2 Supplementary Figures and Methods

Figure S1: a) Event free survival of AML patients from University Hospital Essen with regard to Gfi1 expression.

b) Gene set enrichment analysis (GSEA) of GFI1-low expressing human leukemic cells with resemblance

to gene expression signature in HSCs NSE=2.2, p=0.0001.

100

100

High GFI1, n= 5

Months

EFS (%) AML Essen

0

Medium GFI1 , n=21

p=0.038 Low GFI1, n=4

500

50

HSC-signature enriched in human low GFI1-expressing AML

a b

Figure S2: Influence of GFI1 level on hematopoietic development. a) Schematic representation of the murine Gfi1 locus and the targeted alleles

b) Western blot of nuclear extracts of Gfi1-WT, Gfi1-36S-KI and Gfi1-KD/KD thymocytes.

a

Mac

1

Gr1

WT KD KI 29 31 71

Granulo- cytes

Mono- cytes

35

WT KI KD

α-Lamin

α-Gfi1 62 49

62

kD

hGFI1 cDNA neo

hGFI1 cDNA

WT

GFI1-KD

GFI1-KI

1.2 2 3 4 5 ATG

41 3

Mac

1

Gr1

WT KD KI 29 31 71

Granulo- cytes

Mono- cytes

35

WT KI KD

α-Lamin

α-Gfi1 62 49

62

kD

hGFI1 cDNA neo

hGFI1 cDNA

WT

GFI1-KD

GFI1-KI

1.2 2 3 4 5 ATG

41 3

Mac

1

Gr1

WT KD KI 29 31 71

Granulo- cytes

Mono- cytes

35

WT KI KD

α-Lamin

α-Gfi1 62 49

62

kD

hGFI1 cDNA neo

hGFI1 cDNA

WT

GFI1-KD

GFI1-KI

1.2 2 3 4 5 ATG

41 3

c d

e

b

f

g h

GFI1-cDNA

GFI1-cDNA

ATC

2

c) Cell surface staining of the BM of the indicated mouse cohorts for Mac1 and Gr1.

d) Wright-Giemsa staining of bone marrow cytospins (bar represents 20 µm).

e) Left: percentage of monocytes in bone marrow (N= 4 Gfi1-WT, N=4 GFI1-KI, N=4 GFI1-KD, N=4) ***

p=0.0002. right: total number of monocytes in bone marrow (N= 4 Gfi1-WT, N=4 GFI1-KI, N=4 GFI1-KD);

*** p=0.0003

f) Left: percentage of granulocytes in bone marrow (N= 4 Gfi1-WT, N=4 GFI1-KI, N=4 GFI1-KD) ****

p<0.0001. Right: total number of granulocytes in bone marrow (N= 4 Gfi1-WT, N=4 Gfi1-KI, N=4 Gfi1-

KD); **** p<0.0001

g) Left: percentage of GMPs (Lin-, Kit+, Sca-, CD34+, CD16/32+ bone marrow cells) (N= 6 Gfi1-WT, N=6

GFI1-KI, N=6 GFI1-KD) *** p=0.007. Right: total number of GMPs (Lin -, Kit+, Sca-, CD34+, CD16/32+ bone

marrow cells) (N= 6 Gfi1-WT, N=6 GFI1-KI, N=6 GFI1-KD) * p=0.04.

h) Left: total number of colonies an in semisolid medium (1000 cells seeded) counted after 7 days. One

representative experiment out of four experiments is shown. Right: total number of cells in semisolid

medium (1000 cells seeded) counted after 7 days. One representative experiment out of four experiments

is shown. **** p<0.0001

3

Figure S3:

a) Schematic representation

of isolating lineage negative cells from

the indicated genotypes and

subsequent transduction with a retrovirus encoding MLL-

AF9 and GFP.

h

4

b) About 1000 GFP-positive cells were seeded in semisolid medium. After 10 days, the number

of colonies was determined in three independent experiments with triplicates for each

experiment. Shown are the results from one representative experiment out of three experiments

(*p=0.025).

