Mapping statistics of WGBS analysis and bisulfite conversion efficiency. .... Analysis of non-CpG methylation confirmed that bisulfite conversion efficiencies were ...
1.5
0.25 ng
165 ng
2
3
Tet2/3 DKO
Tet3 KO
Spleen (4 wk after injection)
N.D.
5 μg
Standard
Tet2/3 DKO
Tet3KO
Tet2KO
WT
Tet2/3 DKO
Tet3KO
0.5 ng
e
5 μg
0.5 ng
4
Two-fold serial dilutions
LSK CMP GMP MEP 2 1.5 anti-5hmC Anti-5hm
anti-5hmC Anti-5h
anti-5hmC
1
2
3
N.D.
0
N.D.
0.5
N.D.
1 N.D.
Tet3 mRNA levels (relative to Gapdh)
Tet2 KO
anti-CMS
Two-fold serial dilutions
1
N.D.
N.D.
N.D.
0
Tet2KO
d
0.8
WT
1.2
Standard
1.6
0.4
WT
anti-CMS
Tet2/3 DKO
anti-CMS
Bone marrow Spleen (2 wk after injection)
Tet3KO
LSK CMP GMP MEP
Two-fold serial dilutions
4
Tet2KO
3
165 ng
WT
2
0.25 ng
Two-fold serial dilutions
0.5 1
Standard
Tet2/3 DKO
Tet3 KO
Tet2 KO
WT
c
1
0
Tet2 mRNA levels (relative to Gapdh)
Tet2/3 DKO
DKO
Tet3 KO
WT
2
Tet2 KO
b
WT
Tet1 mRNA levels (relative to Gapdh)
a
Standard
Supplementary Fig. 1
4
LSK CMP GMP MEP
Spleen Spleen (4 wk after injection) (2 wk after injection)
Bone marrow (4 wk after injection)
Supplementary Figure 1. Loss of genomic 5hmC in hematopoietic cells of Tet2/3 DKO mice. (a) Expression of Tet1, Tet2 and Tet3 in sorted LSK and myeloid progenitor cells. Cells were isolated from bone marrow of WT (Tet3fl/fl) or DKO (Tet2-/- Tet3fl/fl Mx1-Cre+) mice at 2 weeks after pIpC administration by flow cytometry, and quantitative RT-PCR was performed. The relative levels of mRNAs after normalization to the level of Gapdh mRNA in the same cell population are shown, with the amount in the WT LSK cells arbitrarily set to 1. N.D., not detected. (b-e) Quantification of 5hmC levels in cells from Tet2/3 DKO mice. (b, c) At two (b) or four (c) weeks following pIpC administration, 5hmC levels in the bone marrow or spleen were quantified by dot blot assay with anti-CMS antibody after treatment of genomic DNA with bisulfite. A synthetic oligonucleotide with a known amount of CMS was used as standard. (d, e) Top panels, At two or four weeks following pIpC administration, 5hmC levels in the spleen (d) or bone marrow (e) were quantified by dot blot assay with anti-5hmC antibody (top panels). Bottom panels, Methylene blue staining was used to monitor equivalent DNA loading. A synthetic oligonucleotide with a known amount of 5hmC was used as standard.
Supplementary Fig. 2
Hematocrit (%)
***
*
*
**
*
**
40 20
7
14 28
Days post-injection
nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l O
0
Pre 0/2
7
Pre 0/2
7
14 28
Days post-injection
Platelets/μl (X 106)
0 nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l O
Pre 0/2
14 28
Pre 0/2
7
14 28
Days post-injection
Days post-injection
g 60
*** ***
0.5 500
Co
14 28
f *
0
Days post-injection
Co
Hemoglobin (g/dL)
e
*
*
h
15
**
**
**
*
**
10 5 0
Pre 0/2
7
14 28
Days post-injection
50 40 30 20
**
10
** *
** *
0 nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l O
Days post-injection
7
1
Lymphocytes/μl (X103)
Pre 0/2
**
2
***
10001
Co
14 28
3
nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l O
nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l O
7
0 Co
Co
Pre 0/2
10
1500 1.5
*
nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l O
0
*
4
Basophils/μl (X103)
5
20
d
Co
10
*
30
Red blood cells/μl (X106)
15
c
Co
**
20
Eosinophils/μl (X103)
b
WT DKO
nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l Co O nt ro DK l O
Monocytes/μl (X103)
a
Pre 0/2
7
14 28
Days post-injection
Supplementary Figure 2. Hematopoietic cell numbers in peripheral blood of WT and Tet2/3 DKO mice. (a-g) Time-course analysis of peripheral blood cell counts. Tet2/3 DKO mice developed progressive monocytosis (a), eosinophilia (b), basophilia (c), thrombocytopenia (d) and anemia (e-g). (h) There was a slight decrease in lymphocyte numbers in the peripheral blood of DKO mice after pIpC injection although the difference at day 28 was not statistically significant (n = 7~10 per each genotype at each time point examined). Means ± SEM are shown. *P < 0.05, **P < 0.005, ***P < 0.0005 (Student's t test).
Supplementary Fig. 3
b
c ** **
50
0/2
DK O
rl ct 4w
k
rl ct 1w
on m 02
k
ct
rl
0 PI
28
DK O
25 o
DK O
rl ct
75
4w
7
Days post-injection
d
100
Percentage
Days post-injection
k
rl 1w
an gr
0/2
2
28
0/
7
DK O
ct
ct
rl
DK O
0
4w
k
DK O
rl ct k
25
PI
0/2
C
0/2
D
50
D
D
11
1w
b+
ctr
l
0
DK O
25
***
75
ct
50
Mac-1+Gr-1-
***
k
Percentage
75
***
100
DK O
***
l
***
ro
***
100
Percentage
Mac-1+Gr-1+
D
Mac-1+
DK O
a
7
28
Days post-injection
e B220+CD19+
DK O
rl ct k 4w
DK O
rl 1w
ce T
4w
ct
0
k
DK O
rl ct k
rl ct k
Days post-injection
02
28
PI
7
WT DKO
10
D
02 PI D
0/2
***
20
B
1w
ce
ll
ct
rl
0
DK O
20
***
DK O
40
30
rl
Percentage
60
***
40
***
ct
***
ll
***
DK O
Percentage
80
CD4+/CD8+
0/2
7
28
Days post-injection
Supplementary Figure 3. Expansion of myeloid lineage cells with decrease in lymphoid lineage cells in Tet2/3 DKO mice. (a-e) Cells in the peripheral blood of WT or Tet2/3 DKO mice were isolated at the indicated time points following pIpC injection and flow cytometric analysis of myeloid (a-c), B-cells (d) and T-cells (e) was performed (n = 7~10 mice per time point examined). Means ± SEM are shown. **P < 0.005, ***P < 0.0005 (Student's t test).
Supplementary Fig. 4
Supplementary Figure 4. Infiltration of livers and lungs of Tet2/3 DKO mice with hematopoietic cells. (a) Enlargement of livers in Tet2/3 DKO mice. Representative photographs of liver from WT and Tet2/3 DKO mice at 4~5 weeks following pIpC injection. Weights of liver at 2~2.5 or 4~5 weeks after pIpC injection were shown at bottom (n = 7~13 per each genotype). Means ± SEM are shown. **P < 0.005, ***P < 0.0005 (Student's t test). (b,c) Hematoxylin and eosin staining of livers at 2~2.5 (b) or 4~5 (c) weeks after pIpC injection. Shown are the loss of normal liver structure and hematopoietic cell infiltration into the livers of Tet2/3 DKO mice. (d) Myeloid cell infiltration into livers of Tet2/3 DKO mice verified by myeloperoxidase staining at 2~2.5 weeks after pIpC injection. (e,f) Histological analysis showing hematopoietic cell infiltration in the lung. Hematoxylin and eosin staining of lungs at 2~2.5 (e) and 4~5 (f) weeks after pIpC injection. For all figures, top panels, 4X magnification; bottom panels, 40X magnification.
Supplementary Fig. 5
Supplementary Figure 5. Histological analysis of bones and spleens. (a,b) Hematoxylin and eosin staining of bones at 2~2.5 (a) or 4~5 (b) weeks after pIpC injection. Note anaemic bones and abnormal morphology of megakaryocytes in Tet2/3 DKO mice. Top panels, 20X magnification; bottom panels, 40X magnification. (c,d) Histological analysis of spleens at 2~2.5 (c) or 4~5 (d) weeks after pIpC injection by hematoxylin and eosin staining. Note disruption of normal splenic structure in Tet2/3 DKO mice. Top panels, 4X magnification; bottom panels, 40X magnification. (e) May-Grünwald-Giemsa-staining of bone marrow aspirates. Note the relatively homogeneous cell population in the DKO sample.
