1985 66: 1202-1207
A Oppenheim, Y Katzir, E Fibach, A Goldfarb and E Rachmilewitz correlates with gene activity of the beta-globin clusterHypomethylation of DNA derived from purified human erythroid cells
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1202 Blood, Vol 66. No 5 (November), 1985: pp 1202-1207
Hypomethylation of DNA Derived From Purified Human Erythroid Cells
Correlates With Gene Activity of the fl-Globin Cluster
By Ariella Oppenheim, VaeI Katzir, Eitan FibaCh, Ada Goldfarb, and Eliezer Rachmilewitz
Analysis of methylation at the $-globin gene cluster was
carried out on DNA derived from nucleated RBCs (ortho-
chromatic normoblasts) isolated from peripheral blood of
patients with /1-thalassemia major or other congenital
hemolytic anemia after splenectomy. A procedure to sepa-
rate these normoblasts from the other nucleated cells of
the peripheral blood was developed. providing us with a
convenient source of DNA for investigating parameters
related to human erythroid differentiation. Blood samples
were obtained from six adult patients who express their
‘y-globin genes at different levels. Inverse correlation
between methylation and gene activity was consistently
R EGULATION of gene expression in higher eukanyotes
is complex and is likely to occur at different hierarchies
and in several steps. Many investigations in recent years have
focused upon changes in chromatin conformation and DNA
methylation that occur during differentiation. The general
view is that activation of tissue-specific genes correlates with
increased sensitivity of the respective chromatin domain to
nucleases’ and with hypomethylation of the DNA.’
The /3-globin gene cluster in mammals and chickens was
one of the first to be studied. It was found that changes in
chromatin conformation and hypomethylation take place
during differentiation and in parallel with the switch from
embryonic to adult gene expression.’4 Methylation of DNA
in vertebrates occurs primarily at cytosine residues in CpG
dinucleotides. Although a causative effect has never been
established, it is generally accepted that globin genes are
hypomethylated in tissues where they are expressed.4
Studies on the human /3-globin gene cluster have been
limited, partly because of inavailability of purified pnepara-
tions of nucleated enythroid cells. In their pioneering work,
Van den Ploeg and Flavell5 have mapped 17 different CpG
sites near the ‘y, tS, and /3 genes that are amenable to analysis
of methylation by restriction endonucleases. The expenimen-
tal approach is based on availability ofenzyme pains that are
sensitive on insensitive to methylation at CpG dinucleotides
embedded in specific recognition sequences. Most of these
sites were fully methylated in DNA prepared from a variety
of tissues that do not express globin genes, such as brain,
sperm, and adult and fetal lymphocytes. Some hypomethyl-
ation was observed in the ‘y gene domain in fetal liver and
near the b and f3 genes in fetal liver and in adult bone marrow
From the Department of Hematology, Hadassah University
Hospital, Hebrew University Hadassah Medical School, Jerusa-
lem, Israel.
Supported in part by a grant from the United States-Israel
Binational Science Foundation and by the Gutwirt Family Founda-
tion.
Submitted Nov 7, 1984; accepted April 29, 1985.
Address reprint requests to Dr Ariella Oppenheim. Department
of Hematology. Hadassah Medical Center, Jerusalem, Israel,
91120.
< I 985 by Grune & Stratton, Inc.0006-4971/85/6605--O010$03.00/0
observed for five of the eight sites analyzed. A site 3’ to the
fi gene was always unmethylated. two sites flanking the e
gene were always found to be methylated. and two sites 5’
to the two ‘1 genes. G7 and A7. were hypomethylated in
correlation with .‘y gene activity of the individual patients. A
site 5’ to the � gene was unmethylated in normoblasts as
well as in WBC. No apparent relation between hypometh-
ylation and gene activity was observed for two additional
sites. The results suggest that methylation at specific
chromosomal locations participate in genetic regulation of
the ft-like globin genes in humans.
