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J. Biol. Chem., Vol. 280, Issue 15, 14413-14419, April 15, 2005
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From the
Departments of Biology, ||Genome Sciences, and **Medicine University of Washington, Seattle, Washington 98195
Received for publication, December 8, 2004 , and in revised form, February 1, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To overcome these inherent limitations of conventional bisulfite methylation analysis for determining methylation patterns on both strands of L1 repeat elements, we have used a double-strand PCR method, "hairpin-bisulfite PCR" (9). With this approach, the limitations of single-strand PCR can be avoided. Strand biases do not occur because both strands are linked prior to PCR amplification. Bisulfite-converted cytosines can be distinguished from evolutionary C-to-T transitions in L1 sequences by the presence, in the former class, of a guanine on the complementary strand, and information from the exact complementary strands of an individual allele is preserved, allowing allele and cell variation to be studied (9).
Our initial hairpin-bisulfite PCR data on L1 sequences using DNAs from two adult human fibroblast lines revealed levels of hemimethylated CpG dyads that were somewhat higher (14.1 and 10.0%, respectively) than levels observed for a single-copy gene, FMR1, in lymphocytes (6.4%) (9) but considerably lower than the 50–80% hemimethylation levels inferred from other published data (11). We now present additional data on the concordance of methylation at CpG dyads within the promoter region of human L1 sequences. Our data (i) distinguish valid from redundant and contaminant sequences by introducing, for repeated sequences, a powerful new method using molecular barcodes, (ii) resolve the controversy on the level of hemimethylation of L1 sequences in fetal fibroblasts, (iii) present data on the concordance of CpG methylation on complementary strands in L1 sequences from other human cell types, and (iv) assess the methylation status of non-CpG cytosines within the regions analyzed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We examined L1 CpG and non-CpG methylation within a 104-bp promoter region of human L1 sequences, a region that contains nine CpG sites corresponding to those between nucleotides 219 and 323 as reported by Woodcock et al. (11). A more limited set of data from a variety of cell types was obtained for a 118-bp promoter region of L1, focusing on the possibility of non-CpG methylation between nucleotides 328 and 446.
We used hairpin-bisulfite PCR to assess methylation status on both strands of individual DNA molecules (9) with the following modifications. 2–5 µg of genomic DNA were cleaved by 25 units of restriction endonuclease BsmA1 for 1 h at 55 °C. For samples of fetal fibroblasts, a molecular-barcoded hairpin linker, either 5'-PO4-ACCAAGCGATGCGTTDDDDDDDCGAGCATCGCT-3' or 5'-PO4-ACCAAGCGATGCDDDDDDDGCATCGCT-3' in which D represents random incorporation of A, G, or T (for review see Ref. 16) was ligated to BsmAI-cleaved genomic DNA for 15 min at 20 °C using 400 units of New England Biolabs T4 DNA ligase in 20 µl with 1x ligation buffer. For DNA samples other than fetal fibroblasts, ligation of hairpin linkers 5'-PO4ACCAAGCGATGCGTTCGAGCATCGCT-3' or 5'-PO4-TGCTAGCGATGCGGTTGAGCATCGCT-3' for the 104- and 118-bp regions, respectively, to BsmAI-cleaved genomic DNA was for 15 min at 20 °C using 400 units of New England Biolabs T4 DNA ligase in 20 µl with 1x ligation buffer.
Bisulfite-conversion cycling conditions were as follows. Hairpin-ligated DNA was denatured in 0.3 M NaOH for 20 min and then heated to 100 °C for 2 min before the addition of 3.4 M sodium bisulfite and 1 mM hydroquinone. The reaction mixture was incubated at 99.9 °C for 1 min followed by 10 cycles of 55 °C for 15 min and 99 °C for 1 min 30 s. Cycling was completed at 55 °C for 1 h and 10 °C for 9 h. A detailed schematic representation of the hairpin-bisulfite PCR method is shown by Laird et al. (see Fig. 2 and supporting Fig. 6 in Ref. 9). Post-bisulfite PCR conditions, TOPO TA cloning reactions, and sequencing of all of the samples were carried out as described previously (9) with the exception of the 118-bp region of the promoter for which PCR primers 5'-CTCCACCCAATTC(A/G)AACTTCCC-3' and 5'-CTAACTTTGGGTAATGG(C/T)GGG(C/T)G-3' were used and optimized with Failsafe buffer F (Epicenter). We also routinely clean the underside of the PCR machine lid with DNA Zap (Ambion). We have confirmed through barcoding that this procedure reduces contamination substantially.
