-R complex. UbcH7 is

homologous to UBC9 with an identity of 29% and the RING domain of the c-Cbl is

homologous to the PML-R with an identity of 37%. A crystal structure of c-Cbl/UbcH7

complex (PDB entry, 1FBV) revealed the binding mode of the RING domain in c-Cbl

with UbcH7 (10). Through computational simulation, we constructed a proposed mode

of the structure of UBC9/PML-R binding mode in the following way: the solution

structure of PML-R (PDB entry, 1BOR) was fitted onto the structure of the RING

domain of c-Cbl and the crystal structure of UBC9 (PDB entry, 1A3S) was fitted onto

the structure of UbcH7 (11, 12). Then the complex structure of UBC9/PML-R was

finally put into energy minimization by fixing backbone atoms.

References

1. E. Kalef, C. Gitler, Methods Enzymol 233, 395 (1994).

2. M. Shi et al., Nat Immunol 9, 369 (Apr, 2008).

3. K. Jensen, C. Shiels, P. S. Freemont, Oncogene 20, 7223 (Oct 29, 2001).

4. X. Zhang et al., Cancer Lett 255, 95 (Sep 18, 2007).

5. T. Pan, J. E. Coleman, Proc Natl Acad Sci U S A 86, 3145 (May, 1989).

6. T. Ressler, J. Synchrotron Rad. 5, 118 (1998).

7. S. E. A., Phys Rev B 48, 9825 (1993).

8. M. Y. e. al., Biochemistry Biophys Res Commun 374, 28 (2008).

9. M. Benfatto, S. Della Longa, J Synchrotron Radiat 8, 1087 (Jul 1, 2001).

10. N. Zheng, P. Wang, P. D. Jeffrey, N. P. Pavletich, Cell 102, 533 (Aug 18, 2000).

11. M. F. Giraud, J. M. Desterro, J. H. Naismith, Acta Crystallogr D Biol Crystallogr 54, 891 (Sep 1,

1998).

12. K. L. Borden, Biochem Cell Biol 76, 351 (1998).

SUPPLEMENTARY FIGURE LEGENDS

fig. S1. As2O3 induces SUMO and ubiquitin conjugations of PML and

PML-RARPML and PML-RAR (B) were immunoprecipitated from extract of

HEK 293T cells expressing FLAG-PML or FLAG-PML-RAR by anti-FLAG M2 beads.

The antibodies used for WB analysis are indicated (anti-S1: SUMO-1; anti-S2/3:

SUMO-2/3; anti-Ub: ubiquitin).

fig. S2. PML-related proteins. (A) Schematic structures of PML-related constructs. (↓),

breakpoints. (B) The sequences of PML-R, PML-B1 and PML-B2. Conserved cysteines

(C) and histidines (H) are numbered.

fig. S3. PAPAO and ReAsH show effects similar to those of As2O3. (A) PAPAO and

As2O3 show similar interactions with BAL (British Anti-Lewisite, an arsenic chelating

agent). The solution of BAL was incubated with proportionally varying concentrations of

As2O3 (■) or PAPAO (▼) at room temperature for 10 min, and then mixed with DTNB

to determine the free sulfhydryl groups by measuring the absorbance at 412 nm. (B and

C) The effects of organic trivalent arsenic compounds (PAPAO and ReAsH) on PML.

(＜) points to parental proteins, (◄) to modification of parental proteins. HeLa cells

stably expressing EGFP-PML were treated with 2 M PAPAO or 2 M ReAsH for 2 hr,

then lysed in RIPA buffer and centrifuged into supernatant (S) and pellet (P) fractions for

Western blotting (WB) assay.

fig. S4. Arsenic specifically targets PML-RARPML. (A) Colocalization between

ReAsH and EGFP-fusion proteinsin transiently transfected HeLa cells. ReAsH

colocalized with EGFP-PML-RAR, but not with EGFP-PLZF-RAR, or EGFP-SP100

or EGFP-TRIM30. PLZF-RAR is a specific fusion protein in a subset of APL patients

who respond poorly to As2O3 and the PLZF moiety in the fusion protein contains two

zinc fingers. SP100 is a component of PML NBs. TRIM30 is a member of the RBCC

protein family. (B) HeLa cells transiently expressing EGFP-fusion proteins were treated

with 2 μM As2O3 for 2 hr, and then lysed in RIPA buffer and centrifuged into supernatant

