• Phospho-serine RUNX1 increases upon megakaryocytic fate specification, and phosphomimetic mutant RUNX1 promotes megakaryocytic fate in MEP.

  • CDK9-induced phosphorylation of RUNX1 in MEPs promotes megakaryocytic fate at the expense of erythroid fate.

Abstract

The specification of megakaryocytic (Mk) or erythroid (E) lineages from primary human megakaryocytic-erythroid progenitors (MEPs) is crucial for hematopoietic homeostasis, yet the underlying mechanisms regulating fate specification remain elusive. In this study, we identify RUNX1 as a key modulator of gene expression during MEP fate specification. Overexpression of RUNX1 in primary human MEPs promotes Mk specification, whereas pan-RUNX inhibition favors E specification. Although total RUNX1 levels do not differ between Mk progenitors (MkPs) and E progenitors (ErPs), there are higher levels of serine-phosphorylated RUNX1 in MkPs than ErPs, and mutant RUNX1 with phosphorylated-serine/threonine mimetic mutations (RUNX1-4D) significantly enhances the functional efficacy of RUNX1. To model the effects of RUNX1 variants, we use human erythroleukemia (HEL) cell lines expressing wild-type (WT), phosphomimetic (RUNX1-4D), and nonphosphorylatable (RUNX1-4A) mutants showing that the 3 forms of RUNX1 differentially regulate expression of 2625 genes. Both WT and RUNX1-4D variants increase expression in 40%, and decrease expression in another 40%, with lesser effects of RUNX1-4A. We find a significant overlap between the upregulated genes in WT and RUNX1-4D–expressing HEL cells and those upregulated in primary human MkPs vs MEPs. Although inhibition of known RUNX1 serine/threonine kinases does not affect phosphoserine RUNX1 levels in primary MEPs, specific inhibition of cyclin dependent kinase 9 (CDK9) in MEPs leads to both decreased RUNX1 phosphorylation and increased E commitment. Collectively, our findings show that serine/threonine phosphorylation of RUNX1 promotes Mk fate specification and introduce a novel kinase for RUNX1 linking the fundamental transcriptional machinery with activation of a cell type–specific transcription factor.

1.
Woolthuis
CM
,
Park
CY
.
Hematopoietic stem/progenitor cell commitment to the megakaryocyte lineage
.
Blood
.
2016
;
127
(
10
):
1242
-
1248
.
2.
Xavier-Ferrucio
J
,
Krause
DS
.
Concise review: bipotent megakaryocytic-erythroid progenitors: concepts and controversies
.
STEM CELLS
.
2018
;
36
(
8
):
1138
-
1145
.
3.
Sanada
C
,
Xavier-Ferrucio
J
,
Lu
Y-C
, et al
.
Adult human megakaryocyte-erythroid progenitors are in the CD34+CD38mid fraction
.
Blood
.
2016
;
128
(
7
):
923
-
933
.
4.
Kwon
N
,
Thompson
EN
,
Mayday
MY
,
Scanlon
V
,
Lu
YC
,
Krause
DS
.
Current understanding of human megakaryocytic-erythroid progenitors and their fate determinants
.
Curr Opin Hematol
.
2021
;
28
(
1
):
28
-
35
.
5.
Lu
YC
,
Xavier-Ferrucio
J
,
Wang
L
, et al
.
The molecular signature of megakaryocyte-erythroid progenitors reveals a role for the cell cycle in fate specification
.
Cell Rep
.
2018
;
25
(
8
):
2083
-
2093.e2084
.
6.
Xavier-Ferrucio
J
,
Scanlon
V
,
Li
X
, et al
.
Low iron promotes megakaryocytic commitment of megakaryocytic-erythroid progenitors in humans and mice
.
Blood
.
2019
;
134
(
18
):
1547
-
1557
.
7.
Warren
AJ
,
Bravo
J
,
Williams
RL
,
Rabbitts
TH
.
