Received: 14 August 2019 | Accepted: 31 January 2020

DOI: 10.1002/jcp.29642

REVIEW ARTICLE

Altering chromatin methylation patterns and the

transcriptional network involved in regulation of
hematopoietic stem cell fate

Mohammad Shokouhian1 | Marziye Bagheri2 | Behzad Poopak3 |

Rouzbeh Chegeni4 | Nader Davari2 | Najmaldin Saki2

1
Department of Hematology and Blood
Transfusion, School of Allied Medical Sciences, Abstract
Iran University of Medical Sciences,
Tehran, Iran Hematopoietic stem cells (HSCs) are quiescent cells with self‐renewal capacity and
2
Thalassemia and Hemoglobinopathy Research potential multilineage development. Various molecular regulatory mechanisms such
Center, Health Research Institute, Ahvaz
as epigenetic modifications and transcription factor (TF) networks play crucial roles
Jundishapur University of Medical Sciences,
Ahvaz, Iran in establishing a balance between self‐renewal and differentiation of HSCs. Histone/
3
Department of Hematology, Faculty of DNA methylations are important epigenetic modifications involved in transcriptional
Paramedical Sciences, Tehran Medical
Sciences, Islamic Azad University, Tehran, Iran regulation of specific lineage HSCs via controlling chromatin structure and
4
Michener Institute of Education at University accessibility of DNA. Also, TFs contribute to either facilitation or inhibition of gene
Health Network, Toronto, Canada
expression through binding to enhancer or promoter regions of DNA. As a result,
Correspondence epigenetic factors and TFs regulate the activation or repression of HSCs genes,
Najmaldin Saki, Research Center of
playing a central role in normal hematopoiesis. Given the importance of
Thalassemia and Hemoglobinopathy, Health
Research Institute, Ahvaz Jundishapur histone/DNA methylation and TFs in gene expression regulation, their aberrations,
University of Medical Sciences, Ahvaz, Iran.
including changes in HSCs‐related methylation of histone/DNA and TFs (e.g.,
Email: najmaldinsaki@gmail.com
CCAAT‐enhancer‐binding protein α, phosphatase and tensin homolog deleted on the
chromosome 10, Runt‐related transcription factor 1, signal transducers and
activators of transcription, and RAS family proteins) could disrupt HSCs fate.
Herewith, we summarize how dysregulations in the expression of genes related to
self‐renewal, proliferation, and differentiation of HSCs caused by changes in
epigenetic modifications and transcriptional networks lead to clonal expansion and
leukemic transformation.

KEYWORDS

epigenetic modifications, gene expression, hematopoietic stem cells, leukemia, transcriptional

regulation

1 | INTRODUCTION equilibrium between self‐renewal and differentiation (Mayani, 2016;

Hematopoietic stem cells (HSCs) are characterized by their quies-
cent state despite preserving self‐renewal and multipotency cap-
abilities. HSCs undergo a hierarchical cascade of differentiation,
which gives rise to different mature cells of myeloid and lymphoid
lineages (Dorritie, Redner, & Johnson, 2014). To ensure their lifelong
maintenance in the bone marrow (BM), HSCs rely on a dynamic
Z. Wang & Bunting, 2013).
HSCs fate is governed by a combination of cell‐intrinsic networks
and extrinsic stimuli (Mayani, 2016). Some intrinsic regulators, in-
cluding epigenetic modifiers, transcription factors (TFs), and signaling
pathways are effective in stem cell function. Indeed, these intrinsic
regulators play a vital role in controlling the balance between self‐
renewal and differentiation of HSCs (Mayani, 2016; Sharma &

J Cell Physiol. 2020;1–20. wileyonlinelibrary.com/journal/jcp © 2020 Wiley Periodicals, Inc. | 1

2 |
Gurudutta, 2016). Modifications in components of chromatin (his-
tones and DNA) cause changes in the structure of nucleosomes,
and make important contributions to gene expression and have a
central role in the determination of HSCs' fate because a decrease in
the expression of self‐renewal genes is correlated to the activation of
genes promoting HSCs differentiation (Mayani, 2016; Ohlsson,
Schuster, Hasemann, & Porse, 2016). Interaction between chromatin
modifications (e.g., histone acetylation/deacetylation and histone/
DNA methylation/demethylation) and TFs plays an essential role in
the modulation of gene expression in HSCs. As processes effective in
chromatin modifications, histone, and DNA methylations are capable
of making genomic DNA accessible for cellular processes such as
transcription (Sharma & Gurudutta, 2016). On the other hand, once
TFs bind to the enhancer or promoter regions of DNA, they facilitate
or inhibit gene expression. When TFs bind to a specific DNA
sequence of target genes, RNA polymerase II is recruited to the
promoter and contributes to transcriptional regulation of HSCs and
lineage‐specific gene expression (Ohlsson et al., 2016; Safaei
et al., 2018).
Aberrant changes or mutations in epigenetic regulators can re-
sult in perturbation of normal hematopoiesis, aberrant clonal hema-
topoietic expansion, and leukemic transformation via altering histone
modifications and gene expression (Carbuccia et al., 2009; Chan &
Majeti, 2013; Kunimoto & Nakajima, 2017). It seems that dysregu-
lations in histone/DNA methylation and TFs activity in HSCs lead to
aberrant gene expression, creating an imbalance between cell self‐
renewal and lineage commitment decisions, leading to abnormal
hematopoiesis and leukemogenesis (myeloid and lymphoid malig-
nancies). Hence, here we discuss how histone/DNA methylation and
TFs modulate gene expression of HSCs in a way that results in the
initiation or progression of leukemia.
2 | H ISTON E A ND DN A M E THY LA TION
I N V O L V E D I N G E N E E X P RE S S I O N O F
NORMA L H EMATOP O IESIS
Epigenetic modifications are vital biological processes for chromatin
remodeling, DNA accessibility and gene expression regulation
(Herviou, Cavalli, Cartron, Klein, & Moreaux, 2016). Histone and DNA
methylations are two types of epigenetic modifications cooperating to
control DNA functions of various cell types such as HSCs via modifying
chromatin and modulating transcription initiation, elongation, splicing,
and termination. These processes play a central role in regulating self‐
renewal, maintenance, and lineage differentiation of HSCs through
specific gene expression patterns (Herviou et al., 2016; Safaei
et al., 2018; Sharma & Gurudutta, 2016). According to Figure 1, pro-
cesses such as histone methylation at lysine 4 of histone H3 and DNA
demethylation are associated with gene transcriptional activation,
while methylation at lysine 27 of histone H3 and DNA methylation
leads to gene transcriptional silencing or stable long‐term repression.
These processes are dependent on one another and their cross‐talk is
essential for controlling gene expression in HSCs.
SHOKOUHIAN ET AL.
2.1 | Histone methylation
Histone modification, a type of posttranslational modification, is
implicated in different biological processes such as cell fate de-
termination via modulating genomic accessibility and gene expres-
sion caused by methylation of lysine or arginine residues. Histone
methylation marks have a central role in epigenetic regulation
through activating or repressing the transcription of genes involved
in either differentiation or proliferation (Asada & Kitamura, 2018;
Katoh, 2013; Madan et al., 2018; P. Zhang, Chen et al., 2018). Epi-
genetic modifiers, which are effective in histone methylation, can
help maintain the stability of specific genomic expression patterns in
HSCs or terminally differentiated cells (Cui et al., 2009).
A family of chromatin‐binding proteins named the additional sex
combs‐like (ASXL) family plays a vital role in histone modifications.
The ASXL1 gene is located on chromosome 20q11 and encodes the
ASXL1 protein as a tumor suppressor, which is crucial for hemato-
poiesis such as regulation of self‐renewal and differentiation of bone
marrow stromal cells and hematopoietic stem and progenitor cells
(HSPCs; Asada, Fujino, Goyama, & Kitamura, 2019; Oak &
Ohgami, 2017; P. Zhang, Chen et al., 2018). The ASXL genes (ASXL1,
ASXL2, and ASXL3) are human homologs of Drosophila Asx recruiting
polycomb group (PcG) repressor complexes (PRC; PRC1 and PRC2)
and trithorax group (TrxG) activator complexes to genes and mod-
ifying chromatin structures. PRC2 recruits PRC1 and catalyzes mono
‐ubiquitination of histone H2A at lysine 119 (H2AK119ub), resulting
in the recruitment of specific genomic loci, trimethylation catalysis of
lysine residue 27 (H3K27; H3K27me3) and consequent transcrip-
tional repression of PcG target genes (Figure 1; Katoh, 2013). TrxG
and PcG are critical proteins of the chromatin‐modification process
with opposing activities in the regulation of gene expression, so that
TrxG is associated with positive regulation of gene expression via
methylation of histone H3 on lysine residue 4 (H3K4), while PcG
represses gene expression through methylation of H3K27 (Krivtsov
& Armstrong, 2007). ASXL1 acts as a mediator for the function of
PRC2 and a cofactor for BRCA‐1 associated protein 1 (BAP1), which
is the deubiquitinase and essential component of the polycomb re-
pressive deubiquitinase complex (P. Zhang, Chen et al., 2018). ASXL1
directly binds to BAP1 and then removes the ubiquitination of
H2AK119ub, which results in transcriptional activation (Asada &
Kitamura, 2018; Asada et al., 2018; Katoh, 2013). On the other hand,
the BAP1/ASXL1 complex causes transcriptional repression by direct
binding to scaffold subunits (EZH2, EED, and SUZ12) of PRC2 (Asada
et al., 2018; Madan et al., 2018) and increasing H3K27me3, which
finally represses further recruitment of PRC1 (Asada et al., 2019;
Katoh, 2013). Histone‐lysine N‐methyltransferase EZH2 is an epige-
netic regulator for catalytic subunit of PRC2, which is effective in
controlling the proliferation and differentiation potential of em-
bryonic stem cells (ESCs) by repressing the INK4B‐ARF‐INK4A locus
and preventing the recruitment of AP1 transcriptional activator from
binding and activating late differentiation genes in ESCs (Ezhkova
et al., 2009). Furthermore, the ASXL1/BAP1 complex has a role in
controlling the expression of INK4B‐ARF‐INK4A locus and is also
SHOKOUHIAN ET AL. | 3

