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Review article
Regulation of human fetal hemoglobin: new players, new complexities
Arthur Bank
The human globin genes are among the
most extensively characterized in the human genome, yet the details of the molecular events regulating normal human
hemoglobin switching and the potential
reactivation of fetal hemoglobin in adult
hematopoietic cells remain elusive. Recent discoveries demonstrate physical
interactions between the ␤ locus control
region and the downstream structural ␥and ␤-globin genes, and with transcription factors and chromatin remodeling
complexes. These interactions all play
roles in globin gene expression and globin switching at the human ␤-globin locus.
If the molecular events in hemoglobin
switching were better understood and
fetal hemoglobin could be more fully reac-
tivated in adult cells, the insights obtained might lead to new approaches to
the therapy of sickle cell disease and ␤
thalassemia by identifying specific new
targets for molecular therapies. (Blood.
2006;107:435-443)
© 2006 by The American Society of Hematology
Introduction
An understanding of the regulation of human fetal hemoglobin
expression is required to rationally approach the problem of
hemoglobin switching and the reactivation of fetal hemoglobin in
adult hematopoietic cells. The human globin loci are among the
best characterized in the human genome at the gene and protein
levels.1,2 The human ␤-globin locus on chromosome 11 contains a
powerful set of enhancer elements, termed the ␤ locus control
region (␤LCR), upstream (5⬘) of the ␤ locus structural genes
(Figure 1).3-5 The ⑀-globin gene is the most 5⬘ globin structural
gene and is active in early fetal life (Figure 1). The ␥-globin genes
(G␥ and A␥) are the major ␤-like genes expressed in most of fetal
life; the ␦- and ␤-globin genes are activated late in fetal life, with
the ␤-globin gene being most highly expressed in erythroid
cells after birth and in adults. At the other major globin locus,
the ␣-globin locus on chromosome 16, by contrast, ␣-globin gene
expression begins in early fetal life and is predominant throughout
fetal and adult life.6 Regulation at the ␣-globin locus differs
greatly from that of the ␤-globin locus and is beyond the scope of
this review.7-10
Human globin gene transcription leads to the expression of
mRNA precursors in the nucleus that are subsequently processed
into mature globin mRNAs in the cytoplasm.1,2 Here, the translational machinery of the cell, including enzymes and ribosomes,
result in the production of globins that combine with heme to form
the normal human hemoglobins. The major hemoglobin in fetal life
is fetal hemoglobin (HbF, ␣2␥2), whereas the major hemoglobin in
adult life is hemoglobin A (HbA, ␣2␤2). Hemoglobin A2 (HbA,
␣2␦2) is a minor hemoglobin, whose expression is usually less than
2% of the total hemoglobin.
The evolution of the human ␥-globin genes and HbF production
in primates is relatively recent; mice have no ␥-globin genes. The
development of ␥-globin genes is most likely related to the
increased oxygen affinity of HbF over HbA, which favors oxygen
delivery to the fetus in the placental circulation. The switch from
human ␥- to ␤-globin gene expression in late fetal life is a major
event in hemoglobin biology. The consequences of this switch lead
to diseases in humans: the hemoglobinopathies such as sickle cell
anemia (SS disease) and ␤ thalassemia.1,2 Although the production
of HbF during fetal life is normal and optimal in patients with these
disorders, the switch to predominantly HbA synthesis around birth
causes pathology. In SS disease, the normal switch to ␤-globin
gene expression results in the accumulation of ␤S, which forms
hemoglobin S (HbS, ␣2␤S2), and leads to HbS aggregation,
hemolytic anemia, and clogging of small vessels.11,12 In human ␤
thalassemia, the switch results in decreased or absent normal
human ␤-globin production and HbA. This leads to excess
␣-globin accumulation and precipitation in early erythroid cells
and causes apoptosis and ineffective erythropoiesis.1,2,6,13
In both SS and homozygous ␤ thalassemia (Cooley anemia),
insufficient HbF is produced in adult hematopoietic cells after the
switch to compensate for the lack of normal ␤-globin production.
Treatments with hydroxyurea and butyrate compounds have resulted in increased levels of HbF and clinical benefit in some
patients with sickle cell disease and ␤ thalassemia.14-18 If the
molecular events in hemoglobin switching were better understood
and HbF could be more fully reactivated in adult cells, the insights
gained might lead to a cure for these disorders.
From the Departments of Medicine and of Genetics and Development,
Columbia University, New York, NY.
Reprints: Arthur Bank, 701 W 168th St, Rm 1604, New York, NY 10032; e-mail:
[email protected].
Submitted June 1, 2005; accepted August 11, 2005. Prepublished online as
Blood First Edition Paper, August 18, 2005; DOI 10.1182/blood-2005-05-2113.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by US Public Health Service grants DK-56635, HL-59887, and DK07373 from the National Institutes of Health.
BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
The players at the ␤-globin locus
At the murine and human ␤-globin loci, gene transcription is
controlled by the complex interactions between (1) cis-acting
sequences: the ␤LCR and downstream globin gene sequences
embedded with histones in nucleosomes, in chromatin (Figure 1),
and (2) trans-acting factors: transcription factors and chromatin
remodeling activities.19-22
© 2006 by The American Society of Hematology
435
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BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
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or as part of chromatin remodeling complexes with HDACs, can
repress and silence specific gene function at many loci and may be
involved in repressing human ␥-globin gene expression.51,60-70
Enzymatic complexes can modify histone N-terminal tails by
methylation, phosphorylation, and ubiquitination as well.71
The complexities: interactions of the players
at the ␤-globin locus
Figure 1. The human ␤-globin locus. (A) The ␤-globin locus on chromosome 11
embedded in chromatin is shown. (B) A linear map with the globin LCR and its
hypersensitive (HS) sites is indicated by the vertical arrows. The structural ⑀-, ␥-, ␦-,
and ␤-globin genes as well as the locations of the olfactory receptor (OR) genes are
shown.
The ␤-globin LCR
The ␤-globin LCR upstream (5⬘) of the globin structural genes is
the major structural component of the murine and human ␤-globin
loci and is required for normal high-level globin gene transcription
(Figure 1). The murine and human ␤LCRs contain 5 critical
DNAase 1 hypersensitive (HS) sites, HS1-5, that are formed in
regions devoid of nucleosomes and that are sites more accessible
than other regions of chromatin to interactions with transcription
factors and downstream gene sequences.3,23,24 Another HS site, 3⬘
HS1, is 3⬘ to the ␤-globin structural globin gene, and olfactory
receptor (OR) sequences are at both the 5⬘ and 3⬘ borders of the
␤-globin locus (Figure 1).24,25
Erythroid cell-specific transcription factors
Several important transcription factors play critical roles in murine
globin gene expression. GATA-1, which binds to the promoters of
many erythroid cell-specific genes and to ␤LCR sequences, is
required for normal erythroid cell differentiation.26-29 Mice deficient in GATA-1 cannot produce mature erythroid cells30-32 and
erythropoiesis can be normalized by restoring GATA-1 function.31,32 Erythroid Krupple-like factor (EKLF) is necessary for
activation of the adult ␤-globin gene and also has important
interactions with the ␤LCR.33-35 Mice with EKLF deficiency die in
utero with a disease resembling severe ␤ thalassemia.36 The
transcription factor NF-E237 is also important in its function at the
human ␤-globin locus although mice deficient in NF-E2 have only
mild erythroid defects.38,39 NF-E4 is a critical transcription factor in
chicken globin switching.40-42 More recently, a human homologue
of chicken NF-E4, NF-E4p22, has been shown to be active in
human fetal globin gene activation.43,44
Chromatin remodeling activities
Acetylation of histones in chromatin leads to increased expression
of specific genes at structural gene loci, including the ␤-globin
locus, by making gene sequences more available to transcription
factors.21,45-52 Specific acetylations of histones H1-H8 in chromatin
can be achieved either by histone acetylase enzymes (HAs) alone
or by adenosine triphosphate (ATP)–driven molecular machines
providing this function, such as SWI/SNF complexes.53-56 SWI/
SNF complexes have been shown to be active at the murine and
human ␤-globin loci.57-60
By contrast, deacetylation of histones in chromatin by histone
deacetylases (HDACs), acting either as enzymes that are HDACs
Many new details about the complex interactions between ␤LCR
elements and downstream globin gene sequences in chromatin
have been elucidated recently in mouse erythroleukemia (MEL)
cells, and in mice at both the murine and human ␤-globin loci, the
latter using human ␤ locus-containing sequences in transgenic
mice.20-23 These recent experiments show that the HS sites at the
␤LCR are required to form a unique 3-dimensional structure at the
chromosomal location of the ␤-globin locus on chromosome 11,
with “looping out” of the locus in a unique spatial configuration, an
active chromatin loop at the ␤-globin locus, termed an active
chromatin hub (ACH).23 The ␤ACH includes the ␤LCR sequences
interacting with downstream globin genes and intergenic sequences, transcriptional factors, and chromatin remodeling elements necessary for globin gene expression and hemoglobin
switching in a special compact configuration present uniquely in
erythroid cells (Figure 1).20-23 The ␤ACH structure is not found in
nonerythroid cells.23
The technology
Studies defining long-range physical contacts between the ␤LCR
and specific downstream gene sequences in the ␤ACH specific to
erythroid cells have used 3 related techniques that have in common:
(1) cross-linking of proteins and DNA in intact cells in their native
chromatin configuration by treatment of the cells with formaldehyde; (2) lysis of the cells, fragmentation of the chromatin, and
removal of protein-DNA cross-links; and (3) identification of
specific ␤ locus DNA sequences interacting with each other by the
use of polymerase chain reaction (PCR) probes representing
specific small defined regions of the locus from 5⬘ of the LCR to 3⬘
of the structural ␤-globin genes. In chromatin immunoprecipitation
(ChIP) analyses, after chromatin fragmentation, antibodies to
specific proteins are used to isolate the DNA sequences associated
with these proteins in vivo, for example, antibodies to acetylated
histones or specific transcription factors.24,45-48,52,72-79 In so-called
chromosome conformation capture (3C) experiments, specific
DNA sequences in contact with each other in vivo are identified by
restriction digestion and controlled religation of DNA fragments
after chromatin cross-linking.20-23,80 In another procedure, tagging
and recovery of associated proteins (TRAP) technology, biotinlabeled probes are used to uniquely extract newly synthesized RNA
associated with protein sequences after cross-linking.81,82