c) Similar to B, number of cells in the semisolid medium was determined 10 days after seeding

(*p=0.035).

d) About 3000 GFP-positive cells were seeded in liquid culture. After 7-10 days, number of cells

was determined in three independent experiments with triplicates for each experiment. Shown

are the results from one representative experiment out of three experiments (*p=0.015).

e) WBCs of GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice in comparison to

healthy mice

f) PLTs of GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice in comparison to healthy

mice (**p<0.0045)

g) HGB of GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice in comparison to

healthy mice (*p=0.018)

h) Western Blot of the nuclear extracts of MLL-AF9 leukemic BM cells from GFI1-KI or GFI1-KD

mice. Upper panel subjected to a GFI1 antibody. Lower panel subjected to a lamin antibody.

i) Percentage of blast in the BM of mice transplanted with GFI1-KD or GFI1-KI leukemic cells

(***p=0.0004)

j) Percentage of blast-cells in the blood of mice transplanted with GFI1-KD or GFI1-KI leukemic

cells (*p=0.05). k) Percentage of cKit+ cells in the BM of mice transplanted with GFI1-KD or GFI1-KI leukemic

cells

l) Number of spleen cells from GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice

compared to control mice (*p<0.01)

m) Percentage of Blasts in spleen GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice

5

Bone marrow of MLL-AF9 transplanted mice

' &/ϭ</ͬ </

' &/ϭ<ͬ <

Figure S4: Bone marrow cytospins of transplanted mice with GFI1KI/KI-MLL-AF9 and GFI1KD/KD-MLL-

AF9

6

Figure S5:

a

gfe

b

h

i j k

l m

0

20

40

60

Dead before

300 days

*

Dead after 300 days

Mean fluorescence intensityn

7

a) Western Blot Analysis of nuclear extracts from BM cells of GFI1-KI (WT/KI) and GFI1-KD

(KD/KD) NUP98-HOXD13 transgene mice. The upper panel subjected to a Gfi1 antibody and

the lower panel to a lamin antibody

b) White blood cell count of peripheral blood of GFI1WT/WTxNUP98/HOXD13,

GFI1KD/KDxNUP98/HOXD13 mice and healthy control mice

c) Platelet number peripheral blood of GFI1WT/WTxNUP98/HOXD13, GFI1KD/KDxNUP98/HOXD13

mice and healthy control mice (*p<0.04); ***p<0.0001)

d) HGB of peripheral blood of GFI1WT/WTxNUP98/HOXD13, GFI1KD/KDxNUP98/HOXD13 mice and

healthy control mice (**p00.0013; ***p<0.0001)

e) Number of spleen cells from GFI1WT/WTxNUP98/HOXD13, GFI1KD/KDxNUP98/HOXD13 mice in

comparison to healthy mice (*p=0.017)

f) Percentage of Blasts in spleen GFI1-KI/WTxNUP98/HOXD13 and

GFI1-KD/KDxNUP98/HOXD13 (**p=0.0085)

g) RT-PCR detecting Gfi1 mRNA expression levels in bone marrow cells from Gfi1-WT/WT

mice or Gfi1-EGFP/WT heterozygous mice

h) Gfi1 protein expression levels in thymocytes from Gfi1 WT/WT mice or Gfi1 KO/WT

heterozygous mice

i) White blood cell count of peripheral blood of GFI1WT/WTxNUP98/HOXD13, GFI1-

EGFPxNUP98/HOXD13mice and healthy control mice

i) Platelet number peripheral blood of GFI1WT/WTxNUP98/HOXD13,

GFI1-EGFPxNUP98/HOXD13 mice and healthy control mice (***p≤0.0001)

k) HGB of peripheral blood of GFI1WT/WTxNUP98/HOXD13, GFI1-EGFPxNUP98/HOXD13 mice

and healthy control mice (***p<0.0001)

l) Number of spleen cells from GFI1WT/WTxNUP98/HOXD13, GFI1-EGFPxNUP98/HOXD13 mice

in comparison to healthy mice (*p=0.0096)

m) Percentage of Blasts in spleen GFI1-WT/WTxNUP98/HOXD13 and

GFI1-EGFPxNUP98/HOXD13

n) Mean fluorescence intensity of GFP in blasts of Gfi1-GFP/WT leukemic according to time of

emergence of leukemia.