F I G U R E 1 Mechanisms of epigenetic modifications and transcription factor (TF) networks in gene expression of HSCs. Epigenetic modifiers
and TFs as regulators of gene expression which play a central role in balancing HSCs fate in normal hematopoiesis. Interaction between
epigenetic modifications and TFs network regulates the activation and repression of genes in HSCs and their aberrant interaction could lead to
leukemogenesis. α‐KG, α‐ketoglutarate; 5mC, 5‐methylcytosine; 5hmC, 5‐hydroxymethylcytosine; ASXL1, additional sex combs‐like; BAP1,
BRCA‐1 associated protein 1; CpG, cytosine‐phosphoguanine; DNMT3A, DNA methyltransferase; H2AK119ub, histone H2A at lysine 119;
H3K27me3, trimethylation catalysis of H3K27; H3K4me3, trimethylated H3K4; SAM, s‐adenosylmethionine; HAT, histone acetyltransferase;
HDAC, histone deacetylases; HSCs, hematopoietic stem cells; IDH1/2, isocitrate dehydrogenase; MLL, mixed‐lineage leukemia; PRC1/2,
polycomb group repressor complexes 1/2; SAH, s‐adenosylhomocysteine; TFs, transcription factors; TET2, ten‐eleven translocation 2

effective in cellular defense against tumorigenesis. It was shown that
high expression of INK4B increases both the recruitment of ASXL1/
BAP1 complex to INK4B locus and deubiquitylation of H2AK119ub,
resulting in cell growth arrest or differentiation (Wu et al., 2015). In
contrast, mutations in ASXL1 contribute to the expansion of hema-
topoietic progenitors in the BM as these mutations decrease the
deubiquitylation of H2AK119ub and increase DNA methylation in
the INK4B promoter followed by a decrease in the expression levels
of p15INK4B (Wu et al., 2015). Interaction of the ASXL1/BAP1
complex with BAP1‐binding partners such as HCFC1 results in the
recruitment of SET1‐like or mixed‐lineage leukemia (MLL) complexes
to specific genomic loci and catalysis of trimethylated H3K4
(H3K4me3), which contributes to transcriptional activation, which is
shown in Figure 1 (Katoh, 2013). The MLL gene is a human homolog
of Drosophila melanogaster TrxG encoding a DNA‐binding protein that
methylates H3K4 and plays an important role in the development
and expression of the Hox gene (Krivtsov & Armstrong, 2007).
Through upregulating the expression of posterior Hox genes in the
hematopoietic cell lineage, MLL triggers the proliferation of
immature hematopoietic progenitors and blocks hematopoietic
differentiation by maintaining the expression of self‐renewal‐related
genes at immature progenitor stages (Akihiko Yokoyama, 2015;
A. Yokoyama, 2017). In fact, MLL is a key factor in controlling the
transcriptional network of HSCs‐specific genes involved in self‐
renewal, proliferation and lineage‐specific differentiation (Artinger
et al., 2013). Previous studies have shown that histone modification
following EZH2 is essential in the cell‐cycle status of HSCs and cell
differentiation by modulating gene expression profiles (Mochizuki‐
Kashio et al., 2011). Given the high level of EZH2 expression in HSCs
and its decrease following differentiation, it has been reported that
EZH2 overexpression helps preserve the long‐term repopulation
potential of HSCs and prevents HSCs exhaustion under replicative
stress conditions (Kamminga et al., 2006). EZH2 has a cooperative
role in managing the balance between self‐renewal and differentia-
tion of HSCs by inhibiting the expression of HSCs differentiation
genes such as the inhibitor of differentiation 2 (ID2) and SOX7 as
well as normal B‐cell differentiation (Herviou et al., 2016; Safaei
et al., 2018). Moreover, EZH2 represses proapoptotic genes such as
NOXA, P21, and WIG1, thereby playing an essential role in protecting
HSCs from cell death (Herviou et al., 2016). EZH2 can increase the
pool of quiescent HSCs and prevent stem cell senescence via in-
hibiting apoptosis and proliferation of HSCs and also regulating
HSCs differentiation (Herviou et al., 2016). In fact,
EZH2‐mediated H3K27me3 leads to PRC1 binding to chromatin,
4 |
mono‐ubiquitinylated H2AK119ub and eventual transcription
repression of target genes (Herviou et al., 2016; Safaei et al., 2018).
EZH2 mutation increases the activity of HSCs, which means a rise in
HSCs proliferation that could result in heterogeneous hematopoietic
malignancies (Herviou et al., 2016; Mochizuki‐Kashio et al., 2015).
Research has provided evidence for the compensatory role of EZH1
for EZH2 (Mochizuki‐Kashio et al., 2011), so that EZH1 may be in-
fluential in the maintenance of hematopoiesis in the absence of EZH2
(Mochizuki‐Kashio et al., 2015). EZH1 is similar to EZH2 in main-
taining primitive hematopoietic cells and balancing HSCs preserva-
tion against senescence and physiological HSCs activation so that a
reduction in its expression is accompanied by the impairment of
HSCs self‐renewal capacity (Hidalgo & Gonzalez, 2013; Hidalgo
et al., 2012). Several studies suggest a connection between changes
in messenger RNA (mRNA) expression of PcG family genes with the
age of HSPCs; for instance, the expression of EZH1 and Bmi1 is
reduced in older HSPCs relative to younger ones, which implies that
the PcG family delays HSPCs senescence (Y. Dong et al., 2018).
According to the above, it can be deduced that PcG and TrxG family
genes and their related chromatin‐binding proteins have indis-
pensable roles in maintaining the mechanism of HSCs cellular
memory and preventing the alteration of HSCs fate determination
through chromatin modifications via promoting or repressing the
expression of target genes. Therefore, defects in these processes
contribute to the senescence, aberrant proliferation, or differentia-
tion of HSCs (defect in hematopoiesis), and development of
leukemogenesis.
2.2 | DNA methylation
DNA methylation/demethylation is another type of epigenetic mod-
ification, which is crucial in cellular processes, such as regulation of
gene expression, genomic stability, and cell lineage commitment.
Dysregulation of these modifications could result in aberrant stem
cell function and tumorigenesis (Brunetti, Gundry, & Goodell, 2017;
Nakajima & Kunimoto, 2014; Sui, Price, Li, & Chen, 2012). In fact,
changes in genomic DNA methylation induce distinctions in different
stages of hematopoietic differentiation as there is a descending
pattern in promoter methylation throughout HSPCs’ differentiation
and aging (Bocker et al., 2011). It was demonstrated that un-
methylated HSCs‐specific genes induce normal hematopoiesis, sup-
porting the role of DNA methylation in silencing the genes affecting
HSCs differentiation (Sharma & Gurudutta, 2016). DNA methyl-
transferase (DNMT) family (DNMT1 and DNMT3A/B) members
cooperate in transcriptional silencing of downstream genes by adding
a methyl group of S‐adenosyl‐methionine, to carbon‐5 (C5) of
cytosine‐phosphoguanine (CpG) dinucleotides in DNA and generating
5‐methylcytosine (5mC; Figure 1; Kunimoto & Nakajima, 2017).
DNMT1 intervenes in methylation of the newly synthesized CpG
island during DNA replication, whereas DNMT3A and DNMT3B in-
tervene in de novo methylation activity (Brunetti et al., 2017). DNA
methylation mediated by DNMT3A represses the expression of
SHOKOUHIAN ET AL.
self‐renewal genes (e.g., NR4A2) as well as the genes involved in
HSCs differentiation (e.g., RUNX1 and GATA3), which contributes to
the maintenance of self‐renewal and normal HSCs development
(Chan & Majeti, 2013; Trowbridge & Orkin, 2011). Two mechanisms
drive differentiation to lineage commitment, one of which is the no
silence gene in HSCs owing to the lack of methylation in DNMT3A‐
null HSCs and the other is elevated H3K4me3 due to receiving
differentiation signals (Challen et al., 2011). Therefore, DNA me-
thylation is an essential director of normal HSCs function.
The ten‐eleven translocation (TET) family (TET1, TET2, and
TET3) of proteins regulates DNA demethylation, making them pivotal
in the normal development of HSCs. Although TET2 and TET3 show
high expression in hematopoietic cells, TET1 has a higher level of
expression in ESCs and serves as a transcriptional repressor in these
cells (Pronier & Delhommeau, 2012). TET1 indirectly mediates the
binding of EZH2 to gene promoters, followed by the recruitment of a
repressive histone mark (H3K27me3) resulting in the repression of
gene expression and ESCs maintenance (Sui et al., 2012). However,
TET1 reduction resulting from PRC2 complex binding to TET1 pro-
motor contributes to cell proliferation (Neri et al., 2015). TET2 is a
tumor suppressor, whose gene is located on chromosome 4q24 and is
essential for DNA hypermethylation prevention and demethylation
promotion (L. Cimmino, Abdel‐Wahab, Levine, & Aifantis, 2011;
Mercher et al., 2012). DNA demethylation involves the conversion of
5mC to 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine (5fC) and
5‐carboxylcytosine (5caC), in which Fe (II) and a‐ketoglutarate (a‐KG)
binding to both the double‐stranded β‐helix fold domain located at
C‐terminus of TET2 protein and oxygen is helpful (L. Cimmino
et al., 2011; Feng, Li, Cassady, Zou, & Zhang, 2019; Mercher
et al., 2012). Indeed, methylated DNA recognition by TET2 in the
CpG island and 5mC insertion into the catalytic cavity facilitate their
reaction with catalytic Fe (II), leading to 5mC oxidation (Hu
et al., 2013). Then, 5hmC becomes deaminated by the activation‐
induced cytosine deaminase/apolipoprotein B mRNA‐editing enzyme
complex protein family, producing 5hmU, after which unmethylated
cytosine is replaced with thymine DNA glycosylase (TDG) via the
base exchange repair (BER) mechanism (L. Cimmino et al., 2011;
Mercher et al., 2012; Nakajima & Kunimoto, 2014). Similar to 5hmC,
5fC and 5caC are detected by TDG and unmethylated cytosine is
replaced with BER (Nakajima & Kunimoto, 2014). On the other hand,
TET2 can have an impact on the regulation of DNA methylation
through passive DNA demethylation, so that this process is per-
formed by replacing 5hmC with unmethylated cytosine (Bowman &
Levine, 2017; L. Cimmino et al., 2011). Moreover, TET protein in-
teraction with some proteins involved in epigenetic mechanisms, in-