Interactions between the ␤LCR and downstream
gene sequences
Although mice lack a true ␥-globin gene homologue, mouse yolk
sac (YS) cells express mouse embryonic globin genes that function
analogous to human ␥-globin genes. In mice transgenic for cosmids
or yeast artificial chromosomes (YACs) containing human ␤-globin
locus sequences, YS cells at embryonic (E) days 9 to 11 express the
human ␥-globin gene, whereas mouse fetal liver (FL) cells at E11
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BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
to E15 express the human ␤-globin gene, recapitulating human ␥to ␤-globin switching to varying extents.20,21,35,59,83-85 Using 3C
technology, recent cross-linking studies have shown physical
interactions specifically between 5⬘ HS sites in the murine ␤LCR
with HS sequences 3⬘ to the ␤ structural genes in FL at E14.5
(adult-type cells), whereas no such interactions are seen in brain
tissue from the same embryos, nor between sequences between the
5⬘ and 3⬘ HS sites.23 In uninduced MEL cells, committed to the
erythroid lineage but not induced to erythroid differentiation, a
␤ACH structure exists that is different from that in brain cells even
when there is only low-level globin synthesis. In these uninduced
MEL cells, however, there are only limited interactions between 5⬘
HS sequences and the 3⬘ HS1.20 On induction of MEL differentiation and the accompanied dramatic increase in globin synthesis, the
interactions between ␤LCR elements and 3⬘ sequences become
much more extensive.20
To further define the elements critical for formation of the active
␤ACH in human erythroid cells, transgenic mice with P1-derived
artificial chromosomes (PACs) carrying a 185-kb fragment of
human ␤-globin locus DNA, the effect of specific deletions at the
locus were studied.21 When the human structural ␤-globin gene
promoter is deleted, there continue to be significant ␤ACH
interactions between the ␤LCR elements upstream, the 3⬘ HS1 and
the human structural genes.21 In mice with deletions of the human
␤-globin promoter and with additional deletions of either human
␤LCR HS2 or HS3, the HS3 deletion disrupts ␤ACH formation,
whereas the HS2 deletion has little additional effect.21 These
studies show that interactions between HS3 and the human
␤-globin promoter are critical for ␤ACH formation.
Interactions of transcription factors with globin sequences
in chromatin
The mouse ␤-globin gene LCR, essential for the activated transcription of genes in the cluster,48 contains multiple binding sites for
transcription factors. ChIP experiments show that in uninduced
MEL cells, the LCR is occupied by small Maf proteins, and, on
erythroid maturation, the NF-E2 complex is recruited to the LCR
and the active ␤-globin promoters. The presence of the of NF-E2
complex is associated with a more than 100-fold increase in murine
␤-globin transcription.48 GATA-1 is associated with murine HS2 in
ChIP experiments as well.76
Other experiments using 3C technology show that there are
specific physical interactions between the transcription factors
GATA-1, FOG-1, and EKLF and the ␤LCR and downstream globin
sequences, again demonstrating the close contact and multiple
interactions of these 2 important regions of the ␤ACH.22,35 In one
study, EKLF-deficient mice were unable to form the ␤ACH,
indicating its critical role in the chromatin configuration in adult
erythroid cells.35 In another, GATA-1–induced loop formation
correlates with the onset of ␤-globin transcription and occurs
independently of new protein synthesis.22 GATA-1 occupies the
murine ␤-globin promoter even in FL erythroblasts of mice lacking
the LCR, suggesting that GATA-1 binding to the promoter and
LCR are independent events that occur prior to loop formation.22
The results show that GATA-1 and FOG-1 are essential for the
formation of the ␤ACH.22
Acetylation of chromatin at the ␤-globin locus
Early ChIP experiments using MEL cells and mouse FL demonstrated that the murine ␤-globin locus is more acetylated when the
␤-globin genes are active than when they are not.45 These studies
HUMAN FETAL HEMOGLOBIN REGULATION
437
show hyperacetylation only at the ␤LCR and the ␤ structural genes
and not of sequences between these regions and were among the
first consistent with looping of chromatin fibers, physical association of these sequences, and formation of the ␤ACH.45 In YS cells,
the murine ␤LCR and both active and inactive promoters are
hyperacetylated.45 The specific inhibition of HDACs in these
studies selectively increased acetylation at a hypoacetylated promoter in FL, suggesting that active deacetylation contributes to
silencing of promoters.45
ChIP studies also show methylation of an individual lysine
residue, H3-meK79, at the active murine ␤-globin gene in adult
erythroid cells that is dependent on the presence of p45/NF-E2.50 In
addition, acetylated H3, and H4 and H3-meK4 are enriched at both
the ␤-globin structural gene and the ␤LCR. An HDAC-dependent
surveillance mechanism may counteract constitutive histone acetyltransferase (HAT) access at chromatin sites.51 There are clearly
many other complex relationships between histone acetylation on
one hand and deacetylation and methylation on the other at the
␤-globin locus.51,52,86-89
RNA polymerase II activity is also associated with active
chromatin remodeling at the ␤-globin locus.75,77,79,82,90 Although
most transcription is of globin structural gene sequences activated
by their association with the ␤LCR, there is also transcription of
intergenic sequences as well.52,90-93 Most intriguing are the unique
developmental stage-specific intergenic transcripts from the region
between the ⑀ and ␥ genes in embryonic-fetal cells and from
between the ␥-and ␦-globin genes in adult cells.93
Human ␥- to ␤-globin switching
The details of the interactions between the cis-acting elements and
trans-acting factors and their relationships in regulating the human
␥- to ␤-globin switch remain elusive. Presumably, in human fetal
life, interactions between transcription factors, chromatin remodeling complexes, and structural gene sequences in chromatin favor
expression of human ␥ genes, and, subsequently in adult-type
hematopoietic cells, ␤-globin gene expression is preferred.
