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The state of DNA methylation of human ϵ-globin gene in erythroid and non-erythroid cells
97
Cell Differentiation and Dev,eelopment.26 (1989) 97-106 Elsevier Scientific Publishers Ireland, Ltd.
CDF 00574
The state of DNA methylation of human <-globin gene in erythroid and non-erythroid cells A.J. Bartzeliotou and G.J. Dimitriadis National Hellenic Research Foundation, 48, Vas. Constantinou Awe., Athens and Department of Biology, Faculty of Sciences, University of Patras, Parras 26000, Greece (Accepted
25 October
1988)
The extent of DNA methylation within the embryonic human c-globin gene domain was studied in erythroid and non-erythroid cell lines. The results obtained show that the human e-globin gene is totally methylated at all sites tested in tissues where it is not expressed, i.e. blood leucocytes. In the erythroid cell lines, KS62 and PUTRO, both forced to embryonic differentiation by induction with haemin, the level of methylation is reduced compared with that observed in blood leucocytes. In the nonerythroid cell lines HeLa and Raji, where the human e-globin gene is not expressed, the overall level of methylation in all sites tested is lower compared with that in erythroid cell lines. DNA methylation;
c-Globin gene; Erythroid cell; Non-erythroid
Introduction DNA in most higher eukaryotes is modified in the form of 5’-methylcytosine (5°C). This modification results from a post replicational methylation of hemimethylated DNA recognized by a CpG sequence specific methylase (Taylor and Jones, 1982). In eukaryotes this modified base occurs predominantly in the sequence 5’ CpG 3’ (Doskosil and Sorn, 1962; Grippo et al., 1968). The distribution of the CpG dinucleotide is nonrandom; about 1-28 of the vertebrate genome (the HTF fraction) consists of stretches very rich in the CpG dinucleotide (Cooper et al., 1983). These stretches occur as ‘islands’ of unmethylated
Correspondence address: Prof. G.J. Dimitriadis, Department of Biology, Faculty of Sciences, University of Patras, Patras 26000, Greece. 0922-3371/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
cell
CpG-rich DNA that are around 1 kb in length (Bird et al., 1985; Gardiner-Garden and Frommer, 1987). There is evidence that transcription of genes with an HTF island is inhibited when the island is methylated (Toni010 et al., 1984; Wolf et al., 1984; Yen et al., 1984). Tissue-specific genes, like human P-globin-like genes which have no obvious HTF character, are usually part of the methylated silent majority of the genome. When activated they appear to be bound to a complex of tissue-specific factors which presumably accomplish what HTF islands can achieve using non-tissue-specific factors (Bird, 1986). However, there is evidence that loss of methylation in the 5’ flanking region of tissue-specific genes has a direct correlation with gene activation. This argument arises from two kinds of experimental results: the inactivity of in vitro methylated globin DNA in transformation experiments Ltd.
98
and the activation of genes by 5’azacytidine. It is worth noting that activation of globin genes by azacytidine has been seen in cells that are already predisposed to differentiate into red cells (Creusot et al., 1982; Charache et al., 1983; Ley et al., 1984), and it can also be induced by agents that are not known to affect methylation. It is interesting that human e-globin gene, although hypomethylated after 5’azacytidine treatment of MEL cells containing human chromosome 11, is not expressed (Ley et al., 1984). In non-erythroid cells demethylation of globin genes by 5’-azacytidine does not lead to their activation (Hsiao et al., 1984). In vivo studies within the human y&?-globin locus have shown that all sites tested are highly modified in sperm DNA. However, DNA from somatic cells is unmodified at some sites in a tissue-specific manner. The regions in and surrounding genes actively expressed in a given tissue show a low level of modification (Van der Ploeg and Flavell, 1980). In the present study we have extended the study of DNA methylation within the e-globin gene domain by using erythroid and non-erythroid cell lines.
insensitive and sensitive to methylation was performed as described (Van der Ploeg and Flavell, 1980). DNA fragments were electrophoresed on 0.8% agarose gels and blotted onto nitrocellulose filters. Hybridization and posthybridization washes were performed as described (Robinson and Vogelstein, 1982) except that we did two additional last washes in 0.1 x standard saline citrate @SC), 0.1% sodium dodecyl sulfate (SDS) at 60°C for 30 min.
DNA probes and labelling As probes we used DNA restriction fragments isolated from a 3.7 kb EcoRI fragment containing the human e-globin gene and subcloned to pBR328 plasmid vector. DNA fragments were labelled by nick translation with a-[32P]dCTP to a specific activity of lo* cpm/pg.
Determination modification
of the extent of “CG or “C.‘“kZ
The determination of the extent of modification in each cleavage site tested was performed by calculating the ratio of the intensity of each band appearing in the slot tested, to the sum of the intensity of all bands present by densitometric scanning.
Materials and Methods Results
Cell cultures K562, PUTKO
and Raji cell lines were grown in RPM1 1640 medium supplemented with 10% heat inactivated fetal calf serum. K562 and PUTKO cells were induced to produce e-globin chains by incubation for 4 days in fresh medium containing haemin to a final concentration of 30 PM. HeLa cells were grown in MEM Eagle’s modified medium supplement with 10% newborn calf serum.
DNA extraction, Southern blot analysis and hybridization Genomic DNA from cultured cells and from peripheral blood leucocytes was prepared as described (Blin and Stafford, 1976; Kan et al., 1977). Cleavage of DNA with restriction endonucleases
In order to study the pattern of DNA methylation in the region of the human c-globin gene we checked all the cleavage sites of the methylationsensitive restriction endonucleases predicted from the sequence map of the gene (Baralle et al., 1982a, b). This region covers a 3919 bp fragment and includes a 2.0 kb 5’ flanking sequence, the entire coding sequence of the human c-globin gene and a very short 3’ flanking sequence (Fig. 6). The 3.9 kb region contains 33 CpGs, 21 of which are found within the 2.0 kb 5’ flanking region; 10 CpGs in the coding region and 2 CpGs in the short 3’ flanking region. The enzymes used were the HhaI, AvaI and the isoschizomeric pair HpaII/MspI, all sensitive to the presence of the 5°C in their recognition sequence (Table I).
99 TABLE
A
I
Restriction
endonucleases
sensitive
Ml234567
to DNA methylation
8
9
10~
23 '?
Endonuclease HpaII MspI HhaI AvaI
Recognition
sequence
cleaved
not cleaved
CCGG CCGG, Cm CGG GCGC CPyCGPuG
CmCGG “CCGG, GGC”CGG G”CGC CPy”CGPuG
!3-!.l-
The six tested CpG methylation sensitive sites represent 18% of the CpG dinucleotides in the 3.9 kb region. Five sites he in the 5’ flanking region, and they account for 24% of the CpGs of this region while the sixth site lies at the 3’ flanking region of the gene. As a methylation insensitive enzyme we used the Hind111 restriction endonuclease. In order to exclude partial digestion, a vast excess of enzyme was used, with internal controls of phage XDNA which was completely digested in all cases.
K562
B H
A
0
3.6 H:Hindlll A:Aval X:Xbal R:EcoRI
Ikb
H
l
X
kb -
1 probe
R
Fig. 2. State of methylation at AvaI site in erythroid and non-erythroid cells. (A) Southern blot of DNA samples double-digested with AvaI/HindIII enzymes. Slots: 1, normal human leucocytes; 2, HeLa cells; 3, Raji cells; 4, induced K562 cells; 5, uninduced K562 cells; 6, induced PUTKO cells; 7, uninduced PUTKO cells; 8, 9, HPFH human leucocytes; 10, P-thalassaemic human leucocytes. (B) Diagrammatic representation of the restriction sites of interest in the region studied. As probe we used the XbaI/EcoRI fragments indicated in panel B.
uninduced inducti
.
