A human modifier of methylation for class I HLA genes (MEMO-1) maps to chromosomal bands 1p35-36.1
A human modifier of methylation for class I HLA genes (MEMO-1) maps to chromosomal bands 1p35-36.1N. C. Cheng, A. J. K. Chan, M. M. Beitsma, F. Speleman1, A. Westerveld and R. Versteeg*
Department of Human Genetics, University of Amsterdam, Academic Medical Centre, PO Box 22700, 1100 DE Amsterdam, The Netherlands and 1Department of Medical Genetics, University of Gent, Gent, Belgium
Received January 2, 1996;Accepted January 4, 1996
Class I HLA genes are expressed in almost all tissues, but expression is low or undetectable in many neuroblastomas. We analysed class I HLA methylation in normal tissues and in 28 neuroectodermal tumour cell lines. HLA-C is hypermethylated in normal adult tissues and 13 cell lines, while 15 cell lines show the hypomethylated phenotype. Hypomethylation of HLA-C strongly correlates with hemizygous deletion of a 9 cM interval on 1p35-36.1, suggesting that this region encodes a modifier of methylation for HLA-C. To test whether hypomethylation of class I HLA genes results from loss of a modifier gene, we fused a hypomethylating neuroblastoma cell line with a hypermethylating cell line. Methylation of class I HLA genes was induced in the hybrids. Furthermore, methylation of HLA-C, -E and -A genes, which are encoded in a 1.4 Mb region on 6p21, is correlated in most cell lines. Our results suggest that 1p35-36.1 encodes a modifier of methylation for class I HLA genes, that is deleted in many neuroblastomas.
One of the mechanisms thought to control tissue-specific and differentiation stage-specific gene expression is CpG methylation (1 ,2 ). Several experiments suggest a role for methylation in transcription regulation. Transfection of in vitro methylated DNA showed that methylation can interfere with gene expression (3 ,4 ), while treatment of cells with the demethylating agent 5-azacytidine can reactivate gene expression (5 ,6 ). Support for the idea that methylation can direct expression came from studies on genomic imprinting: homozygous inactivation of the methyltransferase gene in transgenic mice leads to hypomethylation and the erasure of imprinted expression patterns (7 ).
For most genes, hypomethylation is associated with expression, while silent genes are hypermethylated. However, for some genes of the murine class I major histocompatibility complex (class I MHC) and for the insulin-like growth factor type 2 receptor (Igf2r) gene, expression is associated with methylation of specific regions in the genes, although a causative relationship has not been proven so far (8 -10 ). The expression of the murine class I MHC genes H-2 and Q-10 correlates with methylation of their 3' region. The human class I MHC (called class I HLA) gene cluster maps to a 2 Mb region on chromosomal band 6p21 (see ref. 11 ). It encodes the classical class I HLA antigens, HLA-A, -B and -C, which are highly polymorphic membrane proteins that play an essential role in the immune reaction against virally infected cells and tumour cells. This region also encodes non-classical class I HLA genes with limited polymorphism (e.g. HLA-E, -F and -G) and class I HLA pseudogenes. HLA-A, -B and -C are expressed on most nucleated cells (12 ), but expression is low or undetectable in many neuroblastoma cell lines and tumours (13 ,14 ). We analysed the methylation pattern of the 3' region of class I HLA genes in 28 cell lines of mainly neuroectodermal origin, including 22 neuroblastoma cell lines. Here we show that about half of the cell lines has a hypermethylated pattern, while the other half has a hypomethylated pattern.
Up till now, methyltransferase is the only identified mammalian protein with methylation activity (15 ). Its major function in vivo is probably maintenance methylation of the newly synthesized DNA strand after DNA replication and it is supposed to act in a rather non-specific fashion. However, genetic evidence has been found for so-called `modifiers of methylation', a class of genes that regulates gene-specific methylation (16 ,17 ). Only one modifier of methylation was genetically mapped thus far. The murine Ssm-1 locus which regulates methylation of a certain transgene maps to mouse chromosome 4 (18 ) in a region syntenic to human chromosomal band 1p36 (see ref. 19 ). As this region is often deleted in neuroblastomas (20 ,21 ), we asked whether 1p36 could harbour a modifier of methylation for class I HLA genes. Therefore, we have analysed the 28 cell lines for 1p deletions. Hypomethylation of class I HLA genes strongly correlates with hemizygous deletion of a defined region of 1p35-36, suggesting that this region encodes a modifier of methylation for these genes. The idea that hypomethylation of class I HLA genes results from loss of a modifier gene was further supported by cell fusion experiments. The hypomethylated class I HLA alleles in a neuroblastoma cell line with a 1p deletion became methylated upon fusion with a cell line containing both alleles of the 1p35-36 region and hypermethylated class I HLA genes.