Studies in transgenic mice
Changes in the interactions between the ␤LCR and the downstream
embryonic-fetal and adult globin genes during development and
globin switching have been elucidated recently by 3C studies.20
Cells that express embryonic globin genes have extensive interactions between the structural embryonic gene sequences and HS2
and HS3 sites in the ␤LCR.20 By contrast, cells expressing adult
␤-globins have specific interactions between the adult ␤-globin
genes and HS2 and HS3.20 Transgenic mice carrying the entire
human ␤-globin locus were used in similar studies and, again, show
a switch in the association of HS ␤LCR from contacts with
embryonic and fetal genes in YS cells to contacts with the adult
human ␤-globin gene in adult-type E14.5 FL cells.20 The same
interactions between the adult human ␤-globin gene and 5⬘ HS
elements were obtained by C3 analysis using fresh adult erythroid
cells from human bone marrow as with the E14.5 FL cells,
providing evidence that the same conformational changes occur
between the human ␤LCR and the downstream structural genes at
the native adult human ␤ locus in normal human erythroid cells as
in PACs in transgenic mice.20
To further define the interactions required for switching, the
human ␤-globin promoter and either human HS2 or HS3 were
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BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
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deleted in transgenic mice.21 YS and FL cells doubly mutant for the
␤ promoter and HS2 (⌬␤/⌬HS2) are relatively unaffected.21
However, whereas ⌬␤/⌬HS3 mice express human ␥ genes in YS
cells, there is no formation of the ␤ACH in adult FL.21 These
results suggest that the deletion of HS3 completely destabilizes
normal chromatin structure at the ␤-globin locus in adult-type cells.
Transgenic mice containing cosmids or YACs with deletions of
␤ locus sequences have also been used to study the cis-acting
elements controlling human ␥- to ␤-globin switching.59,83,85,94 The
results in mice with these deletions have been interesting but
inconsistent. Some studies have shown that deletion of intergenic
␥-␦ sequences leads to delayed switching,59,83 whereas others show
either no effects or competing positive and negative effects.83,85,94
Table 1. Hemoglobin defects in homozygotes for thalassemia and
related disorders
State
Anemia
Normal
None
␤⫹ thalassemia
Severe
HbA
HbA2
HbF
Normal
Normal
Normal
Decreased
Normal
Increased*
␤0 thalassemia
Severe
Absent
Normal
Increased*
␦-␤ thalassemia
Mild
Absent
Absent
Increased†
HPFH
None
Absent
Absent
Increased‡
Corfu
Mild
Decreased
Absent
Increased‡
Severe anemia is usually defined as having hemoglobin levels below 70 g/L and
requires frequent transfusions.
*Slightly increased over normal (5%-10% of total normal Hb levels).
†Moderately increased over normal (10%-30% of total normal Hb).
‡Markedly increased, that is, almost completely compensatory (85%-100% of
total normal Hb).
Fetal globin expression in human mutants
Natural mutations at the human ␤-globin locus in patients with
increased HbF have shed important light on the mechanisms
regulating human HbF. Studies of patients with these mutations are
clearly more related to the biology of human HbF regulation than
those in transgenic mice or using cell lines. Transgenic mouse
experiments, even with very large human ␤-globin locuscontaining YACs or PACs, are potentially flawed by the random
position of these sequences and the fact that mice do not have
␥-globin genes. Data have been reported on many individuals with
point mutations in the human ␥ promoter that increase HbF in
adult-type erythroid cells.2,95 These patients are referred to as
having the non–deletion form of hereditary persistence of fetal
hemoglobin (HPFH), as compared to those with deletion-type
HPFH. Mutations have been described in the promoters of both the
G␥ and A␥ genes in these patients, and in most cases, the point
mutations are between ⫺114 and ⫺202 upstream of the human
␥-globin gene transcriptional initiation sites.2,95 Several of these
mutations modify binding sites for GATA-1 or NF-E4 or are at the
distal CAAT box upstream. The level of HbF in heterozygotes for
these mutations varies between 10% and 30% with mutations in the
G␥ promoter and between 4% and 20% in those with A␥
mutations.2,95 A deletion upstream of the A␥ gene between ⫺114 to
⫺102 also leads to about 30% HbF in heterozygotes.96
It is difficult to quantify the effects on ␥-globin transcription
and posttranscriptional events in heterozygotes for these and other
mutations without using the sophisticated techniques described
recently to measure the output of single mutated alleles performed
in the primary erythroid cells of patients.97 As these recent studies
reveal, the levels of HbF in heterozygotes with the same affected ␥
allele are not constant and depend on the effects of the other
allele.97 Thus, only homozygotes for mutations affecting human
␥-globin expression are truly informative.