.:r
Methylation state of the human c-globin gene in elythroid ceI1 lines
HeLa
H
H B:BamWI
57
rB
probe
Fig. 1. Expression of the human r-globin gene in erythroid and non-erythroid cell lines. Total cellular RNA was extracted from the different cell lines and blotted (2 pg/slot) onto nitrocellulose filter using a slot blot apparatus. Filters were hybridized with the BamHI-BamHI probe.
Two erythroid cell lines expressing the human r-globin gene, K562 cells (Loggio and Loggio, 1975; Rutherford et al., 1979) and PUTKO cells (Klein et al., 1980), were used. Both cell lines were induced with haemin to a 2-3-fold increase of transcription (Fig. 1). DNA was isolated from uninduced and induced K562 cells and was double digested with the enzymes Hind111 and AvaI. This digestion generated two fragments which hybridized to XbaI/EcoRI probe (Fig. 2B); Fig. 2A (slots 5, 4) shows a main band, representing the Hind/Hind fragment and a very faint band, 3.6 kb long, representing the Ava/Hind fragment. These results show that the AvaI site was heavily
100 A Ml234567 232 9.7 - I) 6.6 -
2.6 -
%. -
2.3 2.1-
X:Xbal R:EcoRI
r
X
probe R
Fig. 3. State of methylation at HhaI sites in erythroid and non-erythroid cells. (A) Southern blot of DNA samples double-digested with HhaI/HindIII enzymes. Slots: 1, uninduced PUTKO cells; 2, induced PUTKO cells; 3, uninduced KS62 cells; 4, induced K562 cells; 5, Raji cells; 6, HeLa cells; 7, normal human leucocytes; 8, 9, HPFH human leucocytes; 10, P-thalassaemic human leucocytes. (B) Diagrammatic representation of the restriction sites of interest in the region studied. As probe we used the XbaI/EcoRI fragment indicated in panel B.
methylated in K562 cells, and that there was no difference in the state of methylation of this site before and after induction (Fig. 6). The same site was totally methylated in uninduced and induced PUTKO cells as we obtained the Hind/Hind fragment only (Fig. 2A slots 7, 6; Fig. 6). The results of double digestion of genomic DNA with HindIII/HhaI restriction enzymes are shown in Fig. 3. The digestion products of DNA isolated from uninduced and induced K562 cells gave two fragments which hybridized to XbaI/EcoRI probe (Fig. 3B). The Hind/Hind fragment and a 2.6 kb fragment resulted from partial cleavage at the Hh2 site only (Fig. 3A, slots 3, 4). In contrast to Hh2 site which was slightly unmodified, the Hhl site
was totally methylated in both uninduced and induced K562 cells (Fig. 6). In PUTKO cell line both HhaI sites were totally methylated despite induction (Fig. 3A, slots 1, 2; Fig. 6). Double digestion of DNA isolated from K562 and PUTKO cells before and after induction with HindIII/HpaII restriction enzymes resulted in the appearance of three and sometimes four bands hybridizable to the BamHI/BamHI probe (Fig. 4B): the Hind/Hind fragment; the 2.59 kb fragment as a result of digestion at the M2, Hind111 sites; the 1.97 kb fragment as a result of digestion at the M2, M3 sites; and a shorter, unpredicted fragment about 1.4 kb long (Fig. 4A, slots 1, 3, 6, 8). This last fragment was also detected in single digestions with HpaII of DNA from erythroid cells by using e-globin probe, and it may represent cleavage at the M2 site and at a HpaII site not present in the sequencing map. This HpaII site (M4) is located near the end of&the second intron and may be the result of a single base polymorphism at either the CCGT or CCTG sequence present at 1378 or 1440 bp respectively, downstream of the M2 site. As this site did not appear in the non-erythroid cell line (Fig. 5A) we suggest that this polymorphic site is probably a characteristic of erythroid cells. The same polymorphic site has been found in six weeks yolk sac and fetal liver erythroblasts (Mavillio et al., 1983). The M3 site which is located very close to the 3’ end of the human e-globin gene was significantly methylated, but after induction of K562 cells became hypomethylated (Fig. 6). Since M2 site was hypomethylated, we could not get enough information about the state of methylation of Ml site, except if we could be able to detect a 278 bp Ml, M2 restriction fragment. We were not able to detect such a fragment, but this could be due to the limitations of the Southern technique and not due to methylation at the Ml site. We, therefore, performed double digestion of DNA from erythroid cell lines with the EcoRI/ HpaII enzymes, and we used a 5’ flanking region probe, 410 bp long, the EcoRI-BglII fragment (Fig. 4B). If we were able to detect a 1600 bp E-Ml band, then we could get information about the state of methylation of the Ml site. As shown in Fig. 4A (slots l’, 3’), only one band was de-
101
M
1
2
5678
34
9
&M9.8
7
6
4
3’2.
1’
239.79.7-
66-
6.6-
232.11.91.6-
2.32.11.9-
1.31.40.908-
B
H c
E I
M:Mspl/
tkb
cl.9 e1.6e
Hpall
BIBam HI Bg:Bgl II
En&
yqy-l
9% . I
1-u
-
-
cl.97 2.25 42.59
I B
-t -
probes B
E :EcoRI
H HindIll Fig. 4. State of methylation at HpaII/MspI sites in erythroid cells. (A) Left panel: Southern blot of DNA samples double-digested with HpaII/HindIII and MspI/HindIII enzymes. Lanes: 1, uninduced K562 DNA x Hpa/Hind; 2, uninduced K562 DNA x Msp/Hind; 3, induced K562 DNAx Hpa/Hind; 4, induced K562 DNAXMsp/Hind; 5, uninduced PUTKO DNAX Hind; 6, uninduced PUTKO DNAX Hpa/Hind; 7, uninduced PUTKO DNA X Msp/Hind; 8, induced PUTKO DNA x Hpa/Hind; 9, induced PUTKO DNAx Msp/Hind. Right panel: Southern blot of DNA samples double-digested with HpaII/EcoRI and MspI/EcoRI enzymes. Lanes: l’, uninduced K562 DNA X Hpa/ EcoRI; 2’, uninduced K562 DNA X Msp/EcoRI; 3’, induced K562 DNA x Hpa/EcoRI; 4’, induced K562 DNA X Msp/EcoRI; 6’, uninduced PUTKO DNA X Hpa/EcoRI; 7’, uninduced PUTKO DNAXMsp/EcoRI; 8’, induced PUTKO DNAXHpa/EcoRI; 9’, induced PUTKO DNA X Msp/EcoRI. (B) Diagrammatic representation of the restriction sites of interest in the region studied. Slots 1-9 were hybridized to BamHI/BamHI probe and slots 1’ -9’ were hybridized to EcoRI/BglII probe, indicated in panel B.
