The Pharmacology of Hemoglobin Switching: Of Mice and Men

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The Pharmacology of Hemoglobin Switching: Of Mice and Men
By Timothy J. Ley
OW D O RED BLOOD CELLS (RBCs) switch from
fetal to adult-type hemoglobin (Hb)production at
the time of birth? The structures of fetal and adult Hbs and
the genes that encode them have been known for many
years, but this knowledge has not readily yielded the
molecular mechanisms underlying the switch. The mysterious switch is a transcriptional one: the fetal (y) globin genes
are actively transcribed in fetal RBCs, but in adult RBCs,
the P-globin genes are transcribed at a much higher rate
than the y-genes.’
Since the discovery of fetal Hbs, investigators have
proposed that reactivation of fetal Hb (HbF) synthesis
would lessen the severity of diseases caused by P-globin
gene mutations. These hopes were further strengthened
when normal individuals with large amounts of circulating
HbF were identified; these people have “Hereditary Persistance of Fetal Hemoglobin” and no medical problems.’
Preventing or reversing the switch from normal HbF
synthesis to defective adult Hb synthesis could, therefore,
be a therapeutic goal, because high HbF levels do not cause
problems in otherwise normal individuals. But how could
this goal be achieved? Remarkable studies over the past
decade have taken us closer to an answer.
Shortly after the human P-like globin genes were cloned
and sequenced, van der Ploeg and Flavell correlated the
methylation status of DNA sequences near the globin genes
and the activities of these genes in different tissues.’ The
human y-globin genes were undermethylated in tissues
where they were normally expressed (like fetal liver), but
were heavily methylated in adult erythroid cells or in
non-erythroid tissues. This observation led to the formation of a simple hypothesis: if one could cause DNA near
the y-globin genes of adult bone marrow cells to become
hypomethylated, these genes might resume transcription
and HbF production would increase. Joe DeSimone and
Paul Heller seized upon this idea and decided to test it in a
primate model of Hb switching that they had developed.
They knew that the basic structures of the fetal and adult
globin genes of baboons and humans were similar, and
From the Division of Hematology-Oncology,Departments of Medicine and Genetics, Jewish Hospital at Washington [email protected]
Center, St Louis,MO.
Submitted December 20, 1990; accepted December 21,1990.
Suppored by National Institutes of Health Grants DK38682 and
Address reprint requests to Timothy J. Ley, MD, Division of
Hematology-Oncology,Departments of Medicine and Genetics, Jewish
Hospital at Washington University Medical Center, 216 S Kingshighway Blvd, St Louis,MO 63110.
0 1991 by The American Society of Hematology.
proposed that the methylation hypothesis could be directly
tested in this model.
In the late 1970s and early 1980s, a number of investigators had shown that the drug 5-azacytidine could block the
activity of the enzyme that normally methylates newly
synthesized DNA, causing the DNA in dividing cells to
become hypomethylated? Treatment of tissue culture cells
with this drug sometimes activated specific genes and/or
specific developmental “programs.” Throughout the 1970s,
5-azacytidine had also been tested in phase I trials of
patients with acute non-lymphocyticleukemia. The pharmacology and toxicities of 5-azacytidine were, therefore, well
understood in 1981, when this drug was first administered
to baboons. In the first experiments with the drug, 5-azacytidine caused tremendous elevations of HbF levels in the
phlebotimized baboon^.^ The precise mechanism of y-globin
gene activation could not be rapidly determined in baboons,
because the structures of all the globin genes was not yet
In collaboration with DeSimone and Heller, Art Nienhuis and I used the information from the baboon trials to
design treatment protocols for human patients with severe
sickle cell anemia or p-thalassemia; George Dover and Sam
Charache independently treated a group of sickle cell
patients at Johns Hopkins at the same time. To our delight,
relatively non-toxic doses of 5-azacytidine dramatically
increased HbF synthesis in almost all of the individuals who
were tested?.’ The drug indeed caused hypomethylation of
the newly synthesized DNA found in bone marrow cells.5s6
Despite evidence for widespread DNA hypomethylation,
only a restricted cohort of genes (including the y-globin
genes) seemed to be activated by the treatment.’ These
results suggested that either the y-globin genes were
somehow primed to be reactivated in adult bone marrow
cells, or that other mechanisms were partially responsible
for the augmented HbF synthesis.