Figure 2. Mutations and the extent of deletion in thalassemias and HPFH. A ⫻
indicates point mutation. Dotted lines indicate deletions. There is more deletion of ␥-␦
intergenic sequences in HPFH than in ␦-␤ thalassemia, and the 3⬘ extent of the
deletion is greater in HPFH.
Fortunately, homozygotes for some naturally occurring mutations that affect the human ␥-globin locus are available. Some
African homozygotes with an uncommon, benign disorder, deletion HPFH, express ␥-globin genes fully in adult life, have 100%
HbF, and have no anemia.2,95 If human patients with sickle cell
disease or ␤ thalassemia could reactivate their HbF production to
that of HPFH patients, they would be cured. In fact, Saudi patients
doubly heterozygous for ␤S and HPFH, expressing 30% HbF in
most red blood cells, are largely asymptomatic.98,99
In the African form of deletion HPFH, large deletions begin just
3⬘ to the A␥ gene and extend far 3⬘ of the structural ␤-globin gene;
the deletion includes intergenic ␥-␦ sequences and the entire
structural ␦ and ␤ genes (Figure 2; Table 1). The mechanism
leading to the increased level of HbF in HPFH has been shown to
be due, at least in part, to enhancer activity provided by the DNA
sequences from 3⬘ to the deletion brought into proximity of the
structural ␥-globin genes. These human enhancer sequences upregulate human ␥ globin expression in transgenic mice.84,95,100,101
Other deletions at the human ␤-globin locus beginning further
3⬘ to the ␥-globin gene than the HPFH deletion, in the ␥-␦
intergenic region, and extending 3⬘ to delete the ␦- and ␤-globin
genes, result in a lesser increase in ␥-globin than in HPFH in a
syndrome known as ␦-␤ thalassemia (Figure 2; Table 1).1,2,6
Clinically, patients with ␦-␤ thalassemia have increased ␥-globin
and HbF and some anemia and are intermediate in severity between
those with homozygous ␤ thalassemia and HPFH (Table 1).
Patients homozygous for ␤ thalassemia, usually due to point
mutations in the ␤-globin gene leading to either reduced (␤⫹) or
absent (␤0) ␤-globin production have much less ␥-globin compensation, more excess ␣-globin accumulation, and more severe
anemia requiring transfusions than patients homozygous for ␦-␤
thalassemia (Table 1).1,6,13 Patients with homozygous ␦-␤ thalassemia often do not require blood transfusions and are referred to as
patients with thalassemia intermedia. Homozygotes with hemoglobin Lepore have high levels of HbF as well and are of interest.6
However, the decreased expression of the Lepore (fusion ␦-␤)
gene, and the unknown effects of the loss of intergenic sequences
between the normal ␦ and ␤ genes in this condition, make it
difficult to interpret the cause of the increased HbF in these cases.
The differences between the clinical syndromes of ␤ thalassemia on the one hand, and of ␦-␤ thalassemia and HPFH on the
other, associated with different levels of ␥-globin and HbF
compensation as well as differences in the extent of the deletions in
these latter conditions (Figure 2), has long suggested a role for
DNA sequences between the human ␥- and ␦-globin genes
(intergenic ␥-␦ sequences) in ␥-globin gene expression in these
disorders and in normal human globin switching.1,102 However,
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BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
because of the lack of data from suitable homozygotes, and the
negative results of some transgenic mouse experiments, such a role
for these intergenic ␥-␦ sequences has been in question.2,85 In
addition, the enhancer activity from sequences 3⬘ to the HPFH
deletion alluded to earlier provided an alternative explanation for
the increased HbF in HPFH.2
The Corfu deletion
The recent finding that 2 patients homozygous for the Corfu
deletion (Figure 2) have 88% and 90% HbF, only mild anemia, and
no transfusion requirement provide the first strong evidence in
humans that intergenic ␥-␦ sequences alone can regulate the
activity of HbF in adult-type cells, and perhaps play a role in
normal human hemoglobin switching as well.97 The 7.2-kb Corfu
deletion extends from the ␥-␦ region upstream of the ␦ gene to
involve the 5⬘ end of the structural ␦-globin gene (Figure 2).