tected: a 1900 bp band, representing digestion at EcoRI, M2 sites. This means that the Ml site was totally methylated in the erythroid cell lines. The appearance of a faint 3.7 kb band, representing the EcoRI-EcoRI fragment gave the level of methylation at the M2 site, which was in agreement with the level revealed by the HindIII/HpaII digestion and the BamHI-BamHI probe. We conclude that in K562 cell line, Ml site is totally methylated despite induction, site M2 is hypo-
methylated and becomes even more after induction and finally M3 site is heavily methylated before induction and becomes hypomethylated after induction. In PUTKO cell line, the Ml site was also totally methylated (Fig. 4A, slots 6’, 8’), while the overall level of methylation at M2 and M3 sites was higher compared with that observed in K562 cell line (Fig. 4A, slots 6, 8). The effect of induction was similar to that observed in K562 cell line,
102 M
*D
1
2
3
23-
M4567 Lb
664.3-
i..JZ.ll.Q-
aPub
t
b
2.32.1IS-
it
I
H:tiindIII E-EcoRI B.BamHI Bg.BgtII M MspV tipall
I X
E&J
1 E probes
LZI
Fig. 5. State of methylation of HpaII/MspI sites in non-erythroid cells. (A) Left: Southern blot of DNA samples double-digested with HpaII/HindIII and MspI/HindIII enzymes. Lanes: 1, HeLa DNA x Hind; 2, HeLa DNAX Hpa/Hind; 3, HeLa DNA X Msp/Hind; 4, Raji DNA X Hpa/Hind; 5, Raji DNA X Msp/Hind; 6, normal human leucocyte DNA X Hpa/Hind; 7, normal human leucocyte DNA x Hpa/Hind; 8,10, HPFH human leucocyte DNA X Hpa/Hind; 9.11, HPFH human leucocyte DNA x Msp/Hind; 12, p-thalassaemic human leucocyte DNA X Hpa/Hind; 13, b-thalassaemic human leucocyte DNA X Msp/Hind. Right: Southern blot of DNA samples double-digested with HpaII/EcoRI and MspI/EcoRI enzymes. Lanes: 2’, HeLa DNAxHpa/EcoRI; 3’, HeLa DNAXMsp/EcoRI; 4’, Raji DNAXHpa/EcoRI; 5’, Raji DNAXMsp/EcoRI; 6’, normal human leucocyte DNAX Hpa/EcoRI; 7’, normal human leucocyte DNAX Msp/EcoRI; 12’, b-thalassaemic human leucocyte DNAxHpa/EcoRI; 13’, ,!%thalassaemic leucocyte DNAxMsp/EcoRI. (B) Diagrammatic representation of the restriction sites of interest in the region studied. Lanes l-3 were hybridized to the BamHI/ BamHI probe; lanes 4-13 were hybridized to the XbaI/EcoRI probe; lanes 2’-13’ were hybridized to the EcoRI/BglII probe. Probes are indicated in panel B.
but there were no dramatic changes in the level of methylation after induction. This may be due to the fact that PUTKO cells are hybridoma cells between K562 cell line and a human Burkitt lymphoma cell line P3HR-I (Klein et al., 1980). The polymorphic HpaII site was heavily methylated in both PUTKO and K562 cells, despite induction (Fig. 6). Results obtained after double digestion of DNA with HindIII/MspI restriction enzymes agree with those obtained using HpaII. MspI recognizes both unmethylated and methylated bases at the internal C residue of CCGG sequences, but does not cleave if the external C residue is methylated (Table I). However, there is evidence that MspI will not cut
certain sequences in which only the internal C is methylated (Busslinger et al., 1983). In particular, the sequence GGC”CGG is resistant to MspI cleavage. Screening of the e-globin gene sequence did not reveal such a sequence. Analysis with HindIII/HpaII enzymes showed that Ml site was methylated and M2, M3 rather hypomethylated. Double digestion with the HindIII/HpaII enzymes gave two fragments hybridizing to the BamHI-BamHI probe (Fig. 4A, slots 2, 4, 7, 9): the Hind/Hind fragment and the 1.9 kb fragment as a result of digestion at M2, M3 sites. Again we could not get clear information about Ml site, since we were not able to detect the 278 bp MlM2 fragment. Double digestion with EcoRI/MspI en-
103 0
1
2
3
4 7.8 kb 1
8 kb
7
3919 bp
1
-
3.7kb 2.Okb 1.5kb -
0.8kb -_
KS62 uninduced
OQ
0.0
0
99
0
K562 induced
le
000
d
99
0
PUTKOuninduced
00
.a.@
m
0
PUTKO induced
00
-0
ee
9
HcLa
le
oeeo
00
0.0
d
*
@J
*
00
Human Icucocytes normal HPFHO
00
p-Thalassaemic
Fig. 6. DNA methylation pattern in the human r-globin gene domain. (A) Restriction map of the human c-globin gene and its flanking sequences. (B) l , 100%; 0, 70-90%; 0, 40-60s; 0, 10-208, methylated in the CmC form. 0, unmethylated. 0-O represents the mC(m)C modification. The (HpaII/MspI) site located near the end of the second intron is probably polymorphic, because it has not been reported in the c-globin gene sequence. This site is hence marked by an asterisk, when lack of cleavage may have been caused by either its methylation or its absence.
zymes gave the same 1.9 kb band detected after EcoRI/HpaII digestion (Fig. 4A, slots 7’, 9’). This means that MspI cannot cleave at the Ml site, because this site is probably methylated at the external C residue (“C’“‘CGG). The 1.4 kb band was also present with the same intensity as in the HpaII analysis showing that the methylation of this polymorphic site is of the mC’“‘C type. Methylation nonetythroid
state of the human cells
e-globin
gene in
The methylation state of the c-globin gene was examined in DNA isolated from peripheral blood leucocytes, tissue where the (-globin gene is not expressed, from normal individuals and from HPFH and /3-thalassaemic patients. All digestions with “CG methylation sensitive enzyme resulted
in the appearance of the Hind/Hind fragment only, suggesting that all sites checked for methylation were totally modified. Using the MspI restriction endonuclease the degree of “C(“)C modification was defined and was found to be higher than that obtained from K562 (uninduced and induced), PUTKO (uninduced and induced), and HeLa cells (Fig. 5A, slots 6-13). We also used two human cell lines not expressing the e-globin gene, HeLa and Raji cells. As slot blot analysis showed no c-globin, mRNA transcripts were synthesized in these cells (Fig. 1). Thus, we expected to detect a very high level of modification at all sites checked for methylation. However, in the case of HeLa cells, we detected a significantly low level of modification at all sites, the degree of which was much lower than the degree detected in erythroid cell lines expressing
104
the gene. The AvaI site was significantly unmodified; the two HhaI sites were at relatively higher level of modification, but the percentage of methylation was lower compared with that detected in K562 and PUTKO cells (uninduced and induced) (Fig. 2A, slot 2; Fig. 3A, slot 6). Analysis with HpaII and MspI resulted in the appearance of 1.9 kb band only, hybridizing to the BamHIBamHI probe (Fig. 5B) in both enzymatic digestions, meaning that the methylation of the M2 and M3 sites was very low. Ml site was again totally methylated in the “C’“‘C form as shown in Fig. 5A (slots 2’, 3’). In this case the polymorphic HpaII site (M4) was not detected (Fig. 5A, slot 2, 3). Using DNA from Raji cells, a lymphoblastoid cell line, we found the AvaI site to be methylated to an intermediate level, while HhaI sites were heavily methylated with the Hh2 site almost totally methylated (Fig. 2A, slot 3; Fig. 3A, slot 5). HpaII, Ml and M3 sites were heavily methylated, but M2 was hypomethylated. In this case we did not detect the band resulting from cleavage at the polymorphic HpaII site M4 reported for K562 and PUTKO cells (Fig. 5A, slots 4, 5, 4’, 5’). Discussion