Supplementary Fig. 6
a
b WT DKO
Liver (2.5 week after injection)
104
Mac-1
17.9 0 102
105
53.5 103
104
105
53.2
103 102 0 0 102
103
104
105 104
: CD8a
7.51
103 102 0
75 103
104
CD4
: CD4
104
DK O
o M on
l Ctr
Ctr l
CD8+
105
0.152
4
10
103 102 0
17.2
0 102
103
CD4+
: B220
0.245 105 2.16
4
10
CD8
: CD8a
105
B220+ CD19+
0 102
: B220
***
20 0
102 0
105
B220
3.05
103
*
40
B-
104
: CD19
CD19
: CD19
: CD11b: CD11b 105
***
60
DK O
102 0
10.6 103
DK O
1+
103
Ctr l
0 102
Mac-1+ Gr-1-
104
CD 8+
86.8
Mac-1+ Mac-1+ Gr-1+
DK O
2
28.6
CD 4+ -C on tro l
103 10 0
FSC-A
DK O
Gr-1
104
0
50K 100K 150K 200K 250K
2.6 105 4.22e-3
0
: Gr1
105
FSC-A FSC
ac
0
20 M
0 50K 100K 150K 200K 250K 0
l
0
: Gr1
21.4
50K
**
40
DP
1.01
50K
60
l
100K
DK O
150K
***
Ctr
100K
Percentage
SSC-A
SSC-A
150K
80
Ctr
200K
SSC
200K
***
100
DKO 250K
Percentage
WT 250K
105
92.3 0 102
5.43 103
104
: CD4
105
Supplementary Figure 6. Expansion of myeloid lineage cells and reduction of lymphoid cells in the liver of Tet2/3 DKO mice. (a) Flow cytometry was performed to assess myeloid (Gr-1/Mac-1), B lymphoid (B220+ CD19+) and T lymphoid (CD4/CD8) cell populations in the liver of WT or Tet2/3 DKO mice at 2~2.5 weeks after pIpC injection. Hematopoietic cells were gated as shown in the top panel. Note expansion of FSChi and SSChi cells that normally correspond to myeloid cells in the liver of DKO mice. (b) Summary of the percentage of cell populations shown in a (n = 4 per each genotype). Means ± SEM are shown. *P < 0.05, **P < 0.005, ***P < 0.0005 (Student's t test).
Supplementary Fig. 7 Bone marrow
200K
200K
200K
200K
100K
150K 100K
50K
FSC-A FSC
c
Bone marrow
DKO
Ter-119lo
Ter-119+
CD71+
CD71+
2
0 10
3
10
4
10
B220
: B220
5
10
2.37 2
0 10
3
10
4
10
: B220
5
10
ra
D K O
M on
o
C trl
D K O
n
C
trl
D K O
rl ct b+
G
11 D
3×1037
5 0
B220+
D K O
D K O
o
trl C
M on
n ra G
C trl
***
O
4~5 wk
***
2×1027 1×1017
ce
ll
ct
rl
D K O
rl ct P
D K O
C trl
C D 71 +T er 11 9lo
D K O
***
Te r1 19 +C on tro l
0 0
K D
10
O
102 0
14.6
***
K
102 0
0.86 8.0×10
0.0 0
15
-B
103
KO
103
***
0.46 4.0×10
***
20
D
104
C on tro l-B
Percentage
: CD43
: CD43
104
2~2.5 wk
***
Control-DP DKO-DP
D
: Ter-119
C
C
Ter-119+
Control
D K O
11 b
D
K DKO
Mac-1+ Gr-1-
rl
105
Control
Mac-1+ Gr-1+
ct
104
1.27 1.2×10
10
Mac-1+
ll
103
e
20
105
CD43
105
0 102
0 0
***
30
0
1×1017
ce
Ter-119
: Ter-119
40
2×1027
B
105
***
3×1037
rl
104
Mac-1+ Gr-1-
ct
103
Mac-1+ Gr-1+
D K O
0.112
0
4~5 wk
P
2
10 4.52
0 102
2~2.5 wk
D
0.245
5×1057
D K O
10 103
0 0
C trl
102 0
*
C D 71 +T er 11 9lo
103
104
50
C
on
C
: CD71
104
0.235
0.0306
Erythroid cells in BM (%) Percentage
5
10 21.6
2.02
CD71
B
Ter-119+ Ter-119lo Ter-119+ B220+ CD71+ CD71+ D
: CD71
105
*
D K O
Mac-1+ D
105
: Mac-1 : Mac-1
+
11 b
104
+
18 103
11 b
0 102
l-C
Mac-1
6.01
D
105
D
104
D
103
tro
5.93
0 102
4×1047
4×1047
0 l-C
49.7
***
25
2
10 0
FSC-A FSC
1×1017
***
50
O -C
10
2
50K 100K 150K 200K 250K
3×1037
75
tro
10
3
on
Gr-1
104
3
FSC-A
d
***
100
K
74.1
Percentage
: Gr-1
: Gr-1
42.1105 1.86
104
50K
2×1027
DKO
2.27
FSC-A
100K
0 50K 100K 150K 200K 250K 0
WT DKO
(4~5 weeks after injection)
WT
0 50K 100K 150K 200K 250K 0
0
150K
rl
b
FSC-A
100K 50K
0 50K 100K 150K 200K 250K
150K
ct
FSC-A
0 50K 100K 150K 200K 250K 0
FSC-A
b+
0 50K 100K 150K 200K 250K 0
50K
50K 100K 150K 200K 250K
200K
11
0
FSC-A
D
0
100K
50K
50K
200K
150K
100K
Absolute cell number (X107) in bone marrow
50K
150K
FSC-A
200K
SSC-A
100K
SSC-A
SSC-A
150K
FSC-A
200K
150K
0 50K 100K 150K 200K 250K 250K 0
SSC-A
FSC-A
200K
0 50K 100K 150K 200K 250K 250K 0
0 250K 0
50K 100K 150K 200K 250K
100K 50K
50K
SSC-A
0 50K 100K 150K 200K 250K 250K 0
150K
+G r1 D C + 11 on b+ tro G l -C r1 D + 11 D K b+ O G -C r1 D 11 b+ G r1 -
FSC-A
200K
100K
50K
50K 0 50K 100K 150K 200K 250K 250K 0
150K
Absolute cell number (X107) in bone marrow
100K
SSC
100K
150K
O -C
SSC-A
100K
150K
SSC-A
200K
150K
SSC-A
200K
SSC-A
250K
SSC-A
250K
0
10 0
Blood
250K
250K 0
105
Spleen
250K
WT
Blood
250K
50K
SSC-A
Spleen
250K
SSC
SSC-A
Bone marrow
4~5 weeks after injection
DKO
2~2.5 weeks after injection
Te r1 19 +C on tro l
a
Supplementary Figure 7. Dominance of myeloid cells with reduction of erythroid cells and B cells in the bone marrow of Tet2/3 DKO mice. (a) Expansion of FSChi and SSChi cells in Tet2/3 DKO mice. Relative heterogeneity (WT, top) or homogeneity (DKO, bottom) of cells in bone marrow, spleen and blood, as assessed by separating cells according to their side or forward scattering properties. (b) Representative flow cytometry data assessing myeloid (Gr-1/Mac-1), erythroid (CD71/Ter-119) and B lymphoid (B220/CD43) cell populations in bone marrow of WT or DKO mice at 4~5 weeks after pIpC administration. (c) The frequency of cells shown in b. Means ± SEM are shown. **P < 0.005, ***P < 0.0005 (Student's t test). (d,e) Absolute numbers of myeloid (d) or erythroid and lymphoid (e) cell subsets in bone marrow of WT or DKO mice at 2~2.5 or 4~5 weeks after pIpC injection. Means ± SEM are shown. *P < 0.05, **P < 0.005, ***P < 0.0005 (Student's t test).
Supplementary Fig. 8
b
Spleen (4~5 weeks after injection) DKO 12.9105 1.09
105
2
10
102 0
103 102
42.2
5.87e-3
104 103
0 10
10
4
10
3
0 10
4
10
10
B220+
5
10
: CD4
10
2
CD4+
CD8+
2
2.0×10 8
2.0×10 8
2~2.5 wk
*
1.5
4~5 wk
** 1.5
1.5×10 8
1.5×10 8
1.0×10 8
1
1.0×10 8
0.5
5.0×10 7
*
0.5
5.0×10 7
0
KO
8c trl
D
C
D
D
C
ll
D
ce
B
o
M on
G
ra
n
Mac-1+ Mac-1+ Mac-1+ B220+ CD4+ Gr-1+ Gr-1-
CD8+ D
KO
4c trl
rl ct
KO
DK O
trl C
trl C
DK O
rl ct
DK O
b+ 11 D
rl ct
DK O
DK O
CD8+ CD 8
C
D
8
C D 4
CD4+ CD 4
rl
KO
ct
Gr-1-
B220+ D
B
Mac-1+
B
trl C
o
M on
Gr-1+
DK O
trl C
DK O
DK O
ra
n
Mac-1+ Mac-1+ G
b+
ct
rl
0.0
11 D
*
0
0.0
C
**
1
C
Absolute cell number in spleen (X108)
20
2.46 2
10
CD4
***
30
0
0
5
: CD4
40
102
36.7 96.3 3
***
***
trl
: CD8
104
50
105
4-c
104
CD
103
: B220
0.28 105 1.24
2
c
0 102
CD 4-D KO
B220
20.8
CD8
: CD8
: B220
105
B-D KO
104
B-C
103
Percentage
0 102
0
Mac-1+ Mac-1+ Mac-1+ Gr-1+ Gr-1-
103
0
105
11
104
trl
103
2.06
CD
34.3
104
10 0
105
: CD19
: CD19
CD19
105
20
m on o-C trl
104
8-c
Mac-1
103
: Mac1 : Mac1
m on o-D KO
0 102
5
10
CD
4
10
30
CD 8-D KO
3
10
16.3
trl
5.57 2
0 10
68.7
-c
78.9
102 0
40
trl
0
103
DP
102
50
DP -D KO
103
13.8
104
Percentage
4
10
b-c trl
: Gr1
Gr-1
: Gr1
WT 2.64
105
WT DKO
CD 11 b-D KO
a
Supplementary Figure 8. Expansion of myeloid cells and reduction of lymphoid cells in the spleen of Tet2/3 DKO mice. (a) Representative flow cytometry data assessing myeloid (Gr-1/Mac-1), B lymphoid (B220/CD19) and T lymphoid (CD4/CD8) cell populations in spleens of WT or Tet2/3 DKO mice at 4~5 weeks after pIpC administration. (b) The frequency of cells shown in a. Means ± SEM are shown. *P < 0.05, ***P < 0.0005 (Student's t test). (c) Absolute numbers of myeloid and lymphoid cell subsets in spleens of WT or Tet2/3 DKO mice at 2~2.5 (left) or 4~5 (right) weeks after pIpC injection. Means ± SEM are shown. *P < 0.05, **P < 0.005, ***P < 0.0005 (Student's t test).
