Supplementary data
SI Figure 1
A) Multiple amino acid alignment of DP-1 domains and its orthologues in different
species. Amino acid alignment of the DNA binding and dimerization regions of DP-1
is shown across a number of species. Identical residues are shaded in black and
similar residues are shaded in grey. Residues that form dimerization contacts are
shown in red squares, DNA backbone contacts are shown in green circles and DNA
base contacts are shown in blue stars. The residues that are altered by somatic
mutation are highlighted within the human sequence in red. Residues required for
dimerization and DNA binding were taken from Zheng et al 21.
B) Representation of the distribution of DP-1 mutations in tumour derived cell lines and
tissues of origin. Graph detailing the number of DP-1 mutations in different tissues
types. There are 53 mutation events in total that result in either missense, nonsense or
frame-shift mutations within the DP-1 coding region. 24 DP-1 mutations were
identified in the CCLE from 947 cell lines examined. The COSMIC database details
29 DP-1 mutations from 5396 tumours screened.
C) Table detailing the DP-1 mutations and the tumour/cancer cell line in which they were
identified, together with disease stage data where available (n/a= information was not
available). Note that it cannot be excluded that some identified mutations in the
CCLE arise in the cell lines.
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D) cDNA sequence chromatogram comparing HCT15 and HCT116 across the DP-1
cDNA sequence encoding D273 is shown. The heterozygous D273N substitution in
HCT15 is highlighted.
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SI Figure 2
A) DP-1 mutations generated for further analysis. Site-directed mutagenesis was
performed on FLAG-DP-1 to generate the 30 mutants shown in the schematic. Nterminal mutants (N) are shown in red, DNA binding domain mutants (DBD) are
highlighted in green, dimerization domain mutants (DD) are highlighted in blue,
pRb binding domain mutants (RBD) are highlighted purple and C-terminal
mutants (C) highlighted in black.
B) U2OS cells were transfected with FLAG-tagged DP-1 or mutant derivatives in the
presence and absence of HA-tagged E2F-1 as indicated. 48h post-transfection,
cells were fixed and immunostained with HA and FLAG antibodies. DAPI was
used to visualise nuclei.
C) Table detailing the effect of DP-1 mutation on E2F-1 activity in various
transfection-based reporter assays. U2OS cells were transfected with expression
vectors encoding WT E2F-1, WT DP-1 or mutant derivatives, together with the
indicated luciferase reporter construct and pCMV-βgal to monitor transfection
efficiency. 48h post-transfection relative luciferase activity was measured and the
ability of DP-1 to activate transcription when co-expressed with E2F-1 was
assessed. The results from this analysis are summarised as either same/similar
transcriptional activation to wild-type DP-1 (y=E2F-1-dependent transcription is
activated) or reduced ability to activate transcription (n=E2F-1-dependent
transcription is impaired).
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D) U2OS cells were transfected with expression vectors encoding WT E2F-1, WT
DP-1 or mutant derivatives, together with either WT E2F–luciferase or mutant
E2F-luciferase and pCMV-βgal to monitor transfection efficiency. 48h posttransfection, relative luciferase activity was measured and the ability of DP-1 to
activate transcription when co-expressed with E2F-1 was assessed.
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SI Figure 3
A) Overview of the DNA binding segment of the E2F-DP-1 heterodimer. DNA double
helix is indicated in light/dark yellow, E2F as light blue ribbon diagram, DP-1 as
dark grey ribbon diagram. Residues G115 and R168 as indicated through inclusion
of their side-chains. Figure is based on crystal structure (21, PDB1cf7)
B) Environment of G115. Residue atoms are indicated as light spheres against the
phosphate backbone of the DNA indicating surface contacts between DP-1 and
DNA. G115 lies at the beginning of the helical structure (dark grey) of DP-1.
C) Hydrogen bonding between NH2 of the R168 side-chain and the O6 atom of a
guanine base in the DNA strand.
D) The hydrophobic pocket at the dimerisation interface between DP-1 and E2F.
Residue V175 is highlighted in red, other residues are labelled according to their
atoms.
E) Residue environment of I165 showing the hydrophobic interior of DP1 and vicinity
to F119, L142 and F146.
F) Recruitment of pRb to the Cdc6 and DHFR promoters in HCT15 and HCT116 cells.