cluding O‐linked β‐D‐N‐acetylglucosamine (O‐GlcNAc) transferase
(OGT), may be important in gene transcription regulation (Q. Chen,
Chen, Bian, Fujiki, & Yu, 2013; Nakajima & Kunimoto, 2014) as it was
revealed that TET–OGT interaction contributes to the increase in
binding of SET1/COMPASS H3K4 methyltransferase to chromatin
and subsequent promotion of transcriptional activation via increasing
histone H3K4me3 (Deplus et al., 2013; Feng et al., 2019; Nakajima &
Kunimoto, 2014). Prior studies suggest that the catalytic activity of
SHOKOUHIAN ET AL.
TET2 is related to the proteins encoded by other genes such as
metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 (Ko &
Rao, 2011; Pronier & Delhommeau, 2012). IDH1/IDH2 enzymes
convert isocitrate to a‐KG through oxidative decarboxylation
(L. Cimmino et al., 2011; Nakajima & Kunimoto, 2014). As shown in
Figure 1, α‐KG is required for the functioning of the TET family and
some other enzymes such as histone demethylases (JmjC family) and
DNA methyltransferases (ALKBH2/3 and MGMT). It seems that
α‐KG‐dependent reactions are effective in epigenetics and DNA re-
pair of HSCs (Inoue, Lemonnier, & Mak, 2016). IDH1/IDH2 mutant
proteins generate an oncometabolite (i.e., 2‐hydroxyglutarate) that
competes with a‐KG because a‐KG is capable of inhibiting the cata-
lytic activity of TET2 (L. Cimmino et al., 2011; Holmfeldt & Mulligh-
an, 2011; Mercher et al., 2012). The IDH1 mutant promotes clonal
expansion by enhancing self‐renewal in HSCs and expanding lineage‐
specific Sca1+ c‐Kit+ (LSK) cells (Chan & Majeti, 2013). Furthermore,
it has been shown that posttranslational modification (phosphoryla-
tion) of the TET2 enzyme is effective in epigenetic changes during
hematopoiesis. In this context, J. J. Jeong et al. (2019) reported that
Janus kinase 2 (JAK2) results in the interaction of phosphorylated
TET2 with KLF1 (erythroid transcription factor) by phosphorylating
tyrosine's 1939 and 1964 in the catalytic core of TET2. In addition,
increased JAK2 activation caused by erythropoietin (EPO) has a
positive feedback effect on the interaction between TET2 and KLF1,
increasing TET2 activation followed by elevated levels of 5hmC in
the genome and resulting in an increase of erythroid differentiation
(J. J. Jeong et al., 2019). However, the increase of 5hmC in specific
promoter regions of HSPCs caused by TET2 upregulation is accom-
panied with normal hematopoiesis, the decrease in transcriptional
repression of these cells following a reduction in TET2 and 5hmC is
associated with differentiation and increased immature LSK cells
(L. Cimmino et al., 2011; Feng et al., 2019; Ko & Rao, 2011; Pan,
Weeks, Yang, & Xu, 2015). Activating histone marks such as H3K9
acetylation, H3K4me1, and H3K4me3 and the increase in 5hmC on
genomic loci of CD34+ stem/early progenitor cells commitment
during erythropoiesis following opening of condensed chromatin is
accompanied with binding of specific erythroid TFs (e.g., GATA1,
GATA2, and KLF1) to these locations and erythroid differentiation
(Madzo et al., 2014). Epigenetic modifiers DNMT3A and TET2 create
5mC and 5hmC, respectively, subsequently modifying cytosine at the
CpG island that could help activate and repress HSCs genes; there-
fore, the reduction in 5hmC level of HSCs lacking TET2 and DNMT3A
results in differentiation inhibition and self‐renewal promotion of
HSCs through increasing expression of EPOR and KLF1 genes
(X. Zhang et al., 2016). Thus, the balance between 5mC and 5hmC
has an essential role in gene transcription regulation and cell fate
decisions. Increase of 5hmC, 5caC, and 5fC following overexpression
of TET2 can result in the cell‐cycle defect (G1‐S and/or intra‐S
transition) by activating P53 and inducing genetic instability and
mutagenesis in the absence of TDG, which increases C > T muta-
genesis in the CpG island (Mahfoudhi et al., 2016). Also, the increase
of 5hmC following TET2/3 overlapping plays a role in normal HSCs
development by signaling and downstream expression of GATA2B/
| 5
SCL/RUNX1 transcription (C. Li et al., 2015). Given the involvement
of TET2, DNMT3A, IDH1, and IDH2 in the regulation of gene tran-
scription during the development, cellular fate, and function of HSCs,
it appears that dysregulation of these epigenetic modifiers results in
the generation of 5hmC and failure of hematopoiesis.
3 | TRANSCRIPT ION FACTOR N ETWO RKS
AND GEN E EXP RE SSION I N N ORMAL
HEMAT O POIESIS
As primary components of transcriptional regulatory networks, TFs
play an important role in the regulation of gene expression by binding
to specific DNA sequences. Furthermore, the interaction between
transcriptional and epigenetic modifiers is responsible for the control
of gene expression (Gottgens, 2015). Notably, transcriptional
mechanisms play key roles in HSCs fate such as self‐renewal, com-
mitment, and differentiation of specific cell types (Avellino &
Delwel, 2017). In fact, TFs cooperate in managing the balance be-
tween self‐renewal and differentiation of HSCs (Suzuki et al., 2017),
so that lineage‐specific TFs (such as CCAAT‐enhancer‐binding pro-
tein α [C/EBPα], phosphatase and tensin homolog deleted on the
chromosome 10 [PTEN], Runt‐related transcription factor 1
(RUNX1), and signal transducers and activators of transcription
[STAT] family proteins) induce HSCs differentiation through reducing
HSCs self‐renewal and activating lineage‐specific genes (Koschmie-
der, Halmos, Levantini, & Tenen, 2009; Ohlsson et al., 2016). Given
the role of TFs in gene expression of HSCs, it is not surprising that
aberrant expression of HSCs‐related TFs is involved in the initiation
of leukemia transformation by shifting HSCs toward self‐renewal or
differentiation (Suzuki et al., 2017).
3.1 | CCAAT‐enhancer‐binding protein α
CCAAT‐enhancer‐binding protein α (C/EBPα) is a member of the
family of basic region leucine zipper transcription factors (C/EBP‐α,
‐β, ‐γ, ‐δ, ‐ε, and ‐ζ) promoting gene expression via binding to certain
genes (Avellino & Delwel, 2017; Ohlsson et al., 2016). All members of
CEBP have C‐terminal and N‐terminal domains that are essential to
protein interactions (DNA binding and dimerization) and transcrip-
tion control (Avellino & Delwel, 2017). CEBPA is an intron‐less gene
located on chromosome 19q13.1 that encodes two isoforms (42‐kD
and 30‐kD) of a DNA‐binding protein (Avellino & Delwel, 2017;
Ohlsson et al., 2016). C/EBPα is a key factor in differentiation pro-
cesses, such as liver development, adipogenesis, and hematopoiesis
(Ohlsson et al., 2016). There is a high expression of C/EBPα in
myeloid lineage and although its expression is low in HSCs, C/EBPα
has a key role in controlling the expression of genes that keep HSCs
quiescent and maintain long‐term HSCs (LT‐HSCs; Avellino &
Delwel, 2017; Koschmieder et al., 2009; Quintana‐Bustamante
et al., 2012). This transcription factor regulates differentiation, pro-
liferation and cell death of HSCs through preventing their apoptosis
6 |
and maintaining them in a quiescent state (Hasemann et al., 2014;
Ohlsson et al., 2016). Similarly, PU.1, which is an important tran-
scription factor for early myeloid and B‐cells differentiation, has low
expression in HSCs but acts as a regulator for HSC function. It has
been reported that overexpression of C/EBPα and PU.1 can impair
homeostasis of HSCs by reducing self‐renewal capacity, suppressing
proliferation, decreasing mixed colony formation, increasing apop-
tosis, and facilitating differentiation (Fukuchi, Ito, Shibata, Kitamura,
& Nakajima, 2008). On the other hand, C/EBPα results in granulocyte
differentiation via interacting with the DNA‐binding domain of PU.1
and blocking PU.1 function (Reddy et al., 2002). C/EBPα also controls
the survival of HSPCs and maintains their repopulating ability, cell‐
cycle progression, and granulocytic differentiation by affecting the
downstream target of the ecotropic viral integration site 2B
(Zjablovskaja et al., 2017). It was demonstrated that the inhibition of
N‐Myc as a direct target of C/EBPα keeps HSCs in a quiescent state,
while its elevated expression in the complete absence of C/EBPα
results in an increase of proliferation and number of LT‐HSCs in BM
(Ye et al., 2013). C/EBPα converts megakaryocyte/erythroid pro-
genitors and common lymphoid progenitors (CLPs) into the myeloid
lineage and induces myeloid differentiation of HSCs (Fukuchi
et al., 2006). Indeed, the expression of myeloid genes is activated by
C/EBPα binding to promoters or enhancers of myeloid‐related genes
(Avellino & Delwel, 2017). Furthermore, this transcription factor
inhibits the E2A/SCL heterodimer (essential to erythropoiesis) by
inducing the expression of ID1 and suppressing the erythropoietin
receptor (EPOR), resulting in the inhibition of erythroid genes and
induction of myeloid genes (Suh et al., 2006). In addition to the role of
C/EBPα in the initiation of myelopoiesis pathway, its activity is vital
for the functional evolution of myeloid cells such as highly active
migratory behavior (Y. Chen et al., 2009). According to the fact that
the number and function of HSCs and myeloid differentiation are
affected by C/EBPα, we can infer that the deregulation of C/EBPα
expression results in the enhancement of predisposing preleukemic
conditions, especially myeloid leukemogenesis.
3.2 | Phosphatase and tensin homolog deleted on
the chromosome 10
The phosphatase and tensin homolog deleted on the chromosome 10
(PTEN) gene is located on chromosome 10q23 and encodes the PTEN
protein that acts as a tumor suppressor and phosphatase. PTEN has a
vital role in multiple cellular mechanisms such as proliferation, sur-
vival, growth, migration, and differentiation by dephosphorylating
phosphatidylinositol 3, 4, 5‐trisphosphate (PIP3) and inactivating the
phosphatidylinositol 3 kinase (PI3K)/AKT signaling pathway (Fragoso
& Barata, 2014; Morotti et al., 2015). Previous studies have em-
phasized the key role of PTEN in the regulation of quiescence, cell‐
cycle entry, proliferation, and differentiation of HSCs (Fragoso &
Barata, 2014; Morotti et al., 2015). PTEN has two forms: phos-