S 1985 by Grune & Stratton. Inc.
cells. The results were consistent with the notion that
hypomethylation of DNA may be a necessary but insuffi-
cient condition for globin gene expression in humans. How-
even, the hemopoietic tissues analyzed in this study-fetal
liver and adult bone marrow-were heterogeneous in cellular
composition, and did not permit unambiguous interpretation
of the results.
Recently, Mavilio et a16 studied changes in the state of
methylation at the /3-globin gene cluster during ontogeny in
humans. They purified erythroid cells from embryonic yolk
sac, fetal liven, and adult marrow cells. These purified
populations were not homogenous and contained cells at all
stages of erythnopoietic differentiation. An inverse cornela-
tion between methylation and expression of the specific
/3-like globin genes was observed. These authors suggested
that hypomethylation may be part of the mechanism regulat-
ing the globin switch from embryonic to fetal to adult.
Some of the transfusion-dependent patients with f3-thalas-
semia major produce ‘y-globin chains and fetal hemoglobin at
significant levels in adult life. Many of the patients undergo
splenectomy at an early age. Peripheral blood of splenectom-
ized patients is usually rich in immature erythnoid cells,
orthochromatic normoblasts, that are at the last develop-
mental stage before releasing their nuclei and becoming
neticulocytes. We have developed a simple method to sepa-
nate these normoblasts from the other nucleated cells present
in peripheral blood. Thus, we were able to obtain, for the first
time, populations of nucleated enythroid cells that were also
synchronized with respect to differentiation. As subjects, we
used adult patients with varying degrees of spontaneous
‘y-chain production. Our studies on the methylation pattern
showed an inverse correlation between methylation and gene
activity in most of the sites analyzed in the vicinity of the ,
A”y, Gy, t5 and /3 genes.
MATERIALS AND METHODS
Subjects. Patients were chosen for this study according to therate of synthesis of their ‘y globin chains and presence of normoblasts
in their peripheral blood. Healthy individuals were not included
because they lack normoblasts in their peripheral blood. W. D. is a
nonthalassemic patient with congenital hemolytic anemia that may
be due to RBC enzyme defect, and with a normal fl/a globin chain
synthetic ratio. The rest are patients with fJ-thalassemia major. They
are men and women ranging in age from I 3 to 33 years. Rates ofchain synthesis were determined in peripheral blood cells obtained
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METHYLATION OF GLOBIN GENES 1203
prior to transfusion, including normoblasts and reticulocytes, by
urea-carboxymethylcellulose chromatography of ‘4C-leucine-labeled stroma-free hemolysates.’ Some of these data, including
clinical information, have been published previously.8’9
Separation ofnormoblasts. Peripheral blood, 20 to 30 mL, waswithdrawn in hepanin prior to transfusions. As donor blood does notcontain normoblasts, the normoblasts present in the peripheral blood
of the patients are their own. Their level was from 10,000 to 50,000oells/�iL whole blood. The normoblasts were separated from the rest
of the nucleated cells as follows: Volex (starch 2-hydroxyethyl ether,American Hospital Supply Corporation, Evanston, Ill) was mixedwith total blood at a ratio of 1:8 and the mixture was allowed to
separate at 1 x g in plastic pipettes closed at their tips and heldvertically for one to two hours at room temperature. During this
time, two layers were formed. The lower layer contained RBC and
normoblasts, whereas the upper layer contained the WBCs as well as
some RBCs. Under these conditions, good separation was dependent
upon a low hematocrit value of the blood sample (20% to 30%) andwas usually achieved in 90 minutes. To avoid mixing, the upper layer
up to a few millimeters from the separation line was withdrawn fromthe top, the middle part was discarded, and the lower layer was
collected from the bottom. Smears were prepared from both frac-
tions, stained with benzidine and Giemsa, and scored for normo-
blasts and WBCs. In most cases, the nucleated cells in the upperfraction contained <5% normoblasts. Mature RBCs, that were also
present, did not interfere with subsequent procedures. In the lowerfraction, >95% of the nucleated cells were onthochromatic normo-
blasts. When separation was unsatisfactory, the process was repeat-ed. In those cases, the upper fraction was allowed to sediment again
as before. The lower fraction was mixed with saline to obtain a
“hematocrit” value of 20% to 25%, similar to the value of the
original blood. Volex was added as before, and the separation was
repeated. Preparations that were >95% pure were used for DNA
purification. For the methylation studies, the percentage of contami-
nating cells in each preparation is internally controlled, since site
MIS is unmodified in the normoblast DNA and is fully modified inWBC (see below).