Chromatin was extracted and immunoprecipitation was performed using antibodies
against pRb or appropriate non-specific IgG. The binding of pRb to the Cdc6 and
DHFR promoters was assessed by real-time quantitative PCR.
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SI Figure 4
A) FACs profiles for Figure 5A. Wild-type DP-1 or the indicated mutant derivatives,
together with E2F-1 were transfected into U2OS cells. 120h post-transfection
cells were analysed by flow cytometry.
B) HeLa cells were transfected with the indicated siRNA duplexes for 72h. 56h later,
cells were treated with doxorubicin (2M) for 16h, harvested and analysis of
cleaved PARP was performed by immunoblotting.
C) HeLa cells were transfected with FLAG-DP-1 or mutant derivatives, 48h later
cells were treated with doxorubicin (2M) for 16h and subsequently analysed by
immunoblotting with antibodies against cleaved PARP, FLAG (DP-1) and actin.
D) HeLa cells were transfected with FLAG-DP-1 or mutant derivatives, 48h later
cells were treated with doxorubicin (2M) for 16h and subsequently immunoblot
analysis of cleaved PARP was performed. The results of the analysis are
summarised in the table.
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SI Table 1
MUTATION
A2T
A5P
G6V
S23T
P48fs
T66A
V77A
G113S
G115D
G150D
I165M
R168H
V175M
K187del
I189M
N204D
E208*
E225Q
A243S
A243T
V271A
V271F
D273N
C274Y
D295N
D295Y
I297T
K320fs
N366I
G378R
PRIMER (5’-3’)
GCC GCG AAT TCA ATG ACA AAA GAT GCC GGT CTA
AAT TCA ATC GCA AAA GAT CCC GGT CTA ATT GAA GCC AAC
TCA ATG GCA AAA GAT GCC GTT CTA ATT GAA GCC AAC GGA
ATA GAC CAG AAC CTT ACT CCC GGG AAA GGC GTG
GGG AAG CAG CTC TTG CACA AAA ACC TTT GGA CAG TCC
CAA GTG GTA ATT GGT GCG CCT CAG AGA CCG GCA
GCA GCG TCA AAC ACC CTG GCG GTA GGA AGC CCA CAC ACC C
AAA GAA GAG AAG AAT AGC AAG GGC CTA CGG CAT
GA GAG AAG AAT GGC AAG GAC CTA CGG CAT TTC TCC ATG
GAG TTC AGT GCT GCC AAC AAC CAC ATC TTA CCA
TAT GAC CAG AAA AAC ATG AGA CGG CGC GTC TAC
G AAA AAC ATA AGA CGG CAC GTC TAC GAT GCC TTA AAC G
TAC GAT GCC TTA AAC ATG CTA ATG GCC ATG AAC
ATC ATC TCC AAG GAG AAG GAG ATC AAG TGG ATT GG
AAG GAG AAG AAG GAG ATG AAG TGG ATT GGT CTG
GCT CAG GAA TGT CAG GAC TTA GAG GTG GAA AGA
CAG AAC TTA GAG GTG TAA AGA CAG AGG AGA CTT
CAG TCT CAA CTT CAA CAA CTT ATT CTA CAG CAA
CAG AGA AAC CGG CAT TCG GAG CAG CAG GCC AGC
CAG AGA AAC CGG CAT ACG GAG CAG CAG GCC AGC
ACC AGC AAG AAG ACG GCC ATC GAC TGC AGC ATC
ACC AGC AAG AAG ACG TTC ATC GAC TGC AGC ATC
AAG AAG ACG GTC ATC AAC TGC AGC ATC TCC AAT
AAG ACG GTC ATC GAC TAC AGC ATC TCC AAT GAC
ACA TTT GAA ATC CAC AAT GAC ATA GAA GTG CTG
ACA TTT GAA ATC CAC TAT GAC ATA GAA GTG CTG
GAA ATC CAC GAT GAC ACA GAA GTG CTG AAG CGG
TGC TCT GCC GAA GAC CTT AA ATG GCC AGA AGT CTG GTC
GCC AGT GAC CTG ACC ATC GGT GCA GAT GGG ATG
GCC ACA AGC TCC AAT CGG TCT CAG TAC AGC GGC
SI Table 1 Primers used to generate DP-1 mutants shown in SI Figure 2A
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