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WT p53 binding to PUMA promoter (Yu et al., 2001). The
conditions of the experiment were the same as of WAF1
promoter, shown in Fig.2, main text. Changes of amount of
WT p53 occupying PUMA promoter (Sup.2) are less
pronounced at 42°C, than in WAF1, but follow the same
tendency. The lower differences may be caused by the
lower dynamic range of p53 binding to PUMA promoter.
Positive controls are over 9x the level of negatives in
WAF1, while just over 7x in PUMA.
Supplementary Information
Results
Influence of the Hsp90 and Hsp70 inhibition on the WT p53
nuclear mobility
To test effects of chaperone inhibition on general
aspects of WT p53 functionality, we analyzed its mobility
changes in cells using FRAP analysis. H1299 cells were
transfected with plasmids encoding for several variants of
p53-EYFP fusion proteins. Where indicated cells were
additionally transfected with the plasmid encoding for
Hsp70 K71S protein. Cells were analyzed microscopically
24 hours following the transfection. In the case of Hsp90
inhibition cells were treated with 1 μM 17-AAG for 60
minutes prior to imaging. ECFP-EYFP fusion protein was
used as a control, non-interacting protein of a molecular
weight similar to p53. Experiments were performed at
37ºC.
Fitted curves representing fluorescence recovery
are presented in figure Sup. 1. It is clear that not only the
nuclear mobility of any p53 variant is lower than this of
ECFP-EYFP but also that the kinetics of fluorescence
recovery is different. While p53 recovery curves are best
described by the two phase exponential association model
(with the exception of L344P variant), this of ECFP-EYFP
optimally fits one phase exponential model. The result is in
agreement with predicted model of interactions in the
nucleus where p53 interacts with chromatin in both specific
and non-specific manner, while ECFP-EYFP protein is only
subject to molecular sieving effect, resulting from relatively
cluttered nuclear environment. High mobility was also
expected in the case of p53 L344P (Sup. 1A), reflecting its
mostly monomeric quaternary structure as compared to the
other tested tetrameric p53 variants, that additionally results
in the decreased ability to specifically interact with DNA
(Davison et al., 1998). The mobility of p53 variants
changed by oncogenic DBD mutations (R249S and R273H)
is higher that this of WT p53, possibly indicating lower
stability of their interactions with immobile nuclear
components such as chromatin (Sup. 1A). R249S DBD
structure is disturbed, while R273H has the native DBD,
with only the arginine 273 responsible for direct DNA
contact changed (Bullock et al., 2000). Therefore its
increased mobility is very likely due to an impaired ability
to specifically interact with p53 target sequences in gene
promoters.
Upon treatment with 1 μM 17-AAG, the mobility
of WT p53 markedly increases, although it does not reach
the mobility of p53 R249S (Sup. 1B). A less pronounced
change is caused by the cotransfection of Hsp70 K71S,
where WT p53 becomes slightly more mobile in the
nucleus. Thus, at 37°C, a change in the activity of any of
Hsp70 and Hsp90 chaperone systems influences nuclear
mobility of WT p53, most likely reflecting the decrease of
its specific interactions with chromatin.
ATP-dependence of Hsp90 and Hsp70 in the chaperoning
of WT p53 in vitro
Reactions leading to WT p53 specific DNAbinding activity rescue in vitro by Hsp90 and Hsp70/Hdj1
at physiological and heat-shock conditions are ATP
dependent (Sup. 3A, EMSA performed as in main text).
ATPase activities of both chaperone systems at different
temperatures are shown in Sup. 3C and D. The lower ATP
requirement of Hsp90 is reflected in the overall lower
ATPase activity of this system, compared to the
Hsp70/Hdj1.
Materials and methods
Fluorescence Recovery After Photobleaching
For FRAP experiments H1299 cells were
transfected as for conformation IPs (see main text) and
imaged 24 hrs post-transfection.
Images were acquired with Leica DM IRE/TCS
SP2 confocal microscope equipped with full environment
control. Imaging was performed at 32x256 pixels, 5% 514
nm laser power and bleaching (circle of 2 μm diameter)
with one pass at 100% power of both 488 and 514 nm laser
lines. Beam expander was set to 1 and pinhole diameter to 3
AU. 5 pre-bleaching frames, 1 bleach frame and 60 postbleaching frames were acquired in bidirectional “fly” mode.