DNA preparation. To eliminate the hemoglobin, which inter-feres with DNA purification, nuclei were isolated from both theupper and lower Volex fractions. The cells were suspended in 40 mLof reticulocyte standard buffer (RSB) (10 mmol/L ofTris-HCI, pH
7.4, 10 mmol/L of NaCI, 3 mmol/L of MgCI,), and NP4O was
added to a final concentration of 0.5%.b0 The nuclei were pelletedand washed in 0.5% NP4O in RSB. DNA was isolated by amodification of the procedure of Goossens and Kan)1 The isolated
nuclei were digested with proteinase K in 0.5% sodium dodecylsulfate (SDS), followed by RNase digestion and extractions with
phenol and chloroform-isoamyl alcohol. The DNA was precipitated
in ethanol, resuspended in Tnis EDTA (TE) (10 mmol/L of Tnis, Immol/L of EDTA) and dialyzed extensively against TE.
Analysis ofmethylation. DNA (I 5 �g) was digested in 200 pLof reaction mixture using 50-fold excess of the respective restrictionendonuclease according to the manufacturers’ protocols. Undigested
x DNA was included with 10 �iL of the reaction mixture in a paralleltube as an internal standard for completeness of digestion) The
digested DNA was analyzed by electrophoresis on agarose gels and
Southern blotting.”Methylation was studied using the isoschizomers HpaIl (digesting
only unmethylated CCGG sites), and Mspl (insensitive to methyla-
tion of CCGG sequences), or the enzyme pair SaIl (digesting only
unmethylated GTCGAC sites) and TaqI (insensitive to methylation
of TCGA sequences). Methylation at the /3 gene was analyzed byHindlll and Hpall or Mspl double digestion and probing withpflBRI (including BamHI-EcoRl fragment of the second interven-
ing sequence (IVS2) of /3, kindly provided by R. A. Spritz).
Methylation at the h gene was analyzed by double digestion withEcoRI and either SaIl or TaqI, and pbBRI (including BamHI-
EcoRI fragment of IVS2 ofb, obtained from R. A. Spnitz) was used
as a probe. The ‘y genes were analyzed by EcoRI and HpaII/MspI
double digestion, and probing with pyBRI (IVS2 of7, obtained from
R. A. Spritz) and with pH’yI, including the 3.3 kilobase (kb)
HindlIl fragment containing the entire A.�gene.” For the gene, the
DNA was digested with HindIlI and HpaII/MspI and probed with
GSE79, containing 1.3 kb BamHI-EcoRI � gene fragment.’4Hybridization with the respective probes, radioactively labeled by
nick translation, was carried out in 50% formamide in the presence
of 10% dextran sulfate.” Following exposure, the autoradiograms
were scanned, and the level of methylation at each site was quanti-
tated.
RESULTS
Figure 1 depicts the human f3-globin gene cluster. The
sites analyzed for methylation are marked by arrows and
numbered according to Van den Ploeg and FIaveIl.5 The sites
flanking the #{128}gene were not studied by those authors, and are
designated here as Ma and Mb.
DNA methylation at the t gene. In most of the samples
studied, enythroid and nonenythroid, the gene was methyl-
ated at both sites, Ma and Mb. A typical autoradiogram is
presented in Fig 2. DNA, prepared from nonmoblasts of H.