The extent of DNA methylation within the embryonic human c-globin gene domain was studied in erythroid and non-erythroid cell lines. Within the 2.0 kb 5’ flanking sequence of the e-globin gene there are two relatively CpG-rich regions: (a) a 450 bp region located -1750 to -1300 bp upstream of the major cap site. This fragment contains 10 CpG dinucleotides, two of which have been checked for methylation with the AvaI and HhaI enzymes. These two sites are methylated in erythroid (K562, PUTKO) and non-erythroid cells (Raji, blood leucocytes). (b) A 300 bp region located - 650 to - 350 bp upstream of the major cap site. This fragment contains 6 CpG dinucleotides two of which have been checked for methylation with the HhaI and HpaII/MspI enzymes. These two sites are methylated in all cell lines examined as well as in blood leucocytes. The 350 bp region extending upstream of the major cap site contains only two CpG dinucleo-
tides, one of which was checked for methylation with the HpaII/MspI restriction enzymes. This CpG site (M2) is the only site that is significantly unmodified in both erythroid cell lines and becomes even more hypomethylated after induction. It is worth noting that this HpaII site (M2) lies within the region between the two cap sites of the human e-globin gene, which region also contains characteristic promoter elements and a DNAseIhypersensitive site (Alan et al., 1982; Tuan and London, 1984; Zhu et al., 1984). Also, it has been found that although hypomethylation at the M2 site may be necessary for full transcription of the human e-globin gene, it is not sufficient for activation of the e-globin gene in induced MEL cells containing human chromosome 11 (Ley et al., 1984). Therefore, the 1.5 kb region right upstream of the major cap site of human r-globin gene contains at least four methylated CpG dinucleotides when the gene is actively transcribed. This argues against the data presented by Murray and Grosveld (1987) by in vitro transfection experiments with an artificially methylated human yglobin gene. These authors suggest that at least a 1 kb region upstream of the cap site of the y-globin gene should be completely unmethylated to allow full expression of the gene. The short 3’ flanking region 0.7 kb downstream of the poly(A) tail contains only two CpGs, one of which is checked for methylation with the HpaII/MspI enzymes (M3 site). This site is heavily methylated in uninduced K562, uninduced PUTKO, Raji cells and blood leucocytes but is hypomethylated in HeLa cells. After induction of the erythroid cell lines, the level of methylation at M3 site is reduced significantly in K562 cells as well as in PUTKO cells. The human c-globin gene is a tissue-specific gene, has no HTF character and appears to be part of the methylated fraction of the genome. Activation of the gene leads to specific hypomethylation, but this hypomethylation is probably an additional change among all the conformational changes occurring during the process of transcription. The fact that the c-globin gene domain is hypomethylated in two non-erythroid cell lines means that hypomethylation itself cannot cause activation. It has been proposed that specific fac-
105
tors are required for the expression of tissuespecific genes (Chiu and Blau, 1985). Regulatory factors specific for adult, fetal and embryonic globin genes are required for globin genes expression, as has been proved by cell fusion and transient heterokaryon experiments (Anagnou et al., 1985; Papayannopoulou et al., 1985; Baron and Maniatis, 1986). It is worth noting that specific truns-acting factors are not always sufficient to activate a globin gene (Baron and Maniatis, 1986). Finally, our data suggest that there is no clear correlation between methylation and gene expression; if methylation indeed plays a role in gene expression, it must be part of a set of regulatory events that determine the state of gene activation.
Acknowledgments We are grateful to T. Rutherford and G. Klein for providing K562 and PUTKO cell lines, D. Spandidos for providing the human r-globin clone, M. Patrinou-Georgoula, D. Ish-Horowitz and J. Williams for critical reading of the manuscript and N. Lotsari-Andrikopoulou for typing. A.B. holds a NHRF postgraduate fellowship, and this paper is based upon a thesis which will be submitted by her for the doctoral degree at the University of Patras.
References Alan, M., G.J. Grindlay, L. Stefani and J. Paul: Epsilon globin gene transcripts originating upstream from the mRNA cap site in K562 cells and normal human embryos. Nucleic Acids Res. 10, 5133-5147 (1982). Anagnou, N.P., T.Y. Yuan, E. Lii, J. Helder, S. Wieder, D. Glaister, B. Marks, A. Wang, D. Colbert and A. Deisseroth: Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells. Blood 65, 705-712 (1985). Baralle, F.E., C.C. Shoulders, S. Goodboum, A. Jeffreys and N.J. Proudfoot: The 5’ flanking region of human c-globin gene. Nucleic Acids Res. 8, 4393-4404 (1980a). Baralle, F.E., Shoulders, C.C. and Proudfoot, N.J.: The primary structure of the c-globin gene. Cell 21, 621-626 (1980b). Baron, M.H. and T. Maniatis: Rapid reprogramming of globin gene expression in transient heterokaryons. Cell 46,591-602 (1986).
Bird, A.P.: CpG-rich islands and the function of DNA methylation. Nature 321, 209-213 (1986). Bird, A., M. Taggart, M. Frommer, O.J. Miller and D. McLead: A fraction from the mouse genome that is derived from islands of nonmethylated CpG-rich DNA. Cell 40, 91-99 (1985). Blin, N. and D.W. Stafford: A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303 (1976). Busslinger, M., E. de Boer, S. Whight, F.G. Grosveld and R.A. Flavell: The sequence GGCmCGG is resistant to Msp I cleavage. Nucleic Acids Res. 11, 3559-3569 (1983). Charache, S., G. Dover, C. Kirby Smith, T. Conover and M. Moyer: Treatment of sickle cell anemia with 5azacytidine results in increased fetal hemoglobin production and is associated with nonrandom hypomethylation of DNA around the ySg globin gene complex. Proc. Natl. Acad. Sci. USA 80,4842-4846 (1983). Chiu, C.P. and H.M. Blau: 5azacytidine permits gene activation in a previously non inducible cell type. Cell 40,417-424 (1985). Cooper, D.N., M.H. Taggart and A.P. Bird: Unmethylated domains in vertebrate DNA. Nucleic Acids Res. 11, 647-658 (1983). Creusot, F., G. Acs and J.K. Christman: Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2’-deoxycytidine. J. Biol. Chem. 257, 2041-2048 (1982). Doskosil, J. and P. Som: Distribution of 5-methylcytosine in pyrimidine sequence of deoxyribonucleic acids. Biochim. Biophys. Acta 55, 953-959 (1962). Gardiner-Garden, M. and M. Frommer: CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261-282 (1987). Grippo, P., M. Lacarrino, E. Parisi and E. Scarano: Methylation of DNA in developing sea urchin embryos. J. Mol. Biol. 36, 195-208 (1968). Hsiao, W.W., S. Gattoni-Celli, P. Kirschmeier and B. Weinstein: Effects of 5’ azacytidine on methylation and expression of specific DNA sequences in C3H lOT1/2. Mol. Cell. Biol. 4, 634-641 (1984). Kan, Y.W., A. Dozy, R. Trecartin and D. Todd: Identification of a non deletion defect in cu-Thalassemia. N. Engl. J. Med. 297, 1081-1084 (1977). Klein, G.J., I. Zenthen, P. Eriksson, M. Terasakri, A. Bemoco, E. Rosen, S. Masucci and R. Povery: Hybridization of a myeloid leukemia-derived human cell line (K562) with a human Burkitt’s lymphoma line (P3HR-1). J. Natl. Cancer Inst. USA 64, 725-738 (1980). Ley, T.J., Y.L. Chiang, D. Haidaris, N.P. Anagnou, V.L. Wilson and W.F. Anderson: DNA methylation and regulation of the human /3-globin-like genes in mouse erythroleukemia cells containing chromosome 11. Proc. Natl. Acad. Sci. USA 81, 6618-6622 (1984). Loggio, C.B. and B.B. Loggio: Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 45, 321-334 (1975). Mavillio, F., A. Giampaolo, A. Care, G. Migliaccio, M.