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1 unconverted non-CpG cytosine per sequence) were included in our analysis except as described in the next paragraph. The average frequencies of converted non-CpG cytosines in the sequences used for our analysis were 99.4 and 99.8% for the 104- and 118-bp regions, respectively. Sequences with one or more hemimethylated CpG dyads from the 104-bp region had an equally high level of non-CpG conversion efficiency (99.5%) compared with the mean, indicating that hemimethylated dyads are intrinsic to the DNA analyzed and are not a consequence of incomplete conversion of unmethylated CpG dyads. To provide approximately equal representation of data from each of the four passages of fetal fibroblasts analyzed, here we include data from the first 15 well-converted sequences obtained from each passage with the exception of passage 22 from which only 12 well-converted sequences were recovered. To search for possible non-CpG cytosine methylation in the 118-bp region, we also analyzed 14 L1 sequences from human sperm DNA that were chosen because of their lower conversion efficiency. These lower levels of conversion are not often observed with our normal protocol but were generated in some experiments by using fewer denaturation steps or by moving the denaturation steps to the end of the bisulfite procedure.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We analyzed nine of the 22 CpG consensus sites within the L1 promoter (11). For these nine sites, the methylation percentages ranged from 69 to 90% in fetal fibroblast sequences and the two complementary strands had approximately equal levels of methylation (Fig. 2A). The levels of sense-strand and antisense-strand methylation were highly correlated at these nine sites (r2 = 0.78). In contrast to our data presented here, methylation levels of L1 sequences from fetal fibroblasts were reported by Woodcock et al. (11) to be very different between strands. Our analysis of their data indicates that sense-strand methylation averaged 99.4% and that antisense-strand methylation averaged 40% for these CpG sites in their sequences (Fig. 2B). Such discordance in the levels of strand methylation would, if correct, imply high levels of hemimethylation. To search for possible explanations for these differences, we asked whether or not the passaging of fetal fibroblasts in cell culture significantly altered the level of hemimethylation. No systematic differences in the levels of hemimethylation were observed for DNA from cell passages 10, 14, 17, and 22 (Fig. 3). The percentages of CpG dyads hemimethylated in L1 sequences from these four passages were 9.5, 8.5, 7.4, and 7.5%, respectively, values that are not significantly different from the average of 8.3% (p > 0.50).
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We also analyzed DNAs from peripheral blood leukocytes, lymphoblastoid cells, sperm, and previously established adult fibroblasts (9). Unlike fetal fibroblast DNAs, these other DNAs did not have molecular barcodes incorporated within the hairpin linker. Each sequence included in our analysis did, however, have a unique methylation pattern (for example, see Fig. 3 in Ref 9), providing confidence that contamination and limited-template redundancy did not bias these data. DNAs from these other tissues and cell types had levels of hemimethylation ranging from 4.8 to 20.8% (Table I).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Technical Aspects of Hairpin-Bisulfite PCR and Distinguishing Valid L1 Sequences from Redundant and Contaminant Sequences Using Molecular Barcodes—We can be confident that the sense- and antisense-strand methylation patterns presented here represent information from a single allele and locus, even when a sequence is repeated many times in the human genome as is the case for L1 sequences. The hairpin-bisulfite PCR technology provides information on methylation patterns on the complementary strands of individual DNA molecules. The complementary strands remain attached via the hairpin linker throughout the bisulfite conversion and PCR amplification, cloning, and sequencing steps. Thus, this method eliminates the problems of biases in sense and antisense methylation analysis because both strands are amplified in a single step (Fig. 1).
Two technical considerations reinforce the conclusion that the inferred methylation patterns from our hairpin-bisulfite methylation analyses, including the hemimethylated dyads, are an accurate reflection of patterns on both strands of individual L1 sequences. As described under "Materials and Methods," the frequency of hemimethylated dyads is considerably higher than expected from inefficient bisulfite conversion. Additionally, sequences that are the likely result of cross-over PCR are extremely rare. If template switching were to occur during PCR amplification, we would observe sequences in which the sense and antisense strands were not precisely complementary because of the sequence divergence among members of the L1 family. In our initial publication (9) on hairpin-bisulfite PCR, we reported that no case of detectable cross-over PCR had been observed in >200 analyzed L1 sequences. Our most recent analysis of 636 L1 sequences that met our criteria for efficient bisulfite conversion revealed four sequences that were probably the result of cross-over PCR. These four sequences were not included in further analyses. We conclude that the rate of cross-over PCR among analyzed L1 sequences is somewhat <1/100 sequences. Thus, the frequencies of hemimethylated dyads reported here are unlikely to be significantly inflated by problems of inefficient bisulfite conversion or crossover PCR.