Supplementary Fig. 9
b
WT DKO
0
DK O
l ntr co LS k
4 wk
4
4×105
2
2×105
LK k 2w
ntr ol
2 wk
LK -D KO
-C
on
KO
LK D
D KO C on tro l-L K
C on tro l-L SK
0 4w kLK -C o
0
0
LSK
**
6×105
DK O
1
6
l
2
KO
4w
LS k 2w
Number of LK cells (X105)
3
D
K-
C K-
KO
D
*
C on tro l-L in -
2 wk
8
8×105
4
Lin-
DK O
0.0
-L
K l-L C
D
on
3
3.0×104
LK
on
LSK
C
Percentage in bone marrow Percentage of cells
5
tro
KO
l-L
-L
SK
0
SK
20
6
6.0×104
K
***
9
9.0×104
tro
40
1.2×105
l
**
12
tro
60
Number of LSK cells (X104)
80
on
WT DKO 2 weeks after injection
tro
Percentage
- cells Percentage cells among Linwithin ofLin
a
4 wk
Weeks after injection
c
Sorted LSK cells
WT (Tet2+/+ Tet3fl/fl) mice or
or 1st
Tet2-/- Tet3fl/fl ERT2-Cre+ mice
0.75
0.75
0.5
0.5
N.D.
1
1
0.25 0
1
2
Tet3
120
0.25 0
1
Control DKO
Number of colonies / 103 LSK cells
2
1.25
2
DKO
1
Tet2
WT
0
1.25
DKO
2
0.25
DKO
1
DKO N.D.
0.5
0.25 0
Tet3
0.75
0.5
4th
f
After 2nd round of culture
WT
0.75
1
e mRNA levels relative to Gapdh
Tet2
WT
1
3rd
Serial replating in methylcellulose in the presence of 1 μM 4-hydroxytamoxifen
After 1st round of culture
WT
mRNA levels relative to Gapdh
d
2nd
90 60 30 0
1 2 3 4 Number of replatings
Supplementary Figure 9. Altered distribution of hematopoietic stem and progenitor cells upon acute deletion of Tet3 in Tet2-deficient mice. (a) Frequency of LSK (Lin- c-Kit+ Sca1+) and LK (Lin- c-Kit+ Sca1-) cells within Lin- populations (top) or in the total bone marrow (bottom) of WT and Tet2/3 DKO mice at 2 weeks after pIpC injection. Means ± SEM are shown. *P < 0.05, **P < 0.005, ***P < 0.0005 (Student's t test). (b) Absolute numbers of LSK and LK cells in bone marrow of WT and Tet2/3 DKO mice at 2 or 4 weeks after pIpC injection. Means ± SEM are shown. **P < 0.005 (Student's t test). (c) Experimental scheme for colony-forming assay in methylcellulose medium. LSK cells were sorted from bone marrow of WT (Tet3fl/fl) or DKO (Tet2-/- Tet3fl/fl ERT2-Cre+) mice by flow cytometry (n = 4 per each genotype). One thousand cells were serially replated in methylcellulose medium (MethoCult M3534, Stem Cell Technologies) and colonies were counted after 7-9 days. To induce ERT2-Cre recombinase, 1 μM 4-hydroxytamoxifen was added to the cultures. (d,e) Confirmation of Tet2 and Tet3 deletion. Colonies after the first (d) or second (e) round of culture were pooled and quantitative RT-PCR analysis was performed to confirm germline deletion of Tet2 and ERT2-Cre-mediated deletion of Tet3. (f) Combined deficiency of Tet2 and Tet3 leads to increased serial replating capacity in vitro. Colony forming unit assay shows that Tet2/3-deficient, but not WT, LSK cells can be serially replated in methylcellulose medium.
Supplementary Fig. 10
0
75 50 25
2.48
103 2
10 0 3
104
34.8
103
102 0
12.1 3
10 Flt3
104
: c-Kit
51.1
c-Kit
104
100K
4.71e-3
100K
Flt3
103
103
104
CD34 CD34 :
50K 100K 150K 200K 250K
Hoechst Indo-1 (Blue)-ARed
Indo-1 (Blue)-A
*
DKO
103
104
trl -c
D KO
P
P
trl -c
D KO
P
LM
P
ST -D KO
M
D KO
trl
105
: Sca1
105 104
56.3 9.97
103 102 0
32.7 0 102
103
104
: Flt3
105
104 103
LT 27.8
ST 68.9
102 0
105
105
0 102
103
104
: CD34
105
105
104 103
102 0
50 0 102
WT
78.5
102 0 0 102
0 50K 100K 150K 200K 250K0
0.020 0.015 0.010 0.005 0.000
19.3
: Flt3
150K
10
CD48
Side population (%)
0 102
3
4
10 10 CD150
105
104
CD48-CD150+
103 102 0
: CD150
4.44 0 102
103
104
CD34-Flt3- gated
0
2
10 0
105
4
50K
50K 0
103
105
: CD48
0.0134
: Flt3
200K
Hoechst-A
200K
: CD48
Hoechst Blue
250K
LSK 19.4
104
Flt3-/lo gated
250K
ST- Flt3mid Flt3hi HSC (MPP) (LMPP)
105
105
105
DKO
LTHSC
LSK gated
: c-Kit
D KO
ST
-c
trl
D KO
trl -c LT
105
: Flt3
WT
0
DKO
104
10 Sca-1
CD34+ (ST-HSC)
0
105
WT
0 102
150K
104
: Sca1
Ratio of ST-HSC to LT-HSC - WT: 6.517 ± 1.655 - DKO: 4.397 ± 0.756
e
103
: CD34
-c
0 102
105
0 102
CD34(LT-HSC)
Hoechst-A
5
10
LT
4
10
: c-Kit
: c-Kit
100
3
10
CD34 : CD34
f
WT DKO
0
0 10
c-Kit
D KO
-C SC H
d Percentage within LSK Flt3lo/- cells
2
l tro
DKO
on
WT
*
1
1×104
i-C
102 0
2
2×104
Flt 3h
77.7
D KO
20.5
*
3
3×104
trl
ST
-c
102 0
103
LT
id
82.5
4
4×104
ST -D KO
1
1×103
16.4
104
0
105
105
104 103
104
3m
: Flt3
2
2×103
103
: Flt3
Flt
3
0 102
105
ST -C trl
104
105
3×103
0
103
Flt3Flt3 :
*
25
rl
0 102
LMPP
10 0
ct
5
10
**
4
10 0
50
T-
4
10
11.3 2
***
S
3
10
: CD150
10
39.7
2
75
LT -C trl
2
0 10
4×103
Number of LT-HSCs (X103)
LT-HSC
5
10
3
**
LT -D KO
4
10
10
100
trl
3
10
CD150 : CD150
10
10.6
LT -D KO
2
0 10
2.47
3
76.6
Percentage within LSK cells
8.48
10
4
WT DKO
Absolute cell number (X 104)
102 0
9.87
DKO MPP
105
Flt3lo/- gated
102 0
: c-Kit
3
10
49.9
: Flt3
3
10
4
c-Kit
LSK gated
104
CD48
104
105
LSK gated
WT
105
: CD48
105
c
4~5 weeks after injection
DKO : c-Kit
WT : CD48
b
4~5 weeks after injection
Flt3
a
105
: CD150
Supplementary Figure 10. Altered distribution of hematopoietic stem/progenitor cells upon acute deletion of Tet3 in Tet2-deficient mice.
Supplementary Figure 10. Altered distribution of hematopoietic stem/progenitor cells upon acute deletion of Tet3 in Tet2-deficient mice. (a) Acute deletion of Tet3 in Tet2-deficient mice decreases the number of SLAM-marked hematopoietic stem cells (LSK CD150+ CD48-). LSK cells in the bone marrow from WT and DKO mice were analyzed for surface expression of CD48 and CD150. Means ± SEM are shown. **P < 0.005 (Student's t test). (b) LSK cells in bone marrow of WT and Tet2/3 DKO mice were analyzed for surface expression of c-Kit and Flt3, after which the Flt3-/lo fraction was further analyzed based on CD34 expression. CD34- and CD34+ cells in the lower panels represent LT- and ST-HSCs, respectively. (c) Percentage (upper panel) and number (lower panel) of LT-HSCs, ST-HSCs, MPPs and LMPPs in WT and DKO mice. Means ± SEM are shown. *P < 0.05, ***P < 0.0005 (Student's t test). (d) Deficiency of Tet2 and Tet3 results in only a slight decrease in the ratio of ST-HSC to LT-HSCs. (e) Hoechst staining and flow cytometric analysis of the bone marrow of WT and DKO mice. The boxed region in the upper panel indicates the percentage of side population cells, and summary of the results (mean ± SEM) is shown in the lower panel. (f) Increased frequency of CD48+ CD150- population within LT-HSCs (CD34-Flt3- LSK) in DKO compared to WT mice.