Using erythroid cell cultures from patients homozygous for the
Corfu deletion, the authors show that nascent ␥-globin gene
transcripts are greatly increased at each ␥ locus, as compared to
similar cultures from healthy subjects.97 In addition, ␥-globin
mRNA and ␥-globin expression are also extremely high in the
Corfu homozygotes. These results indicate that the Corfu deletion
alone can almost completely reactivate ␥-globin expression in
adult-type cells.97 It is unclear whether the persistence of human
␥-globin and HbF in patients with the Corfu, ␦-␤ thalassemia, or
HPFH deletions require the affected cells to undergo normal
developmental stage-specific switching in late fetal life, and
whether the same deletions occurring later in adult-type cells have
the same effects. Early chromosomal events at the human ␤-globin
locus in late fetal life may normally silence normal ␥-globin gene
expression and limit the extent to which ␥-globin reactivation can
occur in adult human hematopoietic stem cells and their progeny.
Single adult erythroid cells produce both ␥- and ␤-globins, and
adult erythroid cells continue to express some ␥-globin.2 However,
the data from patients with HPFH, ␦-␤ thalassemia, and Corfu
deletion indicate that developmental ␥ silencing, to the extent that
it occurs, can be overcome by the deletion of the sequences
included in these syndromes.
These data also suggest that the intergenic ␥-␦ sequences are
among the critical determinants of the process of human ␥-globin
gene reactivation, and perhaps of normal human ␥- to ␤-globin
switching as well. Although the human ␤LCR and structural gene
sequences are required for high-level globin expression of both the
human ␥- and ␤-globin genes at the ␤-globin locus, changes in
these sequences are not required for continued high-level ␥-globin
gene expression or ␥-globin reactivation in adult-type erythroid cells.
It is possible that the ␥-␦ intergenic region deleted in Corfu
patients contains sequences that both repress and can potentially
activate ␥-globin gene transcription. The presence of both positiveacting and negative-acting sequences and their interactions in
chromatin might explain the differences in ␥-globin gene expression with different extents of deletion of this region, for example, in
different patients with ␦-␤ thalassemia and in transgenic animals.83,103 The previously described and characterized unique RNA
transcripts from the intergenic ␥-␦ region sequences postulated to
play a role in regulating normal human ␥-globin gene expression,93
are disrupted in patients with the Corfu mutation.97 The molecular
mechanisms by which loss of these sequences might affect the
output of the ␥-globin genes is unknown.
HUMAN FETAL HEMOGLOBIN REGULATION
439
Transacting factors in transcriptional
regulation of ␥-globin gene expression
Although interactions between the ␤LCR and downstream globin
genes mediated by transcription factors such as GATA-1, FOG-1,
and EKLF have been documented to occur at the ␤-globin
locus,22,35 these interactions by themselves do not provide clues to
human ␥- to ␤-globin switching because all of these components
are present in both embryonic-fetal and adult cells.34 Normal
human ␥- to ␤-globin switching probably requires developmental
stage-specific changes in transcription factors or chromatin remodeling activities (or both) that lead to either repression of ␥-globin
gene expression or activation of ␤-globin genes (or both).
Whereas GATA-1, FOG-1, and NF-E2 are not differentially
active in fetal versus adult human cells, EKLF is known to be
active predominantly in adult-stage cells, and specifically enhances
human ␤- globin and not ␥-globin gene expression.34 EKLFdeficient transgenic mice do not form the ␤ACH or activate human
␤-globin gene expression.35 In addition, although EKLF interacts
with CBP/p300 and SWI/SNF proteins presumably to activate
murine ␤-globin gene transcription in adult-stage MEL cells,57,104 it
also interacts with Sin3A and HDAC1 corepressors via its zinc
finger domain in K562 embryonic-fetal cells.70 A key lysine that is
both a substrate for CBP acetylation and required for Sin3A
interaction has been identified in these studies.70 Although these
results remain to be confirmed in vivo, the acetylation status of
EKLF may determine its effects at different stages of human globin
switching.70 Fetal Krupple-like factors, FKLF and FKLF-2, have
been described.105,106 FKLF preferentially increases human embryonic-fetal globin gene expression in the human embryonic fetal cell
line, K562.105
Two protein complexes have been described that interact with
␥-globin promoter elements. The direct repeat erythroid-definitive
(DRED) complex contains the nuclear orphan receptors TR2 and
TR4 and binds with high affinity to DR1 (direct repeat) sites in the
human ⑀- and ␥-globin promoters.107,108 The human adult ␤-globin
gene has no DR1-binding sites, and this suggests that ␥-globin
repression in adult cells may be the major function of DRED. This
repression by DRED is supported by the fact that a point mutation
at ⫺117 of the A␥ promoter associated with high HbF disrupts a
DR1-binding site.