phorylated (p‐PTEN) and non‐phosphorylated (non‐p‐PTEN). The
latter is effective in maintaining the quiescent/undifferentiated state
SHOKOUHIAN ET AL.
of HSCs in BM via inhibiting both the PI3K/AKT signaling pathway
and cell contact‐related SRC/FAK/P38 signaling, whereas the former
is related with the promotion of proliferation and differentiation of
HSCs through inhibiting the PI3K/AKT signaling pathway and
enhancing cell contact‐related FAK/P38 signaling activity (J. Li
et al., 2016). As a result, phosphorylation of PTEN could have an
important role in deciding hematopoietic cell fate. It was shown that
PTEN deletion reduced self‐renewal of HSCs, enhancing the level of
HSCs activation and generation of leukemia‐initiating cells, sup-
porting the role of PTEN in the maintenance of quiescence, re-
stricting the activation of HSCs and preventing leukemogenesis
(Yilmaz et al., 2006; J. Zhang et al., 2006). Therefore, PTEN is con-
sidered invaluable in regulating the balance between proliferation
and differentiation of blood cells and its disruption increases HSCs
cycling that might give rise to leukemia.
3.3 | Runt‐related transcription factor 1
Runt‐related transcription factor 1 (RUNX1), known as acute mye-
logenous leukemia‐1 (AML1), is a member of the core‐binding factor
(CBF) family of TFs and a key factor in normal development (D. Hong
et al., 2019). It is helpful in the generation of HSCs from the
endothelium of great vessels including the vitelline and umbilical
arteries as well as from the aorta–gonad–mesonephros region
(Kumano & Kurokawa, 2010; Kurokawa, 2006). RUNX1 expression is
high in myeloid and lymphoid lineage cells, while it has a low ex-
pression in erythroid lineage (North, Stacy, Matheny, Speck, & de
Bruijn, 2004), so that RUNX1 has a central role in the regulation of
HSPCs biology and the establishment of a balance between pro-
liferation and differentiation of myeloid and lymphoid lineages
(D. Hong et al., 2019; North et al., 2004). RUNX1 produces three
alternatively spliced variants, namely RUNX1a, RUNX1b, and
RUNX1c. Researchers have provided evidence for the role of RUN-
X1a in enhancing HSCs self‐renewal and driving the proliferation of
primitive progenitors that enable the expansion of immature pro-
genitors' population (Tsuzuki et al., 2007; Tsuzuki & Seto, 2012).
However, RUNX1b promotes granulocytic differentiation of HSCs
(Tsuzuki et al., 2007). RUNX1 forms a heterodimer complex by
binding to core‐binding factor subunit beta (CBF‐β) and interacting
with the DNA sequence at promoters of target genes, which serve as
either a transcription activator or repressor of target genes involved
in hematopoietic differentiation, cell‐cycle regulation, ribosome bio-
genesis, P53, and transforming growth factor β (TGFβ) pathways
(D. Hong et al., 2019; Sood, Kamikubo, & Liu, 2017). Actually, the
RUNX1/CBF‐β complex represses the expression of a target gene
through binding to the consensus DNA sequence (the PEBP2 site)
and interacting with corepressor mSin3A, which as a regulatory
protein plays a role in stabilization of transcriptional repressor
complex by protecting RUNX1 from proteolytic degradation and
subsequent formation of the transcriptional complex. Phosphoryla-
tion of RUNX1 on Ser249 and Ser266 by an extracellular signal‐
regulated kinase (ERK) in response to growth factor stimuli
SHOKOUHIAN ET AL.
contributes to the release of RUNX1 form mSin3A, leading to re-
cruitment of transcriptional coactivators P300 and CREB‐binding
protein to RUNX1 and conversion of the RUNX1 complex into
an activator of gene transcription (Kurokawa, 2006). In addition, the
interaction between RUNX1/CBF‐β complex and core components of
PRC1 such as Ring1b and Bmi1 recruits PRC1 at site‐specific loci
of target genes in megakaryocytic and lymphocytic cells (Yu
et al., 2012). Furthermore, the increase in the recruitment of TET and
other demethylation‐related enzymes to the CpG island induces DNA
demethylation by RUNX1, which can be effective in unlocking si-
lenced hematopoietic genes and hematopoietic development
(Suzuki et al., 2017). RUNX1 negatively regulates the number of
quiescent HSCs, as there seems to be an inverse correlation between
LT‐HSCs activity and RUNX1 dosage. Overall, the disruption of
RUNX1 function results in the expansion of immature hematopoietic
cells, which develop into a variety of hematological malignancies
(Ichikawa et al., 2008).
3.4 | Signal transducers and activators of
transcriptions
The signal transducers and activators of transcriptions (STATs) family
is composed of seven members of cytoplasmic TFs with a crucial role
in the regulation of proliferation, apoptosis suppression, differentia-
tion, and survival of hematopoietic cells (Dorritie et al., 2014). Acti-
vation of STAT3, by granulocyte‐macrophage‐colony‐stimulating
factor (GM‐CSF) in primary CD34+ BM cells, results in its binding to
DNA sequence of the apoptosis inhibitor (survivin) core promoter
and contributes to the inhibition of apoptosis and stimulation of
CD34+ cell proliferation by increasing the level of survivin
(Gu, Chiang, Zhu, Findley, & Zhou, 2007). An overlap between the
activation of STAT3 and the upregulation of HoxB4 induces gene‐
specific TFs that are responsible for self‐renewal of HSCs such as
Oct‐4 and Nanog, resulting in the promotion of self‐renewal and
repopulating activity of HSCs (S. H. Hong et al., 2014). Notably,
activation of STAT5 by cytokines including stem cell factor, throm-
bopoietin (TPO), granulocyte‐colony‐stimulating factor (G‐CSF),
GM‐CSF, interleukin‐3 (IL‐3), and EPO cooperates in keeping HSCs
quiescent, maintaining their self‐renewal capacity, and promoting
CD34+ cell growth (Dorritie et al., 2014; Z. Wang & Bunting, 2013).
Reduction of STAT5 results in the arrest of B‐cells development in
the pro‐ to pre‐B‐cell stage, supporting its role in the regulation of
lymphoid and myeloid development (Dorritie et al., 2014; Z. Wang &
Bunting, 2013). A study by Z. Wang, Medrzycki, Bunting, and Bunting
(2015) concluded that deficiency of STAT5 causes the upregulation
of proto‐oncogene Myc and apoptosis inhibitor BCL‐2, which con-
tribute to the expansion of preleukemic lymphoid and induce trans-
formation to acute B lymphoblastic leukemia (B‐ALL). It was shown
that the downregulation of this transcription factor could effectively
reduce the number of HSCs and the common myeloid progenitor
(CMP), decreasing their expansion due to the function of STAT5 in
the maintenance and expansion of primitive HSCs (Schepers
| 7
et al., 2007). It was also seen that decreasing expression of
quiescence‐associated genes such as P57 and Tie2 increases cell‐
cycle and apoptosis, which brings about the loss of HSCs quiescence
and reduction of LT‐ HSC numbers (Z. Wang, Li, Tse, & Bunting, 2009;
Z. Wang et al., 2015). In addition, mutation of STAT5 in primitive LSK
cells promotes self‐renewal of HSCs, expansion of multipotential
progenitors and initiation or progress of leukemia such as myelo-
proliferative disorder (MPD; Dorritie et al., 2014; Kato et al., 2005). It
was reported that down‐modulation of C/EBPα in CD34+ cells fol-
lowing activated mutant STAT5A1*6 causes an enhancement of
self‐renewal, expansion of LT‐HSCs, blocked myeloid differentiation,
and a bias toward erythropoiesis (Wierenga, Schepers, Moore,
Vellenga, & Schuringa, 2006). Indeed, the activity of STAT5 on HSCs
self‐renewal and differentiation is dosage‐dependent as low to in-
termediate STAT5 activity is associated with optimal self‐renewal
and LT‐HSCs expansion, impaired myelopoiesis, and induction of
erythropoiesis (Wierenga, Vellenga, & Schuringa, 2008). In general, it
seems that given the association between the activity rate of STAT3
and STAT5 proteins with the regulation of stem cell fate and lineage
commitment, changes in the activity of these STATs may result in
hematologic malignancies.
4 | DYSREGULATION OF HEMATOPOIETIC
STE M CE LLS ‐I N DU C E D LE U K E M I C
TRANSFORMATION BY IMPAIRED
EXPRESSION OF GE NES
Chromatin accessibility and gene expression patterns are controlled
by epigenetic modifications and transcriptional networks, which have
important effects on the regulation of HSCs self‐renewal and their
differentiation into mature cells of myeloid and lymphoid lineages
(Gottgens, 2015; Sharma & Gurudutta, 2016). Any aberration derived
from changes in epigenetic regulators and TFs in HSCs gene ex-
pression results in impaired HSCs fate such as self‐renewal and
lineage choice, as well as an acceleration of leukemic transformation.
4.1 | Additional sex combs‐like 1
Wild‐type (WT) ASXL1 in cooperation with PRC2 inhibits target
genes such as the posterior HoxA gene. In other words, loss of
functional features of this tumor suppressor in ASXL1 truncation
mutation (MT) reduce the inhibition of target genes, which can be
followed by progression and initiation of myeloid malignancies such
as myelodysplastic syndromes (MDS), acute myeloid leukemia (AML),
chronic myelomonocytic leukemia (CMML), and MPD (Asada &
Kitamura, 2018; Asada et al., 2019; Oak & Ohgami, 2017). Alteration
in ASXL1 might induce biased differentiation toward granulocytic/
monocytic lineages, supporting the essential role of ASXL1 in main-
taining HSPCs' functions and differentiation bias (P. Zhang, Chen
et al., 2018). Increased catalytic function and nuclear localization of
BAP1 in ASXL1‐MT results in mono‐ubiquitination at lysine 351 of
8 |
mutant ASXL1. Monoubiquitinated ASXL1‐MT, in turn, increases the
catalytic function of BAP1 and establishes the hyperactive
ASXL1‐MT/BAP1 complex, leading to the decrease in the level of
H2AK119ub in HSCs, aberrant differentiation of these cells and
myeloid progenitor cells (Asada & Kitamura, 2018; Asada et al., 2018;
Nagase et al., 2018). Furthermore, H2AK119Ub at gene promoters
has a repressive effect on gene expression; consequently, its reduc-
tion following the effect of the ASXL1‐MT/BAP1 complex disturbs
gene repression (Balasubramani et al., 2015). Besides this, reduction
of H2AK119Ub mediated by the hyperactive ASXL1‐MT/BAP1