8

Figure S6:

ba

9

a) GO Biological functions overrepresented amongst genes showing increased acetylation in

Gfi1-KD leukemic samples compared to Gfi1-KI samples, as determined using the DAVID

software. Log p value was used to rank the enrichment.

b) KEGG pathways overrepresented amongst Gfi1 target genes showing increased acetylation

and gene expression in GFI1-KD leukemic samples compared to GFI1-KI samples, identified

using DAVID software. Log p value was used to rank the enrichment

c) Scatter plot comparing the log2 fold change in H3K9ac ChIP-seq between Nup98-HoxD13

Gfi1-KD and Gfi1 KI bone marrow AML samples versus change in mRNA levels between

Nup98-HoxD13 Gfi1-KD and Gfi1 KI bone marrow AML samples.

d) GO Biological functions amongst genes showing increased acetylation and mRNA

expression in Gfi1-KD leukemic samples compared to Gfi1-KI samples, as determined using

DAVID software. Log p value was used to rank the enrichment.

e) KEGG pathways overrepresented amongst GFI1 target genes showing increased acetylation

in GFI1-KD leukemic samples compared to GFi1-KI samples, identified using the DAVID

software. Log p value was used to rank the enrichment.

10

Figure S7: Model of pathophysiological function of low GFI1 expression in AML and as a possible therapeutic intervention. Low level of GFI1 cannot recruit sufficiently histone-modifying enzymes to their target genes. This leads

to the increase acetylation of H3K9 of GFI1 target genes involved in leukemia initiation, leading to

expansion of GMPs. Therapeutic intervention with HDACi could potentially worsen the situation. However,

use of Histone-acetylase inhibitors could rebalance the dysregulated epigenetic modifications

Histone H3GFI1KD

HDAC inhibitorHDAC

HAT

HAT inhibitor

Gfi1 target genes

AMLCMP

Accelerated AML development

Delayed AML formation?OncogenesHistone

acetylation

Ac Ac

GMP

11

Figure S8: New epigenetic therapeutic approach for low GFI1/Gfi1 expressing leukemic cells .

a b c

d

e f

h

d

g

12

a) Lineage negative cells from GFI1-KI or GFI-KD cells were transduced with MLL-AF9 and treated with

the indicated doses of Vorinostat (HDACi) or CTK7a (HATi). One representative experiment out of two

experiments is shown.

b) Treatment of the leukemic cells from the indicated mouse strains with Vorinostat (**p<0.00171;

***p<0.0003; ****p<0.00009). One representative experiment out of two experiments is shown.

c) Treatment of the leukemic cells from the indicated mouse strains with CTK7a (*p=0.015; ***p<0.0006).

One representative experiment out of two experiments is shown.

d) Relative GFI1 m-RNA expression level in Kasumi1 and K562 cells. RNA of Kasumi1 and K562 was

isolated and transcribed into cDNA. The relative expression level of GFI1 m-RNA was analyzed via

quantitative real-time PCR.

e) Kasumi 1 (a myeloid cell line with high Gfi1 expression) and K562 (a myeloid cell line with low Gfi1

expression) were treated with different doses of Vorinostat (*p=0.04; ***p=0.0002). One representative

experiment out of three experiments is shown.

f) Kasumi 1 (a myeloid cell line with high Gfi1 expression) and K562 (a myeloid cell line with low Gfi1

expression) were treated with different doses of CTK7a. (**p=0.0025; ***p=0.0007) One representative

experiment out of three experiments is shown.

g) GFI1 expression in AML-cell lines. *p=0.0186 (N=3 for low GFI1-expressing and n=4 for GFI1-high

expressing cell lines 1

h) Fold change of patient samples used for inhibitor treatment. Fold change relative to Gapdh and

normalized to GFI1 loe expressing patient

i) Scheme of isolating leukemic bone marrow cells from the indicated mouse strains and treated with the

indicated doses of Vorinostat (HDACi) or CTK7a (HATi). One representative experiment out of three

experiments is shown. n=3 for each treatment condition.