Another protein complex binding to the human ␥-globin
promoter region is the stage selector protein (SSP) complex.109,110
This complex has recently been shown to have a unique human
p22NF-E4 subunit as its erythroid-specific component111 and to
have a role in globin switching.43,44 Enforced expression of human
p22NF-E4 increases human ␥-globin expression in K562 cells and
delays human ␥- to ␤-globin switching in transgenic mice with a
human ␤-globin YAC.44 In addition, site-specific acetylation of
p22NF-E4 prevents its ubiquitination and reduces the interaction of
p22NF-E4 with HDAC1.43
PYR complex: a possible switch complex
Our group has described a chromatin remodeling complex called
PYR complex found only in adult hematopoietic cells whose
presence suggests the importance of human intergenic ␥-␦- globin
sequences in human ␥ to ␤ switching (Figure 3).112 PYR complex
binds to a 250-bp polypyrimidine (PYR)–rich DNA sequence 1 kb
upstream of the human ␦-globin gene, and the PYR complex
DNA-binding site is included in the Corfu deletion (Figure 2).112
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BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
HDACs in the PYR complex from repressing ␥-globin gene
expression (Figure 3). This hypothesis remains to be directly
studied and confirmed.
A model of human ␥- to ␤-globin switching
Figure 3. The proposed role of the PYR complex in human hemoglobin
switching. The circles indicate unspecified chromatin remodeling complexes and
transcription factors. The details of these interactions in chromatin between the ␤LCR
elements, globin structural genes, erythroid transcription factors, and these chromatin remodeling complexes are unknown. In fetal-embryonic cells, the human ␤LCR is
associated with the ␥-globin gene loci downstream. The blue circles include the
potential activities of FKLF and SSP in this process at the ␥-globin promoter. In
adult-type cells, the ␤LCR associates with and activates ␤-globin gene expression.
New interactions leading to repression of ␥-globin gene expression occur, and PYR
complex binding and its HDACs may contribute to this process. The SWI/SNF
complex subunits, the NURD subunits, and the DNA-binding subunit Ikaros of the
PYR complex are shown.
Deletion of 511 bp of DNA including the PYR binding site
upstream of the human ␦-globin gene delays human ␥ to ␤
switching in mice transgenic for a human ␤-globin locuscontaining cosmid.59,113
More recently, we have shown that the transcription factor
Ikaros, predominantly expressed in adult hematopoietic cells, is the
DNA-binding subunit of the PYR complex.60 Mice that do not
express Ikaros (Ik⫺/⫺) have no PYR complex binding activity.
Ik⫺/⫺ mice with a transgene carrying human ␥- to ␤-globin
sequences have delayed human ␥- to ␤-globin switching as well.114
Taken together, the data suggest that PYR complex has a role in
normal human ␥ to ␤ switching, and that perhaps inhibition of PYR
complex action, for example, by siRNA inhibition of Ikaros
activity, may be an additional therapeutic approach to reactivation
of ␥-globin in adult erythroid cells.
The PYR complex has been isolated from MEL cells and
characterized biochemically.59,60 It is a single complex with Ikaros
as its DNA-binding subunit and contains both positive-acting
protein subunits of the SWI/SNF complex that activate gene
transcription and repressive protein subunits of the NURD complex, an HDAC-containing complex that can repress transcription
(Figure 3).60
Butyrate compounds, known inhibitors of HDAC action,61,69,115,116 increase HbF in patients with SS disease and ␤
thalassemia.17,117,118 Butyrate and other HDAC inhibitors may
function to increase HbF production in humans by preventing the
As indicated earlier, a unique chromosomal domain, the ␤ACH, is
present in all erythroid cells at the human ␤-globin locus establishing a loop-stem structure in the region (Figure 3).20,21 In fetal life,
the ␥-globin genes in chromatin associate with the ␤LCR, ubiquitous, and erythroid-specific and fetal stage-specific transcription
factors, and with putative embryonic-fetal stage-specific chromatin
remodeling complexes or activities including the potential effects
of SSP and FKLF, and favor ␥-globin transcription (Figure 3).20,21
In adult-type erythroid cells, the ␤ACH configuration is changed so
that the ␤LCR now preferentially associates with and activates
human ␤-globin gene expression (Figure 3).20,21 One event in this
process in vivo for inducing conformational changes at the
␤-globin locus in adult-type cells may be the adult stage-specific
formation and action of the PYR complex, by binding to the
intergenic ␥-␦ sequences at the PYR-binding site and repressing
␥-globin transcription by its HDACs (Figure 3). The SWI/SNF
subunits of PYR complex may be involved as well. All of these
proposed roles for PYR complex must await confirmation by
further studies of the role of Ikaros and the PYR complex similar to
those recently described.20,21 In addition, DRED and other currently unknown interactions of ubiquitous and adult stage-specific
transcription factors and chromatin remodeling complexes are
most likely involved in this process, as well as GATA-I, EKLF,
and NF-E2.