S., was digested with HindIlI to yield the expected 8-kb
fragment containing the t gene. Redigestion with HpaII did
not affect that fragment, indicating that both sites Ma and
Mb were fully methylated. Digestion with MspI, which is
insensitive to methylation at CCGG sites, yielded the
expected 2-kb t gene fragment. In nonmoblast samples of two
patients, S. A. and M. A., a faint band of 2.4 kb appeared
following cleavage by Hpall, indicating a low degree of
hypomethylation at site Ma, approximately I 20 base pairs
(bp) upstream from the CAP site (not shown). As expected,
cleavage with MspI resulted in a 2.0-kb fragment. DNA
derived from WBCs of the same patients (S. A. and M. A.)
was methylated at both sites (not shown).
Methylation in the domain ofthe ‘�‘ genes. Methylation
in this region in DNA derived from nonmoblasts was variable
in the diffenent individuals (Table 1 ). The experiments were
repeated at least twice with essentially the same results. The
two extreme cases are presented in Fig 3. Analysis of
methylation of the DNA of W. D. is shown in part A. The
EcoRI fragments of 7.0 and 2.6 kb, representing the G’y and
A’y genes respectively, were not digested with HpaII, indicat-
ing complete methylation at sites M I , M2, and M4. Methyl-
ation at site M6 was also complete (Table I ), as judged by
hybridization of a similar genomic blot to pH ‘yl , containing
MoMb Ml M2 M4M6 S13 M15
1�1 I � ,jAyj � 14 sl-II 1111-
I. � i.
kb 50 40 30 20 10 0
Fig 1 . Genetic map of the /3-globin region and location of themethylation sites studied. linkage map of the structural genes isfrom Proudfoot et al, ref. 1 6 with permission. The arrows point toCpG sites that were analyzed in the present work. Sites Ml
through Ml 5 are the same as in Van der Ploeg and Flavell. ref. 5;Ma and Mb are from Baralle et al, ref. 14. with permission.
For personal use only. by guest on October 14, 2011. bloodjournal.hematologylibrary.orgFrom
(A)
EEE
Hd Hd
Hd �
#{163}lI_ �8.O
Hd T #{128}
5.0 -
2.6- I.
7.0-b’. b’
5.0-. �S
2.6-b �
1.5 - ,.� 1.5-
‘S
8.0I- I
2.4I I
2.0
�; Ml M2
IEIG1 I
M4 M6
EE I EIEI I �AY I
7.0 1.6 .7 2.6 .6I I
5.0
1204 OPPENHEIM ET AL
Fig 2. Methylation at the � gene locus. DNA. purified from
peripheral blood normoblasts of H. S.. was digested with restric-tion nucleases as indicated at top and fragments were separatedby electrophoresis on 1 % agarose gel. The DNA was transferred tonitrocellulose membrane12 and hybridized to �P-Iabeled GSE79.containing a 1 .3 kilobase BamHl-EcoRl fragment including the 3’part of the � gene.” Hd. Hindlll; Hp. Hpall; M. Mspl.
Table 1 . Pattern of DNA Met hylation Along the /3-Globin locus
.Chain
Synthesis
Fraction of Unmethylated DNA
L y iS /3Soixce ‘y/a Ma Mb Ml M2 M4 M6 513 M15
W.D..Nb 0 0 0 0 0 0 0 1
G.Z..Nb .06 0 0 0 0 0 1
G. D., Nb .05 0 0 .3 .06 .06 1
S. A., Nb .12 .01 0 .2 .1 .1 .8 1 1
M. A., Nb .34 .02 0 .2 .2 .2 0 1 1
H. S., Nb .42 0 0 .1 .4 .4 .2 1
WBCt
Nb, normoblasts
0 0 0 0 0 0 1 0
Autoradiograms, like those presented in Figs 2 through 5. were
scanned with a densitometer and the scans were quantitated. To ensure
linearity of optical density to radioactivity for each autoradiogram.
scanning was performed after two different exposure periods. The
experiments on methylation in the y gene domain were repeated twice
with similar results, and the numbers represent an average of the 2
experiments.
Methylation at site M6 was assessed by hybridizing to pHyl . a
genomic A’y probe. ‘3
tDNA was purified from WBC of S. A. Sites near the ‘y genes (Mlthrough M6) were analyzed also in WBC of W. D., S. A.. M. A. and H. S.
and the same results were obtained.