j) Treatment of the leukemic cells from the indicated mouse strains with Vorinostat (***p<0.00011;

****p=0.000062). n=3 for each time point. n=3 for each treatment condition

k) Treatment of the leukemic cells from the indicated mouse strains with CTK7a (**p<0.0046;

***p<0.00024****p<0.000031). n=3 for each treatment condition

13

Figure S9: Lineage negative cells from GFI1-KI or GFI-KD cells were treated with CTK7a (HATi) in the

indicated doses. After 48 hours the cell number was determined. Cells are compared to the

control of each genotype.

14

Suppl. Table 1:Patient cohorts from Essen

% all 13 (n=4) 70 (n=21) 17 (n=5) 87 (n=26) - - -Mean Age 60.5 ± 4.2 56.2 ± 2.9 47.2 ± 9.2 54.5 ± 2.9 0.54 0.27 0.44

Gender (% male) 50 (n=2) 48 (n=10) 60 (n=3) 50 (n=13) 0.94 0.77 1.0Cytogenetic low risk (%) 0 (n=0) 19 (n=4) 40 (n=2) 23 (n=6) 0.35 0.19 0.29

Cytogenetic intermediate risk (%) 100 (n=4) 67 (n=14) 60 (n=3) 65 (n=17) 0.19 0.19 0.17Cytogenetic high risk (%) 0 (n=0) 5 (n=1) 0 (n=0) 4 (n=1) 0.65 n.p. 0.69

NA 0 (n=0) 10 (n=2) 0 (n=0) 8 (n=2) 0.52 n.p. 0.56FAB_M0 0 (n=0) 0 (n=0) 0 (n=0) 0 (n=0) n.p. n.p. n.p.

FAB_M1 25 (n=1) 20 (n=4) 40 (n=2) 23 (n=6) 0.82 0.65 0.93FAB_M2 25 (n=1) 14 (n=3) 20 (n=1) 15 (n=4) 0.59 0.86 0.62

FAB_M4 0 (n=0) 28 (n=6) 20 (n=1) 27 (n=7) 0.24 0.37 0.25FAB_M4E 0 (n=0) 5 (n=1) 0 (n=0) 4 (n=1) 0.65 n.p. 0.69

FAB_M5 50 (n=2) 14 (n=3) 20 (n=1) 15 (n=4) 0.11 0.37 0.11

FAB_M6 0 (n=0) 0 (n=0) 0 (n=0) 0 (n=0) n.p. n.p. n.p.

NA 0 (n=0) 20 (n=4) 0 (n=0) 15 (n=4) 0.34 n.p. 0.41

p value between low

and medium+highGFI1 low GFI1 medium GFI1 high

p value between low and medium

p value between low and

highGFI1 medium

+ high

GFI1 low includes patients who express GFI1 lower than 5%. GFI1 medium includes all patients from 6%

to 60% and GFI1 high includes all patients with 61% to 100% GFI1 expression.

Cytogenetic low risk Cytogenetic low risk: inv(16), t(8;21), t(15;17), poor risk: complex aberrations (≥4

anomalies), chromosome 3q, 5, 7 anomalies, inv(3), t(6;11), t(10;11), t(11q23), t(9;22) Intermediate risk:

all other aberrations 2. Presented is the standard error of mean. Student’s t- test is used for significance

of values. For significance of differences between percentages, two sample t- test were used. n.p= not

possible

15

Suppl. Table 2:

AML Patients Cohort from Verhaak et al.