Observations made in the 1970s by Alter et aI8suggested
that erythroid regeneration was associated with an altered
“program” of HbF synthesis in erythroid progenitor and
precursor cells. Because 5-azacytidine is a cytotoxic compound, Nathan, Stamatoyannopoulos, Papayannopoulou,
and their colleagues argued that cytotoxicity and erythroid
regeneration may have caused 5-azacytidine’s effects on
HbF production: and suggested that alternate cytotoxic
drugs (that did not directly inhibit DNA methylation)
would have the same effects. Indeed, hydroxyurea and
AraC were both shown to increase HbF production in
primates and in humans.’”” “Demethylation” of newly
synthesized DNA was, therefore, not necessary to augment
HbF production. However, in the final analysis, azacytidine
still seems to work by both mechanisms: first, it causes
newly synthesized bone marrow DNA to become hypomethylated; and, secondly, it is cytotoxi~.’~~’~
B/ood,Vol77,No6(March15),1991: pp1146-1152
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These early experiments with pharmacologic “reverse
switching” demonstrated that the switch was “plastic” and
could be manipulated. However, all of the drugs used to
manipulate the switch were cytotoxic, anti-leukemia drugs
with uncertain potentials for causing secondary malignancies if administered over long periods of time. Of the three
drugs tested, hydroxyurea had the least clearcut potential
for causing harm, and already had a proven safety record in
patients with myeloproliferative diseases. For this reason,
clinical trials with hydroxyurea have been initiated in
patients with sickle cell anemia,17s18
and a phase I11 trial is
currently being considered to test its ability to reduce crisis
frequency. Nonetheless, long term administration of cytotoxic compounds will never be an ideal therapeutic option
for patients with hemoglobinopathies. The ideal pharmacologic compound would obviously have little or no toxicity
and would be safe to administer to children during periods
of normal growth and development. The search for such a
compound has continued to demonstrate the usefulness of
large animal models to identify drugs that can alter patterns
of globin synthesis in humans.
Ginder et all9first demonstrated that embryonic chicken
globin genes could be reactivated by administering 5-azacytidine and butyrate compounds to adult chickens. Both
drugs were required for the effect, and a “two-hit’’ model
for embryonic globin gene reactivation was proposed. In
subsequent years, Perrine et almand Bard and Prosmanne”
demonstrated that butyrate metabolites were persistently
elevated in the infants of diabetic mothers, who undergo
delayed HbF switching after birth. Butyrate compounds
were subsequently shown to delay the fetal to adult globin
switch in developing sheep;’ and to augment HbF production in baboons.= Butyrate compounds have not yet been
tested in humans, because safety issues have not yet been
Hematopoietic growth factors can also alter patterns of
Hb synthesis. Because erythropoietin administration is
known to cause a form of erythropoietic “stress,” Al-khatti
et alZ4tested this drug in baboons and detected augmentation of fetal globin synthesis. However, the effect of
erythropoietin on HbF production in sickle cell patients has
been minimal thus far.” Finally, granulocyte-macrophage
colony-stimulatingfactor (GM-CSF) augments HbF production in cultured erythroid progenitors? but its effects on
HbF have not yet been tested in animal models.
Although almost all of the work regarding pharmacologic
manipulation of HbF has been developed in primate or
large animal models, these models remain extremely cumbersome and expensive to use. They are not well suited for
surveys of compounds, and because of differences between
the globin gene clusters of higher primates and humans, not
all of the results can be directly extrapolated to humans. A
better animal model for testing pharmacologic agents is
clearly required; in this issue of Blood, Constantoulakis et
aIz6describe a transgenic mouse model that fulfills many of
these requirements. To understand this model, a brief
review of human globin gene regulation in transgenic mice
is useful.
Early attempts to study the expression of human globin
genes in transgenic mice were at times frustrating, but
ultimately, these studies proved to be very informative.
When an entire human P-globin gene was integrated into
the germline of transgenic mice, only a small proportion of
the founder animals expressed the $-globin gene27*28;
usually, the P-globin transgene was expressed at much lower
levels than the endogenous mouse $-major globin genes.
Even so, human $-globin gene sequences targeted expression exclusively to adult erythroid tissues; that is, the DNA
sequences required for expression in RBCs at the correct
stage of development were present within or near the
P-globin gene.27,28
Experiments performed by three separate
groups, including Kollias et al,29Behringer et al,M and
Trudel et a1,3’ demonstrated that the sequences required
for adult erythroid-specific expression were located within
and immediately 3’ to the P-globin gene.