complex could finally reduce the level of H3K27me3
(Balasubramani et al., 2015) and lead to the promotion of myeloid
transformation via hindering PRC2 recruitment to specific oncogenic
target loci (Abdel‐Wahab et al., 2012; Asada et al., 2019). On the
other hand, through direct binding to the promoter of the HoxA gene
as well as IRF8 and H2AK119ub removal, the ASXL1‐MT/BAP1
complex contributes to the upregulation of posterior HoxA gene and
IRF8. Owing to the role of the HoxA gene and IRF8 in leukemic
transformation and monopoiesis, respectively, their dysregulation is
effective in leukemogenesis (Abdel‐Wahab et al., 2012; Asada &
Kitamura, 2018; Asada et al., 2018). Also, the upregulation of pos-
terior HoxA genes like HoxA9 inhibits PRC2‐mediated methylation
of histone H3K27 in ASXL1‐MT, increasing the expression levels of
microRNA‐125a (miR‐125a) and subsequently reducing the expres-
sion levels of C‐type lectin domain family 5‐member a (Clec5a).
Clec5a has a role in myeloid differentiation, and its reduction influ-
ences the pathogenesis of myeloid malignancies such as MDS de-
velopment (Inoue et al., 2013). The balance between ASXL1 and
BAP1 activity is vital in normal hematopoiesis as increased BAP1
deubiquitinase activity in the ASXL1aa1‐587 truncation mutant in
ASXL1Y588XTg mice promotes myeloid malignancies (Guo
et al., 2018). Recently, a study by Uni et al. (2019) on ASXL1G643fs
knockout in mice revealed that the level of H2AK119ub at the pro-
moter region of p16Ink4a is reduced in HSCs ASXL1‐MT. p16Ink4a is
a major cyclin‐dependent kinase inhibitor, whose function is regu-
lated by PRC1 and has a pivotal role in preventing cell‐cycle pro-
gression by decelerating G1‐S transition and maintaining cellular
senescence (Serrano, Hannon, & Beach, 1993). As a result, p16Ink4a
upregulation, which results from a reduction in PRC1 recruitment to
the promoter region of p16Ink4a in ASXL1‐MT, can accelerate cel-
lular senescence by decreasing the HSCs pool, increasing apoptosis,
cell‐cycle arrest in HSCs fraction, and causing aberrant myeloid dif-
ferentiation (Uni et al., 2019). In another study, it was reported that
decreased levels of H3K27me3 and H3K4me3 in ASXL1+/− and
ASXL1−/− Lin– c‐Kit+ cells, upregulation of apoptotic facilitator
(BCL2l13), and downregulation of antiapoptotic genes (BCL‐2 and
BCL2l12) would decrease HSCs repopulating capacity along with
skewed cell differentiation favoring granulocytic lineage and redu-
cing HSCs pool (Abdel‐Wahab et al., 2013; J. Wang et al., 2014).
Furthermore, by reducing H3K27me3 in the absence of ASXL1, de-
regulation of the transcriptional function of the RNA polymerase II
complex contributes to the downregulation of genes that are critical
for HSPCs maintenance, including vascular cell adhesion molecule
SHOKOUHIAN ET AL.
1, C‐X‐C motif ligand 1 (CXCL1), and CXCL2 (P. Zhang, Chen
et al., 2018). In addition, similar to ASXL1, OGT methylates H3K4/
H3K27 through a direct relationship with HCFC1 and MLL5, thereby
exerting an influence on transcription activation and regulation of
myeloid differentiation. This enzyme directly stabilizes ASXL1
through O‐GlcNAcylation of ASXL1‐S199. Notably, knockdown of
ASXL1, OGT, or MLL5 as suppressors of myeloid malignancies re-
duces H3K4 methylation and blocks myeloid differentiation, which
leads to myeloid malignancies (Inoue et al., 2018). Furthermore,
ASXL1‐MT in human CD34+ progenitor cells decreases the genera-
tion of CD11b+ and CD15+ cells (markers of granulocytes and/or
monocytes or macrophages), subsequently causing a decline in the
quantity of granulocyte, monocyte‐colony‐forming unit (GM‐CFU)
and granulocyte‐colony‐forming unit (G‐CFU) colonies. Eventually,
this mutation both disrupts human granulomonocytic differentiation
and increases the number of multipotent mixed‐lineage colony‐
forming units (GEMM‐CFU; Davies et al., 2013). A recent study by
H. Yang et al. (2018) on an ASXL1Y588XTg mouse model concluded
that the increase in transgenic expression of a truncated form of
ASXL1 in the hematopoietic compartment of mice can be effective in
myeloid differentiation and facilitation of leukemogenesis because
this mutation triggers the increase of LSK and granulocyte‐
macrophage progenitor cells (GMPs) populations with skewed dif-
ferentiation favoring the monocytic/granulocytic lineage in BM. It
was also shown that increased percentage of blast cells in peripheral
blood (PB), BM, and spleen as well as decreased lifespan in this
mouse might reflect the development of myeloid malignancies, in-
cluding MDS, MPD, and AML (H. Yang et al., 2018), which supports
the role of ASXL1‐MT in pathogenesis of myeloid malignancies. A
number of authors have recognized that dysregulation of regulatory
factors for erythropoiesis because of a decline in histone H3K27me3
and H3K4me3 during knockdown of ASXL1 in cord blood CD34+
cells increases apoptosis, induces dysplastic features in the erythroid
lineage, and impairs erythroid commitment and terminal differ-
entiation (Shi et al., 2016). In fact, knockdown of ASXL1 reduces
H3K27me3 on the HoxA and P21 locus in erythroid progenitors,
leading to the apoptosis of erythroid progenitor cells and aberrant
differentiation of the erythroid lineage (Hilgendorf, Folkerts, Schur-
inga, & Vellenga, 2016). ASXL1‐MT in HSCs is mainly responsible for
acquiring additional mutations such as in RUNX1. Consequently, the
simultaneous presence of these mutations could contribute to the
susceptibility of HSCs to leukemic transformation, increased
LT‐HSCs repopulation, reduced apoptosis, and blockage of erythroid
differentiation (Nagase et al., 2018). In addition, coexistence of
ASXL1+/– and NF1+/– (a negative regulator of the RAS signaling
pathway) in HSPCs increases the activity of multiple oncogenic
pathways such as Myc, NRAS, and BRD4, causing aggressive pro-
gression of myeloid malignancies (P. Zhang, He et al., 2018). Prior
studies have suggested that the reduction in acetylation of several
lysine residues on histone H3 and H4 at promoter regions of multiple
TGFβ pathway genes, which is mediated by ASXL1‐MT and SET
binding protein 1 (SETBP1) mutations, results in the repression of
multiple TGFβ pathway genes (Saika et al., 2018). In a study, it was
SHOKOUHIAN ET AL.
revealed that as the dysregulation mechanism of the TGFβ pathway
promotes leukemogenesis (M. Dong & Blobe, 2006), deregulation of
TGFβ signaling in ASXL1‐MT and SETBP1 mutations is effective
for the induction of myeloid neoplasms. This study also addresses the
fact that histone deacetylase inhibitors increase histone acetylation
at promoter regions of TGFβ pathway genes and inhibit leukemic
proliferation of ASXL1‐MT/SETBP1‐mutated cells, which may be
useful as therapeutic drugs for MDS and AML cases affected by these
mutations (Saika et al., 2018). In addition, the recognition of ASXL1
binding partners facilitates the perception of tumor‐suppressive
functions of ASXL1 and its role in myeloid malignancies. A more
comprehensive description can be found in an in‐vitro study by
Z. Li et al. (2017), in which it was demonstrated that ASXL1 inter-
action with the cohesin complex, including RAD21, SMC1A, SMC3,
and STAG1/STAG2, plays a vital role in maintaining normal sister
chromatid separation during cell division and regulation of gene ex-
pression in hematopoietic cells. Furthermore, loss of ASXL1 de-
creases occupancy of RAD21 and SMC1A on target genes, including
STAT3, CBFB, and FUS and impairs telophase chromatin separation
in hematopoietic cells, resulting in the increase of dysplastic myeloid
cells with disrupted chromatin separation and the development of
myeloid malignancies (Z. Li et al., 2017). Therefore, it can be con-
cluded that ASXL1 promotes susceptibility to myeloid transformation
via changing histone modifications and disturbing hematopoiesis.
4.2 | Ten‐eleven translocation 2
Expression and activity of TET2 have a vital role in normal home-
ostasis through controlling the function, self‐renewal, proliferation,
and differentiation of HSCs. Low levels of 5hmC and 5mC accumu-
lation at certain genomic locations in the absence of mutations of
TET2 cause deregulation of DNA methylation (Feng et al., 2019; Ko &
Rao, 2011), and result in a progressive defect of hematopoiesis, in-
cluding increase in HSCs self‐renewal and BM cellularity, abnormal
HSCs proliferation or differentiation, and myeloid (promoted mono-
cyte/macrophage differentiation) or lymphoid neoplasms (Holmfeldt
& Mullighan, 2011; M. Ko et al., 2011; Mercher et al., 2012; Pronier
et al., 2011). In fact, disruption of TET2 function and expression
contributes to the alteration of B‐ and T‐lymphoid differentiation and
expansion of immature progenitors endowed with myeloid colony‐
forming potential (Mercher et al., 2012; Quivoron et al., 2011). TET2
is expressed in low levels in ESCs, but its level increases in hema-
topoietic cells along with differentiation. It has been seen that TET2
knockdown in ESCs reduces the number and cloning capacities of
hematopoietic progenitors, whereas its knockdown in HSCs increases
hematopoietic development (Langlois et al., 2014). Although TET2 is
expressed in high levels among myeloid progenitors, the loss of TET2
catalytic activity could result in myeloid leukemogenesis through
restricting self‐renewal capacity and increasing the proliferation of
premalignant cells (M. Ko et al., 2010; Kunimoto et al., 2014). As a
result, TET2 activity is indispensable for the suppression of myeloid
malignancy (Z. Zhao et al., 2016). Also, it has been observed that
| 9
TET2 plays a role in both the early and late stages of myeloid dif-
ferentiation so that promoter hydroxymethylation along with TET2 is
necessary for gene activation during C/EBPα‐induced trans‐
differentiation. C/EBPα binds to upstream regions of TET2 and
activates TET2, which in turn binds to methylated promoters of
myeloid genes leading to the hydroxylation of these genes and trans‐
differentiation of pre‐B cells into macrophages as TET2 knockdown
inhibits the generation of macrophages (Kallin et al., 2012). Homo-
