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haematologica | 2011 96(5)
EDITORIALS & PERSPECTIVES
635
Erythroid phenotypes associated with KLF1 mutations
Joseph Borg,
1,2
George P. Patrinos,
3
Alex E. Felice
2,4
and Sjaak Philipsen
5
1
Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta;
2
Laboratory of Molecular Genetics,
D
epartment of Physiology & Biochemistry, University of Malta, Malta;
3
U
niversity of Patras, Department of Pharmacy, University
Campus, Patras, Greece;
4
Thalassaemia Clinic, Section of Pathology, Mater Dei Hospital, Msida, Malta;
5
Erasmus MC, Department of
Cell Biology; Netherlands Consortium for Systems Biology; Rotterdam, The Netherlands. E-mail: j.philipsen@erasmusmc.nl
doi:10.3324/haematol.2011.043265
(Related Original Article on page 767)
E
rythroid Krüppel-Like Factor (KLF1; previously known as
EKLF) is an essential erythroid-specific transcription fac-
tor that was first identified by Miller and Bieker in 1993.
1
It binds the CACCC motif, an important DNA binding site in
the regulatory elements of many erythroid genes including the
HBB (β-globin) gene. Mutations in the β-globin CACC box
which prevent KLF1 binding are a cause of β-thalassemia.
2
KLF1 has three zinc finger domains, which mediate sequence-
specific binding to DNA and are, therefore, essential for activa-
tion of KLF1 target genes (Figure 1).
Functions of KLF1: studies in mice
Mouse KLF1 null mutants displayed grossly normal erythro-
poiesis at the embryonic stage when hematopoiesis takes
place in the yolk sac, but they rapidly developed a very severe
form of anemia at the fetal stage, when the site of
hematopoiesis has shifted to the fetal liver. KLF1 null mutants
failed to activate expression of β-globin, which is a fetal/adult
globin in the mouse. Thus, inactivation of KLF1 causes lethal
β-thalassemia. Remarkably, the expression of embryonic β-like
globin genes, εy and βh1, and the α-like globin genes, embry-
onic ζ and α1/α2, appeared to be normal.
3-4
This suggested that
KLF1 has a role in fetal-to-adult globin gene switching as it
occurs in humans. To test this idea, transgenic mice carrying a
complete human β-globin locus were used.
5-6
Such mice
express human γ-globin at the early fetal stages while the
switch to β-globin is completed at the late fetal stages. An aus-
tere reduction in β-globin expression was observed in KLF1 null
fetuses, while expression of γ-globin was not dependent on
KLF1 and even extended in its absence
5-6
(Figure 2). These data
supported a role for KLF1 in globin switching. However, the
anemia of KLF1 null mutants was not rescued by expression of
γ-globin.
7
Genome-wide gene expression profiling studies
revealed a global role for KLF1 in the activation of erythroid-
specific genes
8-10
including globins, membrane- and structural
proteins, heme synthesis enzymes and many other proteins
involved in red cell metabolism. This explained the particularly
severe anemia of KLF1 null mutants (Figure 3).
KLF1 mutations in humans – inhibitor of Lutheran
antigen expression [In(Lu)]
Mutations in human KLF1 were first reported in 2008 by
Singleton and colleagues.
1
1
They described 9 different loss-of-
function KLF1 mutations which were causative to the rare
In(Lu) blood group (Figures 1 and 3). Gene expression profiling
revealed a list of more than 650 putative KLF1 target genes
which considerably overlapped with those reported in KLF1
null mouse studies.
8
-11
These target genes included BCAM that
carries the Lutheran blood group antigens and CD44 that car-
ries the Indian blood group antigens (Figure 3). The expression
of these blood group antigens is suppressed in the In(Lu) indi-
viduals.
11
No other clinical features were reported.
KLF1 mutations in humans – hereditary persistence
of fetal hemoglobin (HPFH)
The direct association of mutations in human KLF1 with
hemoglobin regulation came from the study of a large family
from Malta.
12
Ten out of 27 family members exhibited HPFH
Figure 1. Mutations in human KLF1. All currently
reported mutations are shown; the color code
refers to the original publications as indicated. (A)
Schematic drawing of the KLF1 gene. Exons:
green = non-coding regions; red = coding regions,
orange = zinc fingers (F1, F2, F3). Mutations
between brackets are believed to be neutral sub-
stitutions. (B) Amino acid sequence of the zinc fin-
gers of KLF1 (top line). Bottom line: amino acids
invariable between the zinc finger domains of all
17 human KLF transcription factors (KLF_all).
