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Supplemental Figure Legends
Supplemental Figure 1. BubR1 binds to PCAF but not p300 or CBP.
Plasmids encoding HA-p300, FLAG-CBP, or FLAG-PCAF were co-transfected into
293T cells with plasmids encoding Myc-BubR1. Forty-eight hours after transfection,
cell lysates were subjected to IP with 9E10 and WB analysis with the antibodies
indicated. CBP and PCAF were expressed as FLAG-fusion proteins, and an anti-FLAG
antibody was used to detect CBP and PCAF. Only PCAF was detected in BubR1
immunoprecipitates.
Supplemental Figure 2. Validation of PCAF localization at kinetochores after
siRNA of PCAF. (A, B) HeLa cells were treated with siRNA oligonucleotides against
either GFP or two different PCAF siRNAs. After 48 h, the cells were arrested at
prometaphase by nocodazole (200 ng/ml) treatment for 2 hr and chromosome spreads
were prepared. Metaphase chromosome spreads were pre-extracted and fixed with 4%
PFA. The cells were then immunostained with anti-PCAF and anti-BubR1 antibodies.
CREST was used as centromeric kinetochore markers. The result confirmed the
kinetochore localization of PCAF with BubR1. Furthermore, when PCAF was
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eliminated, BubR1 signal was greatly reduced, indicating that the maintenance of
adequate amounts of BubR1 at prometaphase kinetochores requires PCAF association.
(C) Knockdown of PCAF expression was confirmed by WB using anti-PCAF antibody
(Santa Cruz, H-369). As revealed in the anti-BubR1 blot, knock-down expression of
PCAF resulted in reduced levels of BubR1.
Supplemental Figure 3. BubR1 interacts with Class I HDACs.
Plasmids encoding Myc-BubR1 and FLAG-tagged HDACs 1-6 were cotransfected into
293T cells. Forty-eight hours after transfection, cell lysates were subjected to IP and
WB as indicated. The results show that BubR1 is capable of interacting with HDAC1-3.
Supplemental Figure 4. Mapping the acetylation domain in BubR1.
(A) Schematic illustration of various BubR1-deleted constructs used for mapping the
acetylation site. (B) Wild-type full-length BubR1 (BubR1 FL) or BubR1 deletion
mutants (10 μg each) were co-expressed in 293T cells with 10 μg of the PCAF
expression plasmid as indicated. The acetylated region of BubR1 was determined by IP
with an anti-Ac-K antibody and WB analysis using 9E10 (left panel). The right panel
represents the reciprocal IP and WB analyses as indicated. The N-terminal deletion
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mutants ΔBR1, ΔBR2, and ΔBR3 (amino acids 1–514), as well as full-length BubR1,
were acetylated (asterisks). Similar amounts of each BubR1 fusion protein were
expressed, as shown by the WB analysis of total cell lysates with 9E10 (input, bottom
panel).
Supplemental Figure 5. Mass spectrometry of full length BubR1 purified from
insect cells.
Mass spectrometry of full length BubR1 purified from insect cells before and after in
vitro acetylation utilizing the PCAF HAT (352–832). Before acetylation, mass
spectrometric analysis gave a peak at 1197 Da. This peak is shifted to 1239 Da after in
vitro acetylation with PCAF, indicative of acetylation (+42 Da) at K250.
Supplemental Figure 6. Lysine 250 is the unique acetylation site of BubR1.
(A) In vitro acetylation assays of BR2-1~5 (the region defined to contain acetylated
sites in BubR1) purified from E. coli were performed using 0.05 Ci of [1-14C] AcetylCoA and separated by SDS-PAGE. The gel was dried and exposed to the image plate to
detect radioactivity using a BAS 2500 phosphor Imager (Fujifilm). (B) Sequence
alignment of the acetylated △BR2-3 region. The △BR2-3 region has 6 lysine residues,
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which are marked. Four of these lysine residues (K231, K233, K235, and K250) are
highly conserved in vertebrates. (C) Each lysine was substituted with arginine and
subjected to in vitro acetylation assays with or without active PCAF and then separated
by SDS-PAGE. WB using an anti-Ac-K antibody indicated that every △BR2-3 mutant
except K250R was acetylated, as is the wild-type. These results were also confirmed by
in vitro acetylation assays using [1-14C] Acetyl-CoA. (D) To confirm that K250R is the
acetylated site, the BR2-3 K250R mutant was subjected to in vitro mutagenesis to
destroy other lysines. These five double mutants of △BR2-3 were subjected to in vitro
acetylation assays. WB with anti-GST revealed that similar amounts of proteins were
used in this assay.
Supplemental Figure 7. Validation of anti-Ac-K250 antibody.