When human y-globin genes were tested in transgenic
mice, a different pattern of expression emerged. When
these studies were performed, there was only one recognized murine Hb switch from the embryonic stage (yolk
sac) to the adult stage (fetal liver and adult bone marrow).
Mice were thought to have embryonic $-like globin genes
and adult P-globin genes, but nothing corresponding exactly to the y-globin genes of humans. More recent studies
suggest that there may in fact be embryonic-to-fetal and
fetal-to-adult switches in the mouse”; regardless, the developmental fate of y-globin transgenes was difficult to predict. To the surprise of most workers in the field, human
y-globin transgenes were expressed only at the yolk sac
(embryonic) stage of de~elopment!~~.”
This finding suggested that the embryonic stage is more akin to the fetal
stage of erythroid development in humans, and suggested
that “fetal” stage-specific factors were present in the
embryonic RBCs of developing mice. In contrast to the
human p-globin gene, y-globin sequences required for
targeting to embryonic RBCs are located upstream from
the y-gene~?’.”’~ However, transgenic mice containing
y-globin transgenes behaved similarly to the P-globin transgenics in two regards: first, only a small proportion of the
transgenic mice expressed the y-globin transgenes, and,
secondly, the level of expression was much lower than that
of the embryonic mouse globin gene^."*'^-^^ Certainly all of
the early transgenic studies suggested that sequences within
or near the y- or P-globin genes were responsible for
targeting the expression of these genes to the appropriate
tissue at the appropriate developmental stage. These experiments also suggested that additional DNA sequences
would be required for high level expression of human globin
genes in the erythroid cells of most transgenic mice.
The first clues regarding the nature of these sequences
came from the studies of Tuan et a136and Forrester et al?’
who discovered a series of six DNAse I hypersensitive sites
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located far upstream and far downstream from the human
p-like globin gene cluster (see Fig 1). These DNAse I
hypersensitive sites were erythroid-specific but developmentally stable; that is, these regions of chromatin were “open”
and DNAse I hypersensitive at all stages of erythroid
development. Forrester et aI3*went on to show that these
DNAse I hypersensitive sites were reconstituted when a
non-erythroid human chromosome 11 was transferred into
the mouse erythroleukemia cell environment, suggesting
that the locus itself contains all of the information required
to form an active chromatin domain. They wined the term
“locus activation region” (LAR)to describe this function.
More recently, Dhar et a139have shown that several of these
sites are hypersensitive in non-erythroid and even nonhematopoietic cell lines; only 5’ HS-3 (see Fig 1) is DNAse
I hypersensitive only in erythroid cell lines.
At about the same time, Grosveld et a1 decided to
determine whether DNA sequences derived from this
upstream region contained the “missing” information necessary for high level expression of human globin genes in
transgenic mice. To their surprise, every transgenic founder
line containing a 6-globin gene linked with the far upstream
region expressed its transgene at a level that was similar to
that of the endogenous mouse p-major genes.39The number
of copies of the transgene in the mouse genome was directly
correlated with the level of transgene expression. Furthermore, expression of the transgenes did not seem to be
dependent on the position of transgene integration. The
function of the p-globin transgenes was therefore said to be
“position independent” and “copy-number dependent.”
Because of the powerful effects of the upstream region,
Grosveld called these sequences the dominant cmtrol
region (DCR). (Conferees at the 7th Conference on Hemoglobin Switching agreed upon the consensus name “Locus
Control Region” [LCR] for this region, and further agreed
that the individual DNAse I hypersensitive sites [HS] would
be named 5‘ HS-1,2,3,4, and 5 upstream from the €-globin
gene, and 3’ HS-1 downstream from the p-globin gene, as
indicated in Fig 1).
Because of the powerful and interesting activities of the
LCR in transgenic mice, a number of laboratories rapidly
confirmed and extended these early r e s ~ l f s . ”The
~ ~ ~LCR
was shown to activate a-globin gene expression in transgenic mice,”.” suggesting that the a-globin locus on chromosome 16 might also be under the influence of similar
sequences (see below). Co-injection of ci- and p-globin
5’HS:5 4 3
genes with the LCR has resulted in mice making large
quantities of human Hb44345;
mice producing HbS have been
made by coinjecting a- and p‘-globin genes with the
The importance of the LCR for P-globin gene function in
vivo has also been recently established. Driscoll et ala have
identified a 30-kb deletion that removes 5‘ sites 2-5 in an
Hispanic patient with ySp thalassemia (Fig 1). The entire
p-globin cluster on this chromosome is intact, but none of
the genes in the cluster are functional. Forrester et a149have
recently shown that this deletion closes the normally open
chromatin “domain” of the p-globin gene cluster in erythroid cells, and that it delays the replication of the p-like
genes during the cell cycle.