Blue boxes highlight amino acids involved in coor-
dination of the Zn atom; yellow boxes highlight
amino acids directly involved in DNA binding. The
arrows indicate amino acids that make base-spe-
cific contacts with the DNA double helix.
A
B
Page 1
d
ue to a single point mutation in KLF1 (p.K288X; Figures 1
and 3). This mutation completely abrogates the DNA bind-
ing domain and therefore results in haploinsufficiency for
KLF1. The KLF1 p.K288X carriers displayed high HbF lev-
els, although with considerable variation (mean 8.4%;
range 3.3-19.5%). Part of this variability could be explained
by SNP haplotypes at the BCL11A locus, which encodes a
repressor of γ-globin expression.
13
Importantly, BCL11A
expression was reduced in the KLF1 p.K288X carriers and
KLF1 was shown to be a direct activator of BCL11A expres-
sion
12,14
(Figure 3). Collectively, these data suggested that
attenuation of KLF1 activity could be a fruitful approach to
raise HbF levels in patients with β-type hemoglo-
binopathies.
KLF1 mutations in humans – congenital
dyserythropoietic anemia
A further very interesting twist came from the study of 2
unrelated patients with congenital dyserythropoietic ane-
mia (CDA
15
). On one KLF1 allele, these patients carried a
missense mutation in the second zinc finger (Figures 1 and
3). The mutation, p.E325K, had a dominant effect and
resulted in severe hemolytic anemia. The patients dis-
played 31.6% and 44% HbF, and also expressed embryonic
ζ- and ε-globin.
15
The erythroid cells were deficient for
CD44 and the water channel AQP1, while BCAM expres-
sion was reduced in one case and normal in the other
1
5
(Figure 3). Remarkably, an ethylnitrosourea-induced muta-
tion in the homologous position in mouse KLF1 causes the
dominant Nan (neonatal anemia) phenotype.
16-17
Heterozygous Nan/+ mice displayed hereditary spherocy-
tosis and severe hemolytic anemia. Expression of erythro-
cyte membrane skeleton proteins and β-globin was
reduced, but embryonic globins were present at aberrantly
high levels (Figures 1 and 3). The mutation, p.E339D,
reduced binding to a subset of KLF1 binding sites, and this
selectively affected activation of KLF1 target genes.
1
7
The
variant residues have opposite charges: positive in the CAD
patients (p.E325K, lysine) and negative in the Nan mouse
(p.E339D, aspartic acid). These mutants have different
DNA binding properties that determine the impact on the
e
xpression of KLF1 target genes.
1
5, 17
T
hus, different KLF1
missense mutations that affect DNA binding properties
could well lead to distinct red cell disorders
15
(Figure 3).
KLF1 mutations in humans – zinc protoporphyrin
Significantly raised HbF levels are not always observed in
association with KLF1 mutations. The study of the Maltese
family already noted variability in HbF levels amongst indi-
viduals carrying the p.K288X mutation. SNP haplotyping of
the BCL111A locus accounted for only a small part of the
variability, strongly suggesting that as yet unknown modi-
fiers are involved (Figure 3). This idea is further supported
by data on a family from Sardinia reported in this issue of
Haematologica.
18
Three heterozygous carriers of KLF1
mutations (p.S270X or p.K332Q; Figure 1) had HbF levels
of 0.9-1.2%, which is at the upper end of the normal range.
Interestingly, the two compound heterozygotes
(p.S270X::p.K332Q) displayed very high HbF levels (22.1%
and 30.9%). In addition, these individuals had high levels of
zinc protoporphyrin in their circulation; this was also
observed in the Nan mouse
16-17
(Figure 3).
KLF1 mutations in humans – known unknowns and
unknown unknowns
The recent reports on KLF1 mutations have opened up a
novel research area of human erythropoiesis, with a range
of questions that urgently need answers. The feasibility of
Editorials and Perspectives
636
haematologica | 2011; 96(5)
Figure 2. Activation of a human β-globin locus transgene in KLF1
null mouse mutants. RNA expression levels of the human β-like glo-
bins [embryonic (ε), fetal (γ), adult (β)] were assessed by quantita-
tive S1 nuclease protection assays.