(B)
EEE �
L�_J L��_J L�J
1.5 1.5 .5
Fig 3. Methylation at the domain of the ‘y genes. The experi-ment was performed essentially as described for Fig 2. DNA waspurified from normoblasts of (A) W. D.. and (B) H. S. Followingdigestion by the restriction endonucleases indicated at top, frag-ments were separated on 1 % agarose gel. Hybridization wascarried out using p’yBRl. containing a BamHl-EcoRl fragment ofIVS2 of ‘V. E. EcoRl; Hp. Hpall; M. Mspl.
a Hind III fragment that covers the entire A’y gene” (not
shown). As expected, digestion with MspI reduced most of
the 7.0-kb fragment into 5.0 kb, indicating digestion at site
M I . As noted before by several authors”6”’8 sites M2 and
M4, when methylated, are insensitive to MspI under usual
experimental conditions. Apparently because of flanking
adjacent sequences, these two sites are cleaved, when the
internal C is methylated, only when excessive levels of the
enzyme are applied.’8 In our experiments also, MspI digested
the two sites to the same extent as did HpaII (Fig 3).
The digestion pattern of H. S. is shown in Fig 3B. In this
case, fragments of 5.0 kb and 1 .5 kb appeared following
digestion with HpaII, indicating hypomethylation at the
three sites: Ml, M2, and M4. DNA derived from WBCs of
H. S. showed full methylation at these sites, as did DNA
derived from WBCs ofother patients (W. D., S. A., and M.
A., results not shown). It should be pointed out that because
of the relative location of the sites and the probe, digestion by
HpaII at site Ml could only be detected in those DNA
molecules that had not been cleaved at site M2. The fraction
of DNA unmethylated at sites M2 and M4 (1.5 kb) was
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- -7.6
�
Hd Hd
7.6
-2.3
‘s�-1.5
METHYLATION OF GLOBIN GENES 1205
assumed to result from equal hypomethylation at both sites
(Table I).
Methylation 5’ to the b gene. Methylation at site SI 3
was studied with EcoRI and SaIl or TaqI. The pattern
obtained with DNA prepared from normoblasts of S.A. is
shown in Fig 4. Double digestion with EcoRI and either SalI
or Taql gave the 1 .5-kb fragment indicative of cleavage at
the 513 site. DNA derived from WBCs of S. A. was also
cleaved by Sail, indicating that the site was unmethylated in
WBCs as well.
Methylation 3’ to the /3 gene. Analysis of methylation at
site MIS was performed with HindlIl and HpaII/MspI. In
the six samples of normoblasts studied, the 7.6-kb HindIlI
fragment encompassing the f3 gene was completely digested
with HpaII to yield a 4.7-kb fragment, suggesting that this
site was nonmethylated. Typical results are presented in Fig
5 (DNA of S. A.). Control experiments with WBC DNA
showed that the 7.6-kb HindIll fragment was resistant to
digestion by 1-Ipall and therefore fully methylated at this
site.
Hd HdHd #{149} +
Hp M
M 15
/1 .j,
DISCUSSION
In the present study, we have used peripheral blood
normoblasts to analyze the degree of DNA methylation
EE
ST
S13
El E
I ‘I’ ci IE I
L I
2.3I -I
1.5
Fig 4. Methylation at the h gene locus. The experiment was
performed essentially as described for Fig 2. DNA was purifiedfrom normoblasts of S. A. Following digestion by the restrictionendonucleases indicated at top. fragments were separated on1 .4% agarose gel. Hybridization was carried out with pbBRl.containing a BamHl-EcoRl fragment of IVS2 of h. E. EcoRl; S. SaIl;T. TaqI.