Calandrini, G. Russo, G.L. Dagliardi, G. Mastroberardino, M. Marinucci and C. Pesche: Molecular mechanisms of human hemoglobin switching: selective undermethylation and expression of globin genes in embryonic, fetal and adult erythroblasts. Proc. Natl. Acad. Sci. USA 80, 6907-6911 (1983). Murray, E.J. and F. Grosveld (1987) Site specific demethylation in the promoter of human y-globin gene does not alleviate methylation mediated suppression. EMBO J. 8, 2329-2335 (1987). Papayannopoulou, T.H., P. Lindsley, S. Kurachi, K. Lewison, T. Hemenway, M. Melis, N.P. Anagnou and V. Najfeld: Adult and fetal human globin genes are expressed following chromosomal transfer into MEL cells. Proc. Natl. Acad. Sci. USA 82, 780-784 (1985). Robinson, S. and B. Vogelstein: The association of transcriptionally active genes with the nuclear matrix of the chicken oviduct. Cell 28, 99-106 (1982). Rutherford, T.R., J.B. Clegg and D.J. Weatherall: K562 human leukemic cells synthesize embryonic hemoglobin in response to hemin. Nature 280, 164-165 (1979). Taylor, S.M. and P.A. Jones: Mechanism of action of eukaryotic DNA methyltransferase: use of 5-azacytosinacontaining DNA. J. Mol. Biol. 162, 679-692 (1982). Toniolo, D., M. D’Urso, G. Martini, M. Persico, V. Tufano, G.
Battistuzzi and L. Luzzatto: Specific methylation pattern at the 3’ end of the human housekeeping gene for glucose 6-phosphate dehydrogenase. EMBO J. 3,1987-1995 (1984). Tuan, D. and I.M. London: Mapping of DNAse I-hypersensitive sites in the upstream DNA of the human embryonic e-globin gene in K562 leukemia cells. Proc. Natl. Acad. Sci. U.S.A. 81, 2718-2722 (1984). Van der Ploeg, L.H.T. and R.A. Flavell: DNA methylation in the human y&%globin locus in erythroid and non erythroid tissues. Cell 19, 947-958 (1980). Wolf, S.F., S. Dintzis, D. Toniolo, G. Persico, K.D. Lunnen, J. Axelman and B.R. Migeo: Complete coordinance between glucose 6-phosphate dehydrogenase activity and hypomethylation: implications for X chromosome dosage compensation. Nucleic Acids Res. 12, 9333-9348 (1984). Yen, P.H., P. Patel, A.C. Chinault, T. Mohandas and J. Shapirol: Differential methylation of hypoxanthine phosphoribosyltransferase genes on active and inactive human X chromosomes. Proc. Natl. Acad. Sci. U.S.A. 81, 1759-1763 (1984). Zhu, J., M. Allan and J. Paul: The chromatin structure of the human c-globin gene: nuclease hypersensitivity correlate with multiple initiation sites of transcription. Nucleic Acids Res. 12, 9191-9204 (1984).
Cell Differentiation and Dev,eelopment.26 (1989) 97-106 Elsevier Scientific Publishers Ireland, Ltd.
CDF 00574
The state of DNA methylation of human <-globin gene in erythroid and non-erythroid cells A.J. Bartzeliotou and G.J. Dimitriadis National Hellenic Research Foundation, 48, Vas. Constantinou Awe., Athens and Department of Biology, Faculty of Sciences, University of Patras, Parras 26000, Greece (Accepted
25 October
1988)
The extent of DNA methylation within the embryonic human c-globin gene domain was studied in erythroid and non-erythroid cell lines. The results obtained show that the human e-globin gene is totally methylated at all sites tested in tissues where it is not expressed, i.e. blood leucocytes. In the erythroid cell lines, KS62 and PUTRO, both forced to embryonic differentiation by induction with haemin, the level of methylation is reduced compared with that observed in blood leucocytes. In the nonerythroid cell lines HeLa and Raji, where the human e-globin gene is not expressed, the overall level of methylation in all sites tested is lower compared with that in erythroid cell lines. DNA methylation;
c-Globin gene; Erythroid cell; Non-erythroid
Introduction DNA in most higher eukaryotes is modified in the form of 5’-methylcytosine (5°C). This modification results from a post replicational methylation of hemimethylated DNA recognized by a CpG sequence specific methylase (Taylor and Jones, 1982). In eukaryotes this modified base occurs predominantly in the sequence 5’ CpG 3’ (Doskosil and Sorn, 1962; Grippo et al., 1968). The distribution of the CpG dinucleotide is nonrandom; about 1-28 of the vertebrate genome (the HTF fraction) consists of stretches very rich in the CpG dinucleotide (Cooper et al., 1983). These stretches occur as ‘islands’ of unmethylated
Correspondence address: Prof. G.J. Dimitriadis, Department of Biology, Faculty of Sciences, University of Patras, Patras 26000, Greece. 0922-3371/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
cell
CpG-rich DNA that are around 1 kb in length (Bird et al., 1985; Gardiner-Garden and Frommer, 1987). There is evidence that transcription of genes with an HTF island is inhibited when the island is methylated (Toni010 et al., 1984; Wolf et al., 1984; Yen et al., 1984). Tissue-specific genes, like human P-globin-like genes which have no obvious HTF character, are usually part of the methylated silent majority of the genome. When activated they appear to be bound to a complex of tissue-specific factors which presumably accomplish what HTF islands can achieve using non-tissue-specific factors (Bird, 1986). However, there is evidence that loss of methylation in the 5’ flanking region of tissue-specific genes has a direct correlation with gene activation. This argument arises from two kinds of experimental results: the inactivity of in vitro methylated globin DNA in transformation experiments Ltd.
98
and the activation of genes by 5’azacytidine. It is worth noting that activation of globin genes by azacytidine has been seen in cells that are already predisposed to differentiate into red cells (Creusot et al., 1982; Charache et al., 1983; Ley et al., 1984), and it can also be induced by agents that are not known to affect methylation. It is interesting that human e-globin gene, although hypomethylated after 5’azacytidine treatment of MEL cells containing human chromosome 11, is not expressed (Ley et al., 1984). In non-erythroid cells demethylation of globin genes by 5’-azacytidine does not lead to their activation (Hsiao et al., 1984). In vivo studies within the human y&?-globin locus have shown that all sites tested are highly modified in sperm DNA. However, DNA from somatic cells is unmodified at some sites in a tissue-specific manner. The regions in and surrounding genes actively expressed in a given tissue show a low level of modification (Van der Ploeg and Flavell, 1980). In the present study we have extended the study of DNA methylation within the e-globin gene domain by using erythroid and non-erythroid cell lines.
insensitive and sensitive to methylation was performed as described (Van der Ploeg and Flavell, 1980). DNA fragments were electrophoresed on 0.8% agarose gels and blotted onto nitrocellulose filters. Hybridization and posthybridization washes were performed as described (Robinson and Vogelstein, 1982) except that we did two additional last washes in 0.1 x standard saline citrate @SC), 0.1% sodium dodecyl sulfate (SDS) at 60°C for 30 min.