6
Days of mouse embryonic
development are indicated (E10.5, E12.5, E14.5). The status of the
mouse KLF1 alleles is shown: wt = wild-type; het = heterozygous for
KLF1 null allele, null = homozygous for KLF1 null allele. Data cour-
tesy of Drs. Beatriz Nuez and Frank Grosveld.
Figure 3. Phenotypes caused by KLF1 mutations The inner ring dis-
plays KLF1 mutations and potential modifiers. The number of differ-
ent mutants reported is shown. The middle ring displays critical KLF1
target genes/loci whose expression is affected by the KLF1 muta-
tion(s). The outer ring displays phenotypes. Clinical conditions are in
the boxes; the colors refer to the publications shown below. HBA =
α-globin locus; HBB = β-globin locus; EMS = erythrocyte membrane
skeleton, ZnPP = zinc protoporphyrin.
Page 2
a
ttenuating KLF1 activity to raise HbF levels in patients
with β-type hemoglobinopathies remains to be deter-
mined. Related to this, we have only begun to understand
the phenotypes associated with the different KLF1 muta-
tions, and more in-depth analyses might very well reveal
that there is significantly more overlap between these phe-
notypes than currently appreciated (Figure 3). The impact
of missense mutations on the phenotypic outcome also
warrants further investigation. For instance, missense
mutations of critical residues in the zinc finger domains of
KLF1 affect its DNA binding properties and may have a
dominant phenotype, as exemplified by the p.E325K muta-
tion in the CDA patients
15
and the p.E339D mutation in the
Nan mouse.
16-17
In contrast, the p.K332Q mutation, which
also affects the DNA binding properties of KLF1, does not
have a dominant effect.
18
Genome-wide assessment of the
in vivo binding sites of these mutant KLF1 proteins may help
to understand the phenotypic variability. In addition, it
appears that some target genes, such as BCL11A, EPB4.9
and CD44, are very sensitive to perturbations in KLF1 activ-
ity, while the effects on others, such as BCAM and γ-globin,
are much more variable. Studies in the Nan mouse indicate
that this might depend on the class of KLF1 binding site
responsible for activation of KLF1 target genes.
17
In addi-
tion, modifier genes may affect the expression of the “vari-
able response” genes, as exemplified by the intricate inter-
play of KLF1 and BCL11A on γ-globin expression.
1
2,14
Identifying these modifier genes and characterizing the
molecular properties of KLF1 mutants are important chal-
lenges for further research. In addition, we expect that
many novel cases of individuals with KLF1 mutations will
be discovered in the near future. Novel mutations may pro-
vide insight into the functional domains of KLF1. Most of
the reported mutations affect the DNA binding domain
(Figures 1 and 3). KLF1 is known to interact with co-factors
such as the chromatin remodeling SWI/SNF complex, and
it undergoes post-translational modifications such as phos-
phorylation, acetylation and SUMOylation (M Siatecka
and JJ Bieker, submitted manuscript, 2011). Mutations
affecting these biochemical properties may provide impor-
tant clues to the functional roles of protein-protein interac-
tions and post-translational modifications. Furthermore,
given the broad impact of KLF1 on erythroid-specific gene
expression in the mouse,
8-10
the array of human erythroid
phenotypes associated with KLF1 mutations will likely be
expanded as more cases are described in detail. We note
that mutations without an obvious phenotype are equally
important to report, but difficult to publish in the peer-
reviewed literature. To keep track of all KLF1 mutations
and associated phenotypes, we have implemented the
microattribution process
19
and initiated a collaborative
effort for functional analysis of KLF1 mutants
(http://www.ithanet.eu/eklf-klf1). We strongly call upon
the hematology community to participate in these initia-
tives, to ensure that valuable information on KLF1 muta-
tions is systematically collected and accessible to both clin-
ical and research scientists.
Acknowledgments: this work has been supported by institution-
al funding of the University of Malta, and the Malta Department
of Health (AEF and JB), a fellowship of the Malta Government
Scholarship Scheme (JB), European Commission grants
(
GEN2PHEN; FP7-200754 and ITHANET; FP6-026539) to
GPP, and the Netherlands Genomics Initiative (NGI), Erasmus
MC (MRace; 296088), the Landsteiner Foundation for Blood
Transfusion Research (LSBR; 1040), and the Dutch organization
for scientific research (NWO; DN 82-301 and 40-00812-98-
08032) to SP. We apologize to our colleagues whose work could
not be cited due to space constraints.