(A) BubR1 purified from insect cells was subjected to in vitro acetylation with or
without activated PCAF and probed via WB with the indicated antibodies to assess
whether anti-Ac-K250 polyclonal antibodies specifically recognize the acetylated form
of BubR1. (B) 293T cells were transfected with the indicated plasmids, treated with
nocodazole and MG132 to prevent degradation of BubR1 in mitosis, and subjected to IP
with 9E10 to verify whether anti-Ac-K250 antibodies detect ectopically expressed
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BubR1. The result shows that anti-Ac-K250 antibodies detect Myc-BubR1 and –K250Q
but not –K250R, indicating that K250Q mimics the acetylated BubR1 and K250R is
deficient in acetylation in vivo. To verify the anti-Ac-K250 antibody, we detected the
blot with the anti-Ac-K250 antibody and re-probed with an anti-Ac-K antibody. (C)
TSA treatment stabilizes the levels of wild-type BubR1, but not the levels of BubR1K250R. HeLa cells were transfected with Myc-tagged BubR1 or K250R, the
acetylation-deficient mutant.
The cells were then synchronized by nocodaozole
treatment and mitotic shake-off. Nocodazole treated cell lysates were subjected to
western blot analysis of BubR1.
Wild-type BubR1 levels decrease in mitosis (+CHX,
compare 0 h with 2 and 5 h), and are elevated by treatment with TSA (+TSA and TSA +
CHX).
In contrast, K250R is barely detectable after 2 h of nocodaozole treatment.
Therefore, the levels of K250R are not stabilized by treatment with TSA.
Supplemental Figure 8. Effects of BubR1 acetylation on localization and protein
levels in different mitotic stages. (A) Plasmids encoding the acetylation-defective form
(K250R), acetylation-mimic form (K250Q), and wild-type form of BubR1 tagged with
Myc were transfected into HeLa cells and co-immunostained with anti-tubulin
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antibodies and 9E10 to discriminate endogenous BubR1. The level of the acetylationdefective form (K250R) was significantly lower at kinetochores in mitosis than that of
wild-type BubR1 under the same exposure and contrast conditions. The level of the
acetylation-mimic form (K250Q) was significantly higher at kinetochores throughout
mitosis. Photographs represent merged images of 20 consecutive 0.5-μm optical
sections taken at 1,000 X magnification using DeltaVision RT (AppliedPrecision). (B)
Supporting data for Figure 6C.
treatment with MG132.
K250R intensities at kintochores increased after
HeLa cells were transfected and treated as in Figure 6C.
Five hours after monastrol teatment, cells were treated with MG132 (+MG132).
Cells
were then fixed and co-stained with 9E10, anti-alpha-tubulin antibodies (-Tub), and
CREST antibodies.
Chromosomes were stained with DAPI before taking pictures.
Supplemental Figure 9. BubR1 acetylation does not affect Bub3 binding.
293T cells were transfected with BubR1-GFP- and Myc-Bub3-encoding plasmids and
subjected to IP with 9E10. Rabbit serum was used as a negative control. The result
indicates that the binding of Bub3 to BubR1 is not affected by the acetylation status of
BubR1. This result suggests that the kinetochore localization of BubR1 is not directly
affected by BubR1 acetylation.
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Supplemental Figure 10. BubR1 depletion results in shortened mitotic timing and
premature entry into anaphase.
The HeLa cells stably expressing histone H2B-GFP (HeLa-H2B) were transfected with
synthetic siBubR1 targeting the 3’UTR and subjected to time-lapse microscopy.
Compared with control untransfected HeLa cells (Control, Supplemental movie 4), the
timing of entry into anaphase was premature. Also noteworthy is that the chromosomes
segregated without proper alignment at the metaphase plane (siBubR1, Supplemental
movie 5).
Supplemental Figure 11. BubR1 acetylation regulates mitotic progression.
In order to explain further the data described for mitotic timing in relation to
fluorescence intensity in Figure 7, two different methods of plotting were employed.
(A) Cumulative frequency plots of NEBD to anaphase onset times in siBubR1 and
DsRed-tagged BubR1 cotransfected cells, as described by Sorger and colleagues
(McAinsh et al., 2006). NEBD=T0, as determined from live-cell movies.
(B) Time
from NEBD to anaphase onset was measured for each cell and plotted as a percentage
of cells, as described by Wolthius and Pines (Wolthuis et al., 2008).
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Supplemental Figure 12. BubR1 is ubiquitinated by APC/C. HeLa cells were
cortansfected with siRNA to APC3 or control and DsRed-BubR1 encoding plasmids.
BubR1 levels were analyzed by fluorescence intensity measurements, coupled with
mitotic timing by time-lapse microscopy, using Image J software (National Institute of
Health). The cells were analyzed are representatives of 6 different cells in two
independent experiments. The result shows that BubR1 is destroyed by APC/C
immediately before the onset of anaphase (Supplemental movie 7).
Supplemental Figure 13. Western blot analysis of the protein levels in mitosis in the
presence of CHX.
(A) HeLa cells were pre-synchronized in S phase by 2 mM
thymidine and subsequently arrested at prometaphase by 200 ng/ml of nocodazole.