The expression of several murine class I MHC genes correlates with methylation of the 3' region of the genes (8 ,9 ). To study a possible role of methylation in the regulation of human class I MHC genes, we have analysed the methylation pattern of the 3' region of HLA-C in nine normal tissues and 28 tumour cell lines. DNAs were digested with MspI and the methylation-sensitive HpaII isoschizomer. Southern blot filters were hybridized with a probe from the 3' untranslated region (UTR) of HLA-C (see Fig. 1 ). This probe recognizes polymorphic HLA-C MspI fragments of 2.3 or 2.4 kb and cross-hybridizes with polymorphic HLA-B fragments in the 5 kb range. The normal tissues show hypermethylation of HLA-C as the 2.3 and 2.4 kb MspI fragments are found as larger, methylated fragments in the HpaII digests (Fig. 2 A). Only in lung and spleen a very faint unmethylated band is seen. Hybridization with a CpG island control probe shows that HpaII digestions were complete (Fig. 2 B). Also 13 tumour cell lines show the hypermethylated HLA-C phenotype (Fig. 3 ), including one Wilms' tumour (sample 1), one melanoma (sample 2), four primitive neuroectodermal tumour (PNET, samples 3-6) and seven neuroblastoma (samples 7-13) cell lines. However, 15 other neuroblastoma cell lines have HLA-C alleles that are, at least partially, unmethylated, as shown by the appearance of the 2.3/2.4 kb fragments in HpaII digests (Fig. 3 , samples 14-28). The intensity of the unmethylated fragments varies strongly, indicating that partial methylation still can occur: e.g. cell lines UHG-NP, N206, LA-N-5 and NMB have completely unmethylated HLA-C fragments, while the unmethylated bands are less pronounced in SJNB-8, SJNB-12 and SK-N-BE and hardly detectable in SJNB-10.
The class I HLA gene cluster encompasses about 2 Mb on chromosomal band 6p21. We also analysed the cell lines for methylation of other class I HLA genes in this cluster. HLA-E maps ~700 kb distal from HLA-C (see Fig. 1 ). The cell line filters were hybridized with a 3' UTR probe of HLA-E recognizing a 1.7 kb MspI fragment (Fig. 4 ). Most cell lines with a hypermethylated HLA-C pattern (samples 1-13) also have a hypermethylated HLA-E pattern. Exceptions are LA-N-6 and, to a lesser extent, SK-N-SH. Most cell lines showing the unmethylated HLA-C fragments also have an unmethylated HLA-E fragment. Also this group shows exceptions: SJNB-12 and SJNB-8. Just like for HLA-C, the intensity of the unmethylated band varies among samples 14-28. These results suggest that methylation of HLA-E is co-regulated with methylation of HLA-C, but the correlation does not hold for all cell lines.