Joseph Borg is an academic staff member of the University of
Malta, Faculty of Health Sciences, Department of Applied
Biomedical Science, and a researcher on the Thalassaemia
Project in the Laboratory of Molecular Genetics. George P.
Patrinos is Assistant Professor of Pharmacogenomics at the
University of Patras, Department of Pharmacy. He has been
involved in globin gene regulation research for over 15 years. His
research interests include pharmacogenomics of fetal hemoglobin
augmenting agents. Alex. E. Felice is the founder of the
Thalassaemia Project at the University of Malta where he is
Professor, and the Malta Department of Health, Mater Dei
Hospital where he is Visiting Consultant. His interests are in
hemoglobin epidemiology and globin gene control. Sjaak
Philipsen is Professor of Genomics of Cell Differentiation at the
Department of Cell Biology in Erasmus University Medical
Center in Rotterdam. His main research interest is how tran-
scription factor networks control erythropoiesis.
Financial and other disclosures provided by the author using the
ICMJE (www.icmje.org) Uniform Format for Disclosure of
Competing Interests are available with the full text of this paper at
www.haematologica.org.
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haematologica | 2011; 96(5)
JAK2
V617F
/TET2 mutations: does the order matter?
Elodie Pronier,
1
Cyril Quivoron,
2
Olivier A. Bernard,
2
Jean-Luc Villeval
1
1
INSERM U1009;
2
INSERM U985; Institut Gustave Roussy, Université Paris Sud11, France. Correspondence: Jean-Luc Villeval.
E-mail: villeval@igr.fr doi:10.3324/haematol.2011.042846
(Related Original Article on page 775)
E
P and CQ contributed equally to this article
A
ccording to the World Health Organization classifi-
cation, myeloproliferative neoplasms (MPN) include
chronic myelogenous leukemia, also known as
BCR-ABL1–positive MPN, classic BCR-ABL1-negative
MPN including polycythemia vera (PV), essential thrombo-
cythemia (ET) and primary myelofibrosis (PMF), and non-
classic forms (i.e. systemic mastocytosis, chronic
eosinophilic leukemia not otherwise specified, chronic neu-
trophilic leukemia and unclassifiable MPN). All these sub-
types are stem cell-derived clonal myeloproliferation, asso-
ciated with the overproduction of mature blood elements
and variable rates of transformation to acute myeloid
leukemia (AML).
1
JAK2
V617F
activating mutation is the most prevalent abnor-
mality observed in BCR-ABL1-negative MPN, found in vir-
tually all cases of PV and in about half of ET and PMF (96%,
55% and 65%, respectively). This mutation lies in the
pseudokinase-domain of JAK2 and disrupts its regulatory
activity. Another mutation affecting JAK2 exon 12 is
observed in 3% of all PV cases. Mutations affecting W515
of the thrombopoietin receptor MPL are detected in PMF
and ET patients. Additional mutations have been identified
in MPN (reviewed in
1
). Defects in the control of intracellu-
lar signaling involve mutations in LNK and CBL genes.
Genetic abnormalities affecting epigenetic regulation, and
possibly responsible for disease initiation, concern the
ASXL1, EZH2 and TET2 genes. Finally, mutation in IKZF1
and IDH1/2 may be implicated in MPN transformation. In
PV, among these additional mutations to JAK2
V617F
, TET2
mutations are those most frequently reported (16%); the
others are only described in small subsets of patients.
1
TET2 belongs to a family of three conserved genes in
mammals: TET1, TET2 and TET3. The founding member of
the family, TET1, has been identified as a fusion partner of
MLL in the t(10;11)(q22;q23) translocation of acute
leukemia.
2,3
The involvement of TET3 in hematologic disor-
der has not yet been described. The TET proteins are mem-
bers of the 2-oxoglutarate (2-OG)- and Fe(II)-dependent
dioxygenase that are able to convert 5-methyl-cytosine (5-
mC) to 5-hydroxymethyl-cytosine (hmC).
4,5
Recent reports
indicate an important role for TET1 and TET2 (and, there-
fore, hmC) in the control of ES cell self-renewal and differ-
entiation.
6
TET3 might be involved in genome reprogram-
ming following fecundation.
7
5-hydroxymethylation: a novel major player
in the epigenetic field
For decades, the implications and impact of 5-mC in
human genome has been extensively studied and it is
known to be associated with low gene expression. In con-
trast, little is known about the recently identified hmC.