At least 10 cells for each experimental variant were imaged.
Fluorescence intensity in the bleached region was
corrected for total specimen bleaching. Recovery curves
were normalized and each set of replicates was fitted to one
or two phase exponential association equation with an R2 of
no less than 0.95. The differences between all curves shown
in Sup. 1 are statistically significant (larger than 95%
confidence intervals, not shown for the chart clarity).
PUMA promoter ChIP
The experiment was carried out as described for
WAF1 promoter ChIP in the main text, with PUMA-specific
starters used for RT-PCR. The starters were described
previously (Shi et al., 2007).
Protein purification
Human WT p53 and its oncogenic variants were
purified as described (Nichols and Matthews 2002), with
minor modifications. p53 proteins were overproduced from
the pT7-7-Hup53 vector (a kind gift from T. Hupp), in
BL21 RIL E.coli strain (mutations introduced by sitedirected mutagenesis).
The effect of Hsp90 and Hsp70 inhibition on the WT p53
binding to the PUMA promoter in cells
To extend the ChIP analysis to a promoter of proapoptotic, p53-dependent gene, we performed analysis of
1
Human Hsc70 (Hsp70-8) and Hdj1 (human DjB1)
were overexpressed and purified as described previously
(King et al., 2001). Human recombinant Hsp70 (Hsp70-1a)
was purified exactly as Hsc70, after overexpression in
BL21 RIL E.coli strain frrm pET11b-Hsp70 construct, a
kind gift from R. Morimoto.
MBP-His-Hdj2 protein (human DjA1) was
purified and tags removed as described (Kanazawa et al.,
1997), after overexpression from pMALc2x-hdj2 vector, a
kind gift from K. Terada.
Human MBP-Hsp90α was purified and the tag
removed as described previously (Walerych et al., 2004).
Human Hsp90β protein was purified as follows:
Human Hsp90 beta WT was overexpressed from pBAD24Hsp90β vector in BL-21 RIL E.coli strain at 20°C for 12
hours after induction with 10g/l of arabinose. Cells were
harvested by centrifugation at 8 000 g for 10 min and
frozen in liquid nitrogen. Bacteria pellet was lysed in buffer
A (50 mM Tris-HCl pH 8.0, 10% glycerol, 1 mM PMSF, 1
mM EDTA, 5 mM DTT) containing 1 mg/ml lysozyme for
1 hour at 4°C . The cell suspension was briefly heat
shocked in 42ºC water bath for 3 min, then sonicated with
10 2-s pulses. Afterwards the suspension was centrifuged
at 100 000 g for 1 hour at 4°C. Ammonium sulfate (till 30%
of saturation) was added slowly with stirring to the cleared
supernatant and the precipitate was separated by
centrifugation at 100 000g for 1 hour at 4°C. The pellet was
discarded and the supernatant was applied onto a ButylSepharose (Amersham) column equilibrated with buffer B
(50 mM Tris-HCl pH 7.6, 30% Ammonium sulfate, 2 mM
DTT, 1 mM PMSF). The protein which bound to the
column was eluted via gradient of decreasing ionic strength
and increasing glycerol concentration. The fractions
containing Hsp90 beta protein were pooled and loaded onto
a Hydroxyapatite (BioRad) column equilibrated with buffer
D (25 mM HEPES pH 7.6, 1.2 M KCl, 10% glycerol, 2
mM DTT, 1 mM PMSF). Bound proteins were eluted with
a 0-500 mM gradient of potassium phosphate (pH 7.2).
Peak fractions were pooled, dialyzed against buffer E (25
mM HEPES pH 7.6, 25 mM KCl, 10% glycerol, 2 mM
DTT) and applied onto linearly connected Heparin and
Rescue Q6 FPLC (Amersham) colums. The bound proteins
were eluted separately from each column by means of ionic
strength gradient (from 50 mM to 800 mM KCl in buffer
E). Fraction containing Hsp90 beta protein were pooled,
frozen in liquid nitrogen and kept at -80ºC for further
experiments.
After purification, all recombinant chaperones
were tested for the ability to participate in the luciferase
refolding assay, as described previously (Walerych et al.,
2004). This allows to make sure if activities of different
batches of purified chaperones are similar, before applying
them to p53 activity rescue reactions.