Supplementary Fig. 11
b
M C
CMP
D
-C
trl
KO
KO
EP
D
GMP
KO D
0
MEP
c
MEP
d 105
FSC FSC-A
WT
2
0 10
3
10
4
10
CD27 : CD27
5
10
2×1024
0 10
3
10
4
10
5
10
IL-7Ra : IL-7Ra
KO
4~5 wk D
w
k-
C
KO
2~2.5 wk
-4
2
trl
00 D
4.59
102 0
1×1014 trl
103
C
104
w
102 0
105
-2
103
104
: IL-7Ra
k-
103
***
3×1034
LP
50K 100K 150K 200K 250K
105
4.9
104
0 102
5
10
*
Weeks post-injection C
0
4
10
4×1044
LP
0.724
2
3
10
: CD27
Flt3
103
5
10
16
2
10 0
WT DKO
C
FSC-A
104
10 0
2
0 10
50K 100K 150K 200K 250K
: Flt3
0 105
102 0
3
10
Number of CLP cells (X104)
Lin-
: Flt3
3.29
102 0
103
CLP
104
Flt3
103
11.2
104
: Flt3
: Flt3
104
DKO
105
5
10
Lin/CD11c
: Lin/CD11c/7AAD : Lin/CD11c/7AAD
M
G M PC trl
trl
25 trl
O
50
Co
nt ro
l-M
GMP
***
75
DK
O
EP
DK
P
O Co
nt ro l
CMP
Co
nt ro
l-C
M
P
0.00
4~5 wk
-C
0.25
MEP
*
100
KO
**
0.50
GMP
D
0.75
CMP
EP
WT DKO
0
M
: CD34
105
G M PC trl
104
KO
103
D
0 102
D
8.17
1.00
DK
Percentage in bone marrow
CD34 : CD34
105
C
104
P-
103
25
M
0 102
47
CMP
MEP
***
50
C
40.4
102 0
2~2.5 wk ***
75
trl
32.1
-G M
102 0
103
100
C
103
GMP
4
10
Percentage of cells among Lin-
: FcgR
4
10
31.7
LK gated
19.110
10
FcγRII/III
: FcgR
DKO 5
WT DKO
P-
WT 5
Percentage within LK cells Percentage of cells among Lin-
2~2.5 weeks after injection
KO
a
Supplementary Figure 11. Distribution of myeloid and lymphoid progenitors in the bone marrow. (a) Representative flow cytometric analysis of myeloid progenitor cell subsets in the bone marrow of WT and Tet2/3 DKO mice at 2~2.5 weeks after pIpC injection, as assessed by the expression of FcγRII/III and CD34 within LK population (top). Percentage of each myeloid progenitor in bone marrow is shown at bottom. Means ± SEM are shown. **P < 0.005 (Student's t test). (b) Frequency of myeloid progenitor cell subsets within LK populations of WT and Tet2/3 DKO mice at 2~2.5 or 4~5 weeks after pIpC injection. Means ± SEM are shown. *P < 0.05, ***P < 0.0005 (Student's t test). (c) Gating strategy to identify common lymphoid progenitor (CLP) cells in bone marrow. CLPs are defined as Lin- Flt3+ CD27+ IL-7Rα+. (d) Absolute number of CLPs in the bone marrow of WT and Tet2/3 DKO mice at 2~2.5 or 4~5 weeks after pIpC injection. Means ± SEM are shown. *P < 0.05, ***P < 0.0005 (Student's t test).
Supplementary Fig. 12
a
Tet2-/- Tet3fl/fl Mx1-Cre+
WT
or Testers (CD45.2+)
Chimerism 4 wk
WT vs
+
Bone marrow
Blood
200K
200K
200K
SSC-A
SSC-A
250K
SSC-A
250K
150K
150K
150K
100K
100K
100K
50K
50K
50K
0 250K 0
0 50K 100K 150K 200K 250K 250K 0
FSC-A
0 50K 100K 150K 200K 250K 0 250K
FSC-A
200K
SSC
150K
100K
100K
100K
50K
50K 0 0
0 50K 100K 150K 200K 250K 0
FSC FSC-A
FSC-A
SSC-A
150K
150K
50K 100K 150K 200K 250K
200K
SSC-A
200K
SSC-A
Spleen
250K
WT
b
DKO
Competitors (CD45.1+)
DKO
pI-pC i.p.
Recipients (CD45.1+)
50K 0 50K 100K 150K 200K 250K 0
FSC-A
50K 100K 150K 200K 250K
FSC-A
Supplementary Figure 12. Competitive engraftment assay and tumor transfer assay. (a) Experimental scheme for competitive repopulation assay. CD45.2+ bone marrow cells from Tet2+/+ Tet3fl/fl Mx1-Cre- (WT) or Tet2-/- Tet3fl/fl Mx1-Cre+ (DKO) mice were mixed with equal number of CD45.1+ competitor cells and transplanted into lethally irradiated CD45.1+ congenic mice. At 4 weeks after transplantation, chimeric mice were injected with pIpC five times intraperitoneally (week 0) and peripheral blood was examined for donor chimerism at the indicated time. (b) FSChi and SSChi cells in the recipients of WT and DKO splenocytes. Relative heterogeneity (WT, top) or homogeneity (DKO, bottom) of cells in the bone marrow, spleen, blood, as assessed by separating cells according to their side or forward scattering properties.
Supplementary Fig. 13
b
Splenocyte transfer (Tet2fl/fl Tet3fl/fl Mx1-Cre+)
Percent survival
Percent survival
100
c
d
WT DKO
WT
80
150 100 50 0 WT
40
Days post pIpC injection Days post-injection
0 WT
DKO
g
15.9 3.23
0 102
103
104
: Mac1
105
3.91e-3
10
3
10
103
104
27.5105 0.859
4
10
1.77103
2
2
10
0 2
0 10
3
10
4
0
5
10
10
2.41 39.2105 : Ter119
CD71
4
10
0.596
10
1.59 : CD71
5
10
: CD71
4
10
3
10
0.25
0.157 2
0 10
3
10
10
102 0 2
0 10
3
10
4
10
4
Ter-119
: Ter119
0.279
102
5
10
0 102
10 0
18.3 103
104
: Mac1
Mac-1
2
105
9.33 0 102
83.4 103
104
: Mac1
105
102 0 5
Lin
10
43.4
2
10
0 10
102 0 2
3
4
4
10
103 2
10 0
4
10
67.1 0 102
6.09e-3
3
10
102 0 2
10
0 10
3
10
104
CD34
: CD34
4
5
10
: Sca1
10
1.6
4
10
103 102 0
18.1 103
5
10
LK
23.3
5.92105
5
10
4
10
5
5
10 10 Sca-1
: Sca1
3
10
: c-Kit
10
0.488
103
Lin-
48.8
105
84.7 0 102
2.69 103
104
: CD34
105
5
10
1.14
56.1
103
3
10
10 10 27.1 6.73 : Ter119
1.33103
3
4
2
4
0 10
7.3
10
3 4 10 10 c-Kit
105
105
4
3.11 2
10.8 104
10 0
4
10
: c-Kit
102
103
2
0 10
1.42
: Mac1
103
105
103
0 102
4
10
5
10
0
Spleen
: CD71
: CD71
2.59
Bone marrow
WT 5
103 10 0
104
104
105
: Gr1
DKO
103
: Mac1
25.6 105 0
0
4
10
Gr-1
: Gr1
: Mac1
105
0 102
13.20 87.8
74.8 0 102
105
94.9
11.9 105 7.78e-3
4
102 0
102 0
: Lin
44.6
WT DKO : Gr1
WT DKO
102 0
103
DKO
105
: c-Kit
WT
3
10
104
Bone marrow
0
DKO
104
WT 1.83
: FcgR
WT
39.5 105 1.95e-3
Spleen
0
0.5
0
Blood
0.2
1
5
10
Spleen
DKO : Gr1
0.4
1.5
: Gr1
0.6
WT
***
2
Liver weight (g)
0.8
DKO
i
: Gr1
***
DKO
WT
h
Spleen weight (g)
f
50
: Lin
30
c-Kit
20
100
: c-Kit
10
e
*
FcgRII/III
0
150
LK gated
20
DKO
Lin- gated
Neutrophils /μl (X103)
DKO
: FcgR
40
10
DKO
WT
60
0
j
***
200
WBCs /μl (X103)
a
0.149 2
0 10
3
10
4
10
: Ter119
5
10
Supplementary Figure 13. Transfer of splenocytes from Tet2/3 DKO mice leads to myeloid leukemia in secondary recipients.
Supplementary Figure 13. Transfer of splenocytes from Tet2/3 DKO mice leads to myeloid leukemia in secondary recipients. (a) Kaplan-Meier curve representing the percent survival of recipient mice transplanted with 2 x 106 splenocytes from WT (Tet2fl/fl Tet3fl/fl) and diseased Tet2/3 DKO (Tet2fl/fl Tet3fl/fl Mx1-Cre+) mice (n = 10 per each group). (b) May-Grünwald-Giemsa-stained peripheral blood smears of recipient mice. (c) Recipients of Tet2/3 DKO splenocytes developed progressive leukocytosis with neutrophilia. Means ± SEM are shown. *P < 0.05, ***P < 0.0005 (Student's t test). For a summary of other hematopoietic parameters, see Supplementary Table 2b. (d) Enlargement of spleens of mice that received Tet2/3 DKO splenocytes. (e) Representative photographs of femurs and tibiae from recipients of WT or Tet2/3 DKO splenocytes. (f) Weights of spleen or liver. Means ± SEM are shown. ***P < 0.0005 (Student's t test). (g) Hematoxylin and eosin staining of livers show loss of normal liver structure and infiltration with hematopoietic cells. 4X magnification. (h) A representative flow cytometric analysis of myeloid-lineage cells (Gr-1+/Mac-1+) in the bone marrow, spleen and blood of recipient mice. (i) Increase in the frequency of Lin- populations in the spleen from recipients of Tet2/3 DKO splenocytes. (j) A representative flow cytometric analysis of erythroid-lineage cells (Ter-119+/CD71+) in the bone marrow and spleen of recipient mice.