Structural basis for the heterodimeric interaction between the acute leukaemia-associated transcription factors AML1 and CBFb
.
EMBO J
.
2000
;
19
(
12
):
3004
-
3015
.
8.
Collins
A
,
Littman
DR
,
Taniuchi
I
.
RUNX proteins in transcription factor networks that regulate T-cell lineage choice
.
Nat Rev Immunol
.
2009
;
9
(
2
):
106
-
115
.
9.
Growney
JD
,
Shigematsu
H
,
Li
Z
, et al
.
Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype
.
Blood
.
2005
;
106
(
2
):
494
-
504
.
10.
Owen
CJ
,
Toze
CL
,
Koochin
A
, et al
.
Five new pedigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy
.
Blood
.
2008
;
112
(
12
):
4639
-
4645
.
11.
Kuvardina
ON
,
Herglotz
J
,
Kolodziej
S
, et al
.
RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation
.
Blood
.
2015
;
125
(
23
):
3570
-
3579
.
12.
Zhou
L
,
Wu
D
,
Zhou
Y
, et al
.
Tumor cell-released kynurenine biases MEP differentiation into megakaryocytes in individuals with cancer by activating AhR-RUNX1
.
Nat Immunol
.
2023
;
24
(
12
):
2042
-
2052
.
13.
Biggs
JR
,
Peterson
LF
,
Zhang
Y
,
Kraft
AS
,
Zhang
D-E
.
AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex
.
Mol Cell Biol
.
2006
;
26
(
20
):
7420
-
7429
.
14.
Illendula
A
,
Gilmour
J
,
Grembecka
J
, et al
.
Small molecule inhibitor of CBFβ-RUNX binding for RUNX transcription factor driven cancers
.
EBioMedicine
.
2016
;
8
:
117
-
131
.
15.
Zhang
L
,
Fried
FB
,
Guo
H
,
Friedman
AD
.
Cyclin-dependent kinase phosphorylation of RUNX1/AML1 on 3 sites increases transactivation potency and stimulates cell proliferation
.
Blood
.
2008
;
111
(
3
):
1193
-
1200
.
16.
Skene
PJ
,
Henikoff
S
.
An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites
.
Elife
.
2017
;
6
:
e21856
.
17.
Xie
Z
,
Bailey
A
,
Kuleshov
MV
, et al
.
Gene set knowledge discovery with Enrichr
.
Curr Protoc
.
2021
;
1
(
3
):
e90
.
18.
Draper
JE
,
Sroczynska
P
,
Leong
HS
, et al
.
Mouse RUNX1C regulates premegakaryocytic/erythroid output and maintains survival of megakaryocyte progenitors
.
Blood
.
2017
;
130
(
3
):
271
-
284
.
19.
Morita
K
,
Suzuki
K
,
Maeda
S
, et al
.
Genetic regulation of the RUNX transcription factor family has antitumor effects
.
J Clin Invest
.
2017
;
127
(
7
):
2815
-
2828
.
20.
Bluteau
D
,
Glembotsky
AC
,
Raimbault
A
, et al
.
Dysmegakaryopoiesis of FPD/AML pedigrees with constitutional RUNX1 mutations is linked to myosin II deregulated expression
.
Blood
.
2012
;
120
(
13
):
2708
-
2718
.
21.
Ichikawa
M
,
Asai
T
,
Saito
T
, et al
.
AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis
.
Nat Med
.
2004
;
10
(
3
):
299
-
304
.
22.
Wang
L
,
Huang
G
,
Zhao
X
, et al
.
Post-translational modifications of Runx1 regulate its activity in the cell
.
Blood Cells Mol Dis
.
2009
;
43
(
1
):
30
-
34
.
23.
Zhang
Y
,
Biggs
JR
,
Kraft
AS
.
Phorbol ester treatment of K562 cells regulates the transcriptional activity of AML1c through phosphorylation
.
J Biol Chem
.
2004
;
279
(
51
):
53116
-
53125
.
24.