Plates were resolved in 1M LiCl: 1M HCOOH, dried and
spots corresponding to non-hydrolyzed ATP and free
phosphate were scanned for radioactive signals on STORM
Phosphoimager (Amersham) and quantified using
ImageQuant software (Amersham). All results were
corrected for the spontaneous ATP hydrolysis.
References:
Bullock AN, Henckel J, Fersht AR. (2000). Quantitative
analysis of residual folding and DNA binding in mutant p53
core domain: definition of mutant states for rescue in cancer
therapy. Oncogene 19: 1245-1256.
Davison TS, Yin P, Nie E, Kay C, Arrowsmith CH. (1998).
Characterization of the oligomerization defects of two p53
mutants found in families with Li-Fraumeni and LiFraumeni-like syndrome. Oncogene 17: 651-656.
Kanazawa M, Terada K, Kato S, Mori M. (1997). HSDJ, a
human homolog of DnaJ, is farnesylated and is involved in
protein import into mitochondria. J Biochem (Tokyo) 121:
890-895.
King FW, Wawrzynow A, Hohfeld J, Zylicz M. (2001).
Co-chaperones Bag-1, Hop and Hsp40 regulate Hsc70 and
Hsp90 interactions with wild-type or mutant p53. Embo J
20: 6297-6305.
Nichols NM, Matthews KS. (2002). Human p53
phosphorylation mimic, S392E, increases nonspecific DNA
affinity and thermal stability. Biochemistry 41: 170-178.
Shi X, Kachirskaia I, Yamaguchi H, West LE, Wen H,
Wang EW et al. (2007). Modulation of p53 function by
SET8-mediated methylation at lysine 382. Mol Cell 27:
636-646.
Walerych D, Kudla G, Gutkowska M, Wawrzynow B,
Muller L, King FW et al. (2004). Hsp90 chaperones wildtype p53 tumor suppressor protein. J Biol Chem 279:
48836-48845.
Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B.
(2001). PUMA induces the rapid apoptosis of colorectal
cancer cells. Mol Cell 7: 673-682.
ATPase assay
ATPase activity of Hsp90 chaperones was
measured as described earlier (Walerych et al., 2004).
ATPase of Hsp70/Hdj chaperones was measured
with radioactively labeled phosphate method. 5 µM Hsp70
with or without 2,5 µM Hdj1 was incubated in 50 µl buffer:
40 mM Hepes pH 7.5, 150 mM KCl, 5 mM MgCl2, 1 mM
ATP, 0,5 µCi [33P]γATP in 100 µl reaction buffer.
Reactions were carried out at 25°C-42°C. At time points 060 min 1 µl samples were spotted on PEI-cellulose plates.
2
Figure legends
Sup. 1 The influence of Hsp90 and Hsp70 on nuclear mobility of p53
H1299 cells were transfected with plasmids encoding for indicated proteins and 24 hours post-transfection subjected to the
FRAP analysis (description of the experimental procedure in Materials and methods).
A. Nuclear mobility of indicated p53 variants compared directly.
B. Influence of dominant-negative Hsp70 K71S overexpression or Hsp90 inhibition (1h of 1 µM 17-AAG treatment) on WT
p53 nuclear mobility.
Sup.2 p53 binding to the PUMA promoter upon Hsp70 and Hsp90 inhibition in H1299 cells.
Description as for Fig.2, main text, with PUMA replacing WAF1.
Sup. 3 ATP-dependence of p53 chaperoning reactions by Hsp90 or Hsp70-Hsp40 and their ATPase activity.
A. ATP dependence of Hsp90β and Hsp70-Hdj1 were compared by the incubation of indicated molecular chaperones with WT
p53 (protein concentrations as in main text, Fig. 3), in the presence of 0, 5 and 7 mM of ATP, at temperatures and time periods
that allow to distinguish specific activities of both chaperones (see main text, Fig.3).
B. The Hsp90β ATP hydrolysis assay (ATPase assay; see Materials an methods) was performed at indicated temperatures for
90min. Means presented as bars and standard deviation values were calculated from 3 repeats of the measurement.
C. The Hsp70 and Hsp70-Hdj1 ATP hydrolysis assays were performed at indicated temperatures for 60 min. Error bars are
calculated from results of 3 repeats of the measurement.
3