Because the region containing the LCR sequences spans
a large stretch of DNA, investigators immediately tried to
identify the critical functional sequences within this region.
Initial studies focused on the DNAse I hypersensitive sites
themselves. Accordingly, DNA sequences between the
hypersensitive sites were removed in successive deletions
creating “mini-LCR” and “micro-LCR”
seemed to retain all of the detectable LCR functions in
studies performed with transgenic mice or mouse erythroleukemia cells. DNA sequences near 5‘ HS-2 and -3 have
most of the activity in these
but 5‘ HS-1 and -4
also seem to have more subtle functions. The combination
of all four 5‘ sites is required apparently for full function of
the LCR in transgenic mice.”1353
How does the LCR normally function in erythroid
precursors? The answer to this question is not yet known,
but intense investigations in a number of laboratories have
begun to find a number of clues. First, when LCR sequences are integrated into genomic DNA, they seem to be
able to open a chromatin domain and keep it open, isolating
sequences within the domain from potentially negative
influences nearby. The LCR, therefore, seems to increase
the number of “productive integration events”; hence,
nearly every transgenic mouse containing a P-globin gene
linked to an LCR expresses the transgene. Secondly, the
LCR seems to have classical enhancer function that is
relatively specific for erythroleukemia cells. Tuan et
were the first to demonstrate this enhancer effect with 5’
HS-2; these results have been confirmed and extended by
other groups within the past year.55s56
The LCR also seems
to make a variety of globin and non-globin promoters
inducible in erythroleukemia cells;@59suggesting that at
3’HS: I
-21.4 -16-14.7 -10.9 -6.1
Hispanic Y&BThal,
Fig 1. Map of the human p-globin gene cluster. The locations and names of the 5’ and 3’ HS are shown. The numbers above the arrows refer to
the distances (in kilobases)of the 5’ sites upstreamfrom the €-globingene, or of the 3’ site downstreamfrom the p-globin gene. The 3’ breakpoint
of the.Hispanlc ySp thalassemia is shown; this deletion removes -30 kb of DNAfrom this region.” 5‘ HS 1 through 4 are now called the LCR.”
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least some of this inducibility function lies within the LCR
The precise molecular mechanisms that determine
these effects are not yet understood in detail. However, Ney
et al,55*60
Talbot et a1,61 and Moi and Kad6 have suggested
that sequences near a directly repeated AP-l/NF-E2 site
within 5‘ HS-2 are absolutely required for several of the
functions listed above, and that specific transcription factors bind to these sites and influence the function of the
LCR sequences. A region with different primary DNA
motifs appears to be responsible for the activity of 5‘ HS-367;
perhaps different transcription factors may regulate the
function of this site.
Additional proof of the importance of 5’ HS-2 sequences
has recently come from experiments that detected evolutionary conservation of the critical regions in this site. Because
mice have the ability to recognize human LCR sequences,
Anne Moon and 163 reasoned that mice might also have
LCRs. Indeed, 5’ HS-1 and -2 are highly conserved upstream from the mouse P-globin cluster. The region within
5’ HS-2 known to be important for the function of this site is
absolutely conserved between mice and humans, even
though these sequences do not encode proteins. This
remarkable degree of conservation is similarly preserved in
the goat: Li et a1 have simultaneously demonstrated that 5‘
HS-1, -2, and -3 are all conserved in location and position
upstream from the goat @-likeglobin gene cluster.” Reports of evolutionary conservation of LCR structure and
function are beginning to emerge in other animal species as
Finally, Higgs et [email protected]’have now shown that the a-globin
gene cluster also has a locus control region located some 30
kb upstream from the t;-globin gene in a DNAse 1hypersensitive site that contains several small motifs related to those
found in the P-LCR. The importance of this upstream
region for function of the a-globin gene cluster has been
established in vivo. Hatton et a167and Liebhaber et alS have
described deletions that remove the a-LCR region but
leave the a-globin genes intact. The a-like globin genes on
both of the chromosomes bearing these deletions are
With the discovery of the LCR, investigators in several
laboratories added LCR sequences to the E-, y-, and
P-globin genes, and repeated the earlier transgenic experiments (see Table 1). Surprisingly, the LCR seemed to
disrupt the “normal” developmental expression of the
individual y- and @-globingenes in transgenic mice. For
example, while the y-globin gene alone is expressed only in
embryonic RBCs, y-globin genes linked with the LCR are
expressed in both embryonic and adult RBCS!~~.~’
p-globin gene alone is normally expressed only in adult
RBCs; the @-globin gene attached to the LCR is also
expressed in both embryonic and adult RBCS.~’In contrast,
the human €-globin gene is not expressed in any mouse
tissue when injected alone; €-globin genes plus the LCR are
expressed in embryonic but not adult RBCS.’~,” The €-globin
gene is, therefore, “autonomously” regulated in the presence of the LCR, but the y- and p-genes are not.