The fetal fibroblast sequences presented here (Fig. 1) have unique barcodes, thus verifying that they are appropriately amplified sequences rather than re-amplified products or contaminants. We introduced molecular barcodes for hairpin-bisulfite PCR of a single-copy gene, FMR1, to distinguish valid sequences from redundant examples of an allele that had previously been obtained as well as from contaminant sequences (16). Similar use of molecular barcodes is reported here for the family of repeated L1 elements. The molecular barcodes have in most cases identified valid sequences that also had distinctive methylation patterns. Contamination from previous PCR reactions is identified by redundant matching barcodes in PCR products and, in most cases, by the identity of the inferred methylation pattern. For completely methylated or unmethylated sequences, however, the molecular barcodes are indispensable in determining whether a sequence is from an L1 allele not previously sampled. This information is crucial for accurate quantification of various methylation states among alleles and cells, as exemplified in Fig. 1, and described more fully under "Results."
Levels of Hemimethylation in L1 Sequences from Fetal Fibroblasts in Cell Culture—The most frequently recovered L1 sequences from fetal fibroblasts were densely methylated with primarily concordant methylation patterns at consensus CpG dyads (Fig. 1). From our larger data set of 471 CpG dyads (57 L1 sequences) from fetal fibroblast DNA, 91.7% (n = 432) were concordantly methylated and 8.3% (n = 39) were hemimethylated. Among these 57 sequences, only two (3.5%) were observed that did not fit the pattern of hypermethylation. Distinctive barcodes indicated that these two sequences were derived from different genomic L1s (Fig. 1). CpG dyads in these two sequences were strikingly concordant in their methylation status in that all of the dyads were fully unmethylated. Site-specific variations in methylation were observed in fetal fibroblast L1s, but the two complementary strands had approximately equal and highly correlated levels of methylation (Fig. 2A). Thus, we observe primarily concordant methylation in both hypermethylated and hypomethylated L1 sequences and low levels of hemimethylation at all of the nine CpG sites analyzed.
Our data on levels of hemimethylation in L1 sequences of fetal fibroblasts conflict with data reported by Woodcock et al. (11). These authors analyzed the methylation patterns of sense and antisense strands independently using bisulfite conversion and concluded that there were large differences between strand methylation. Specifically, Fig. 3 in Ref. 11 indicates that sense-strand methylation in their analyzed sequences was close to 100%, whereas antisense-strand methylation averaged 
40% (see Fig. 2B). If correct, these data would indicate that most of the CpG sites in this region (59%) were hemimethylated. In contrast, our data indicate that sense- and antisense-strand methylation in this region are highly concordant with an average level of hemimethylation of only 8.3% (Fig. 2A). The levels of hemimethylation in L1s from these fetal fibroblasts were not significantly different between samples from the four passages. Thus, it seems unlikely that different stages of passaging of fetal fibroblasts in tissue culture would explain these different estimates of L1 hemimethylation (Fig. 3).
The validity of conclusions drawn from the independent analysis of the two complementary strands of DNA relies on the assumption that the independent PCR amplification and cloning of the complementary strands is unbiased and that the separate amplifications of sense and antisense strands draw from the same population of previously double-stranded molecules. This assumption is not always valid. Warnecke et al. (18), for example, provided evidence of strand bias for the retinoblastoma tumor suppressor gene when using single-strand PCR. Further studies indicated that unmethylated T-rich DNA had been amplified preferentially, thus leading to a PCR bias and to an inaccurate estimate of methylation (19). Variations in methylation among individual L1 elements, including a low percentage of hypomethylated alleles (Fig. 1), provide ample opportunities for observing apparent strand difference under conditions of strand-biased PCR. Efforts to control for methylated versus unmethylated bias using cloned sequences (11) may not be sufficient within the context of single-strand DNA amplification of repeated sequences that vary in sequence as well as methylation status.
Methylation of L1 Sequences from Different Cell Types—We asked whether or not levels of hemimethylation of CpG dyads in L1s from different cell types are comparable with those that we observe from cultured fetal fibroblasts. L1 sequences from DNAs of other human cells had levels of hemimethylation that spanned the average observed for fetal fibroblasts ranging from 4.8% in sperm to 20.8% in a leukocyte sample. Although these hemimethylation values show considerable variation, none approaches the high level implicit in data from Woodcock et al. (11).