I I
4.7
Fig 5. Methylation at the /3 gene locus. The experiment wasperformed essentially as described for Fig 2. DNA was purifiedfrom normoblasts of S. A. Following digestion by the restrictionendonucleases indicated at top. the fragments were separated on1 % agarose gel. Hybridization was carried out with p$BRI. contain-ing a BamHl-EcoRI fragment of IVS2 of /3. Hd. HindlIl; Hp. Hpall; M.Mspl.
along the f3-globin gene cluster. Mature onthochnomatic
nonmoblasts are readily available fnom many of the patients
with thalassemia major or with hemolytic anemia who have
been previously splenectomized. They provide a homogenous
population of nucleated cells at the same stage of erythroid
maturation. Therefore, this system is a useful and convenient
source for studying aspects of human erythnoid diffenentia-
tion.
Hypomethylalion and gene activity in adult normo-
blasts. The degree of DNA methylation in nonmoblasts of
six adult patients with varying levels of spontaneous -y-chain
production are summarized in Table 1.
Sites Ma and Mb, flanking the t gene, were essentially
fully methylated in all the patients studied, except for two
patients who showed a very low degree of hypomethylation at
site Ma. To the best of our knowledge, expression of the
gene has never been observed in adults, thalassemic or
nonthalassemic. Therefore, methylation at these two sites
appears to be inversely related to gene expression, in agree-
ment with an earlier report.6 A different situation probably
occurs following administration of 5-azacytidine. When an
adult patient with j3-thalassemia major was treated with the
drug, the t gene became unmethylated, although mRNA
production was minimal.’9
The level of hypomethylation at sites M2 and M4, 53 bp
upstream from the CAP site of the G’y and A’y genes,
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REFERENCES
1206 OPPENHEIM ET AL
8. Cividalli G, Kerem H, Ezeckiel E, Rachmilewitz EA: /3#{176}-
thalassemia intermedia. Blood 52:345, 1978
correlates with the relative activity of those genes in the
different patients as assayed by globin chain synthesis (Table
I ). Hypomethylation at these two sites appears to correlate
tightly with transcriptional activation of the two genes
during human ontogeny as well,6 and also after phanmaco-
logic induction of fetal hemoglobin production.’9”#{176} In con-
trast, hypomethylation at site M 1 , 3.6 kb 5’ to G’y, and at site
M6, 0.2 kb 3’ to A-y, do not appear to be related to activities
of those genes.
We questioned whether the hypomethylation at sites M2
and M4 observed in normoblasts was related to gene activity
and not to some other mutations that impair methylation at
those sites per se. Therefore, DNA was extracted from WBC
samples of W. D. and of the three patients with significant
hypomethylation at this region (S. A., M. A. and H. S.), and
methylation at the ‘y gene domain was analyzed. In all cases,
the DNA derived from WBCs was fully methylated at the
four sites (Table I).
The site upstream to the b gene, SI 3, was not methylated
in the two samples of normoblasts and in the WBC sample
studied (Table I ). It appears that this site becomes unmeth-
ylated in some early plunipotent marrow stem cells that give
rise to both erythroid and nonerythroid hemopoietic precur-
sons. This site has been previously reported to be fully
methylated in human sperm cells.5 It has not been analyzed
in other hemopoietic tissues.
Site M I 5, approximately 550 bp downstream to the /3gene, was completely unmethylated in the six patients stud-
ied. It should be pointed out that although five of these
patients have mutated f3-globin genes, none of them is known
to be defective in its transcriptional control, therefore, these
genes should behave as “active” at the DNA level. The WBC
sample studied was fully methylated at this site (Table 1).