Supplementary Fig. 14
10
20
30
40
3
10
102 0
51.8
1
0 10
2
3
10
4
g
105
10
3.25
103
72.2 0 102
103
104
i
103
67.9 104
Mac-1
0.584
7.83
103 2
10 0
103 102 0
0.191
1.54 0 10
3
10
4
10
2.68
10
4
10
103
103
104
: Ter119
30.9105 15.3
CD71
1.87
0 102
5
: Ter119
12.2
10 0
1.11
102 0
0.723 0 102
103
104
105
Ter-119 : Ter119
103 102 0
105
60
4.98 103
104
: Mac1
105
* ***
20
91.1 100
4
10
103
105
*
40
0
52.4 0 102
102 0
***
25
4.71 0 102
4.12 103
104
: Mac1
105
***
75 50
***
25 0
105
4
103
104
***
50
15.4
10
7.06
2
10
41.4
***
75
0
5
10
100
Bone marrow
9.67
4
10
10
4
DKO
Spleen
10
2
: CD71
: CD71
4
105
DKO 26.8105 0.972
1.17
: CD71
: CD71
WT 5
0 10
3
: Mac1
5.61 103
: Mac1
10
2.12 2
10
: Gr1
Gr-1
: Gr1
10
0 102
15.6
24.6105 0.0563
1.88
4
102 0
102 0
13
: Mac1
105
10
11.6105 1.27
104
102 0
3
5
: Mac1
WT DKO
104
3.7
2
0
WT DKO
104
78.4
Spleen
0.5
WT
DKO : Gr1
1
20 0
43105 3.88
1.46
e
***
Blood
2
1.5
105
: Gr1
1
2
WT
: Gr1
0.4 0.3 0.2 0.1 0
h
***
: Gr1
***
Liver weight (g)
f
Spleen weight (g)
Days post-injection
80 60 40
DKO
W TM ac 1+ D K O -M ac 1+ W TTe r1 19 D K + O -T er 11 9+ W TC D 19 D + K O -C D 19 W + TC D 4/ D C K D O 8 -C D 4/ C D 8
0
WT
W TM ac 1+ D K O -M ac 1+ W TC D 4/ C D 8 D K O -C D 4/ C D 8 W TC D 19 + D K O -C D 19 +
0
Neutrophils /μl (X103)
25
***
W TM ac 1+ D K O -M ac 1+ W TTe r1 19 D K + O -T er 11 9+ W TC D 19 D + K O -C D 19 W + TC D 4/ D C K D O 8 -C D 4/ C D 8
DKO
100 75 50 25 0
Percentage (bone marrow)
50
WT DKO
Percentage (spleen)
75
d
Percentage (blood)
WT
c
Bone marrow
100
Percent survival
b
Bone marrow transfer (Tet2fl/fl Tet3fl/fl ERT2-Cre+)
WBCs /μl (X103)
a
0.0838 0 102
103
104
: Ter119
105
Supplementary Figure 14. Transfer of bone marrow cells from Tet2/3 DKO mice leads to myeloid leukemia in secondary recipients.
Supplementary Figure 14. Transfer of bone marrow cells from Tet2/3 DKO mice leads to myeloid leukemia in secondary recipients. (a) Kaplan-Meier curve representing the percent survival of recipient mice transplanted with 2 x 106 nucleated bone marrow cells from WT (Tet2fl/fl Tet3fl/fl) and diseased Tet2/3 DKO (Tet2fl/fl Tet3fl/fl ERT2-Cre+) mice (n = 5 per each group). (b) May-Grünwald-Giemsa-stained peripheral blood smears of recipient mice. (c) Recipients of Tet2/3 DKO bone marrow cells developed progressive leukocytosis with neutrophilia. Means ± SEM are shown. ***P < 0.0005 (Student's t test). For a summary of other hematopoietic parameters, see Supplementary Table 2c (d) Enlargement of spleens of mice that received Tet2/3 DKO bone marrow cells. (e) Representative photographs of femurs and tibiae from recipients of WT or Tet2/3 DKO bone marrow cells. (f) Weights of spleen or liver. Means ± SEM are shown. ***P < 0.0005 (Student's t test). (g) Hematoxylin and eosin staining of livers show loss of normal liver structure and infiltration with hematopoietic cells. 4X magnification. (h) A representative flow cytometric analysis of myeloid-lineage cells (Gr-1+/Mac-1+) in the bone marrow, spleen and blood of recipient mice. Summary of the percentage of each cell population is also shown in the right panel. (i) A representative flow cytometric analysis of erythroid-lineage cells (Ter-119+/CD71+) in the bone marrow and spleen of recipient mice.
Supplementary Fig. 15
WT
40
0
20
40
Days post-injection
0.1 0
1
1 0.5 0
2
WT DKO
1
0.357
3
10
55.2 0 102
2
WT DKO
g
50 0
30 105 0.0442
104
102 0
100
3.25
104 3
10
102 0
14.4 4.93 103
104
WT
105
0 102
103
104
105
: Mac1 : Mac1 105
0.0668
5.06 105 0.1
4
10
103 102 0
86.7 0 102
4.49
4
10
103
104
105
*** 100
***
75
***
50 25 0
100
***
***
75
***
50 25 0
102 0
8.18 27.2 103
DKO
91.8
Spleen
0.2
1.5
105
e
***
DKO : Gr1
0.3
2
WT
: Gr1
Liver weight (g)
0.4
h
**
: Gr1
2.5
***
: Gr1
0.5
Spleen seight (g)
f
150
0 102
W TM ac 1+ D K O -M ac 1+ W TTe r1 19 D K + O -T er 11 9+ W TC D 19 D + K O -C D 19 W + TC D 4/ D C K D O 8 -C D 4/ C D 8
0
Neutrophils /μl (X103)
DKO
20
DKO
W TM ac 1+ D K O -M ac 1+ W TTe r1 19 D K + O -T er 11 9+ W TC D 19 D + K O -C D 19 W + TC D 4/ D C K D O 8 -C D 4/ C D 8
60
Percentage (bone marrow)
Percent survival
WT DKO
80
200 150 100 50 0
d
***
Percentage (spleen)
WT
100
c
Bone marrow
b
Mac-1+ cell transfer (Tet2fl/fl Tet3fl/fl Mx1-Cre+)
WBCs /μl (X103)
a
68.2 103
104
105
: CD71
33.5 105
10
0.259
0.0802
10
10
102 0
1.35
103 102 0
0.533 0 102
103
104
105
0.326Ter119 18.9105 :
CD71 104
0.284 0 102
103
104
102 0 103
104
2.12
103 102 0
105
Ter-119
: Ter119
104
105
0 D K O -M ac 1+ TC D 4/ C D 8 D K O -C D 4/ C D 8 W TC D 19 + D K O -C D 19 +
103
W
0 102
1+
105
ac
104
25
M
103
Mac-1
82.3
T-
10 0
13.7 7.2
50
W
2
***
105
104
1.3 0 102
72.2
10
***
75
1.56 Ter119 5.2 :
2.61
103
10 0
3
: Mac1 : Mac1
4
4.78
3
105
: CD71
2.41
4
2
0 102
DKO
: CD71
: CD71
105
10
104
Bone marrow
WT
3
***
100
Spleen
i
104
9.61
Percentage (blood)
14.1 105 0.89
Blood
0.0186
: Gr1
105
Gr-1
: Gr1
: Mac1 : Mac1
0.804 0 102
103
104
: Ter119
105
Supplementary Figure 15. Transfer of Mac-1+ cells from Tet2/3 DKO mice leads to myeloid leukemia in secondary recipients.
Supplementary Figure 15. Transfer of Mac-1+ cells from Tet2/3 DKO mice leads to myeloid leukemia in secondary recipients. (a) Kaplan-Meier curve representing the percent survival of recipient mice transplanted with 2 x 106 Mac-1+ cells from WT (Tet2fl/fl Tet3fl/fl) and diseased Tet2/3 DKO (Tet2fl/fl Tet3fl/fl Mx1-Cre+) mice (n = 9 per each group). (b) May-Grünwald-Giemsa-stained peripheral blood smears of recipient mice. (c) Recipients of Tet2/3 DKO Mac-1+ cells developed progressive leukocytosis with neutrophilia. Means ± SEM are shown. ***P < 0.0005 (Student's t test). For a summary of other hematopoietic parameters, see Supplementary Table 2d. (d) Enlargement of spleens of mice that received Tet2/3 DKO Mac-1+ cells. (e) Representative photographs of femurs and tibiae from recipients of WT or Tet2/3 DKO Mac-1+ cells. (f) Weights of spleen or liver. Means ± SEM are shown. ***P < 0.0005 (Student's t test). (g) Hematoxylin and eosin staining of livers show loss of normal liver structure and infiltration with hematopoietic cells. 4X magnification. (h) A representative flow cytometric analysis of myeloid-lineage cells (Gr-1+/Mac-1+) in the bone marrow, spleen and blood of recipient mice. Summary of the percentage of each cell population is also shown in the right panel. (i) A representative flow cytometric analysis of erythroid-lineage cells (Ter-119+/CD71+) in the bone marrow and spleen of recipient mice.