Long
MW
,
Heffner
CH
,
Williams
JL
,
Peters
C
,
Prochownik
EV
.
Regulation of megakaryocyte phenotype in human erythroleukemia cells
.
J Clin Invest
.
1990
;
85
(
4
):
1072
-
1084
.
25.
Martin
P
,
Papayannopoulou
T
.
HEL cells: a new human erythroleukemia cell line with spontaneous and induced globin expression
.
Science
.
1982
;
216
(
4551
):
1233
-
1235
.
26.
Murate
T
,
Saga
S
,
Hotta
T
, et al
.
The close relationship between DNA replication and the selection of differentiation lineages of human erythroleukemia cell lines K562, HEL, and TF1 into either erythroid or megakaryocytic lineages
.
Exp Cell Res
.
1993
;
208
(
1
):
35
-
43
.
27.
Papayannopoulou
T
,
Nakamoto
B
,
Kurachi
S
,
Nelson
R
.
Analysis of the erythroid phenotype of HEL cells: clonal variation and the effect of inducers
.
Blood
.
1987
;
70
(
6
):
1764
-
1772
.
28.
Koh
CP
,
Wang
CQ
,
Ng
CEL
, et al
.
RUNX1 meets MLL: epigenetic regulation of hematopoiesis by two leukemia genes
.
Leukemia
.
2013
;
27
(
9
):
1793
-
1802
.
29.
Bailey
TL
.
STREME: accurate and versatile sequence motif discovery
.
Bioinformatics
.
2021
;
37
(
18
):
2834
-
2840
.
30.
Elagib
KE
,
Racke
FK
,
Mogass
M
,
Khetawat
R
,
Delehanty
LL
,
Goldfarb
AN
.
RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation
.
Blood
.
2003
;
101
(
11
):
4333
-
4341
.
31.
Shrivastava
T
,
Mino
K
,
Babayeva
ND
,
Baranovskaya
OI
,
Rizzino
A
,
Tahirov
TH
.
Structural basis of Ets1 activation by Runx1
.
Leukemia
.
2014
;
28
(
10
):
2040
-
2048
.
32.
Zheng
R
,
Wan
C
,
Mei
S
, et al
.
Cistrome Data Browser: expanded datasets and new tools for gene regulatory analysis
.
Nucleic Acids Res
.
2019
;
47
(
D1
):
D729
-
D735
.
33.
Tijssen
MR
,
Cvejic
A
,
Joshi
A
, et al
.
Genome-wide analysis of simultaneous GATA1/2, RUNX1, FLI1, and SCL binding in megakaryocytes identifies hematopoietic regulators
.
Dev Cell
.
2011
;
20
(
5
):
597
-
609
.
34.
Wilson
NK
,
Foster
SD
,
Wang
X
, et al
.
Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators
.
Cell Stem Cell
.
2010
;
7
(
4
):
532
-
544
.
35.
Pantano
L
.
DEGreport: Report of DEG Analysis. R package version. 2023;1.36.0
. Accessed 31 March 2023. http://lpantano.github.io/DEGreport/.
36.
Fujiki
H
,
Kimura
T
,
Minamiguchi
H
, et al
.
Role of human interleukin-9 as a megakaryocyte potentiator in culture
.
Exp Hematol
.
2002
;
30
(
12
):
1373
-
1380
.
37.
Xu
XR
,
Carrim
N
,
Neves
MAD
, et al
.
Platelets and platelet adhesion molecules: novel mechanisms of thrombosis and anti-thrombotic therapies
.
Thromb J
.
2016
;
14
(
suppl 1
):
29
.
38.
Zou
S
,
Teixeira
AM
,
Yin
M
, et al
.
Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation
.
Thromb Haemost
.
2016
;
116
(
3
):
506
-
516
.
39.
Ji
M
,
Li
H
,
Suh
HC
,
Klarmann
KD
,
Yokota
Y
,
Keller
JR
.
Id2 intrinsically regulates lymphoid and erythroid development via interaction with different target proteins
.
Blood
.