Prometaphase-arrested cells were collected by mitotic shake-off and then washed. Cells
were subsequently released for mitotic progression in the presence of CHX, and lysates
were collected at the indicated time points. The levels of BubR1, acetylated BubR1 (AcK250), Cdc20, Cyclin B, CENP-E, Aurora A, Plk1, and Mad2 were analyzed by WB.
(B) Graph representing the levels of proteins at indicated time points, as shown in (A).
Band intensities were measured using Multi Gauge software (Fujifilm).
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Supplemental Figure 14. BubR1 acetylation inhibits D-box- or KEN box-mediated
destruction of BubR1. (A) Schematic illustration of BubR1 constructs used for the
assay in (B) D-box and two KEN boxes (KEN1 and KEN2) are marked. To destroy the
degrons, RXXL of the D-box was substituted with AXXA and KEN was substituted
with AAA, respectively, in each construct. (B) Wild-type, K250R, and K250Q
expression constructs, tagged with Myc at their N-termini, were in vitro mutagenized to
delete the KEN boxes (KEN1KEN2) or the D-box (D-box) in all three BubR1
constructs. HeLa cells transfected with the constructs indicated were synchronized at
prometaphase by treatment with 200 ng/ml nocodazole, followed by mitotic shake-off.
Cells were then released into the cell cycle in the presence of cycloheximide (CHX) to
avoid de novo protein synthesis. Lysates were prepared at the time points indicated and
subjected to WB with the antibodies indicated. The same blots were reprobed with antiLamin A/C antibodies as loading controls. Because arrest and synchronization of HeLa
cells after elimination of endogenous BubR1 is not possible, WB in these experiments
was carried out in the presence of endogenous BubR1. Therefore, the blot only shows
the level of ectopically expressed BubR1. (C) In order to confirm that BubR1 is
destroyed after prometaphase, which was not clear in (B), endogenous BubR1 was
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depleted from HeLa cells with siRNA and Myc-BubR1 encoding plasmids were
transfected. Then the cells were synchronized at prometaphase by nocodazole treatment
and mitotic shake-off and released in the presence of CHX. Lysates were prepared at the
indicated time points after release and subjected to WB with 9E10 or anti-actin
antibodies.
Supplemental Figure 15. Yeast Mad3 is not acetylated. (A) Schematic drawings of
the constructs used. Full length (Mad3_FL), N-terminus (Mad3p_N), and C-terminus
(Mad3p_C) Mad3 were fused to GST and purified from E. coli. Mad3 and Bub1
homology regions 1 and 2 are marked. (B) Recombinant proteins were subjected to in
vitro acetylation assays with or without purified PCAF enzyme. ΔBR2-3 of BubR1, the
domain that is acetylated, was included as a control. Ponceau staining in the lower panel
reveals that similar amounts of proteins were loaded for each assay. After the assay, the
samples were loaded onto a gel for SDS-PAGE and subjected to WB with anti-Ac-K
antibodies. The result shows that Mad3 is not acetylated, whereas ΔBR2-3 is. The
higher molecular weight bands detected in all lanes, where PCAF was included, are
autoacetylated PCAF (refer to Fig. 3).
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References
McAinsh AD, Meraldi P, Draviam VM, Toso A, Sorger PK (2006) The human
kinetochore proteins Nnf1R and Mcm21R are required for accurate chromosome
segregation. EMBO J 25(17): 4033-4049
Wolthuis R, Clay-Farrace L, van Zon W, Yekezare M, Koop L, Ogink J, Medema R,
Pines J (2008) Cdc20 and Cks direct the spindle checkpoint-independent destruction of
cyclin A. Mol Cell 30(3): 290-302
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Supplemental movies
Movies
Supplemental Movie 1. Timing of mitosis in WT-expressing cells after depletion of
endogenous BubR1.
DsRed tagged BubR1-WT and siBubR1 (3'UTR) were co-expressed in HeLa cells
stably expressing H2B-GFP.
Live images were captured every 5 minutes.
Supplemental Movie 2. Timing of mitosis in K250R-expressing cells after
endogenous BubR1 depletion.
Supplemental Movie 3. Timing of mitosis in K250Q-expressing cells after
endogenous BubR1 depletion. Chromosomes do not segregate for the recorded times
and experience death.
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Supplemental Movie 4. Timing of mitosis in a normal HeLa cell.
Supplemental Movie 5. Timing of mitosis in a BubR1-depleted cell.
Supplemental Movie 6. Timing of mitosis in a cell with BubR1 destroyed of KEN1
and KEN2 [BubR1_KEN1,2 (AAA)]. Note that chromosomes segregate in only 15
minutes from NEBD but the red fluorescence remains even after chromosome
segregation.
Supplemental Movie 7. Timing of mitosis in wild-type BubR1 expressing cells after
endogenous BubR1 and APC3 was depleted with siRNA. Chromosomes do not
segregate in recorded times (5 hours).
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