DNA probes and labelling As probes we used DNA restriction fragments isolated from a 3.7 kb EcoRI fragment containing the human e-globin gene and subcloned to pBR328 plasmid vector. DNA fragments were labelled by nick translation with a-[32P]dCTP to a specific activity of lo* cpm/pg.
Determination modification
of the extent of “CG or “C.‘“kZ
The determination of the extent of modification in each cleavage site tested was performed by calculating the ratio of the intensity of each band appearing in the slot tested, to the sum of the intensity of all bands present by densitometric scanning.
Materials and Methods Results
Cell cultures K562, PUTKO
and Raji cell lines were grown in RPM1 1640 medium supplemented with 10% heat inactivated fetal calf serum. K562 and PUTKO cells were induced to produce e-globin chains by incubation for 4 days in fresh medium containing haemin to a final concentration of 30 PM. HeLa cells were grown in MEM Eagle’s modified medium supplement with 10% newborn calf serum.
DNA extraction, Southern blot analysis and hybridization Genomic DNA from cultured cells and from peripheral blood leucocytes was prepared as described (Blin and Stafford, 1976; Kan et al., 1977). Cleavage of DNA with restriction endonucleases
In order to study the pattern of DNA methylation in the region of the human c-globin gene we checked all the cleavage sites of the methylationsensitive restriction endonucleases predicted from the sequence map of the gene (Baralle et al., 1982a, b). This region covers a 3919 bp fragment and includes a 2.0 kb 5’ flanking sequence, the entire coding sequence of the human c-globin gene and a very short 3’ flanking sequence (Fig. 6). The 3.9 kb region contains 33 CpGs, 21 of which are found within the 2.0 kb 5’ flanking region; 10 CpGs in the coding region and 2 CpGs in the short 3’ flanking region. The enzymes used were the HhaI, AvaI and the isoschizomeric pair HpaII/MspI, all sensitive to the presence of the 5°C in their recognition sequence (Table I).
99 TABLE
A
I
Restriction
endonucleases
sensitive
Ml234567
to DNA methylation
8
9
10~
23 '?
Endonuclease HpaII MspI HhaI AvaI
Recognition
sequence
cleaved
not cleaved
CCGG CCGG, Cm CGG GCGC CPyCGPuG
CmCGG “CCGG, GGC”CGG G”CGC CPy”CGPuG
!3-!.l-
The six tested CpG methylation sensitive sites represent 18% of the CpG dinucleotides in the 3.9 kb region. Five sites he in the 5’ flanking region, and they account for 24% of the CpGs of this region while the sixth site lies at the 3’ flanking region of the gene. As a methylation insensitive enzyme we used the Hind111 restriction endonuclease. In order to exclude partial digestion, a vast excess of enzyme was used, with internal controls of phage XDNA which was completely digested in all cases.
K562
B H
A
0
3.6 H:Hindlll A:Aval X:Xbal R:EcoRI
Ikb
H
l
X
kb -
1 probe
R
Fig. 2. State of methylation at AvaI site in erythroid and non-erythroid cells. (A) Southern blot of DNA samples double-digested with AvaI/HindIII enzymes. Slots: 1, normal human leucocytes; 2, HeLa cells; 3, Raji cells; 4, induced K562 cells; 5, uninduced K562 cells; 6, induced PUTKO cells; 7, uninduced PUTKO cells; 8, 9, HPFH human leucocytes; 10, P-thalassaemic human leucocytes. (B) Diagrammatic representation of the restriction sites of interest in the region studied. As probe we used the XbaI/EcoRI fragments indicated in panel B.
uninduced inducti
.
.:r
Methylation state of the human c-globin gene in elythroid ceI1 lines
HeLa
H
H B:BamWI
57
rB
probe
Fig. 1. Expression of the human r-globin gene in erythroid and non-erythroid cell lines. Total cellular RNA was extracted from the different cell lines and blotted (2 pg/slot) onto nitrocellulose filter using a slot blot apparatus. Filters were hybridized with the BamHI-BamHI probe.
Two erythroid cell lines expressing the human r-globin gene, K562 cells (Loggio and Loggio, 1975; Rutherford et al., 1979) and PUTKO cells (Klein et al., 1980), were used. Both cell lines were induced with haemin to a 2-3-fold increase of transcription (Fig. 1). DNA was isolated from uninduced and induced K562 cells and was double digested with the enzymes Hind111 and AvaI. This digestion generated two fragments which hybridized to XbaI/EcoRI probe (Fig. 2B); Fig. 2A (slots 5, 4) shows a main band, representing the Hind/Hind fragment and a very faint band, 3.6 kb long, representing the Ava/Hind fragment. These results show that the AvaI site was heavily
100 A Ml234567 232 9.7 - I) 6.6 -
2.6 -
%. -
2.3 2.1-
X:Xbal R:EcoRI
r
X
probe R
Fig. 3. State of methylation at HhaI sites in erythroid and non-erythroid cells. (A) Southern blot of DNA samples double-digested with HhaI/HindIII enzymes. Slots: 1, uninduced PUTKO cells; 2, induced PUTKO cells; 3, uninduced KS62 cells; 4, induced K562 cells; 5, Raji cells; 6, HeLa cells; 7, normal human leucocytes; 8, 9, HPFH human leucocytes; 10, P-thalassaemic human leucocytes. (B) Diagrammatic representation of the restriction sites of interest in the region studied. As probe we used the XbaI/EcoRI fragment indicated in panel B.
methylated in K562 cells, and that there was no difference in the state of methylation of this site before and after induction (Fig. 6). The same site was totally methylated in uninduced and induced PUTKO cells as we obtained the Hind/Hind fragment only (Fig. 2A slots 7, 6; Fig. 6). The results of double digestion of genomic DNA with HindIII/HhaI restriction enzymes are shown in Fig. 3. The digestion products of DNA isolated from uninduced and induced K562 cells gave two fragments which hybridized to XbaI/EcoRI probe (Fig. 3B). The Hind/Hind fragment and a 2.6 kb fragment resulted from partial cleavage at the Hh2 site only (Fig. 3A, slots 3, 4). In contrast to Hh2 site which was slightly unmodified, the Hhl site
was totally methylated in both uninduced and induced K562 cells (Fig. 6). In PUTKO cell line both HhaI sites were totally methylated despite induction (Fig. 3A, slots 1, 2; Fig. 6). Double digestion of DNA isolated from K562 and PUTKO cells before and after induction with HindIII/HpaII restriction enzymes resulted in the appearance of three and sometimes four bands hybridizable to the BamHI/BamHI probe (Fig. 4B): the Hind/Hind fragment; the 2.59 kb fragment as a result of digestion at the M2, Hind111 sites; the 1.97 kb fragment as a result of digestion at the M2, M3 sites; and a shorter, unpredicted fragment about 1.4 kb long (Fig. 4A, slots 1, 3, 6, 8). This last fragment was also detected in single digestions with HpaII of DNA from erythroid cells by using e-globin probe, and it may represent cleavage at the M2 site and at a HpaII site not present in the sequencing map. This HpaII site (M4) is located near the end of&the second intron and may be the result of a single base polymorphism at either the CCGT or CCTG sequence present at 1378 or 1440 bp respectively, downstream of the M2 site. As this site did not appear in the non-erythroid cell line (Fig. 5A) we suggest that this polymorphic site is probably a characteristic of erythroid cells. The same polymorphic site has been found in six weeks yolk sac and fetal liver erythroblasts (Mavillio et al., 1983). The M3 site which is located very close to the 3’ end of the human e-globin gene was significantly methylated, but after induction of K562 cells became hypomethylated (Fig. 6). Since M2 site was hypomethylated, we could not get enough information about the state of methylation of Ml site, except if we could be able to detect a 278 bp Ml, M2 restriction fragment. We were not able to detect such a fragment, but this could be due to the limitations of the Southern technique and not due to methylation at the Ml site. We, therefore, performed double digestion of DNA from erythroid cell lines with the EcoRI/ HpaII enzymes, and we used a 5’ flanking region probe, 410 bp long, the EcoRI-BglII fragment (Fig. 4B). If we were able to detect a 1600 bp E-Ml band, then we could get information about the state of methylation of the Ml site. As shown in Fig. 4A (slots l’, 3’), only one band was de-
101
M
1
2
5678
34
9
&M9.8
7
6
4
3’2.