Our results on peripheral blood nonmoblasts differ from
those of Mavilio et al,6 who reported only partial hypometh-
ylation at site M I 5 in erythnoid cells purified from adult
bone marrow. We have analyzed a homogenous population of
onthochnomatic nonmoblasts, at the last stage of differentia-
tion before losing their nuclei, whereas Mavilio et a16 studied
a mixture of differentiating enythnoid cells at several devel-
opmental stages (pnonormoblasts, basophilic, polychromatic,
and orthochromatic normoblasts). Apparently, hypometh-
ylation at this site is taking place at the very last cell
divisions. Therefore, it appears that the onset of transcription
I . Conklin K, Groudine M: Chromatin structure and gene
expression, in Razin A, Cedar H, Riggs AD (eds): DNA Methyl-ation and Its Biological Significance. New York, Springer Verlag,
1984, p 293
2. Razin A, Cedar H: DNA methylation in eukaryotic cells. Int
Rev Ctyobiol 92:1�9,19843. Groudine M, Kohwi-Shigematsu T, Gelinas R, Stamatoyan-
nopoulos G, Papayannopoulou T: Human fetal to adult hemoglobin
switching: Changes in chromatin structure of the /3-globin gene
locus. Proc NatI Acad Sci USA 80:7551, 1983
4. Shen CKJ: DNA methylation and developmental regulation ofeukaryotic globin gene transcription, in Razin A, Cedar H, Riggs
AD (eds): DNA Methylation and Its Biological Significance.New York, Springer Verlag, 1984, p 249
of the /3-globin gene may precede complete hypomethylation
at this site. It should be pointed out that also for the chicken
vitellogenin gene hypomethylation at the 5’ end region has
been shown to occur after transcriptional activation by
estrogen.”
Hypomethylation and transcriptional activation of the -y
and f3-globin genes. Remarkable correlation emerges
between our results on hypomethylation at specific CpG sites
and chromosomal regions that undergo conformational
changes at the chromatin level as assayed by hypersensitivity
to DNase I.’ Sites M2 and M4, whose hypomethylation
correlates with activity of the ‘y genes in adult patients
(Table I ), are very close to two major hypersensitive sites.’
Likewise, the CpG sites Sl3, 5’ to the t5 gene, and MIS, 3’ to
the /3 gene, are also located near major hypersensitive sites.
At all these regions, hypomethylation and hypersensitivity to
DNase I were observed in tissues where those genes were
actively transcribing. The CpG sites Ml and M6, whose
hypomethylation did not correlate with gene activity, are
located in regions where minor sites of DNase I sensitivity
were found.
Recent experiments, utilizing DNA that was methylated
in vitro and introduced into cultured cells, also demonstrated
that methylation at the 5’ region of the ‘y gene prevented its
transcription.” One can therefore conclude that the 5’
regions of the G� and A, genes are involved in their regula-
tion and probably also in some aspects of the globin switch.
However, in the /3 gene, the situation may be different. Site
M I 5, whose hypomethylation coincides with the last stages
of erythroid differentiation, is located at the 3’ flanking
region. Therefore it appears that the /3-globin gene may be
regulated, at least in part, by its 3’ region. This observation is
consistent with recent findings on sequences required for
regulated expression of human /3-globin in mouse erythnoleu-
kemia cells.22” The processes by which demethylation at
specific chromosomal locations take place during differentia-
tion, and their fine relation to gene regulation, are yet to be
resolved.
ACKNOWLEDGMENT
We wish to thank Dr H. Cedar for helpful discussions and reviewof the manuscript; Dr A. Razin for helpful discussion; Mrs V. Levi,
Mrs A. Treves, and Mrs A. Karsai for competent technical assis-tance.
5. Van der Ploeg LHT, Flavell RA: DNA methylation in the
human -yb/3-globin locus in erythroid and nonerythroid tissues. Cell19:947, 1980
6. Mavilio F, Giampaolo A, Care A, Migliaccio G, Calandrini M,Russo G, Pagliardi GL, Mastroberardino G, Maninucci M, Peschle
C: Molecular mechanisms of human hemoglobin switching: Selec-tive undermethylation and expression of globin genes in embryonic,
fetal and adult erythroblasts. Proc NatI Acad Sci USA 80:6907,1983
7. Cividalli G, Kerem H, Rachmilewitz EA: Homozygous /3#{176}and/3+ thalassemia in Kurdish Jews and Arabs. Hemoglobin 1:333,
1977
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METHYLATION OF GLOBIN GENES 1207
9. Cividalli G, Kerem H, Rachmilewitz E: Globin synthesis in
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