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© 1995 Oxford University Press 3196-3198

Restriction endonuclease isoschizomers ItaI, BsoFI and Fsp4HI are characterised by differences in their sensitivities to CpG methylation

Restriction endonuclease isoschizomers ItaI, BsoFI and Fsp4HI are characterised by differences in their sensitivities to CpG methylation B. H. Ramsahoye*, A. K. Burnett and C. Taylor1

Department of Haematology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK and 1Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, Bristol, UK

Received June 13, 1997; Accepted June 24, 1997

ABSTRACT

BsoFI, ItaI and Fsp4HI are isoshizomers of Fnu4HI (5'-GC <=> NGC-3'). Both Fnu4HI and BsoFI have previously been shown to be inhibited by cytosine-specific methylation within the recognition sequence. Fnu4HI is inhibited if either the internal cytosine at position 2 or the external cytosine at position 5 of the restriction sequence is methylated, but the precise nature of the methylation sensitivity of BsoFI is unclear from the literature. The methylation sensitivities of ItaI and Fsp4HI have not previously been reported. By methylating the plasmid pUC18 with M.SssI (a DNA cytosine-5'-methyltransferase with a specificity for CpG), we have determined that ItaI is sensitive only to methylation of internal CpG sites within the restriction sequence. The methylation sensitivity of Fsp4HI is identical to that of Fnu4HI, being inhibited by methylation of either internal CpG sites or overlapping CpG sites. BsoFI, like the other isoschizomers tested, is sensitive to a combination of internal and overlapping CpG methylation. BsoFI is also sensitive to overlapping CpG methylation (in the absence of internal CpG methylation) if CpG overlap with both sides of the recognition sequence. Sites containing one overlapping CpG (in the absence of internal CpG) are cut when methylated but show marked individual variation in their rates of cleavage. Considerable variation in the rate of cleavage by BsoFI is also observed at sites containing only internal methylated CpG. Some sites are cut slowly,whilst others fail to cut even after prolonged incubation with excess of enzyme.

Restriction enzymes that cut in the sequence 5'-GCNGC-3' have been used to study 5'-d(CGG)n-3' repeats. Such repeats are unstable and are prone to expansion (1 -3 ). Expanded repeats have been described in a variety of neurological disorders, the fragile X syndrome being the best characterised. Trinucleotide repeat expansion has now been described in a number of other conditions such as Huntington's disease [5'-d(CAG)n-3'] (4 ), and myotonic dystrophy [5'-d(CAG)n-3'] (5 ,6 ). In fragile X syndrome, abnormal methylation is associated with expansion of the 5'-d(CGG)n-3' repeats in the 5' untranslated region of the FMR1 gene (3 ,7 ). This may lead to transcriptional silencing of the gene. Methylation of these repeats has been detected by genomic sequencing and the use of restriction endonucleases such as Fnu4HI. This enzyme recognises the sequence 5'-GCNGC-3' but is inhibited by methylation of either the internal cytosine at position 2 of the recognition sequence or the external cytosine at position 5 (8 ).

ItaI, BsoFI and Fsp4HI are isoschizomers of Fnu4HI and are therefore potentially useful tools in such studies (9 ). However, at present the sensitivities of ItaI and Fsp4HI to cytosine methylation are unknown. New England Biolabs (D. Robinson, NEB, unpublished observations) report that BsoFI is sensitive to overlapping CpG methylation (methylation of the external cytosine), but all possible combinations have not been studied. Conversely, a study using methylated and unmethylated pBluescript SK(+) DNA as well as oligonucleotides containing trinucleotide repeats, has shown that restriction is only inhibited by methylation if both the internal and the external cytosines within the recognition sequence are methylated (10 ).

We have studied the sensitivities of ItaI, BsoFI, Fsp4HI and Fnu4HI to cytosine-5 methylation by in vitro methylation of pUC18 DNA with the methylase SssI (a DNA cytosine-5'-methyltransferase with a specificity for CpG). The subset of 5'-GCNGC-3' sites containing an internal CpG (i.e. 5'-GCSGC-3' sites, where S = G or C) or overlapping CpG(s) (where the restriction site is flanked on the 5' side by a C and/or on the 3' side by a G), can be methylated with this enzyme. Using this system, as the CpG dinucleotide is a palindrome, methylation at position 2 on one strand of the restriction site is always accompanied by reciprocal methylation at position 3 in the complementary strand. Whilst this pattern of methylation at internal cytosines is not likely to resemble that induced by a corresponding cognate methylase (such a methylase would be likely to methylate the position 2 cytosine on both strands), it is likely to be the most frequently encountered in mammalian DNA, where the vast majority of methylcytosines occur in the sequence CpG. Therefore, the methylation sensitivities determined by this method can be applied directly to studies on mammalian DNA.