% all 11 (n=32) 82 (n=233) 7 (n=20) 89 (n=253) - - -Mean Age 41.5 ± 2.2 43.24 ± 0.8 38.8 ± 2.3 42.9 ± 0.77 0.43 0.46 0.55

Gender (% male) 47 (n=15) 56 (n=130) 65 (n=13) 57 (n=143) 0.21 0.34 0.28

Cytogenetic low risk (%) 8 (n=2) 19 (n=36) 65 (n=11) 22 (n=47) 0.0006 0.36 0.066

Cytogenetic intermediate risk (%) 44 (n=11) 64 (n=125) 29 (n=5) 61 (n=130) 0.5 0.045 0.066Cytogenetic high risk (%) 48 (n=12) 16 (n=31) 6 (n=1) 15 (n=32) 0.0001 <0.0001 <0.0001

KRAS_mutation 3 (n=1) 1 (n=3) 0 (n=0) 1 (n=3) 0.44 0.34 0.33

IDH1_mutation 3 (n=1) 10 (n=23) 5 (n=1) 10 (n=24) 0.71 0.2 0.2

IDH2_mutation 9 (n=3) 9 (n=21) 5 (n=1) 9 (n=22) 0.6 1.0 1.0

FLT3_TKD_mutation 0 (n=0) 14 (n=32) 5 (n=1) 13 (n=33) 0.21 0.025 0.03

NRAS_mutation 22 (n=7) 9 (n=20) 10 (n=2) 9 (n=22) 0.27 0.026 0.024

FLT3_ITD_mutation 3 (n=1) 33 (n=76) 15 (n=3) 31 (n=79) 0.12 0.0006 0.001

CEBPA_mutation 22 (n=7) 9 (n=22) 0 (n=0) 4 (n=22) 0.029 0.026 0.0001

NPM1_mutation 6 (n=2) 37 (n=87) 15 (n=3) 36 (n=90) 0.26 0.0006 0.0001

EVI1_expression 22 (n=7) 4 (n=9) 0 (n=0) 4 (n=9) 0.029 0.0001 0.0001

FAB_M0 19 (n=6) 2 (n=5) 0 (n=0) 2 (n=5) 0.43 0.0097 <0.0001

FAB_M1 34 (n=11) 22 (n=52) 20 (n=4) 22 (n=56) 0.28 0.13 0.13

FAB_M2 3 (n=1) 22 (n=52) 55 (n=11) 25 (n=63) 0.0001 0.012 0.005

FAB_M4 9 (n=3) 22 (n=51) 10 (n=2) 21 (n=53) 0.9 0.089 0.11

FAB_M4E 6 (n=2) 0 (n=0) 0 (n=0) 0 (n=0) 0.27 0.0002 0.0001

FAB_M5 19 (n=6) 22 (n=52) 15 (n=3) 22 (n=55) 0.71 0.7 0.7

FAB_M6 0 (n=0) 1 (n=2) 0 (n=0) 0 (n=0) n.p. 0.57 n.p.

RAEB 0 (n=0) 2 (n=4) 0 (n=0) 0 (n=0) n.p. 0.42 n.p.

RAEB-T 0 (n=0) 4 (n=10) 0 (n=0) 0 (n=0) n.p. 0.25 n.p.

NA 10 (n=3) 3 (n=7) 11 (n=2) 5 (n=9) 0.91 0.055 0.25

p value (between low and medium +

high)

p value (between low and medium)GFI1 low GFI1 high

p value (between low and

high)GFI1 medium

GFI1 medium +

high

GFI1 low includes patients who express GFI1 lower than 5%. GFI1 medium includes all patients from 6%

to 60% and GFI1 high includes all patients with 61% to 100% GFI1 expression.

Cytogenetic low risk: inv(16), t(8;21), t(15;17), poor risk: complex aberrations (≥4 anomalies),

chromosome 3q, 5, 7 anomalies, inv(3), t(6;11), t(10;11), t(11q23), t(9;22) Intermediate risk: all other

aberrations 2. Presented is the standard error of mean. Student’s t- test is used for significance of values.