In mice, a locus able to induce methylation of a certain transgene construct was genetically mapped to a region syntenic with human chromosomal band 1p36 (18 ). As this region is often deleted in neuroblastomas, we analysed whether a modifier of methylation for class I HLA could map to 1p36. We previously investigated chromosome 1p deletions of 16 neuroblastoma cell lines by a combination of Southern blot analysis with highly informative VNTR probes and FISH analysis (22 ). In the present study, we add the chromosome 1p deletion mapping data of 12 more cell lines (Fig. 6 , see legends to the figure for methods). We identified 18 cell lines with deletions in one allele of the short arm of chromosome 1. All cell lines with no loss of chromosome 1p material or with small distal 1p deletions show the hypermethylated HLA-C phenotype. The hypomethylated HLA-C phenotype is only seen in cell lines with relatively large 1p deletions, all being neuroblastoma cell lines (Fig. 6 ). One cell line, SJNB-10, is ambiguous, as it has a large deletion, but the unmethylated HLA-C band is hardly detectable (see Fig. 3 , sample 25). SJNB-1 has the largest 1p deletion among the cell lines with the hypermethylated phenotype, while GI-ME-N and UHG-NP have the smallest 1p deletions among cell lines from the hypomethylated group (Fig. 6 ). These three cell lines have deletion breakpoints that map between D1S7 (1p35-36.1) and ID3 (1p36.12-13). The switch between the hypo- and hypermethylated phenotype apparently maps between these markers. From the 28 cell lines, 27 are consistent with these data and one is ambiguous. Our results strongly suggest that a locus which regulates methylation of the HLA-C gene maps between D1S7 and ID3. The genetic distance between these markers is less than 9 cM (Fig. 7 ). We have named this locus Methylation Modifier-1 (MEMO-1).
As methylation of HLA-C and E is correlated, it is likely that the MEMO-1 locus also influences HLA-E methylation. However, in four of the 28 cell lines HLA-E does not follow the HLA-C methylation pattern and in these four cell lines there is no correlation with the presence or absence of the MEMO-1 region. Therefore, it is likely that HLA-E methylation is also affected by other factors than MEMO-1.
Ploidy, MEMO-1 copy number and HLA-C methylation pattern in neuroblastoma cell lines
Cell lines
Ploidy
No. of chromosomes
HLA-C methylation
1 with MEMO-1
regiona
NN-1
2n+ (47-51)b
2
+
SK-N-SH
2n+/- (44-46)
2
+
SK-N-FI
2n+/- (45-51)
2
+
LA-N-2
3n- (65)
3
+
SK-N-AS
2n (46)
2
+
LA-N-6
2n- (43)
2
+
SJNB-1
4n- (86-89)
2c
+
GI-ME-N
4n- (89-92)
2c
-
UHG-NP
3n+ (73-79)
2c
-
AMC-106
2n+/- (33-53)
1
-
SJNB-12
4n-- (83)
3
-
IMR32
2n+ (47-48)
1
-
SJNB-8
2n- (42)
1
-
KCNR
2n (46)
1
-
TR14
3n- (63)
2
-
N206
2n+ (47-50)
2
-
SK-N-BE
2n- (44)
1
-
SJNB-6
2n+/- (47)
1
-
LA-N-1
4n- (79-87)
3
-
LA-N-5
2n (46-47)
1
-
NMB
4n- (79-84)
3
-
SJNB-10
2n- (42-46)
1
+/-
aPresence of the MEMO-1 region was shown by colocalization of markers defining the MEMO-1 region on the same chromosome 1 by FISH (see legend to Fig. 6 for method). The proximal border of MEMO-1 is represented by probes for either D1S60 or D1S201 and the distal border by ID3 (see Materials and Methods for positions of probes).bMode or range of chromosome number.cThese three cell lines define the MEMO-1 region, as their deletion chromosomes have breakpoints between the markers D1S60 and ID3.
Cell lines with the hypomethylated HLA-C pattern show allelic loss of the MEMO-1 region. This could suggest that hypo- methylation results from a strict gene dosage effect of MEMO-1. However, translocations and duplications of chromosome 1 regions have often been observed in neuroblastoma cell lines. To analyse the possibility that hypomethylation of HLA-C results from a reduced copy number of the MEMO-1 locus, we analysed all neuroblastoma cell lines for ploidy and copy number of the MEMO-1 region (see Table 1 ). Copy number was determined by double colour FISH with two probes flanking the MEMO-1 region (see Fig. 6 ). All neuroblastoma cell lines with the hypermethylated HLA-C pattern have one copy of the MEMO-1 region per haploid genome. Cell lines with the hypomethylated HLA-C pattern generally have less than one copy of the MEMO-1 region per haploid genome. However, several cell lines with the hypomethylated phenotype and allelic loss of the MEMO-1 region have duplications of the intact chromosome 1. Therefore, their MEMO-1 copy number is only slightly less than one per haploid genome. These are cell lines SJNB-12, LA-N-1 and NMB (three copies of the MEMO-1 region in a near-tetraploid background), TR14 and UHG-NP (two copies in a near-triploid background) and N206 (two copies in a diploid background). All these cell lines show a good correlation between HLA-C hypomethylation and allelic loss of the MEMO-1 region, but the correlation between hypomethylation and copy number of the MEMO-1 region is less evident. These data suggest that hypomethylation of HLA-C in neuroblastoma cell lines with allelic loss of 1p35-36.1 does not exclusively result from a gene dosage effect. Impaired MEMO-1 activity of the 1p35-36 region on the remaining chromosome 1 could also play a part (see Discussion).