By studying the published sequence of pUC18 (GenBank accession number A02710) it has been possible to determine the exact sequences of all the cleavage sites using DNASTAR sequence analysis software (DNASTAR Inc., Fig. 2 ). Complete CpG methylation of the plasmid was confirmed by the total failure of HpaII to restrict the DNA [Fig. 1 HpaII (+) digest]. It could be determined that only methylation of internal CpG inhibited restriction by ItaI. Thus after methylation, all 5'-DGCGGCH-3' sites and 5'-DGCSGCG-3' sites could not be cleaved, but 5'-DGCWGCG-3' and 5'-CGCWGCG-3' sites were completely cleaved (D = A, T or G, W = A or T, H = A, T or C and S = G or C-the recognition sequence is underlined and potentially methylatable cytosines are emboldened). Cleavage occurred in the absence of internal CpG methylation even when the external cytosines of both strands were methylated at CpG. The findings with respect to Fnu4HI were consistent with those previously reported. Restriction was inhibited by methylation of either internal CpG or overlapping CpG. The methylation sensitivity of Fsp4HI was found to be identical to that of Fnu4HI.


Figure 1. Restriction digests of unmethylated and methylated pUC18 plasmid. An aliquot of 2 [mu]g of pUC18 plasmid was incubated (37oC for 2 h) with 4 U of M.SssI (New England Biolabs) in a 50 [mu]l reaction mix containing 320 [mu]M S-adenosylmethionine and 1* NEB buffer 2 (New England Biolabs). At the end of the reaction the DNA was precipitated in ethanol and resuspended in 50 [mu]l water. Aliquots of 0.4 [mu]g were digested in 20 [mu]l reaction volumes with either 10 U (25 times excess) of MspI (New England Biolabs, cleavage site 5'-C <=> CGG-3' and 5'-C <=> MGG-3'), HpaII (methylation sensitive isoschizomer of MspI, Boehringer Mannheim), ItaI (Boehringer Mannheim), BsoFI (New England Biolabs), Fnu4HI (New England Biolabs) or 4 U (10 times excess) of Fsp4HI (SibEnzyme) in the manufacturers' recommended buffers. The same quantities of unmethylated plasmid were also digested with these enzymes. BsoFI incubations were for 4 h at 55oC. All other restriction digests were for 4 h at 37oC. The digests were electrophoresed on a 5% non-denaturing polyacrylamide gel. The results for Fsp4HI are not shown. Abbreviations: M, 1 kb marker (Gibco BRL); -, unmethylated pUC18; +, methylated pUC18. Notes: (i) In the BsoFI (+) and Fnu4HI (+) digests, the bands migrating just faster than the 298 bp marker are not identical. The band in BsoFI (+) is 273 bp and results from partial digestion of the 484 bp fragment (sum of the 211 and 273 bp fragments), whereas the band in Fnu4HI (+) is 286 bp and results from complete failure of restriction at the 3rd and 4th 5'-GCNGC-3' sites (Fig. 2). This has been confirmed by additional digestion of the BsoFI and Fnu4HI digests with BspLU11I, which cuts the 273 bp fragment but not the 286 bp fragment (data not shown). (ii) The 287 bp product of unmethylated plasmid digests has migrated marginally slower than the 298 bp marker. This difference is due to the base composition of this fragment rather than an insertion mutation in our plasmid. This phenomenon is not infrequently observed with non-denaturing polyacrylamide gels. We have confirmed this by digesting the related pUC19 plasmid (which differs from pUC18 solely in the orientation of the multiple cloning site) with Fnu4HI. The 287 bp fragment of pUC19 migrates to exactly the same position (data not shown). We have also sequenced this region in our plasmid and have excluded the presence of an insertion mutation that might have accounted for this discrepancy (data not shown).

The result in the case of BsoFI was less clear. There were four sites at which it was possible to examine the effects of overlapping CpG methylation in the absence of internal CpG methylation (two 5'-DGCWGCG-3' sites and two 5'-CGCWGCG-3' sites, Fig. 2 ). Restriction was observed at both sites where the restriction sequence was overlapped by CpG on one side (5'-DGCWGCG-3' sites) but was completely inhibited when CpG overlapped with the restriction sequence on both sides (i.e. the 5'-CGCWGCG-3' sites). There were three sites in pUC18 where internal and overlapping CpG methylation occurred together (5'-DGCSGCG-3' sites). BsoFI failed to cut at all of these sites after methylation of the plasmid. The independent effect of internal CpG methylation could also be examined at four sites which lack overlapping CpG (5'-DGCGGCH-3' sites). Here, two of the four sites were not cleaved when methylated. Of the other two, one site was partially cleaved and the other was fully cleaved when methylated (Fig. 2 ).


Figure 2. ItaI, BsoFI, Fsp4HI and Fnu4HI restriction map of linearised pUC18 before and after methylation with M.SssI. The positions of 5'-DGCGGCH-3', 5'-DGCWGCG-3', 5'-CGCWGCG-3' and 5'-DGCSGCG-3' are also shown (D = A, G or T, H = A, C or T, W = A or T, S = C or G). Footnotes: a, overlapping 5'-DGCWGCH-3' sites; b, overlap between a 5'-DGCSGCG-3' site and a 5'-DGCWGCG-3' site; c, complete cutting of a 5'-DGCGGCH-3' site by BsoFI; d, early cutting at a 5'-DGCWGCG-3' site; e, late cutting at a 5'-DGCWGCG-3' site. The dotted line (BsoFI digest) indicates partial cutting of a 5'-DGCGGCH-3' site as suggested by the reduced intensity of the bands at 273 and 484 bp in lane 10 of Figure 1.