Therapeutic approaches to reactivation of
␥-globin and HbF in adult erythroid cells
Expression of HbF can be controlled at the cellular level by the
increased production of cells synthesizing large amounts of HbF by
erythropoietin, other cytokines, changes in cell cycle kinetics, and
other unknown factors.2,119,120 In situations of “stress erythropoiesis” HbF is produced preferentially by early erythroid progenitors
and this can lead to increased HbF levels.2,119,120 However, even
with severe stress, the number of HbF-producing clones and their
output is limited and does not compensate for the deficit or lack of
HbA-producing cells in disorders such as human homozygous ␤
thalassemia.2,119,120
Intracellularly, human ␥-globin gene expression during development and in reactivation in adult-type cells can be controlled at the
levels of posttranscriptional processing of ␥ mRNA transcripts,
mRNA stability, translation, and posttranslational events, as well as
at the level of transcription. In transgenic mice, the relative
half-lives of human ␥- and ␤-globin gene transcripts are similar.19
Agents that increase human HbF in patients may work at one or
more levels.115,118,121 For example, hydroxyurea and 5-azacytidine
kill dividing cells preferentially and may increase ␥-globin expression indirectly through this effect.115,118,122 5-Azacytidine is also a
demethylating agent and may reactivate ␥ expression by this
mechanism as well.118,122 Butyrate may work both by HDAC
inhibition and by increasing ␥-globin translation on ribosomes.121
The combination of erythropoietin and hydroxyurea has synergistic
effects in increasing HbF in some patients.115,118,123
Several important signal transduction pathways have been
shown to be associated with increases in ␥-globin in K562 cells and
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BLOOD, 15 JANUARY 2006 䡠 VOLUME 107, NUMBER 2
primary human CD34⫹ cells, and modified by hydroxyurea and
butyrate therapy.124-128 This signaling includes (1) modulation of
soluble guanylate cyclase and of cyclic AMP and GMP124,128; (2)
increased nitric oxide production126; (3) changes in protein kinases
modulated by stem cell factor127; and (4) inhibition of Stat3
phosphorylation.125 New therapies to increase HbF levels in
humans may result from exploiting these observations.
Inhibition of DRED complex activity, or, as mentioned
earlier, of Ikaros and PYR complex action by siRNAs or other
small molecules in adult erythroid cells may add to the
therapeutic regimen for increasing HbF. Similarly, increasing
the expression of proteins such as NF-E4, and Id2 and of other
proteins shown to be up-regulated with increased HbF may be
useful as well.44,129
Last, there appears to be an inverse relationship between human
␥- and ␤-globin accumulation in adult erythroid cells.97 In patients
doubly heterozygous for Corfu and ␤ thalassemia genes or
heterozygotes for Corfu alone when there is more ␤-globin and
HbA present, there are less than expected levels of ␥-globin and
HbF accumulation.97 In contrast, this inverse relationship is not
seen when measuring primary globin transcripts in the erythroid
cells of these patients.97 Primary transcripts from a single Corfu
locus in patients doubly heterozygous for Corfu and ␤ thalassemia
genes or Corfu heterozygotes (with one Corfu locus) are shown to
be expressed, as expected, at about half the level of ␥ transcripts of
the Corfu homozygotes (with 2 Corfu loci). These data indicate
that posttranscriptional events may determine the limits to
which ␥-globin production and HbF can increase in adult human
cells producing ␤-globin and HbA.97 This effect may be due to
the preferential stabilization of human ␤ mRNA over ␥ mRNA
mediated by differential binding to limiting amounts of ribonucleoprotein complexes.97,130 Thus, down-regulating human
␤-globin expression may be a useful additional approach to
optimizing HbF production in patients being treated with
HbF-inducing agents.
HUMAN FETAL HEMOGLOBIN REGULATION
441
Summary
Although many of the events controlling the activity of the
␤-globin locus are known, the details of those regulating normal
human hemoglobin switching and reactivation of HbF in adult
hematopoietic cells remain to be elucidated. If the molecular events
in hemoglobin switching or ␥-globin gene reactivation were better
understood and HbF could be fully reactivated in adult cells, the
insights obtained might lead to a cure for these disorders. An active
chromatin configuration with interactions between the ␤LCR and
downstream genes by looping of chromatin fibers has been
demonstrated in all erythroid cells studied. These interactions are
modulated by transcription factors and chromatin remodeling
activities. In human fetal cells, the ␥-globin gene is preferentially
activated by these interactions, and ␥-globin and HbF predominate.
In adult-stage cells, these interactions result in the ␤LCR enhancing human ␤-globin gene expression by associating specifically
with sequences at the ␤-globin gene instead of the ␥-globin gene.
The potential role of intergenic ␥-␦ sequences in this process has
recently been strengthened by studies in patients homozygous for
the Corfu deletion who have very high HbF levels, only mild
anemia, and no transfusion requirements. In addition, an adult
stage-specific hematopoietic cell-specific chromatin remodeling
complex, PYR complex, may be involved in switching, and
suggests new approaches to reactivating HbF such as preventing
Ikaros action by siRNAs in adult erythroid cells. Similarly,
increased activity of FKLF and SSP, and repression of DRED
complex, may increase ␥-globin and HbF in adult erythroid cells as
well. Identification and characterization of other chromatin remodeling complexes and their interactions with transcription factors,
the ␤LCR, and downstream sequences in chromatin should provide
further details of the dynamic processes of ␥-globin gene regulation and human hemoglobin switching and will hopefully lead to
new combination therapies to enhance fetal hemoglobin expression
in adult erythroid cells.
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From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
2006 107: 435-443
doi:10.1182/blood-2005-05-2113 originally published online
August 18, 2005
Regulation of human fetal hemoglobin: new players, new complexities
Arthur Bank
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