Supplementary Fig. 16 4 3 2 1 0
NES: -5.467 FDR < 0.0001 P < 0.0001
-0.1 -0.2 -0.3
12
Color Key and Histogram
Pre-MegE
NES: 9.382 0 FDR < 0.0001 P < 0.0001 -0.1
DE genes in LSK_DKO Vs LSK_Ctrtl
NES: -8.58 FDR < 0.0001 P < 0.0001
-0.2
-0.2
-0.3
Color Key
Color Key
(Ivanova et al., Science, 2002)
8
CLP 0 -0.1
NES: -2.34 FDR < 0.0001 P < 0.0001
(Sanjuan-Pla et al., Nature, 2013)
4
Meis1 Gata2 Nap1l3 Rarb Klf12 Prdm16 Hoxa5 Lmo2 Zfp37
Genes
Irf6
Color Key
Mycn Hlf Msi2 Hoxa9
−1
0
1
Row Z−Score
Runx1t1
LSK_DKO_B
Zfp532
LSK_DKO_A
LSK_Ctrtl_A
Mecom
LSK_Ctrtl_B
Zfp612
Ndn
-1
0
LSK_Ctrtl_A
Z-score
1
LSK_DKO_B
Z Scores of RPKM values
Z Scores of RPKM values
LSK_DKO_A
HSC-specific genes (Riddle et al., Cell, 2014)
LSK_Ctrtl_B
LSK_DKO_A
LSK_Ctr tl_A
Tcf15
Early progenitors (s-mpp)
Nr3c2
Epb4.1l3 Mecom Gabbr1 Il17re Nkx2−3 Hoxa9 Msi2 Hlf Stxbp4 Phxr4 Padi4 Hnf4a Eya1 Prkd2 Tanc2 Tcf7l2 Nfatc2 Prkce Zfp287 BC031353 Nlgn2 Arhgef5 Dab2ip Rnf122 Nrgn Pglyrp2 Itih5 Gcnt2 Fam69b Socs5 Meis1 Rbpms Zfp608 Ddx58 Sfi1 2900026A02Rik Vezf1 Tnfsf10 Dnmt3b Nfkbiz Gramd1a Oas2 Csad Alcam Arrdc3 Ctla2b Cand2 Ppp1r13b Fchsd2 Hmga2 Samd9l Cxxc5 Zfhx3 Impact Pde4b Ifi44 Chd3 Trp53inp1 Abcg1 Tmbim4 S100a10 Ffar2 Ly6a Tmem176a Atg7 Dnase2a Oas1a Ifitm3 Ifitm1
(Ng et al., Immunity 2009)
Color Key
−1
0
1
Row Z−Score
Z Scores of RPKM values
Color Key
−1
0
1
Row Z−Score
Z Scores of RPKM values
Rhd Bex4 Nckap1 Slc40a1 Pla2g4c Igf2r Trib2 Ermap Dmd Gata1 Shank3 Lphn2 Arhgef12 Car2 Abcb4 Rab4a Grb10 Ppp1r9a Mtap7 Tnik Aqp9 Hemgn Mllt3 Gnb4 Sorbs1 Nfia Slc12a2 Car1 Slc22a3 Casc4 Vamp5 Tgfbr3 Aldh7a1 Mast4 Xrcc5 Tal1 9830001H06Rik Mta3 Lpin2 Igf1r Ssbp3 Cd81 Tspan32 Foxn3 Creg1 Snap23 Cd97 Apoe St8sia6 Mt1 Scin
(Ng et al., Immunity 2009)
Z Scores of RPKM values
Supplementary Figure 16. Tet2 and Tet3 differentially modulate stem cell- and lineage-affiliated transcriptional programs. Z Scores of RPKM values
Papss2 Cybb Cx3cr1 Csf1r Igsf6 C3 Pld4 Pmaip1 Sema4a Ms4a3 Tifab Clec7a Ccl6 Mtus1 Hdc Emr1 Pik3r6 Kmo Lgals3 Cldn15 Nr p1 Fcgr3 Ptpro Alas1 Dmkn Lpxn Csf2rb2 5430435G22Rik Sepx1 Ap3s1 Idh1 Fcgr2b Trem3 1190002H23Rik Dram1 Cfp C1galt1c1 Ctsz Tmem38b Plod3 Psap Lta4h Ccbl2 Csf2rb Acot9 Mlkl Mapkapk3 Sorl1 Nucb2 Sdf2l1 Kctd20 Lamp2 2010111I01Rik H47 Sh2b2 Cd48 Erlin1 Mtm1 Gng12 Edem1 Ostf1 Fabp5 Zeb2 2810417H13Rik Nnt Dpp4 Ube2w Actr3 Wdr1 Sgpl1 Idh2 Sgk3 5430427O19Rik Hsp90b1 Itpr1 Pde4d
Genes
Rbpms
Genes
Egr1
Genes
Hoxb3
LSK_DKO_B
Igf2bp2
LSK_DKO_B
Pbx1
LSK_DKO_B
Prdm5
LSK_DKO_A
Nfix
LSK_DKO_A
Zfp521
HSC-specific genes (stem)
Zfp467
Csf1r Csf2rb Csf2rb2 Csf3r Itgam Ly6c2 Mpo Irf8 Sfpi1 Mnda Ifi205 CD14 Fcgr2b Ncf1 Ncf2 Ncf4 Ctsg Lyz1 Lyz2 Tcf3c Dntt Satb1 Notch1 Sox2 CD72 Ets1
LSK_DKO_A
Glis2
Mnda Cd14 Ccr2 Csf1r Slfn2 Mpo Tyrobp Ctsg Ms4a6c Prtn3 Ctsh Gria3 Lyz2 Ccl9 Rassf4 Irf8 Plac8 Lyz1 Mgst1 Csf2rb2 Ahnak 4632428N05Rik Scpep1 H2−DMb2 Tgfbi S1pr3 Ms4a6b Fcer1g Anxa2 Idh1 Tcn2 Ctss Fcgr2b Tcfec Unc93b1 Anxa1 Ifi205 Grn Naga Ap1s2 Hfe Cst3 Ms4a4b Soat1 Napsa BC064078 Ugt1a7c Asah1 BC013712 Tnfaip8l2 Gatm Arhgap26 Cd52 Ly6e S100a6 Ralb Gm885 Myo1f Tubb6 Emb Lta4h Hvcn1 Gpr160 Atp8b4 1600014C10Rik Sla Rab3d H2−DMb1 Tmem50b Mgst2 Mapkapk3 Serpinb1a Sell Cotl1 Tax1bp3 Sorl1 Fgl2 Rab32 0610031J06Rik Twf2 Gramd3 Arpc1b Slc29a3 Cd300a Tmsb4x Lbp Lst1 Plxnb2 Rps6ka1 Hexa Anxa6 Coro1a Litaf Ceacam1 Rab44 Ncf4 Sfpi1 Diap2 Glipr1 Tpm4 Il6st Spns3 Abcd1 Pgam1 Hk3 Trf Fgd2 Fes Cnn2 Cd68 Arhgdib Cd53 Man2b1 Pkm2 Ncf2 Serpinf1 Lass6 S100a11 Vim Alox5ap Rac2 Itgb7 Dhrs7 Ncf1 Pold4 Hk2 Laptm5 Edem2 Myo1g Bex6 Rab31 Lcp1 Tmem173 H2−M3 Sirpa Tnfrsf1a Rin3 Sh3bgrl3 Tmem176a Gpi1 Hpcal1 Rgs19 H2−DMa Gbp3 Ptpn6 Fcgrt Ffar2 Vwa5a Krtcap2 Lsp1 Gm2a Ninj1 Lrrc33 Myadm Rpl13 Tmem205 Parp8 Lpcat2 Arpc2 Flnb Acpl2 Ivd Ifi47 Skap2 Dpp4 Ube2w Tpd52 Pak1 Scamp2 Kctd12 Hexb Csf3r Fam107b Fam102b Msra Myl12b Psmb8 Psmb10 Swap70 Ramp1 Plin3 Sypl Parl Prkcd Idh2 Lpar6 St8sia4 A630001G21Rik Fxyd5 Wls Tmem176b 1810058I24Rik Map3k1 Arl6ip1 Akap13 Ptpre Sh2d5 Macf1 Smad5 Elmo1 Trp53bp1 Gabpb1 Slco3a1 Znrf1 Arid1b Sesn1 Gnaq Map4k4 Cdk19 Mast3 H2−Ob Atp2b4 Camk2d Tsc22d1 1110028C15Rik Satb1 Hmga2 Wdfy1 Fmnl2 Notch1 Adamts10 AI504432 Eltd1 Camk2g Sox4 Pag1 Xpo7 Marcks Anks1 Alcam Lonrf3 9030619P08Rik 4931406C07Rik Nfkbiz Aff3 Dusp2 Ets1 Nfkbid Snn Calcrl Epb4.1l4b Arl4c Cd72 Ttpa P2ry14 Fam134b Repin1 Tmprss3 Gpsm1 Ccnd1 Hdgfrp3 Esr1 Jakmip1 AW555464 Dntt Il18r1
LSK_Ctrtl_B
Hoxb4
DKO
1
LSK_Ctrtl_B
Etv6
0
LSK_Ctrtl_B
Runx1
WT −1
Row Z−Score
LSK_Ctrtl_A
Ndn Dsg2 2510009E07Rik Prkaa2 Zfp532 Fhl1 Mecom Crim1 Cuedc1 Nbea Gucy1a3 Cdkn1c Bgn Msi2 Sdpr Pf4 Hlf Cadps2 Cdk14 Mylk Fgd5 Chrnb1 Dip2c Hoxa10 Mycn Crispld1 Fnbp1l Mllt4 Gkap1 Ppp1r9a Gimap8 Hdgfrp3 Mmrn1 Asah2 Angpt1 Prdm16 Zbtb4 Gimap1 Nrxn1 Per3 Pear1 Itsn1 Pglyrp2 Mamdc2 Nfat5 Armcx1 Rhobtb3 Gata2 Trove2 Parp12 Csgalnact1 Meis1 Mpzl1 Inadl Dst Smarca2 Psd3 Serpinb9 Spns2 Med12l Egr1 Gimap5 Rgs1 Dach1 Maml2 Pdzk1ip1 Lass4 Atp10d Utrn Pbx1 6720401G13Rik Osbpl1a Igf1r Ocrl Diap1 Ikzf2 Sgms1 Rbbp6 Ppp1r15a Akap13 Sord P2rx1 Stat1 Ubl7 S100a1 Ptgs1 Selp
Vdr
Hoxb5
d
DKO
1
LSK_Ctrtl_A
0
Row Z−Score
LSK_Ctrtl_A
WT −1
Genes
DKO
Lymphoid and myeloid genes primed in HSCs (s-myly)
WT
Row Z−Score
Genes
−1.5 −0.5 0.5 1.5
GMP-specific Erythroid genes primed in HSC (s-ery) Genes (d-my)
c
0
b
LSK_DKO_B
Count
Pre-GM
HSC 0
LSK_Ctr tl_B
Enrichment score
a
Supplementary Figure 16. Tet2 and Tet3 differentially modulate stem cell- and lineage-affiliated transcriptional programs. a, Gene set enrichment analysis (GSEA) of RNA-Seq data shows that the pre-granulocyte/ macrophage progenitor (pre-GM) gene signature is significantly enriched for genes up-regulated in Tet2/3 DKO compared with WT LSK cells, whereas the HSC, pre-megakaryocyte/ erythrocyte progenitor (Pre-MegE) and common lymphoid progenitor (CLP) gene signatures are enriched in genes downregulated in Tet2/3 DKO compared with WT LSK cells. b-d, Heatmap representation showing differential expression of genes highly expressed in HSC and their immediate downstream progenitors (b, c), as well as lineage-affiliated genes (d) in Tet2/3 DKO LSK cells compared with WT LSK cells. The gene sets (including stem, s-mpp, s-ery, d-my and s-myly) have been described. Only genes with a p-value ≤ 0.05 and fold change > 1.5 or < 0.67 were considered. Asterisks in b indicate genes used to induce reprogramming of differentiated hematopoietic cells into induced HSCs. The color key for all heatmaps indicates row-wise scaled RPKM values (z-score).