Because of the variable pattern observed with BsoFI at methylated 5'-DGCGGCH-3' sites, we investigated whether variable rates of cleavage could account for the observations with this enzyme. This phenomenon is well described at sites of non-cognate methylation with other restriction enzymes and may depend on the particular sequences adjacent to the restriction sites. Methylated plasmid was incubated with 10 times excess of BsoFI and the digest sampled at 1, 2, 4, 8 and 16 h. The results are presented in Figure 3 A. In the 1 h digest, the bands at 206, 286, 303, 328 and 366 bp arise from cleavage at unmethylated (non-methylatable) sites. A band at 102 bp is also present at this stage. This band results from cleavage at a methylated 5'-DGCWGCG-3' site (also see d, Fig. 2 ). As the digest proceeds, the band at 102 bp increases in intensity and there is evidence of cleavage at a further three methylated sites. The appearance of bands at 73 and 213 bp result from cleavage at the second methylated 5'-DGCWGCG-3' site (also see e, Fig. 2 ). Finally, in the 16 h digest, additional bands (see arrows, Fig. 3 A) can be seen at 104 and 109 bp. These result from cleavage at a methylated 5'-DGCGGCH-3' site (see c, Fig. 2 ). Consequently, there is loss of the fragment at 286 bp but this is replaced by a faint band at 273 bp which runs in an almost identical position. The latter results from partial cleavage at a 5'-DGCGGCH-3' site within the 484 bp fragment (see dotted line, Fig. 2 ).


Figure 3.(A) Time course for the digestion of CpG methylated pUC18 in 10 times excess of BsoFI. An aliquot of 2.4 [mu]g of pUC18 DNA was methylated according to the protocol in Figure 1. An aliquot of 2 [mu]g of the methylated plasmid was digested with 20 U of BsoFI in a 100 [mu]l reaction mix at 55oC under oil. Aliquots (20 [mu]g) were removed at the times shown, placed on ice and precipitated immediately in ethanol. Aliquots of 0.4 [mu]g of the unmethylated and methylated pUC18 were also digested with 4 U of HpaII for 16 h to demonstrate completion of the CpG methylation. The digests were resuspended in water and electrophoresed on a 5% non-denaturing polyacrylamide gel with a 1 kb ladder (Gibco BRL). The arrows mark the positions of bands at 104 and 109 bp. These bands are present only in the 16 h digest. The result of the time course experiment is discussed in the text. (B) Table depicting the sequences surrounding the four 5'-DGCGGCH-3' sites within pUC18. These sites show marked differences in their susceptibility to cleavage by BsoFI.

Hence the time course demonstrates that CpG methylation results in slower rates of cleavage with BsoFI at certain restriction sites. The two methylated 5'-DGCWGCG-3' sites in our plasmid were cleaved, but at variable rates. Also, two of the four methylated 5'-DGCGGCH-3' sites were cut slowly by BsoFI, cleavage being apparent only after >8 h incubation with 10 times excess of enzyme. The remaining two sites failed to cut even after prolonged digestion in excess of enzyme. Conversely, all methylated 5'-CGCWGCG-3' sites and 5'-DGCSGCG-3' sites failed to cut with BsoFI. Digestion of the methylated plasmid to completion (25 times excess of enzyme for 20 h) with each of the restriction enzymes studied did not result in any additional digestion over that observed in Figure 1 (data not shown).

Variation in the rate of cleavage by BsoFI at sites containing non-cognate methylation probably depends on the particular sequences surrounding these sites. However, we have been unable to identify a common sequence motif surrounding the 5'-DGCGGCH-3' sites which confers additional resistance to BsoFI when the restriction site is methylated. To illustrate this, the sequences surrounding these sites are presented in Figure 3 B.

In summary, the methylation sensitivity of ItaI is distinct from that of its isoschizomers. ItaI is inhibited solely by internal CpG methylation. Fnu4HI and Fsp4HI are inhibited by either internal CpG or overlapping CpG methylation. BsoFI demonstrates a differential rate-of-cleavage effect at sites containing internal methylated CpG (5'-DGCGGCH-3' sites) and sites containing a single overlapping CpG (5'-DGCWGCG-3' sites), but is consistently sensitive both to bilateral overlapping CpG methylation (5'-CGCWGCG-3' sites) and a combination of internal and overlapping CpG methylation (5'-DGCSGCG-3' sites).

ACKNOWLEDGEMENTS

Dr B. Ramsahoye is supported by the Medical Research Council of the UK. We would like to thank Dr Derek Bowen for advice in the preparation of this manuscript.

REFERENCES

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*To whom correspondence should be addressed. Tel: +44 1222 744528; Fax: +44 1222 744523; Email: ramsahoye@cardiff.ac.uk
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