Mouse embryos homozygous for mutations in the methyltransferase gene die at the mid-gestation stage, which illustrates the essential role of methylation in the developing organism (24 ). The observations of transgene-specific (16 ,17 ) and parental-allele-specific (10 ,25 ,26 ) methylation patterns indicate the existence of gene-specific modifiers of methylation. However, no such modifiers were characterized thus far. The murine Ssm-1 locus is the only mammalian modifier of methylation that is genetically mapped (18 ). This locus regulates the methylation of a transgene construct. The natural target of this modifier, however, is unknown. Here we report the mapping of a human modifier of methylation with HLA-C and probably other class I HLA genes, as endogenous targets.
In neuroblastoma cell lines and tumours, class I HLA expression is often low or absent (13 ,14 ). As the expression of some murine class I MHC genes is associated with methylation, we analysed the methylation pattern of class I HLA genes in nine normal human tissues and 28 human neuroectodermal tumour cell lines. The 3' region of HLA-C is heavily methylated in normal tissues, but hypomethylated in about half of the cell lines. These cell lines all have hemizygous deletion of a small region on chromosome 1p35-36.1. Upon somatic fusion between cell lines with hyper- and hypomethylated class I HLA genes, the unmethylated class I HLA alleles become methylated. The hypomethylated phenotype therefore probably results from the impaired function of a modifier of methylation. Taken together, our data strongly suggest that a human modifier of methylation (MEMO-1) maps on chromosome 1p and regulates the methylation of HLA-C and probably other class I HLA genes. Deletions in three cell lines define the map position of MEMO-1 on 1p35-36.1 between the markers D1S7 and ID3. The genetic distance between these markers is less than 9 cM. Owing to the limited number of cell lines available, the fine mapping on 1p is based on three cell lines only. Therefore, some caution should be taken in drawing definite conclusions on the precise MEMO-1 location.
Allelic loss of the MEMO-1 region has no effect on the methylation pattern of a series of control genes. This implies that MEMO-1 does not randomly catalyse gene methylation, but specifically acts on class I HLA genes. It is not excluded though that MEMO-1 can modify other target genes as well. The finding that MEMO-1 and Ssm-1 map to the same mouse/human syntenic chromosomal region could suggest that these loci are identical. However, it is also possible that this region encodes multiple modifiers of methylation.
Some cell lines with hemizygous deletion of the MEMO-1 region show the fully unmethylated pattern (UHG-NP, N206, LA-N-5 and NMB), while several other cell lines with 1p deletions show only weak unmethylated HLA-C bands (SJNB-8, SJNB-12, SK-N-BE and SJNB-10). It is unlikely that this variation is caused by cell culture conditions, as differential methylation of polymorphic HLA-C alleles is seen within one cell line. The 2.4 kb HLA-C alleles in IMR32 and SJNB-6 are heavily methylated, while the 2.3 kb alleles are unmethylated (Fig. 9 ). This could suggest that HLA-C is imprinted, i.e. the paternally and maternally inherited alleles could be differentially methylated. However, cell lines KCNR and SJNB-8 have two polymorphic alleles which are equally hypo- or hypermethylated (Fig. 9 ), thus contradicting the idea of class I HLA imprinting. A more plausible explanation might be that the various class I HLA alleles have a different `susceptibility' to methylation. There are at least 20 HLA-C alleles (see ref. 27 ), which probably all generate a 2.3 or a 2.4 kb MspI fragment. Some alleles could be more susceptible to methylation, even in the context of only one MEMO-1 allele. Also HLA-E alleles may differ in their susceptibility to MEMO-1. Although polymorphism of HLA-E is less well studied, at least four different alleles have been identified (27 ).