Supplementary Fig. 17
All 0.6 Frequency
Fraction of CpGs
d
CpGs covered ≥ 10X (n = 8,244,600)
0.8
0.4
0.2
1
0.2
0.8
0.15
0.6
0.1
0.4
0.05
0.2 0
0
0
Hypermethylated
b
Frequency
%(5mC + 5hmC)/ total C
CpGs covered ≥ 20X (n = 422,570)
0.2
0.8
0.15
0.6
0.1
0.4
0.05
0.2 0
0
Frequency
% methylation in DKO
Hypomethylated 1
0.2
0.8
0.15
0.6
0.1
0.4
0.05
0.2 0
c
0 Genome
% methylation in WT
Random genome fragments
All genes
% 5mC + % 5hmC
1
DMRs
a
100
100
80
80
60
60
40
40 WT
20
WT 20
DKO
0
0 TSS -2 kb
DKO
TTS Gene body
-40 + 2 kb
-20
0
20
40
Relative position in random genome fragments
Supplementary Figure 17. Loss of Tet2 and Tet3 results in aberrant gene expression and DNA methylation.
Supplementary Figure 17. Loss of Tet2 and Tet3 results in aberrant gene expression and DNA methylation. a, Histogram of genome-wide CpG modification (5mC + 5hmC) in WT (blue) and Tet2/3 DKO (red) LSK cells at CpGs covered by at least ten reads. The histogram shows a typical bimodal distribution where the majority of CpGs are highly methylated. b, Scatter plot of genome-wide CpG methylation comparing WT LSK (x-axis) and Tet2/3 DKO LSK (y-axis) cells at individual CpGs (black dots) covered by at least 20 reads in each condition. The graph indicates the genome-wide increase of DNA methylation in Tet2/3 DKO LSK cells, and the Lowess curve confirms that the majority of CpGs in both WT and DKO genomes are methylated. c, Left, Average DNA methylation (5mC+5hmC) along gene regions, including 2 kb upstream of the transcription start sites (TSS) and 2 kb downstream of the transcription termination sites (TTS). Considering all genes, the average level of DNA methylation is slightly but consistently increased in Tet2/3 DKO LSK samples (red lines) compared to the control LSK samples (blue lines); note the close correlation between the triplicate biological samples in each case. Similar results were obtained when considering upregulated or downregulated genes (see Figure 5d). Right, Average DNA methylation (5mC+5hmC) in randomly chosen genome fragments of similar size. d, DMRs are significantly enriched in promoters and exons.
Supplementary Fig. 18
a
CGI shores 12005 171 (1.42%) 3 7 1539 116 135 53 (0.44%) 5
Downregulated
3
No. of CGI shores less methylated in DKO Upregulated Downregulated
224 20 16
Total no. of canyons
1,135
- Downregulated
- Downregulated
67
No. of canyons that expand in DKO
4
- Downregulated
3
Genes associated with shrinkingLSK_DKO canyons Vs LSK_Ctrtl(n=525) 10
LSK_DKO Up Regulation −−−−>
5 0
●
● ●
●
● ●
● ●
●
●
●
● ●
●
●
●
●
●
● ●
●
● ●
WT
5
●
DKO
●
●
● ●
●
0
● ●
Car2
-5
Gcnt2
67
WT
-5
67
3
●
-5 −5
0 0
5 5
10
Mean of Normalized Counts
10
15 15
Mean of normalized counts
-5
0
5
33.26%
19.46%
17.79%
0.2073
10
15
Downregulated genes with shrinking canyons 50
●
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●
19
20
e
4
●
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● ●● ● ● ●● ● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ●●● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●●● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●●● ● ● ● ●● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ●● ● ● ●● ●● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ●● ●●● ● ● ● ● ● ●● ●● ●● ● ●● ●● ● ●● ● ● ● ●● ● ●●●● ● ● ●● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ●● ●●● ● ●● ● ● ● ●● ● ● ● ● ● ●●● ● ● ● ● ●● ● ● ●● ●● ●● ● ● ● ● ● ● ● ● ●● ●● ● ●● ● ● ● ●● ● ● ●●●●● ● ● ●● ● ● ●● ● ● ●●●● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ●●●●● ● ●● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ●● ● ●●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●● ● ●● ● ● ● ● ●● ● ● ●●● ● ●● ● ● ● ●● ●● ● ●● ●●● ●● ● ● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ● ●● ● ● ●● ● ● ● ● ●●● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ●● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ●● ●● ● ●●● ● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ●● ●● ● ●● ● ● ●● ● ● ● ●● ●● ●●● ●● ●●● ●● ●● ● ●● ● ● ●●● ● ●● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ●●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ●● ● ● ●● ●●● ● ● ● ● ● ●● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ●● ●●●● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●●●● ● ● ● ● ● ●● ● ● ●●● ● ● ●● ● ●● ● ● ● ●● ●● ● ● ● ●● ● ●● ● ●● ● ● ● ●● ● ●● ●● ●● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ●● ● ●● ●● ● ●● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●
Genes associated with expanded canyons (n=70)
●
● ●
● ● ● ●
●
●
3.838e−31
29.8%
0 is canyon center
●
● ●
● ●
●
−5
Log2FoldChange(LSK−DKO/LSK−Ctrl)
● ● ● ● ●
0 )
70
- Upregulated
20
100
59 (5.20%)
Genes associated with expanded canyons
0
100
525 19
10
c
77
- Upregulated
-20
Methylation difference (DKO-WT)
465 (40.97%)
Genes associated with canyons that shrink
1539
-40
28
No. of canyons that shrink in DKO
d
224
846 (74.54%)
- Upregulated
135
-5
% 5mC + %5hmC % Methylation (5mC + 5hmC)
No. of canyons close to a gene
16
0
0
b
116
80
Downregulated No. of CGIs less methylated in DKO Upregulated
20
60
No. of CGI shores more methylated in DKO Upregulated
5
40
Downregulated
Differentially expressed genes
20
No. of CGIs more methylated in DKO Upregulated
Log2 fold change (DKO LSK/control LSK)
Total no. of promoter CpG islands (CGIs)
0 100 0
Mean of normalized counts
WT DKO
DKO
0 10 0 100 0 100 0
Supplementary Figure 18. DNA methylation at CpG islands and CGI shores and differentially methylated regions (DMRs).
WGBS
WT
Gcnt2
RNA-seq
10
Supplementary Figure 18. DNA methylation at CpG islands and CGI shores and differentially methylated regions (DMRs). a, Left panel, Summary of the number of CpG Islands (CGIs) and CGI shores within or at the promoter regions (± 2 kb) of genes that show altered methylation in WT compared to Tet2/3 DKO LSK cells. Right panel, lack of a clear relation between the DNA modification change in CGI shores at promoters (n=12,005) and the change in expression level of the associated genes. The left, central and right sets of dots show CGI shores with decreased, unchanged or increased methylation (methylation differences < -10, -5 to +5 and >+10 respectively). Of 1539 CGI shores that show increased DNA modification, only 251 genes are differentially expressed (red dots), and of these, 116 genes are upregulated whereas 135 genes are downregulated. b, Summary of the number of canyons in WT LSK cells, including the number of canyons that shrink or expand in Tet2/3 DKO LSK cells compared to WT. c, Average DNA methylation at canyons that decrease in length in Tet2/3 DKO LSK compared to WT LSK cells. The plot illustrates the overall absence of DNA methylation in the canyon center and the increase of DNA methylation at the canyon borders in Tet2/3 DKO LSK compared to control LSK cells. There is a tendency for the gain of methylation in Tet2/3 DKO LSK cells to be more pronounced at the right side of figure, which corresponds to the canyon border towards or within the downstream promoter region of the nearest gene. d, The figure shows the MA plot of Fig. 5a, with the red dots indicating all genes differentially expressed between WT and Tet2/3 DKO LSK cells, and the green dots the genes associated with canyons that shrink (left panel; n=525) or expand (right panel; n=70) in Tet2/3 DKO LSK cells versus WT LSK cells. Black dots, genes that are both differentially expressed and associated with canyons that shrink or expand. Genes associated with shrinking canyons are biased towards downregulation in Tet2/3 DKO LSK cells compared to WT. e, Examples of RNA-seq (top) and WGBS (bottom) results for two downregulated genes (Car2 and Gcnt2) associated with canyons that shrink in Tet2/3 DKO LSK (red) compared with WT LSK (blue) cells.