Methylation Status of Non-CpG Cytosines in the L1 Promoter—Methylated cytosines that are not within CpG contexts are occasionally reported (11). Consistent methylation at such non-CpG cytosines, if verified, would be interesting in part because the mechanism of methylation inheritance would be different from that for CpG methylation, which depends on the symmetry of CpG/CpG dyads (4–6). For the 104-bp region of the human L1 promoter, we confirmed the observation of Woodcock et al. (11) that non-CpG cytosines in this region are rarely, if ever, methylated in human fetal fibroblasts (Fig. 4A).
These authors (11) did, however, report that two non-CpG cytosines within the 118-bp region at nucleotides 432 and 436 of the human L1 promoter are consistently unconverted and hence are possibly methylated. We find no support for this conclusion in our hairpin-bisulfite PCR data. In 64 well-converted sequences (>98.6% conversion), from two adult fibroblast lines, leukocytes, and lymphoblastoid cells, we found that these two cytosines were always converted and hence unmethylated (Fig. 4B). To explore the possibility that we missed non-CpG cytosine methylation by selecting only well-converted sequences, we examined a small number of less-well converted L1 sequences from human sperm DNA. In 14 sequences with 90–98% conversion of non-CpG cytosines, only one instance of non-conversion at site 432 was observed for these two sites (Fig. 4C). Other unconverted cytosines in these poorly converted sequences occurred most often in the region with three closely spaced CpGs (Fig. 4C). Regions with high CG content, especially when hypermethylated, are refractory to denaturation, which will inhibit bisulfite conversion (17). Although our data on these two cytosines were not from fetal fibroblasts, DNAs from two adult fibroblasts lines and from two other cell types were examined. It is possible that L1s in fetal fibroblasts are different from other L1s, but we consider it more likely that incomplete conversion at these sites and subsequent PCR redundancy explain the previous conclusion of methylation of several specific non-CpG cytosines (11). Therefore, we conclude that non-CpG cytosines in these regions are rarely, if ever, methylated including at nucleotides 432 and 436.
Concluding Remarks—For human L1 promoter sequences, our results resolve a controversy in the literature. Our data are consistent with high levels of concordant CpG methylation on complementary strands, hence with low levels of hemimethylation inferred by Hansen (3) and Laird et al. (9), and are inconsistent with high levels of hemimethylation reported by Woodcock et al. (11). Data from a variety of cell types, including cultured fetal fibroblasts, support this conclusion. We also report that two non-CpG cytosines within the L1 promoter reported by Woodcock et al. (11) to remain unconverted during bisulfite conversion are routinely converted in our hairpin-bisulfite PCR procedure in DNAs from different cell types. We conclude that these and all of the other non-CpG cytosines in the regions analyzed are rarely, if ever, methylated. The methods of analysis used here for L1 sequences are applicable to other repeated sequences for which methylation analyses on complementary strands are especially difficult. The application of these experimental approaches will likely provide insights into the origins and consequences of variations in methylation patterns.
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FOOTNOTES |
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¶ Present address: Dept. of Biological Sciences, Stanford University, Stanford, CA 94305.
To whom correspondence may be addressed: Dept. of Biology, University of Washington, Box 351800, Seattle, WA 98195. Tel.: 206-616-9385; Fax: 206-543-3041; E-mail: afb4{at}u.washington.edu (A. F. B.) or cdlaird{at}u.washington.edu (C. D. L.).
1 The abbreviation used is: L1, LINE1.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and antisense-strand (
) sequences were 80.4 and 81.3%, respectively. B, methylation levels reported by Woodcock et al. (
A/T transitions. In examples of divergence, information on the complementary strands of individual DNA duplexes distinguishes evolutionary changes from PCR errors (
98.2% conversion of non-CpG cytosines (representing zero or one non-conversion event per sequence) were used for analysis. Dashes within the sequence indicate sequencing errors resulting from uncalled bases. The first four analyzed sequences from passage 10 (top four) and passage 22 (next four) are shown here. Additionally, two fully unmethylated sequences from passage 14 were observed (bottom two). Distinctive barcodes indicate that these 10 sequences were derived from different genomic L1s. For example, sequence AB307 had a barcode of 5'-AATGGCA-3' and sequence AB313 had a barcode of 5'-TTAGGGT-3'. For the former barcode, the C is either a PCR error (because cytosines were not included in the barcodes (
) representing the earliest sample and DNA from passage 22 (x) representing the last sample from these cells. Hemimethylation data for DNA from intermediate passages 14 (