Cell lines were cultured at 37oC in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum, 20 mM L-glutamine, 10 U/ml penicillin and 10 µg/ml streptomycin under 5% CO2. For primary references of the cell lines used in this study, see Cheng et al. (22 ), Reynolds et al. (29 ), Johnson et al. (30 ) and Versteeg et al. (31 ). The Wilms' tumour cell line AP10 was kindly provided by R. Slater. Some of the PNET cell lines are originally described as neuroblastoma cell lines, but were reclassified as PNETs based on their t(11;22) translocation (29 ).
CHP100 and N206 cells were transfected with the pSV2gpt and pSV2neo markers, respectively, and selected with mycophenolic acid (MPA) or G418 medium. Resistant clones were isolated and cell fusion was performed with 50% polyethylene glycol-1450 in suspension. Hybrids were selected by G418 plus MPA medium. Two to 3 weeks after selection, hybrid clones were picked and further expanded. The same procedure was performed for the fusion between GI-ME-N and IMR32.
The HLA-A probe was a 485 bp PvuII-MspI fragment isolated from HLA-A2 (32 ). The HLA-C probe was a 432 bp BamHI-SspI fragment derived from HLA-Cw2 (33 ). The HLA-E probe is a 244 bp PCR product as described by Boucraut et al. (34 ). Chromosome 1 probes used for deletion analysis include CEB15 (1p36.33), D1Z2 (1p36.33), CEB88 (1p36.3), PND (1p36.23-31), D1S56 (1p36.13), D1S482, ID3 (formerly designated Heir-1) and FUCA (1p36.12-13), D1S60 and D1S7 (1p35-36.11), D1S201, D1S57 (1p34.3), MYCL (1p32-34), D1S62, CEB82, D1S17 (1p32-pter), NRAS (1p13), D1Z1 (1q12 heterochromatic region) and MUC1 (1q21-23). Map positions of chromosome 1 markers were according to Van Roy et al. (35 ), Dracopoli et al. (36 ), Murray et al. (37 ) and Gyapay et al. (38 ). D1S62 maps proximal of MYCL (39 ) and CEB82 maps to the interval of GLUT1 and D1S15 (G. Vergnaud, personal communication), which are proximal to MYCL (36 ). For D1S201 we used CEPH YAC 909-f-6 and for the markers ID3/FUCA/D1S482 we used CEPH YAC 777-g-2.
Ten µg of genomic DNAs were digested with 10 U/µg DNA of the appropriate enzyme overnight to completion and electrophoresed on a 0.8% agarose gel. For the methylation studies, DNAs were digested with 10 U MspI or HpaII per µg DNA overnight and incubated further for 4 h with addition of 5 U enzyme per µg DNA. As control for completeness of HpaII digestions, DNAs were digested with an excess of enzyme (40 U/µg DNA). Identical hybridization patterns were observed compared with digestions with 15 U/µg DNA (data not shown). Also hybridizations with a CpG island probe confirm the HpaII digestions to be complete. Southern blotting on Hybond-N+ membrane (Amersham) by the alkali blotting method was performed as according to the manufacturer. Hybridization was carried out overnight in 0.5 M NaHPO4, pH 6.8, 7% SDS, 1 mM EDTA and 50 µg/ml sonicated herring sperm DNA at 65oC, with the exception of HLA locus-specific hybridizations which required temperatures of 75-80oC.
We thank N. van Roy and P. van Sluis for their contribution to the FISH analysis of chromosome 1p deletion mapping. We thank G. Vergnaud and A. Weith for providing probes and R. Bernards, T. Look, R. C. Seeger, G.M. Brodeur, M. Schwab, R. Slater, P. Cornaglia-Ferraris and P. van der Saag for cell lines. This study is supported by grants of the Stichting Kindergeneeskundig Kankeronderzoek (SKK), Vereniging Voor Kankerbestrijding and the European Concerted Action on Molecular Cytogenetics of Solid Tumors (PL920156).
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