Supplementary Fig. 19
a
b WT
DKO
c
DKO
γH2AX γH2AX DAPI γH2AX DAPI
γH2AX
WT γH2AX
9 0 3 6 9
Bone marrow
Time (h) 0 3 6
WT
0h
0h
1h
1h
3h
3h
14 h
14 h
22 h
22 h
DKO
γH2AX γH2AX DAPI γH2AX DAPI
γH2AX Actin
Spleen
γH2AX Actin 1 week after injection
GMP cells (ERT2-Cre)
LSK cells (ERT2-Cre)
d
WT
mRNA levels (relative to Gapdh) 18"
16"
14"
5"
***
4" 6"
WT DKO
7" 8"
53bp1
12"
3"
0h
N.D.
8"
N.D.
10"
2"
***
1"
Tet3
6"
4"
0 2 4 6 8 10 12 14 16 18
Tet2
2"
γH2AX γH2AX DAPI γH2AX DAPI
e
0"
γH2AX
DKO
9" 10" 11"
Rad51
12"
1h
13" 14"
Rad54
15" 17"
**
16"
Xrcc2
HR
18"
3h
19"
* 20"
Xrcc3
21" 22" 23"
Rpa1
24"
14 h
25"
* 26"
Xrcc6
27" 28"
NHEJ
29"
Xrcc5
30"
22 h
31"
* 32"
Prkdc Mac-1+ cells (ERT2-Cre)
Supplementary Figure 19. Loss of Tet2 and Tet3 results in accumulation of DNA damage and impaired DNA repair. (a) Combined loss of Tet2 and Tet3 leads to accumulation of γH2AX. Control and Tet2/3 DKO mice were irradiated (6 Gy) at 1 week after pIpC injection and bone marrow (top panel) or spleen (bottom panel) were harvested at the indicated time points. Whole cell lysates were prepared and analyzed for the expression of γH2AX. Actin serves as a loading control. (b) Efficient DNA repair in DKO LSK cells. LSK cells were isolated from control or DKO mice (Tet2-/- Tet3fl/fl ERT2Cre+) at 3 weeks after pIpC or tamoxifen injection, respectively, and DNA repair kinetics in response to 6 Gy of ionizing radiation were assessed by immunocytochemistry. (c,d) DNA damage repair is impaired in myeloid lineage cells upon loss of Tet2 and Tet3. GMP (c) or Mac-1+ cell (d) were sorted from the bone marrow of control and Tet2-/- Tet3fl/fl ERT2-Cre+ DKO mice at 3 weeks after tamoxifen injection and DNA repair kinetics in response to 6 Gy of ionizing radiation were assessed by immunocytochemistry. (e) TET proteins control the expression of DNA repair genes in myeloid cells. Mac-1+ cells were sorted from the bone marrow of WT (Tet2+/+ Tet3fl/fl) and DKO (Tet2-/- Tet3fl/fl Mx1-Cre+) mice at 3 ~ 4 weeks after pIpC injection, and quantitative RT-PCR (PCR with reverse transcription) was performed to assess the expression of genes implicated in homologous recombination (HR) and non-homologous end-joining (NHEJ). Results are expressed as fold change compared with WT cells (arbitrarily set to 1). Data from three independent experiments are shown (Means and SEM). N.D., not detected. *P < 0.05, **P < 0.005, ***P < 0.0005 (Student's t test).
Supplementary Fig. 20
Supplementary Figure 20. Chromosomal aberrations in bone marrow cells from Tet2/3 DKO mice. Red cell-depleted bone marrow cells were isolated from WT mice and sick Tet2/3-deficient DKO mice and subjected to G-banded karyotyping. In one of four bone marrow samples, 6/20 DKO cells examined displayed a structural abnormality of chromosome 3 – an unbalanced translocation of distal chromosome 6 to distal chromosome 3, resulting in an extra copy of the translocated sequences of chromosome 6 and a loss of copy of chromosome 3 distal to the breakpoint on 3.
Supplementary Fig. 21 Original blots for the main Fig. 6a,b,c, h and Supplementary Fig. 19a Spleen DKO
WT
1 2 3 4
MW (kDa)
Weeks:
* n.s. γH2AX
20 15
Actin MW (kDa)
Pre
1 2 3 4
Pre
Weeks:
Pre
Bone marrow DKO WT
1 2 3 4
Pre
Fig. 6a
1 2 3 4
MW (kDa)
* n.s. γH2AX
20 15
Actin
MW (kDa) 50 37
50 37
50 37
* n.s. Actin
37
* n.s.
20
20
γH2AX
15
Long exposure * n.s.
50
Actin
37
* n.s.
20
γH2AX
15
Short exposure
50
Tet2/3 DKO *
n.s. Actin
*
n.s. γH2AX
15
MW (kDa)
MW (kDa)
WT Tet2 KO Tet3 KO
MW (kDa)
WT Tet2 KO Tet3 KO
Spleen Tet2/3 DKO
WT Tet2 KO Tet3 KO
Tet2/3 DKO
WT Tet2 KO Tet3 KO
MW (kDa)
Bone marrow
Tet2/3 DKO
Fig. 6b
Long exposure *
50
n.s. Actin
37
*
20
n.s. γH2AX
15
Short exposure
Fig. 6c Bone marrow DKO
WT Time (h): 0
3
6
Spleen
9
0
3
6
9
Time (h): 0
MW (kDa)
6
9
0
3
6
9
* n.s. γH2AX
20
γH2AX
15
3
MW (kDa)
* n.s.
20
DKO
WT
15
MW (kDa) MW (kDa) 50
50
Actin
37
Actin
37
Suppl. Fig. 19a Bone marrow
20 15
DKO
WT Time (h): 0 3
WT DKO
WT DKO
MW (kDa)
WT DKO
Fig. 6h
6
9
0 3
6
9
MW (kDa) 20
* n.s. γH2AX
n.s.
*
15
γH2AX
MW (kDa) 50 37
Actin MW (kDa)
* n.s. * n.s. Actin
50 37
Spleen DKO
WT Time (h): 0 3 MW (kDa) 20
6
9
0 3
6
9
* n.s. γH2AX
15
MW (kDa) 50 37
Actin
Supplementary Tables Supplementary Table 1. Long-term monitoring of health status of WT, Tet2-deficient and Tet3-deficient mice. WT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Genotype Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet2fl/fl Tet3fl/+ Tet3fl/+ Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl Tet3fl/fl
Days post injection (DPI) 15 months 15 months 15 months 15 months 15 months 14.5 months 14.5 months 14.5 months 12.8 months 12.8 months 12.8 months 12.8 months 12.8 months 10.7 months 10.7 months 10.7 months 10.7 months 8.4 months 7.3 months 7.3 months 7.3 months
Health status healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy
T2KO 1 2 3 4 5 6 7 8 9 10 11 12
Genotype Tet2-/- Tet3fl/fl Tet2-/- Tet3fl/fl Tet2-/- Mx1-Cre Tet2-/- Tet3fl/fl Tet2-/- Tet3fl/fl Tet2-/- Mx1-Cre Tet2-/- Mx1-Cre Tet2-/- Tet3fl/fl Tet2-/- Tet3fl/fl Tet2-/- Mx1-Cre Tet2-/- Tet3fl/fl Tet2-/- Tet3fl/fl
Days post injection (DPI) 15 months 12.2 months 13.2 months 10.7 months 10.7 months 10.7 months 10.7 months 10.7 months 8.4 months 8.4 months 7.3 months 7.3 months
Health status healthy Found dead Found dead healthy healthy healthy healthy healthy healthy healthy healthy healthy
T3KO 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Genotype Days post injection (DPI) Tet3fl/fl Mx1-Cre 15 months Tet3fl/fl Mx1-Cre 15 months Tet3fl/fl Mx1-Cre 15 months Tet3fl/fl Mx1-Cre 15 months Tet3fl/fl Mx1-Cre 14.5 months Tet3fl/fl Mx1-Cre 10.7 months Tet3fl/fl Mx1-Cre 10.7 months Tet3fl/fl Mx1-Cre 10.7 months Tet3fl/fl Mx1-Cre 10.7 months Tet3fl/fl Mx1-Cre 10.7 months Tet3fl/fl Mx1-Cre 10.7 months Tet3fl/fl Mx1-Cre 10.7 months Tet3fl/fl Mx1-Cre 7.3 months Tet3fl/fl Mx1-Cre 7.3 months
Health status healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy healthy
Supplementary Tables Supplementary Table 2. Hematopoietic parameters after cell transfer experiments a. Haematopoietic parameters in recipients of splenocytes (Tet2-/- Tet3fl/fl Mx1-Cre+). Parameter
WT
DKO
P value
Abnormality
1.95±0.33
98.14±27.54
**, 0.0008
Leukocytosis
RBC (10 /µl)
6
6.27±0.64
3.4±0.28
*, 0.0063
Anaemia
Hemoglobin (g/dL)
8.93±0.78
5.6±0.6
*, 0.0119
Anaemia
Hematocrit (%)
32.69±3.09
21.62±2.46
*, 0.0288
Anaemia
450.25±41.67
240.8±34.63
*, 0.005
Thrombocytopenia
1.16±0.36
6.372±1.07
**, 0.0002
Lymphocytosis
0.67±0.08
79.9±22.96
**, 0.0009
Neutrophilia
0.1±0.012
7.89±2.98
*, 0.0492
Monocytosis Basophilia
3
WBC (10 /µl)
3
Platelet (10 /µl) 3
Lymphocyte (10 /µl) 3
Neutrophil (10 /µl) 3
Monocyte (10 /µl) 3
0.003±0.0016
0.54±0.23
*, 0.0112
7
5.15±0.54
2.59±0.43
*, 0.0059
7
9.92±1.27
31.56±0.35
***,
