Post on 06-Jul-2020
transcript
GO term # of genes% PValue
response to extracellular stimulus 50 2.243158 7.35E-06
response to nutrient 35 1.570211 2.79E-05
response to nutrient levels 44 1.973979 4.35E-05
cellular response to stress 99 4.441454 5.22E-05
regulation of apoptosis 132 5.921938 6.07E-05
regulation of programmed cell death 132 5.921938 9.57E-05
regulation of cell death 132 5.921938 1.13E-04
cell cycle 126 5.652759 1.48E-04
response to DNA damage stimulus 69 3.095559 1.57E-04
programmed cell death 103 4.620906 1.59E-04
positive regulation of apoptosis 77 3.454464 1.84E-04
death 118 5.293854 2.10E-04
apoptosis 101 4.53118 2.20E-04
positive regulation of programmed cell death 77 3.454464 2.33E-04
cell death 117 5.248991 2.39E-04
positive regulation of cell death 77 3.454464 2.66E-04
Supplemental Table 1. GO terms of doxorubicin responsive genes reverted by all three siDINo siRNAs.
0
2
4
6
8
10
12
- + - + - + - +
H1299 control H1299 + P53 H1299 + P53V272C
H1299 + P53R273H(dn)
Fo
ld ∆
Do
x/c
trl
0
5
10
15
20
25
30
- 16 h 22 h - 16 h 22 h
MDA-MB SW636
Fold
∆do
x/Ct
rl DINOCDKN1A
DINOCDKN1A
dox
0
5
10
15
20
25
30
0 4 8 12 16 20 24
fold
∆ D
ox/C
trl
U20S
doxorubicin (hours)
A
B C
Supplementary Figure S1a. RT-PCR of DINO across a time course of DNA damage in U20S cell line.b. RT-PCR of DINO in H1299 p53 null cells complemented with indicated p53 variants.c. RT-PCR of DINO in cancer cell lines with known p53 mutations.
- +
dox
p53
KO
w
ildty
pe
Supplementary Figure S2(A) Genome browser snapshot of 3-seq in wildtype or p53 null MEFs +/- doxorubicin. Red box indicates region syntenic to human DINO. (B) Genomic context of zebrafish CDKN1A. Grey bars indicate PCR primer amplicons.(C) RT-PCR of TP53 and CDKN1A in zebrafish embryos treated with UV at 18 hpf and harvested at 24 hpf. (D) RT-PCR of regions along the zebrafish CDKN1A promoter +/- UV. Letters correspond to grey bars in (B).
PhastCons Fish Conservation
1 _
0 _
500 bp
zebrafish CDKN1A
0
0.5
1
1.5
2
2.5
- +
CDKN1A
+UV/
-UV
zebrafish CDKN1A
zebrafish TP53
UV0
1
2
3
4
5
6
7
8
- + - + - + - + - + - + - + - + - + - + - +
a b c d e f g h i j k
+UV/
-UV
UV
A
B
D
k j i h g f e d c b a
zDINO
C
-+-+-+
-+-
+
mouse CDKN1A
Inpu
tIP
-IgG
IP-p
53
p53LSD1
MDM2SET7
WB:
Supplementary Figure S4Immunoprecipitation of p53 followed by western blot of known interacting proteins LSD1 and MDM2. Identical conditions did not detect SET7.
00.0050.01
0.0150.02
0.0250.03
0.0350.04
0.0450.05
Norm
aliz
ed E
xpre
ssio
n
CDKN1A
00.010.020.030.040.050.060.070.080.090.1
Norm
aliz
ed E
xpre
ssio
n
RRM2b
0
0.5
1
1.5
2
2.5
Norm
aliz
ed E
xpre
ssio
n
DDB2
Supplementary Figure S5293 Cells transfected with vector, DINO, or two DINO fragments (0-300 and 450-951) missing the CLIP hairpin. RT-PCR for p53 target genes
SET7
DINO LACZ
lysine methyl
silver stainSET7TAF10
Supplementary Figure S3Under identical enzyme/substrate/RNA conditions as Fig. 3G, no TAF10 methylation was detectable via anti lysine methyl antibody (Abcam Ab23366) (detects TAF10 methylation in Antigone, 2004) or anti pan-methyl Lysine antibody (Abcam Ab7315).
siCTRL siSET7
- + - +SET7
B-actin
02468
10121416
- + - +
siCTRL siSET7
Rela
tive
expr
essi
on
CDKN1A
0
0.2
0.4
0.6
0.8
1
1.2
- + - +
siCTRLsiSET7
Rela
tive
expr
essi
on
SET7
0
1
2
3
4
5
6
7
- + - +
siCTRL siSET7
Rela
tive
expr
essi
on
TP53a b
c
0
0.5
1
1.5
2
2.5
- + - +
siCTRL siSET7R
elat
ive
Exp
ress
ion
TP53
0
1
2
3
4
5
6
7
8
- + - +
siCTRL siSET7
Rel
ativ
e E
xpre
ssio
n
CDKN1A
0
0.2
0.4
0.6
0.8
1
1.2
1.4
- + - +
siCTRL siSET7
Rel
ativ
e E
xpre
ssio
n
SET7c
dox dox dox
dox dox dox
Supplementary Figure S6a. Western blot of knockdown by SET7 siRNA in U20S cells. b. RT-PCR of indicated genes in U20S cellsc. RT-PCR of indicated genes in primary human fibroblasts