For significance of differences between percentages, two sample t- test were used. n.p= not possible

16

Suppl. Table 3: Cox-regressional analysis on prognostic factors

p-Value

Hazard

Ratio

Lower 95%

Confidence

Interval

Higher95%

Confidence

Interval

Caryotype (Good prognosis

caryotype)

,17 0,67 0,4 1,2

NRAS_mutation (present) ,9 1,03 0,6 1,7

NPM1_mutation (present) ,02 0,6 0,4 0,9

EVI1_expression (present) ,09 1,7 0,9 3,2

CEBPA_mutation (present) ,04 0,5 0,27 0,98

Gfi1 expression (low

expression)

,05 3,1 1,01 9,2

Cox regressional multivariate analysis was performed on the cohort presented by Verhaak et al.

(see Supplementary Table 2) including low GFI1 expression.

17

Suppl. Table 4: Patient characteristics of examined samplesName Genotype Gfi1 origin Description

Gfi1-WT WT/WT murine normal WT Gfi1

GFI1-KI KI/KI human murine Gfi1 is replaced by human GFI1

GFI1-KD KD/KD human

murine Gfi1 is replaced by human Gfi1, only 5-15%

expression

Gfi1-EGFP-KI WT/GFP murine one Gfi1 allele is replaced by GFP

Overview about the used mice strains

The table gives an overview about the different mice strains, which were used in these studies. It is

depicted where the Gfi1 is originated from (murine or human). Furthermore a short description about the

exact genotype is given.

For References please see Material and Methods.

18

Suppl. Table 5: Characteristics of patients treated with Vorinostat and CTK7a

Patient Age Sex Genotype Cytogenetic Mutations SourceGfi1 level

1 39 M Gfi1 36S high normal karyotype none bone marrow

2 22 F Gfi1 36S low normal karyotype none bone marrow

F=female, M= male, high= high GFI1 expression, low= low GFI1 expression

For detailed experiment information please see Material and Methods. The Gfi1 mRNA expression was 7-

fold higher in patient 1.

19

Supplementary Material and Methods

Statistical methods

Statistical analysis was done with Graph-Pad Prism 4 or 6 software (La Jolla, CA, USA)

or SPSS version 19 (IBM, Düsseldorf, Germany). The survival rate was calculated using

the Kaplan-Meier method. P values below 0.05 were considered statistically significant.

Differences in percentage were determined using the two-sample t-test. Other statistical

analyses were done using either paired or unpaired two-sided Student´s t-test. The EFS

was assessed unadjusted by stratifying only for expression levels of GFI1. The Cox

proportional-hazards regression modeling was used for age, sex and cytogenetic

findings. For survival of the human cohorts the Log-rank and for murine cohorts the

Mantel Cox test was used. All p values reported are two-sided and considered

significant at 0.05. Due to the exploratory nature of our work, no adjustments were

made for multiple hypothesis testing.

Isolation and culture of hematopoietic progenitors

Lineage-negative (Lin-) cells were isolated from 8-12 week old mice using a Lineage

Cell Depletion Kit (130-090-858, Miltenyi Biotec, Bergisch Gladbach, Germany). For in

vitro studies, Lin- cells were cultured at a density of 1*106 cells/ml in SCM medium

(IMDM (GIBCO) 20% FBS (PAN Biotech GmbH), 1% penicillin-streptomycin (Sigma-

Aldrich, Taufkirchen, Germany) and 20 ng/ml SCF (Miltenyi Biotec Biotec, Cologne,

Germany), 10 ng/ml Interleukin-3 (Miltenyi) and 10 ng/ml Interleukin-6 (Miltenyi Biotec)).

Lin- cells for transplantation were retrovirally transduced using Marrow Max Bone

20

Marrow Medium (GIBCO, Darmstadt, Germany) (20 ng/ml SCF, 10 ng/ml Interleukin-3

and 10 ng/ml Interleukin-6.

Retrovirus production, transduction and transplantation of murine hematopoietic

progenitors

Retroviral transduction of Lin- cells with MLL-AF9 onco-fusion protein-encoding

vectorswas performed at day 3 and 4 after isolation of the cells. Retroviral supernatants

were collected after transient calcium phosphate transfection of 293Tmyc cells with 2.25

μg pCL-Eco retroviral packaging vector (IMGENEX) and 20 µg retroviral plasmid DNA.