1’
239.79.7-
66-
6.6-
232.11.91.6-
2.32.11.9-
1.31.40.908-
B
H c
E I
M:Mspl/
tkb
cl.9 e1.6e
Hpall
BIBam HI Bg:Bgl II
En&
yqy-l
9% . I
1-u
-
-
cl.97 2.25 42.59
I B
-t -
probes B
E :EcoRI
H HindIll Fig. 4. State of methylation at HpaII/MspI sites in erythroid cells. (A) Left panel: Southern blot of DNA samples double-digested with HpaII/HindIII and MspI/HindIII enzymes. Lanes: 1, uninduced K562 DNA x Hpa/Hind; 2, uninduced K562 DNA x Msp/Hind; 3, induced K562 DNAx Hpa/Hind; 4, induced K562 DNAXMsp/Hind; 5, uninduced PUTKO DNAX Hind; 6, uninduced PUTKO DNAX Hpa/Hind; 7, uninduced PUTKO DNA X Msp/Hind; 8, induced PUTKO DNA x Hpa/Hind; 9, induced PUTKO DNAx Msp/Hind. Right panel: Southern blot of DNA samples double-digested with HpaII/EcoRI and MspI/EcoRI enzymes. Lanes: l’, uninduced K562 DNA X Hpa/ EcoRI; 2’, uninduced K562 DNA X Msp/EcoRI; 3’, induced K562 DNA x Hpa/EcoRI; 4’, induced K562 DNA X Msp/EcoRI; 6’, uninduced PUTKO DNA X Hpa/EcoRI; 7’, uninduced PUTKO DNAXMsp/EcoRI; 8’, induced PUTKO DNAXHpa/EcoRI; 9’, induced PUTKO DNA X Msp/EcoRI. (B) Diagrammatic representation of the restriction sites of interest in the region studied. Slots 1-9 were hybridized to BamHI/BamHI probe and slots 1’ -9’ were hybridized to EcoRI/BglII probe, indicated in panel B.
tected: a 1900 bp band, representing digestion at EcoRI, M2 sites. This means that the Ml site was totally methylated in the erythroid cell lines. The appearance of a faint 3.7 kb band, representing the EcoRI-EcoRI fragment gave the level of methylation at the M2 site, which was in agreement with the level revealed by the HindIII/HpaII digestion and the BamHI-BamHI probe. We conclude that in K562 cell line, Ml site is totally methylated despite induction, site M2 is hypo-
methylated and becomes even more after induction and finally M3 site is heavily methylated before induction and becomes hypomethylated after induction. In PUTKO cell line, the Ml site was also totally methylated (Fig. 4A, slots 6’, 8’), while the overall level of methylation at M2 and M3 sites was higher compared with that observed in K562 cell line (Fig. 4A, slots 6, 8). The effect of induction was similar to that observed in K562 cell line,
102 M
*D
1
2
3
23-
M4567 Lb
664.3-
i..JZ.ll.Q-
aPub
t
b
2.32.1IS-
it
I
H:tiindIII E-EcoRI B.BamHI Bg.BgtII M MspV tipall
I X
E&J
1 E probes
LZI
Fig. 5. State of methylation of HpaII/MspI sites in non-erythroid cells. (A) Left: Southern blot of DNA samples double-digested with HpaII/HindIII and MspI/HindIII enzymes. Lanes: 1, HeLa DNA x Hind; 2, HeLa DNAX Hpa/Hind; 3, HeLa DNA X Msp/Hind; 4, Raji DNA X Hpa/Hind; 5, Raji DNA X Msp/Hind; 6, normal human leucocyte DNA X Hpa/Hind; 7, normal human leucocyte DNA x Hpa/Hind; 8,10, HPFH human leucocyte DNA X Hpa/Hind; 9.11, HPFH human leucocyte DNA x Msp/Hind; 12, p-thalassaemic human leucocyte DNA X Hpa/Hind; 13, b-thalassaemic human leucocyte DNA X Msp/Hind. Right: Southern blot of DNA samples double-digested with HpaII/EcoRI and MspI/EcoRI enzymes. Lanes: 2’, HeLa DNAxHpa/EcoRI; 3’, HeLa DNAXMsp/EcoRI; 4’, Raji DNAXHpa/EcoRI; 5’, Raji DNAXMsp/EcoRI; 6’, normal human leucocyte DNAX Hpa/EcoRI; 7’, normal human leucocyte DNAX Msp/EcoRI; 12’, b-thalassaemic human leucocyte DNAxHpa/EcoRI; 13’, ,!%thalassaemic leucocyte DNAxMsp/EcoRI. (B) Diagrammatic representation of the restriction sites of interest in the region studied. Lanes l-3 were hybridized to the BamHI/ BamHI probe; lanes 4-13 were hybridized to the XbaI/EcoRI probe; lanes 2’-13’ were hybridized to the EcoRI/BglII probe. Probes are indicated in panel B.
but there were no dramatic changes in the level of methylation after induction. This may be due to the fact that PUTKO cells are hybridoma cells between K562 cell line and a human Burkitt lymphoma cell line P3HR-I (Klein et al., 1980). The polymorphic HpaII site was heavily methylated in both PUTKO and K562 cells, despite induction (Fig. 6). Results obtained after double digestion of DNA with HindIII/MspI restriction enzymes agree with those obtained using HpaII. MspI recognizes both unmethylated and methylated bases at the internal C residue of CCGG sequences, but does not cleave if the external C residue is methylated (Table I). However, there is evidence that MspI will not cut
certain sequences in which only the internal C is methylated (Busslinger et al., 1983). In particular, the sequence GGC”CGG is resistant to MspI cleavage. Screening of the e-globin gene sequence did not reveal such a sequence. Analysis with HindIII/HpaII enzymes showed that Ml site was methylated and M2, M3 rather hypomethylated. Double digestion with the HindIII/HpaII enzymes gave two fragments hybridizing to the BamHI-BamHI probe (Fig. 4A, slots 2, 4, 7, 9): the Hind/Hind fragment and the 1.9 kb fragment as a result of digestion at M2, M3 sites. Again we could not get clear information about Ml site, since we were not able to detect the 278 bp MlM2 fragment. Double digestion with EcoRI/MspI en-
103 0
1
2
3
4 7.8 kb 1
8 kb
7
3919 bp
1
-
3.7kb 2.Okb 1.5kb -
0.8kb -_
KS62 uninduced
OQ
0.0
0
99
0
K562 induced
le
000
d
99
0
PUTKOuninduced
00
.a.@
m
0
PUTKO induced
00
-0
ee
9
HcLa
le
oeeo
00
0.0
d
*
@J
*
00
Human Icucocytes normal HPFHO
00
p-Thalassaemic
Fig. 6. DNA methylation pattern in the human r-globin gene domain. (A) Restriction map of the human c-globin gene and its flanking sequences. (B) l , 100%; 0, 70-90%; 0, 40-60s; 0, 10-208, methylated in the CmC form. 0, unmethylated. 0-O represents the mC(m)C modification. The (HpaII/MspI) site located near the end of the second intron is probably polymorphic, because it has not been reported in the c-globin gene sequence. This site is hence marked by an asterisk, when lack of cleavage may have been caused by either its methylation or its absence.