2008
;
112
(
4
):
1068
-
1077
.
40.
Uoshima
N
,
Ozawa
M
,
Kimura
S
, et al
.
Changes in c-Kit expression and effects of SCF during differentiation of human erythroid progenitor cells
.
Br J Haematol
.
1995
;
91
(
1
):
30
-
36
.
41.
Love
MI
,
Huber
W
,
Anders
S
.
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2
.
Genome Biol
.
2014
;
15
(
12
):
550
.
42.
Tanaka
T
,
Kurokawa
M
,
Ueki
K
, et al
.
The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability
.
Mol Cell Biol
.
1996
;
16
(
7
):
3967
-
3979
.
43.
Wang
S
,
Zhang
Y
,
Soosairajah
J
,
Kraft
AS
.
Regulation of RUNX1/AML1 during the G2/M transition
.
Leuk Res
.
2007
;
31
(
6
):
839
-
851
.
44.
Wee
HJ
,
Voon
DC
,
Bae
SC
,
Ito
Y
.
PEBP2-beta/CBF-beta-dependent phosphorylation of RUNX1 and p300 by HIPK2: implications for leukemogenesis
.
Blood
.
2008
;
112
(
9
):
3777
-
3787
.
45.
Johnson
JL
,
Yaron
TM
,
Huntsman
EM
, et al
.
An atlas of substrate specificities for the human serine/threonine kinome
.
Nature
.
2023
;
613
(
7945
):
759
-
766
.
46.
Elagib
KE
,
Mihaylov
IS
,
Delehanty
LL
, et al
.
Cross-talk of GATA-1 and P-TEFb in megakaryocyte differentiation
.
Blood
.
2008
;
112
(
13
):
4884
-
4894
.
47.
Qing
Y
,
Wang
X
,
Wang
H
, et al
.
Pharmacologic targeting of the P-TEFb complex as a therapeutic strategy for chronic myeloid leukemia
.
Cell Commun Signal
.
2021
;
19
(
1
):
83
.
48.
Ni
Z
,
Schwartz
BE
,
Werner
J
,
Suarez
J-R
,
Lis
JT
.
Coordination of transcription, RNA processing, and surveillance by P-TEFb kinase on heat shock genes
.
Mol Cell
.
2004
;
13
(
1
):
55
-
65
.
49.
Yoshimi
M
,
Goyama
S
,
Kawazu
M
, et al
.
Multiple phosphorylation sites are important for RUNX1 activity in early hematopoiesis and T-cell differentiation
.
Eur J Immunol
.
2012
;
42
(
4
):
1044
-
1050
.
50.
Starck
J
,
Cohet
N
,
Gonnet
C
, et al
.
Functional cross-antagonism between transcription factors FLI-1 and EKLF
.
Mol Cell Biol
.
2003
;
23
(
4
):
1390
-
1402
.
51.
Pencovich
N
,
Jaschek
R
,
Tanay
A
,
Groner
Y
.
Dynamic combinatorial interactions of RUNX1 and cooperating partners regulates megakaryocytic differentiation in cell line models
.
Blood
.
2011
;
117
(
1
):
e1
-
e14
.
52.
Bacon
CW
,
D'Orso
I
.
CDK9: a signaling hub for transcriptional control
.
Transcription
.
2019
;
10
(
2
):
57
-
75
.
53.
Jiang
H
,
Zhang
F
,
Kurosu
T
,
Peterlin
BM
.
Runx1 binds positive transcription elongation factor b and represses transcriptional elongation by RNA polymerase II: possible mechanism of CD4 silencing
.
Mol Cell Biol
.
2005
;
25
(
24
):
10675
-
10683
.
54.
Guo
H
,
Friedman
AD
.
Phosphorylation of RUNX1 by cyclin-dependent kinase reduces direct interaction with HDAC1 and HDAC3
.
J Biol Chem
.
2011
;
286
(
1
):
208
-
215
.
You do not currently have access to this content.
Sign in via your Institution