zygous TET2 mutations have a role in increasing self‐renewal and
capacity of HSCs, expansion of LSK and CMP in BM and the fetal
liver (FL; Nakajima & Kunimoto, 2014). TET2 increases the conver-
sion of 5mC to 5hmC at target HoxA cluster regions, which increases
the activation of HoxA transcription and plays an important role in
the maintenance of genome stability (M. T. Bocker et al., 2012). In
contrast, increased phosphorylation of histone H2AX (as a DNA da-
mage marker), increased DNA methylation and decreased HoxA
transcription in the loss of TET2/3 function cause genome instability,
uncontrolled cell proliferation, and rapid progression of myeloid
leukemogenesis (An et al., 2015). Moreover, enhancing long‐term
repopulating and self‐renewal capacity of LSK cells can result in the
expansion of common myeloid progenitors in FL by inactivating TET2
in FL (Kunimoto et al., 2012). TET2 is not only effective in BM but has
an impact on restricting the expansion and function of HSCs in FL.
Besides this, TET2 is vitally important in erythropoiesis because
upregulation of c‐Kit as well as AXL and ERK tyrosine kinases re-
sulting from knockdown of TET2 cause clonal expansion of the ery-
throid progenitor (Qu et al., 2018). In addition, knockdown studies of
TET2 or TET3 have distinct consequences, so that TET2 knockdown
causes defects in early stages of erythropoiesis, resulting in hyper-
proliferation and impaired differentiation of erythroid progenitors,
while defects in the late stages of erythropoiesis caused by TET3
knockdown lead to rising apoptosis, generation of bi/multinucleated
polychromatic/orthochromatic erythroblasts, and impaired enuclea-
tion (Yan et al., 2017). Cooperation of TET2 mutations with other
gene mutations promotes the development/transformation of var-
ious types of hematological malignancies. This fact was successfully
described by Jin et al. (2018) when they revealed that NRASG12D/+
and TET2−/+ cause expansion of HSCs pool and multipotent pro-
genitors with promoting cytokine hypersensitivity, highly preserved
self‐renewal capacity, and competitiveness, which contribute to the
acceleration of preleukemia (e.g., CMML‐like disease; Jin et al., 2018).
It was also reported that the expression of lineage‐specific TFs in-
creases as HSCs differentiate, including upregulation of erythroid
regulators (e.g., KLF1 and EPOR) if there is a double knockout of
TET2 and DNMT3A (X. Zhang et al., 2016). In addition, given the
protective role of TET2 in HSPCs genomes (susceptibility to the ac-
quisition of DNA mutagenicity), increased accumulation of numerous
mutations, including APC, NF1, FLT3, CBL, Notch1, and MLL2 has
been observed when there is TET2−/− (Pan et al., 2017). There is a
lower frequency of TET1 mutation in hematopoietic malignancies
than TET2/3 mutation. Overexpression of BCL‐2 decreases 5hmC
and increases 5mC in loss of TET1, increasing self‐renewal of pro-
genitor B‐cells and the development of B‐cell lymphoma (L. Cimmino
10 | SHOKOUHIAN
et al., 2015). Considering that TET2 mutations play a role in myeloid
and lymphoid neoplasms, restoring TET2 function is likely to be
useful in therapeutic strategies and supporting evidence in this
respect was provided by Cimmino et al. (2017), who showed that
restoration of TET2 could inhibit aberrant self‐renewal of pre-
leukemic stem cells because treatment with vitamin C as a cofactor
of Fe (II) and a‐KG is accompanied with increased restoration of TET
activity in leukemia cells (Cimmino et al., 2017; Pan et al., 2015).
Vitamin C acts as a pharmacologic mimic of TET2 restoration and is
beneficial in promoting DNA demethylation, reversing aberrant
HSPCs self‐renewal and enhancing the sensitivity of leukemia cells to
PARP inhibition (Cimmino et al., 2017).
4.3 | DNA methyltransferase 3A
DNA methylation of HSCs by DNMT is vital in protecting the self‐
renewal of HSCs and multipotent progenitors by silencing pre-
dominant differentiation programs. Lack of methylation, elevated
H3K4me3, and incomplete repression of HSCs‐specific genes in the
mutation of DNMT3A activate the self‐renewal pathway and expand
the number of HSCs in BM (Challen et al., 2011). Indeed, upregula-
tion of the Hox gene and myeloid ecotropic viral integration 1 (Meis1)
due to DNMT3A mutations could be followed by enhanced HSCs self
‐renewal (Tan, Liu, & Chen, 2015). These mutations are effective both
on LT‐HSCs expansion and on increasing differentiation blockage of
HSCs into all hematopoietic lineages (Brunetti et al., 2017; Chan &
Majeti, 2013). A strong relationship was reported between loss of
DNMT3A and initiation of the premalignant condition of clonal
hematopoiesis with indeterminate potential by M. Jeong et al. (2018),
in which it was shown that loss of DNMT3A significantly increased
the self‐renewal capacity of HSCs and produced HSCs with indefinite
longevity and immortality. It was also demonstrated that loss of
DNMT3A causes BM failure with MDS features such as BM hy-
percellularity, the arrest of erythroid progenitors' development,
proliferation enhancement of myeloid progenitors, and an increase in
apoptosis of mature myeloid cells (Celik et al., 2015). Loss of
DNMT3A not only increases myeloproliferation in BM but expands
LSK cells and multipotent progenitors in the liver (Guryanova
et al., 2016). On the other hand, given the influence of interaction
between DNMT3A and PcG on expression regulation of their target
genes, it has been observed that mutation of DNMT3A causes
aberrant recruitment of PRC1 to specific differentiation‐associated
gene loci and plays a role in blocking myeloid differentiation of HSCs
and promoting monoblastic transformation (Koya et al., 2016). In
fact, mutation of this methyltransferase leads to a transformation of
HSCs to myeloid (e.g., MDS, AML, and primary myelofibrosis) and
lymphoid malignancies (e.g., T‐ and B‐ALL; Mayle et al., 2015).
Furthermore, the combined loss of DNMT3A and other mutations
such as c‐Kit, Notch1, NRASG12D, and KRAS in HSCs is effective in
leukemic transformation (Celik et al., 2015; Mayle et al., 2015).
Activation of the Wnt/β‐catenin signaling pathway in simultaneous
deletion of DNMT3A and DNMT3B enhances HSCs self‐renewal and
ET AL.
blocks differentiation (Challen et al., 2014). As primitive mutations in
HSPCs, DNMT3A, TET2, and ASXL1 mutations can be involved in
clonal hematopoiesis and generation of clonal preleukemic stem cells
(pre‐LSCs; Kunimoto & Nakajima, 2017; Sato, Wheat, Steidl, &
Ito, 2016). Pre‐LSCs are similar to HSCs in their differentiation and
self‐renewal capacity and can produce LSCs following the second
mutations (Chan & Majeti, 2013; Tan et al., 2015). Clonal hemato-
poiesis caused by DNMT3A mutations and FLT3‐ITD may lead to
leukemogenesis in multiple lineages, as it was shown that hetero-
zygous and homozygous DNMT3A mutations with FLT3‐ITD could be
effective in myeloid and lymphoid leukemias, respectively (L. Yang
et al., 2016). Also, coexistence of DNMT3A mutations with the ac-
quisition of mutant nucleophosmin plays a role in the development of
MPD and final transformation to AML (Loberg et al., 2019). DNMT1,
which is a member of DNMT, has a crucial role in HSCs' self‐renewal
and differentiation of HSCs to myeloid progenitors. In contrast, loss
of DNMT1 can increase cycling and differentiation of myeloid‐
restricted progenitors (Trowbridge, Snow, Kim, & Orkin, 2009). Dif-
ferentiation of myeloid‐erythroid progenitors increases when the
expression level of myeloid‐erythroid genes is increased following
hypomethylation of GATA1, ID2, CD48, and C/EBPα transcription
factors in HSCs lacking DNMT1 (Broske et al., 2009). Also, increased
expression of C/EBPα through hypomethylation of CpG islands in
DNMT1 mutant HSPCs results in defective HSPCs maintenance and
induced HSPCs proliferation (X. Liu et al., 2015). Therefore, the
DNMT family is critical in controlling differentiation and self‐renewal
of the hematopoietic system where their mutations can initiate and
maintain leukemogenesis.
4.4 | CCAAT‐enhancer‐binding protein α
C/EBPα is responsible for the maintenance of HSCs self‐renewal and
proliferation of myeloid progenitors and its deficiency increases
HSCs self‐renewal, blocks differentiation of the common myeloid
progenitor, and increases proliferation of myeloid blasts (Avellino &
Delwel, 2017; Koschmieder et al., 2009; Ohlsson et al., 2016).
Moreover, increasing expression of Bmi1 as a direct target of C/EBPα
in C/EBPα deficiency contributes to the enhanced competitive re-
populating activity of HSCs (P. Zhang et al., 2004). It was found that
mutations in C‐terminal C/EBPα cause premalignant HSCs expansion
and increase the proliferation of LT‐HSCs (Bereshchenko
et al., 2009). Indeed, mutations in C‐terminal are effective in blocking
terminal differentiation (Quintana‐Bustamante et al., 2012). If
mutations in C‐terminal are homozygous, there is a decrease in
myeloid gene expression and an increase in erythroid‐specific gene
expression (Bereshchenko et al., 2009). However, mutations in
N‐terminal C/EBPα are associated with the silencing of HSCs ex-
pansion and the formation of committed myeloid progenitors
(Quintana‐Bustamante et al., 2012). In fact, mutations in the
N‐terminal are effective in maintaining immature progenitors. The
combination of N‐ and C‐terminal C/EBPα mutations expands
premalignant stem cells and accelerates disease development
SHOKOUHIAN ET AL.
(Bereshchenko et al., 2009). As a transcription factor involved in
blocking terminal differentiation of granulocytes, C/EBPα represses
E2F and has an important role in controlling the proliferative capa-
city of early myeloid progenitors. Mutation of C/EBPα impairs E2F
repression, which results in BM transformation with blocking
granulocyte differentiation and expansion of myeloid progenitors
(Porse et al., 2005). In addition, lack of inhibition of E2F activity and
downregulation of c‐Myc following deletions of C/EBPα amino
terminus and basic region result in granulocytic development
(D'Alo et al., 2003). Given the role of C/EBPα in balancing differ-