Nontissue-treated 48 well plates were coated with 1mg/ml retronectin (Takara Bio Inc.,

Saint-Germaine en Lye, France) overnight at 4°C and blocked with 2% bovine serum

albumin, fraction V (BSA, Sigma) for 30 minutes at room temperature. The virus

supernatant was centrifuged two times for 90 min onto retronectin-coated plates and

Lin- cells were centrifuged for 10 min at 1200 rpm and 4°C. 2µg/ml of polybrene

reagent, Millipore (TR-1003-G) was added and the cells maintained at 37°C in 5% CO 2.

Transduced cells (1*105 GFP-positive cells) were harvested at day 5 after isolation and

injected i.v. into lethally irradiated mice together with 1.5*105 congenic competitive BM

cells.

Flow cytometry analysis

The following antibodies were used for FACS staining: Ly-6G/Ly-6C(Gr-1) (108407),

CD11b (101227), CD8a (100707), CD4 (100433), TER-119/Erythroid Cells Antibody

(116207), CD45R/B220 (103233), CD117(c-Kit) (135105), all from Biolegend (San

21

Diego, CA, USA). Apoptosis was measured by staining the cells with Annexin V

(556419, BD Bioscience, Heidelberg, Germany). For GMP staining, 3*106 BM cells were

stained with CD16/32 PE-Cy7 (101318), Biotin mouse lineage depletion cocktail, Sca-1

FITC (108105 ), CD34 PE (119308), cKit APC (105811), SAv PerCP (45-4317-82),

(ebioscience), Cells were analyzed using FACScan and LSR-II (Becton-Dickinson).

Cell culture

Kasumi1, HL-60 and 293Tmyc cells were obtained from ATCC (Manassas, VA, USA)

and cultured according to supplier’s instructions.

Western Blot

Gfi1 (2.5D17) (1:1000; G6670); Sigma-Aldrich; Anti-Mouse IgG (Goat) (1:10000;

NEF822001EA; Perkin Elmer), Lamin B (C-20) (1:1000; sc-6216; Santa Cruz) and

donkey anti-goat IgG-HRP (1:13000; sc-2020; Santa Cruz) antibodies were used for

Western Blot.

Inhibitoradministration

Vorinostat (SAHA, cat. nr. SML0061-25MG, Sigma-Aldrich) and CTK7a (382115-10mg,

Millipore) were dissolved in DMSO. Approximately 7*104-1*105 cells of peripheral blood

or BM aspirates of AML patients were seeded in 96 well non-tissue culture plates in

IMDM (20% FBS, 1% Pen/Strep and 10 ng/ml human SCF, 10 ng/ml FLT3-Ligand and

10 ng/ml human TPO (Miltenyi Biotec)).

22

RNA-Sequencing and ChIP-Sequencing:

RNA-sequencing:

Sequencing libraries were prepared from RNA extracts using the Illumina TruSeq

Stranded mRNA Kit according to the manufacturer’s instructions, and sequenced using

the TruSeq PE Clusterkit v3-cBot-HS on an Illumina HiSEq 2000 system. Sequencing

reads were aligned to the mm10 genome using Tophat v2.0.10 3. Reads were

processed with Samtools 4 and then mapped to Ensembl transcripts using HTSeq.

Differential expression was tested using the DESeq R package (R Coding Team).

A genome coverage file was generated and scaled to RPM using Bedtools 5 (GEO

accession no. GSE72671).

ChIP-sequencing:

Sequencing libraries were prepared from immunoprecipitated chromatin samples using

the TruSeq DNA kit from Illumina according to the manufacturer’s instructions and

sequenced using the TruSeq PE Clusterkit v3-cBot-HS on an Illumina HiSeq 2000

system.Reads were aligned to the mouse reference mm10 genome using Bowtie2

v2.10 5. Reads were processed and duplicates were removed using Samtools, and

promoter coverage for heatmaps was generated using the annotatePeaks.pl function of

the Homer Software. The CuffDiff R package was used to quantify changes in

acetylation levels on gene promoters.

23

References

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