(S) and pellet (P) fractions for WB assay.

fig. S5. EXAFS data (inset) and k3-weighted Fourier transforms of As-PML-R and

Zn-PML-R. A single-shell model was used for the best fit of the raw data. Black curves

represent experimental data and red lines, the best fit.

fig. S6. The mutants of PML C77/C80A and C88/C91A show an attenuated affinity

to arsenic and have no response to As2O3 modulation. (A) Mass Spectrometry of

PML-R and PML-R C77/C80A titrated with As2O3. 10 M PML-R or PML-R C77/C80A

was incubated with 10 M As2O3. The mutant of PML-R C77/C80A exhibited a

significant reduction in arsenic binding. (B) PML C77/C80A and PML C88/91A showed

no response to As2O3 in terms of pellet transfer and high-molecular-weight modification.

HEK 293T cells ectopically overexpressing FLAG-PML, FLAG-PML C77/C80A or

FLAG-PML C88/C91A were treated with 2 M As2O3 for indicated time points, and then

lysed in RIPA buffer and centrifuged into supernatant (S) and pellet (P) fractions for WB

assay. (C) The colocalization signals of PML C77/C80A or PML C88/C91A with ReAsH

were attenuated. Scale bar, 10 m.

fig. S7. As2O3 induces aggregation and detergent resistance of PML NBs. (A)

Time-lapse confocal microscopy of PML NBs. EGPF-PML HeLa cells were treated with

2 M As2O3 and the time-lapse images of the same cells were captured under confocal

microscopy at 37 0C for indicated times. Scale bar, 10 m. (B) As2O3 induces detergent

resistance of PML NBs. EGFP-PML HeLa cells were treated without (ø) or with 2 M

As2O3 for 30 min. The cells were collected and lysed in 1% Triton X-100 buffer, RIPA

buffer or 3 M urea buffer, and the smears of the cell lysates were directly observed under

fluorescent microscope. Scale bar, 5 m. (C) The effects of As2O3 on stably expressed

PML in cells. EGFP-PML HeLa cells were treated with 2 μM As2O3 for indicated time

points, and then lysed in RIPA buffer and centrifuged into supernatant (S) and pellet (P)

fractions for WB assay.

fig. S8. Predicted structural model of the UBC9/PML-R complex. (A) Binding mode

of UBC9 and PML-R. UBC9 is shown in cyan in the cartoon view. PML-R is displayed

as a blue cartoon structure. The two zinc ions in PML-R are indicated by two red balls.

The cysteines and histidine coordinated with zinc ions are shown as colored sticks. (B)

Putative local structure of the binding interface between UBC9 and the ZF1 of PML-R.

Table S1. Best fits to the EXAFS for Zn-PML-R and As-PML-R samples.

Samples Bond Coordinated

Number N

Bond length

R (Å)

Debye-Waller

Factor σ2×10

-3(Å

2)

Fit Residuate of

R space Rfactor

Zn-PML-R

Zn-N 0.5 2.04±0.02 1.23± 0.20

5.52 Zn-S 3.5 2.32±0.02 5.48±0.20

As-PML-R As-S 3.2±0.4 2.27 ± 0.02 4.87±0.20 4.74

Table S2. Bond lengths between arsenic or zinc and the conserved cysteine/histidine

residues of PML-R.

ZF1 ZF2

Conserved Residues Cys57 Cys60 Cys77 Cys80 Cys72 His74 Cys88 Cys91

Zn-PML-R (Å) 2.32 2.34 2.31 2.33 2.34 2.00 2.30 2.35

As-PML-R (Å) 3.70 2.27 2.23 2.27 2.34 3.27 2.21 2.23

fig. S1

fig. S2

fig. S3

fig. S4

fig. S5

fig. S6

fig. S7

fig. S8