entiation and self‐renewal, dysregulation of this transcription factor
can play a causative role in the neoplastic process.
4.5 | Mixed‐lineage leukemia
MLL has a vital role in controlling HSCs' gene expression such as self‐
renewal, proliferation, and lineage‐specific differentiation, and
translocations or fusion of this transcription coactivator are asso-
ciated with leukemogenesis (Yokoyama, 2015). The SET domain of
MLL in MLL translocations increases the expression of Hox and other
genes, which transform hematopoietic cells into LSC (Krivtsov &
Armstrong, 2007). In addition, MLL fusion proteins activate the Hox
gene and promote the self‐renewal of HSCs (Yokoyama, 2015). MLL
fusion proteins bind to CpG islands within the Hox locus and facil-
itate TF binding, upregulating HoxA7, A9, and Meis1 and causing
immortalization of hematopoietic cells, blocking myeloid differ-
entiation and inducing hematopoietic progenitors to leukemia
(Martin et al., 2003; Yokoyama, 2015). For instance, MLL‐AF4 fusion
protein activates transcriptional programs that play a role in leuke-
mia through occupying regions of the genome affecting the devel-
opment and self‐renewal of HSCs and changing chromatin structure
and histone modifications in these regions (Guenther et al., 2008).
ENL binding to CBX8 (a component of PRC1) in MLL‐ENL fusion and
neutralization of PRC1 activate gene loci necessary for transforma-
tion to leukemia (Maethner et al., 2013). Furthermore, MLL‐PTD
fusion enhances HSCs self‐renewal, reducing the number of HSCs
because of increased apoptosis, clonal expansion with lineage bias,
and blockage of myeloid differentiation (Y. Zhang et al., 2012). As the
interaction between MLL and RUNX1c/EBFβ prevents RUNX1 from
ubiquitin‐mediated degradation, recruits MLL to regulate down-
stream target genes and causes HSCs differentiation (X. Zhao
et al., 2014), it was observed that downregulation of RUNX1c/EBFβ
as a result of CXXC domain of MLL fusion proteins promotes
self‐renewal, differentiation blockade, and leukemogenesis (X. Zhao
et al., 2014). TET1 as a fusion partner of MLL has been detected in t
(10;11) (p12;q23) translocation (L. Cimmino et al., 2011; Mercher
et al., 2012). MLL fusion proteins directly bind to the promoter region
of TET1, increase the expression of TET1 and subsequently raise
5hmC level. Then, TET1 along with MLL fusion proteins increases
transcriptional activation in critical cotargets including HoxA9,
Meis1, and pre‐B‐cell leukemia homeobox 3 to inhibit apoptosis and
cell differentiation and promote cell proliferation, which finally
| 11
results in leukemogenesis (Huang et al., 2013). It was reported that
deficiency of MLL in HSCs has a role in exiting HSCs from a quiescent
state and increasing HSCs populations that undergo cell‐cycle entry.
However, deficiency of MLL in myeloid‐erythroid progenitors re-
duces the proliferation and frequency of these progenitors (Jude
et al., 2007). Actually, deficiency of MLL reduces the expression of
Hox gene and antiapoptotic BCL‐2, which plays a role in blockage of
frequency and growth of hematopoietic colonies (Ernst, Mabon,
Davidson, Zon, & Korsmeyer, 2004). Deletion of MLL results in a
defect in the hematopoietic stem and progenitor pool, including re-
duced numbers of quiescent HSCs, LT‐HSC, and ST‐HSC (McMahon
et al., 2007). Therefore, dysregulation of HSCs self‐renewal and
expansion of progenitors following fusion or translocations of MLL
might contribute to leukemogenesis.
4.6 | Phosphatase and tensin homolog deleted on
the chromosome 10
Phosphatase PTEN has a role in the regulation of quiescence, pro-
liferation, and differentiation of HSCs. PIP3 accumulation and hy-
peractivation of AKT mediated by PTEN deletion promote HSCs
proliferation, increase levels of cycling HSCs and HSCs exhaustion,
reducing LT‐HSCs, and leading to myeloid leukemogenesis (Fragoso
& Barata, 2014; Morotti et al., 2015). Activation of mTOR in HSCs via
interaction of the P110B isoform of PI3K with RAC in PTEN defi-
ciency can result in the expansion of myeloid cells (Yuzugullu
et al., 2015). In addition, increase in PI3K signaling with mutation of
PTEN arrests HSPCs differentiation into mature cells, enhances
HSPCs proliferation and homing of HSPCs from BM to the caudal
hematopoietic tissue and increases the numbers of mitotic cells in
this tissue (Choorapoikayil, Kers, Herbomel, Kissa, & den
Hertog, 2014). Moreover, hyperactivation of the PI3K/AKT pathway
in Gr‐1+CD11b+ BM cells (myeloid cells) with PTEN deletion pro-
duces high levels of G‐CSF, IL‐8, and CXCL12, which promote HSCs
mobilization from BM to the spleen, splenomegaly, and leukemo-
genesis (Tesio et al., 2013). It has been observed that both G‐CSF and
inflammatory cytokines (e.g., interferon‐α) trigger HSCs mobilization
and expansion in the spleen through hyperactivation of the
PI3K/mTOR pathway in PTEN‐deficient HSCs (Porter et al., 2016).
The PTEN mutant with activated β‐catenin promotes self‐renewal
and expansion of HSCs with long‐term functional capacity without
differentiation. In fact, activation of β‐catenin blocks differentiation
through upregulation of ID2 and reduces multiple differentiation‐
inducing TFs, and the PTEN mutant enhances HSCs self‐renewal and
expansion through upregulation of antiapoptotic MCL1 (Perry
et al., 2011). It seems that the activation of Wnt/β‐catenin and PTEN/
PI3k/AKT pathways is effective in inducing proliferation and LT‐HSC
expansion with inhibiting apoptosis and blocking differentiation.
Therefore, this combination of findings provides some support for
the conceptual promise that the inhibition of PI3K signaling in PTEN
deficiency would be efficacious in therapeutic practices by decreas-
ing hyperproliferation and inducing differentiation of cells.
12 | SHOKOUHIAN
4.7 | RAS proteins family
RAS proteins belongs to GTPase family and transfer signals from
various receptors (e.g., receptor tyrosine kinases, G protein‐coupled
receptors, and cytokine receptors) to activate multiple downstream
signaling pathways (including RAF/MEK/ERK and PI3K/AKT), there-
by playing a central role in the regulation of cellular growth and
proliferation, survival, and gene expression (Khan et al., 2019). Mu-
tation or aberrant activation of RAS proteins in humans (e.g., KRAS
and NRAS) might lead to leukemogenesis via uncontrolled tran-
scriptional gene expression (Khan et al., 2019). Loss of KRAS‐WT
causes HSC depletion and increases the proliferation of myeloid
precursors by triggering the activation of all RAS isoforms and hy-
peractivation of cytokine signaling (such as GM‐CSF in myeloid cells),
eventually inducing the mechanisms of leukemogenic activity (Kong
et al., 2016). Also, loss of KRAS reduces TPO‐evoked ERK1/2 acti-
vation in HSCs, resulting in a decline of HSCs self‐renewal, LT‐HSCs
compartments and bias toward myeloid differentiation that could
reduce myeloid progenitors and neutrophil survival by reducing GM‐
CSF‐evoked ERK1/2 activation in myeloid cells (Damnernsawad
et al., 2016). However, increased activity of KRAS‐ERK signaling in
KRAS mutant induced pluripotent stem cells (iPSCs) as a result of
reduced basic fibroblast growth factor retains self‐renewal of un-
G12D
differentiated cells in human iPSCs (Kubara et al., 2018). KRAS
elevates the level of phosphorylated STAT5, ERK, and S6 in HSPCs
and sensitizes these cells to GM‐CSF stimulation, thereby increasing
the proliferation of HSPCs (Van Meter et al., 2007). Moreover,
G12D
KRAS induces the initiation of leukemias via reducing the num-
ber of HSCs, increasing proliferation of primitive HSCs with en-
hanced repopulating ability, and increasing the entry of HSCs to the
cell cycle by upregulating cyclin D1 expression (Sabnis et al., 2009).
As the first genetic hit in HSCs, KRAS mutations have a role in
preserving the extensive proliferation capacity and survival of HSCs
and may induce leukemic transformation in cooperation with sec-
ondary genetic hits (J. Zhang et al., 2009). For instance, a combina-
G12D
tion of KRAS with secondary Notch1 mutations in thymocytes is
effective in initiating acute T‐lineage leukemia/lymphoma (Sabnis
et al., 2009). On the other hand, the coexistence of KRASG12D/+ and
DNMT3A mutations could promote myeloid malignancies. As the
first genetic hit, DNMT3A drives clonal hematopoietic expansion and
leukemogenesis after a prolonged latency, while it depletes HSCs and
increases self‐renewal of myeloid progenitors in combination with
KRASG12D/+, which finally results in the initiation and promotion of
myeloid malignancies such as juvenile myelomonocytic leukemia,
CMML, and AML (Chang et al., 2015). It has also been shown that
mutations of NRAS increase the proliferation of HSPCs and the
quantity of myeloid lineage cells (S. W. Shen et al., 2004). Besides
this, the upregulation of cell‐cycle inhibitors, namely P21CIP1/WAF1
and p16INK4a in NRAS mutations is associated with t growth sup-
pression of HSPCs, an increase in myelomonocytic colonies, and
decrease in erythroid differentiation (S. Shen, Passioura, Symonds, &
Dolnikov, 2007). In fact, NRAS mutation increases early erythro-
blasts, inhibits differentiation in early erythroid cells, and upregulates
ET AL.
the expression of CD34 and megakaryocyte antigens (CD41/CD61)
on erythroblasts by promoting the expression and activation of
protein kinase C, which leads to dyserythropoiesis in preleukemia
(Darley et al., 2002). On the other hand, hyperactivation of MAPK,
AKT, and STAT5 pathways and increasing monocyte‐colony‐forming
unit (M‐CFU) colony formation in NRASG12D plays a role in the dif-
ferentiation of HSCs to myelomonocytic lineage cells and initiation of
MPD (T. Wang et al., 2015). Hyperactivity of MEK/ERK1/2 signaling
in NRASG12D/+ causes HSCs hyperproliferation along with enhanced
self‐renewal capability, increases the number of LT‐HSCs and ex-
pansion of myeloid progenitors so that downregulation of MEK/ERK
signaling by pharmacologic and genetic approaches can be beneficial
in decreasing these processes (J. Wang et al., 2013). It was reported