zymes gave the same 1.9 kb band detected after EcoRI/HpaII digestion (Fig. 4A, slots 7’, 9’). This means that MspI cannot cleave at the Ml site, because this site is probably methylated at the external C residue (“C’“‘CGG). The 1.4 kb band was also present with the same intensity as in the HpaII analysis showing that the methylation of this polymorphic site is of the mC’“‘C type. Methylation nonetythroid
state of the human cells
e-globin
gene in
The methylation state of the c-globin gene was examined in DNA isolated from peripheral blood leucocytes, tissue where the (-globin gene is not expressed, from normal individuals and from HPFH and /3-thalassaemic patients. All digestions with “CG methylation sensitive enzyme resulted
in the appearance of the Hind/Hind fragment only, suggesting that all sites checked for methylation were totally modified. Using the MspI restriction endonuclease the degree of “C(“)C modification was defined and was found to be higher than that obtained from K562 (uninduced and induced), PUTKO (uninduced and induced), and HeLa cells (Fig. 5A, slots 6-13). We also used two human cell lines not expressing the e-globin gene, HeLa and Raji cells. As slot blot analysis showed no c-globin, mRNA transcripts were synthesized in these cells (Fig. 1). Thus, we expected to detect a very high level of modification at all sites checked for methylation. However, in the case of HeLa cells, we detected a significantly low level of modification at all sites, the degree of which was much lower than the degree detected in erythroid cell lines expressing
104
the gene. The AvaI site was significantly unmodified; the two HhaI sites were at relatively higher level of modification, but the percentage of methylation was lower compared with that detected in K562 and PUTKO cells (uninduced and induced) (Fig. 2A, slot 2; Fig. 3A, slot 6). Analysis with HpaII and MspI resulted in the appearance of 1.9 kb band only, hybridizing to the BamHIBamHI probe (Fig. 5B) in both enzymatic digestions, meaning that the methylation of the M2 and M3 sites was very low. Ml site was again totally methylated in the “C’“‘C form as shown in Fig. 5A (slots 2’, 3’). In this case the polymorphic HpaII site (M4) was not detected (Fig. 5A, slot 2, 3). Using DNA from Raji cells, a lymphoblastoid cell line, we found the AvaI site to be methylated to an intermediate level, while HhaI sites were heavily methylated with the Hh2 site almost totally methylated (Fig. 2A, slot 3; Fig. 3A, slot 5). HpaII, Ml and M3 sites were heavily methylated, but M2 was hypomethylated. In this case we did not detect the band resulting from cleavage at the polymorphic HpaII site M4 reported for K562 and PUTKO cells (Fig. 5A, slots 4, 5, 4’, 5’). Discussion
The extent of DNA methylation within the embryonic human c-globin gene domain was studied in erythroid and non-erythroid cell lines. Within the 2.0 kb 5’ flanking sequence of the e-globin gene there are two relatively CpG-rich regions: (a) a 450 bp region located -1750 to -1300 bp upstream of the major cap site. This fragment contains 10 CpG dinucleotides, two of which have been checked for methylation with the AvaI and HhaI enzymes. These two sites are methylated in erythroid (K562, PUTKO) and non-erythroid cells (Raji, blood leucocytes). (b) A 300 bp region located - 650 to - 350 bp upstream of the major cap site. This fragment contains 6 CpG dinucleotides two of which have been checked for methylation with the HhaI and HpaII/MspI enzymes. These two sites are methylated in all cell lines examined as well as in blood leucocytes. The 350 bp region extending upstream of the major cap site contains only two CpG dinucleo-
tides, one of which was checked for methylation with the HpaII/MspI restriction enzymes. This CpG site (M2) is the only site that is significantly unmodified in both erythroid cell lines and becomes even more hypomethylated after induction. It is worth noting that this HpaII site (M2) lies within the region between the two cap sites of the human e-globin gene, which region also contains characteristic promoter elements and a DNAseIhypersensitive site (Alan et al., 1982; Tuan and London, 1984; Zhu et al., 1984). Also, it has been found that although hypomethylation at the M2 site may be necessary for full transcription of the human e-globin gene, it is not sufficient for activation of the e-globin gene in induced MEL cells containing human chromosome 11 (Ley et al., 1984). Therefore, the 1.5 kb region right upstream of the major cap site of human r-globin gene contains at least four methylated CpG dinucleotides when the gene is actively transcribed. This argues against the data presented by Murray and Grosveld (1987) by in vitro transfection experiments with an artificially methylated human yglobin gene. These authors suggest that at least a 1 kb region upstream of the cap site of the y-globin gene should be completely unmethylated to allow full expression of the gene. The short 3’ flanking region 0.7 kb downstream of the poly(A) tail contains only two CpGs, one of which is checked for methylation with the HpaII/MspI enzymes (M3 site). This site is heavily methylated in uninduced K562, uninduced PUTKO, Raji cells and blood leucocytes but is hypomethylated in HeLa cells. After induction of the erythroid cell lines, the level of methylation at M3 site is reduced significantly in K562 cells as well as in PUTKO cells. The human c-globin gene is a tissue-specific gene, has no HTF character and appears to be part of the methylated fraction of the genome. Activation of the gene leads to specific hypomethylation, but this hypomethylation is probably an additional change among all the conformational changes occurring during the process of transcription. The fact that the c-globin gene domain is hypomethylated in two non-erythroid cell lines means that hypomethylation itself cannot cause activation. It has been proposed that specific fac-
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tors are required for the expression of tissuespecific genes (Chiu and Blau, 1985). Regulatory factors specific for adult, fetal and embryonic globin genes are required for globin genes expression, as has been proved by cell fusion and transient heterokaryon experiments (Anagnou et al., 1985; Papayannopoulou et al., 1985; Baron and Maniatis, 1986). It is worth noting that specific truns-acting factors are not always sufficient to activate a globin gene (Baron and Maniatis, 1986). Finally, our data suggest that there is no clear correlation between methylation and gene expression; if methylation indeed plays a role in gene expression, it must be part of a set of regulatory events that determine the state of gene activation.
Acknowledgments We are grateful to T. Rutherford and G. Klein for providing K562 and PUTKO cell lines, D. Spandidos for providing the human r-globin clone, M. Patrinou-Georgoula, D. Ish-Horowitz and J. Williams for critical reading of the manuscript and N. Lotsari-Andrikopoulou for typing. A.B. holds a NHRF postgraduate fellowship, and this paper is based upon a thesis which will be submitted by her for the doctoral degree at the University of Patras.
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