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)

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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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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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