by J. Wang et al. (2011) that tumor transformation through en-
dogenous oncogenic NRAS is related to the dose of NRAS and cell
type. NRASG12D/G12D in myeloid progenitors and T‐cell lineage pro-
motes the proliferation of myeloid progenitors by hyperactivating
ERK signaling and increasing the development of acute T‐ALL, re-
spectively, leading to acute MPD. However, NRASG12D/+ results in
the development of CMML (95% of BM cells) and T‐ALL (8% of BM
cells) (J. Wang et al., 2011). Furthermore, the co‐occurrence of
NRASG12D and loss of TET2 in HSCs promotes TPO hypersensitivity
in HSCs through activation of JAK/STAT signaling and preserves
competitiveness and self‐renewal of HSCs, accelerating HSCs pro-
liferation and promoting leukemogenesis (Jin et al., 2018). Moreover,
the cooperation of NRASG12D with the ASXL1 mutant can promote
myeloid leukemogenesis (Abdel‐Wahab et al., 2012). Therefore, RAS
mutation in HSCs dysregulates the expression and function of sig-
naling cascade mechanisms involved in HSCs maintenance, thereby
playing a causative role in leukemia development.
4.8 | Runt‐related transcription factor 1
RUNX plays a central role in regulating the interaction between
HSCs and the BM niche, so that the loss of RUNX1 leads to hy-
persensitivity of HSCs to G‐CSF, contributing to the enhanced mo-
bilization of HSCs and LSK cells into the spleen and PB (Lam
et al., 2016). G‐CSF hypersensitivity of RUNX1+/− cells reduces the
expression of C‐X‐C chemokine receptor type 4 and increases the
activation of STAT3 signaling by reducing the expression of protein
inhibitor of activated STAT3, which leads to the expansion and mo-
bilization of HSCs as well as blockage of myeloid differentiation (Chin
et al., 2016). It has been shown that the loss of RUNX1 is effective in
increasing the number of HSCs having high self‐renewal capacity and
that the clonal maintenance of leukemia‐initiating cells suppresses
cellular fail‐safe mechanisms induced by oncogenic NRAS such as
apoptosis via upregulating BCL‐2 expression, senescence by elevat-
ing Bmi1 expression, and differentiation (Motoda et al., 2007). Loss of
RUNX1 downmodulates apoptosis and elevates the percentage of
cells in the G1 cell cycle and increases LT‐HSCs (Cai et al., 2011).
Selective deletion of RUNX1 results in increased activation and
quiescence of HSCs, rising number of LT‐HSCs, CMP, GMP, LSK cells
SHOKOUHIAN ET AL.
expansion, and decreasing number of CLP, pro‐B, pre‐B, and mature
B‐cells, leading to abnormalities in the development of T‐cells and
thrombocytopenia (Kumano & Kurokawa, 2010). Similarly, a study by
Behrens et al. (2016) showed that RUNX1 knockout could increase
granulocytic/monocytic commitment and myeloid progenitors with
skewing toward megakaryocyte progenitors. RUNX1 mutations in-
crease self‐renewal, enhance LT‐HSCs, and deregulate the expression
of critical genes involved in granulocytic differentiation on specific
loci (e.g., C/EBPα), which drive the inhibition of GMP differentiation
and leukemogenesis, including myeloid and lymphoid leukemias
(Gerritsen et al., 2019). In addition, RUNX1 mutations might lead to
the creation and expansion of preleukemic stem cells in BM by
lowering growth and biosynthesis, shrinking cell phenotype, reducing
ribosome biogenesis, and reducing apoptosis (Cai et al., 2015).
Therefore, RUNX1 deficiency may drive the progression of leukemia
via dysregulating apoptosis and proliferation. As P53 is effective in
steady‐state hematopoiesis, it regulates HSCs quiescence and self‐
renewal by blocking cell‐cycle entry (Asai, Liu, Bae, & Nimer, 2011; Y.
Liu et al., 2009). P53 reduction (P53+/− and P53−/−) downmodulates
apoptosis and increases the quantity of proliferating HSCs, leading to
the initiation and progression of leukemic cells (Asai et al., 2011;
Dumble et al., 2007). Also, P53 mutation enhances HSCs' self‐
renewal and promotes HSCs' expansion, leading to clonal expansion
of HSCs and leukemic transformation (S. Chen et al., 2018; Dumble
et al., 2007; Nabinger et al., 2019). Besides this, P53 mutation in
combination with second hits (e.g., FLT3‐ITD) could be effective in
the expansion of myeloid progenitor cells (Nabinger et al., 2019).
Interestingly, the oncogenic fusion protein of RUNX1 has a causative
role in leukemogenesis. RUNX1‐TEL fusion increases the number of
HSCs while retaining their self‐renewal capacity, blocking differ-
entiation of B‐cells, and enhancing self‐renewal of B‐cell precursors,
resulting in the induction of B‐lineage‐committed leukemic cells
(Morrow, Horton, Kioussis, Brady, & Williams, 2004; Schindler
et al., 2009). Another RUNX1 fusion, namely, RUNX1–ETO, enhances
the self‐renewal of HSPCs, inhibits colony formation, and blocks the
proliferation of committed progenitors, leading to leukemogenesis,
and the accumulation of both immature hematopoietic blast cells and
myeloid cells (Mulloy et al., 2002). This fusion is able to change he-
matopoietic cell fate so that RUNX1–ETO downregulates SCL and
GATA1 expression and upregulate PU.1 in myeloid‐erythroid pro-
genitors, which suppress erythropoiesis and promote myelopoiesis
(Yeh et al., 2008). The increased COX/β‐catenin signaling pathway
caused by this fusion enhances hematopoietic self‐renewal and leu-
kemogenesis (Y. Zhang et al., 2013). Increased self‐renewal of pro-
genitor cells, decreased G‐CFU, and increased M‐CFU as a result of
RUNX1–ETO fusion inhibits the differentiation of granulocytic cells,
which promote the long‐term growth of myeloid cells as well as an
expansion of immature granulocytes and myeloblasts in BM (Tonks
et al., 2004). Also, an increase in the motility of preleukemic cells and
a decrease in the adhesion of these cells to stromal cells can drive the
migration of preleukemic cells across the BM barrier and cause dis-
ease progression in RUNX1–ETO cases (Saia et al., 2016). RUNX1–
ETO in association with ASXL1 mutations results in promoted
| 13
RUNX1–ETO‐induced myeloid leukemogenesis (Asada & Kita-
mura, 2018; Asada et al., 2018). On the other hand, given the over-
lapping of ASXL2 target genes with transcriptional targets of RUNX1
and RUNX1–ETO, it has been observed that ASXL2 deletion co‐
operates with RUNX1–ETO in the promotion of leukemogenesis
(Micol et al., 2017). Taken together, regarding the vital role of
RUNX1 in the regulation of hematopoietic development, disruption
of RUNX1 may lead to the initiation or progression of
leukemogenesis.
5 | D IS C U S S I O N
Normal hematopoiesis is controlled by gene expression patterns
that guard the balance between self‐renewal and differentiation of
HSCs; so that several epigenetic modifiers and TFs are involved in
the regulation of gene expression and fate in these cells by
activation or repression of specific genes. The activation of a
specific gene locus depends on the ability of the accessibility of
DNA to TFs. TFs facilitate gene expression through binding to the
enhancer or promoter regions of DNA via modifying the chromatin
structure and making genes available for transcription. These
processes occur following modification of chromatin structures via
histone methylation, enrichment of specific histone marks
(H3K4me3) by histone modifiers such as the ASXL1/BAP1 complex
and MLL, acetylation of histone proteins by transferring the acetyl
group from acetyl‐CoA to specific lysine residues, as well as DNA
demethylation via producing 5hmC by the TET family. Further-
more, transcription silencing in specific genes of HSCs can occur
via chromatin compaction and repressive epigenetic modifications
such as the H3K27me3 histone marker, histone deacetylation, and
DNA methylation at CpG islands via producing 5mC by DNMTs.
On the other hand, aberrant interaction in these regulators might
lead to leukemogenesis via altering the expression of functional
genes that manage self‐renewal, lineage commitment, differ-
entiation, and cell‐cycle progression of HSCs (Figure 1). For
instance, some primitive mutations such as DNMT3A, TET2, and
ASXL1 in HSPCs cause clonal hematopoiesis of indeterminate
potential, which combined with secondary mutations, contribute
to leukemic transformation, especially myeloid leukemias. In ad-
dition, a decline in the repression of genes involved in maintenance
and differentiation bias of HSCs, caused by decreased levels of
H3K27me3 and 5hmC in mutations of ASXL1 and TET2, may
trigger the differentiation of HSCs toward myeloid and lymphoid
lineages. Furthermore, dysregulation in chromatin modifications
and signaling pathways of HSCs as a result of aberrant TFs ex-
pression related to the maintenance and differentiation of HSCs
could contribute to the initiation or acceleration of leukemias. It
appears that epigenetic regulators and TFs have important roles in
protecting self‐renewal of both HSCs and LSCs. Therefore, the
evaluation of changes in TFs and epigenetic regulators as well as
their functional pathways in genomic instability of HSCs could
yield high‐value targets for leukemia therapy.
14 | SHOKOUHIAN
A C K N O W L E D GM E N T
We wish to thank all our colleagues in Shafa Hospital and Allied
Health Sciences School, Ahvaz Jundishapur University of Medical
Sciences.
CO NFLICT OF I NTERE STS
The authors declare that there are no conflict of interests.
A UT HO R C ONT RI BU TIO NS
N. S. conceived the manuscript and revised it; M. Sh., M. B., B. P., R.
Ch., and N. D. wrote the manuscript and prepared the figures.
E TH I C A L A P P R O V A L
This article does not contain any studies with human participants or
animals performed by any of the authors.
OR CID
Mohammad Shokouhian http://orcid.org/0000-0001-5061-7058
Marziye Bagheri http://orcid.org/0000-0003-3479-0277
Najmaldin Saki http://orcid.org/0000-0001-8494-5594
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How to cite this article: Shokuhian M, Bagheri M, Poopak B,
Chegeni R, Davari N, Saki N. Altering chromatin methylation
patterns and the transcriptional network involved in
regulation of hematopoietic stem cell fate. J Cell Physiol.
2020;1–20. https://doi.org/10.1002/jcp.29642