Indeed, the first study reporting a hydroxylated form of 5-
mC in mammalian DNA was described in the early 70s.
8
However, this modified base did not receive full attention
until 2 reports demonstrating that hmC accounts for 0.6%
and 0.03% of the total nucleotides in Purkinje cells
9
and
murine ES cells,
5
respectively.
The function of hmC is not yet clear. Several reports have
indicated that hmC prevent the binding of proteins inter-
acting with 5-mC and hmC and may represent a step
toward demethylation.
8
Using a novel chemical method,
Song et al.
10
showed an enrichment of hmC in maturing
murine brain cells that increases with age and is associated
with gene expression. Contrasting results have been report-
ed in human samples: TET2 mutation or inhibition of its
catalytic activity affect the level of hmC in myeloid malig-
nant samples, but was associated with a decrease of 5-mC
in MDS (myelodysplastic syndromes) samples and with an
increase of DNA methylation in AML.
11,12
TET2 function in normal hematopoiesis and MPN
TET2 is expressed in a wide range of tissues, such as kid-
ney, brain and the hematopoietic system.
13
Recent analyses
using short-hairpin RNA in murine stem cells from bone
Page 4
  • Source
    • "transcription factor genes in both primitive and definitive erythroid cells (Siatecka and Bieker, 2011; Tallack and Perkins, 2010; Yien and Bieker, 2013). Relatedly, links have been established between mutant or haploinsufficient levels of EKLF and altered human hematology and anemia (Borg et al., 2011; Helias et al., 2013; Siatecka and Bieker, 2011; Singleton et al., 2012). Comparative analysis of expression arrays between EKLF wildtype and EKLF-null fetal liver cells show that a number of genes involved in execution of the terminal erythroid differentiation program are downregulated in the absence of EKLF (Drissen et al., 2005; Hodge et al., 2006; Pilon et al., 2006, 2011; Tallack et al., 2012, 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: The erythroblastic island provides an important nutritional and survival support niche for efficient erythropoietic differentiation. Island integrity is reliant on adhesive interactions between erythroid and macrophage cells. We show that erythroblastic islands can be formed from single progenitor cells present in differentiating embryoid bodies, and that these correspond to erythro-myeloid progenitors (EMPs) that first appear in the yolk sac of the early developing embryo. Erythroid Krüppel-like factor (EKLF; KLF1), a crucial zinc finger transcription factor, is expressed in the EMPs, and plays an extrinsic role in erythroid maturation by being expressed in the supportive macrophage of the erythroblastic island and regulating relevant genes important for island integrity within these cells. Together with its well-established intrinsic contributions to erythropoiesis, EKLF thus plays a coordinating role between two different cell types whose interaction provides the optimal environment to generate a mature red blood cell.
    Full-text · Article · Jun 2014 · Development
  • Source
    • "Several factors have been identified to be involved in the suppression of the g-globin genes. For example, BCL11A and KLF1 were recently shown to lead to elevated g-globin gene expression when their activity is suppressed (Borg et al., 2011; Sankaran et al., 2008; Xu et al., 2010 Xu et al., , 2011). Interestingly, the promoters of the g-globin genes were previously identified as the regions responsible for their suppression (Berry et al., 1992; Dillon and Grosveld, 1991; Li et al., 2001; Starck et al., 1994; Yu et al., 2006), whereas the BCL11A protein binds to a region downstream of the g-globin genes. "
    [Show abstract] [Hide abstract] ABSTRACT: Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells in vivo. Using targeted chromatin purification (TChP), a double-pull-down strategy with a tetracycline-sensitive "hook" bound to a specific promoter, we identify transcription factors bound to the repressed γ-globin gene-associated regulatory regions. After validation of the binding, we show that, in human primary erythroid cells, knockdown of a number of these transcription factors induces γ-globin gene expression. Reactivation of γ-globin gene expression ameliorates the symptoms of β-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients.
    Full-text · Article · Jul 2013 · Cell Reports
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    • "doi:10.1371/journal.pgen.1003612.g010 found in mutant mice [36,37], and the homologous mutation in humans causes human disease [38]. Similar to our case, the Klf1 mutation changed the central of 3 amino acids predicted to contact DNA in the zinc finger of a KLF protein. "
    [Show abstract] [Hide abstract] ABSTRACT: KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3(H275R) ). This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3(H275R) mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3(H275R) mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans.
    Full-text · Article · Jul 2013 · PLoS Genetics
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