Table 1. DevelopmentalRegulation of Human p-Like Globin Gene6 in
Transgenic Mice
p + LCR
Y+# BY+
7 - 8
Simplified summary of human p-like globin gene activities in transgenic mice. + and - symbols indicate the presence or absence of
transgene activity in the indicated erythroid tissues. ”Embryonic“
erythroid cells are yolk-sac derived (day 9 to 10); ”adult“ erythroid cells
may be derived from late gestational stage fetal livers (day 12 to 14).
adult bone marrow, or adult reticulocytes.No attempt has been made in
this table to indicate the relative activities of the transgenes. In general,
constructions containing the LCR are expressed at high levels in all
transgenic mice. Modifiedwith permi~sion?~
To address this quandary of developmental disregulation
with the LCR, Behringer et a17’ and Enver et aln both
decided to add the LCR to the intact ySp region of the
p-globin cluster and repeat the experiments. Both groups
demonstrated that appropriate y + P switching did occur in
this context. As shown in Table 1, the y-globin gene was
expressed at high levels in embryonic RBCs and the P-gene
was not. In contrast, the y-gene was “silenced” in adult
erythroid tissues, and the P-gene was fully active. These
experiments suggested a competitive model of regulation
for the y- and P-globin genes in transgenic mice; this model
gained further credibility in an additional experiment in
which the P-globin gene was deleted from the large ySp
fragment.m With this construction, the y-gene was again
inappropriately expressed in adult erythroid tissues. A
model where the y- and P-globin genes compete for an
LCR function best explains these results, but more recent
experiments from Fraser, Grosveld, and colleagues suggest
that the y-globin gene can, in fact, be silenced “autonomously” in adult erythroid tissues if additional 3’ y-globin
gene flanking sequences and 3’ HS-1 are added to the
transgenic construction^.^^ Further experiments will be
required to sort out the precise DNA sequences that cause
the differences between these models. Nonetheless, an
accurate recapitulation of human y- --* P-globin gene
switching has now been accomplished in transgenic mice,
opening the door for the testing of pharmacologic agents in
a mouse model.
In this issue of Blood, Constantoulakis et aIz present data
from the first small animal model designed to test the
effects of pharmacologic agents known to augment HbF
synthesis in humans. Three different kinds of transgenic
mice were tested. In the first, transgenic mice with the
y-globin gene alone (expressed only in embryonic RBCs)
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did not respond to any of the pharmacologic agents known
to increase human HbF synthesis. In the second group of
mice, a y-globin gene was linked with a “micro-LCR”
cassette. This y-globin gene is now “disregulated” and
expressed in embryonic and adult erythroid cells (see Table
1). However, when adult mice from this founder line were
treated with 5-azacytidine, hydroxyurea, butyrate, or erythropoietin, a striking increase in the number of HbFcontaining reticulocytes and in y-globin mRNA levels were
observed. These results are specific for the y-globin gene,
because a P-globin-LCR mouse did not respond to these
agents. These results seem to indicate that the y-globin
gene and the LCR sequences somehow interact with each
other to produce this effect,52259
and suggest that all of the
DNA sequences required for a response to these drugs
must lie within the “micro-LCR’ and/or the y-globin gene
itself. The y-LCR model for pharmacologic manipulation is
extremely powerful, but perhaps transgenic mice containing the entire y80-globin gene cluster plus the LCR will be
even more useful, because these animals have a more
“physiologic” switching mechanism in play. Regardless, a
small animal model that can detect the effects of drugs on
human y-globin gene activity is now ready for further
testing. The availability of such a model should greatly
enhance our ability to identify pharmacologic agents for the
treatment of patients with P-thalassemia or sickle cell
The author thanks Diana Coleman for preparing this manuscript.
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1991 77: 1146-1152
The pharmacology of hemoglobin switching: of mice and men
TJ Ley
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