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Genome-wide quantitative assessment of variation in DNA methylation patterns.

Xie H, Wang M, de Andrade A, Bonaldo Mde F, Galat V, Arndt K, Rajaram V, Goldman S, Tomita T, Soares MB - Nucleic Acids Res. (2011)

Bottom Line: However, little is known about genome-wide variation of DNA methylation patterns.We further identified 12 putative allelic-specific methylated genomic loci, including four Alu elements and eight promoters.Lastly, using subcloned normal fibroblast cells, we demonstrated the highly variable methylation patterns are resulted from low fidelity of DNA methylation inheritance.

View Article: PubMed Central - PubMed

Affiliation: Falk Brain Tumor Center, Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago IL 60614-3394, USA. hxie@childrensmemorial.org

ABSTRACT
Genomic DNA methylation contributes substantively to transcriptional regulations that underlie mammalian development and cellular differentiation. Much effort has been made to decipher the molecular mechanisms governing the establishment and maintenance of DNA methylation patterns. However, little is known about genome-wide variation of DNA methylation patterns. In this study, we introduced the concept of methylation entropy, a measure of the randomness of DNA methylation patterns in a cell population, and exploited it to assess the variability in DNA methylation patterns of Alu repeats and promoters. A few interesting observations were made: (i) within a cell population, methylation entropy varies among genomic loci; (ii) among cell populations, the methylation entropies of most genomic loci remain constant; (iii) compared to normal tissue controls, some tumors exhibit greater methylation entropies; (iv) Alu elements with high methylation entropy are associated with high GC content but depletion of CpG dinucleotides and (v) Alu elements in the intronic regions or far from CpG islands are associated with low methylation entropy. We further identified 12 putative allelic-specific methylated genomic loci, including four Alu elements and eight promoters. Lastly, using subcloned normal fibroblast cells, we demonstrated the highly variable methylation patterns are resulted from low fidelity of DNA methylation inheritance.

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The methylation status of CpG/CpG dyads in subcloned human normal lung fibroblast determined by hairpin-bisulfite PCR. (A) Bisulfite sequencing results for genomic locus 1 (chr9:139174924-139175041). (B) Bisulfite sequencing results for genomic locus 2 (chr10:134480046-134480230). The methylated cytosines are indicated with filled circles while unmethylated cytosines are indicated with open circles. The bold curved lines represent hairpin linkers connected to both complementary strands. The symmetrical methylation statuses of CpG/CpG dyads indicate an accurate methylation inheritance, while asymmetrical methylation status (hemimethylated) indicates a failure in the transmission of methylation status or a de novo methylation event.
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Figure 5: The methylation status of CpG/CpG dyads in subcloned human normal lung fibroblast determined by hairpin-bisulfite PCR. (A) Bisulfite sequencing results for genomic locus 1 (chr9:139174924-139175041). (B) Bisulfite sequencing results for genomic locus 2 (chr10:134480046-134480230). The methylated cytosines are indicated with filled circles while unmethylated cytosines are indicated with open circles. The bold curved lines represent hairpin linkers connected to both complementary strands. The symmetrical methylation statuses of CpG/CpG dyads indicate an accurate methylation inheritance, while asymmetrical methylation status (hemimethylated) indicates a failure in the transmission of methylation status or a de novo methylation event.

Mentions: Using a hairpin-linker ligated to restriction-enzyme-digested genomic DNA, Laird and colleagues successfully obtained methylation information for the two complementary DNA strands simultaneously (18). In this study, genomic DNA isolated from subcloned normal fibroblast cells was first digested with the methylation-insensitive TaqI restriction endonuclease, and then ligated to hairpin linkers. After bisulfite conversion and PCR cloning, sequencing reactions were conducted to generate methylation profiles for two genomic loci that exhibited high methylation entropies in normal cerebellum and in ependymomas (Supplementary Figure S3). More detailed methylation patterns of these two genomic loci can be visualized at http://cmbteg.childrensmemorial.org/cgi-bin/gbrowse/btech (32). To ensure an accurate calculation of the fidelity of inheritance of DNA methylation, the sequence reads with incomplete bisulfite conversion (containing unconverted cytosines at non-CpG dinucleotides) were discarded. A total of 27 sequence-reads comprising 12 distinct methylation patterns and 30 sequence-reads comprising 10 distinct methylation patterns were obtained for genomic locus 1 (chr9:139174924-139175041) and genomic locus 2 (chr10:134480046-134480230), respectively. Both genomic loci exhibited high methylation variations, similarly to those observed in normal cerebellum and in ependymomas (Figure 5).Figure 5.


Genome-wide quantitative assessment of variation in DNA methylation patterns.

Xie H, Wang M, de Andrade A, Bonaldo Mde F, Galat V, Arndt K, Rajaram V, Goldman S, Tomita T, Soares MB - Nucleic Acids Res. (2011)

The methylation status of CpG/CpG dyads in subcloned human normal lung fibroblast determined by hairpin-bisulfite PCR. (A) Bisulfite sequencing results for genomic locus 1 (chr9:139174924-139175041). (B) Bisulfite sequencing results for genomic locus 2 (chr10:134480046-134480230). The methylated cytosines are indicated with filled circles while unmethylated cytosines are indicated with open circles. The bold curved lines represent hairpin linkers connected to both complementary strands. The symmetrical methylation statuses of CpG/CpG dyads indicate an accurate methylation inheritance, while asymmetrical methylation status (hemimethylated) indicates a failure in the transmission of methylation status or a de novo methylation event.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105398&req=5

Figure 5: The methylation status of CpG/CpG dyads in subcloned human normal lung fibroblast determined by hairpin-bisulfite PCR. (A) Bisulfite sequencing results for genomic locus 1 (chr9:139174924-139175041). (B) Bisulfite sequencing results for genomic locus 2 (chr10:134480046-134480230). The methylated cytosines are indicated with filled circles while unmethylated cytosines are indicated with open circles. The bold curved lines represent hairpin linkers connected to both complementary strands. The symmetrical methylation statuses of CpG/CpG dyads indicate an accurate methylation inheritance, while asymmetrical methylation status (hemimethylated) indicates a failure in the transmission of methylation status or a de novo methylation event.
Mentions: Using a hairpin-linker ligated to restriction-enzyme-digested genomic DNA, Laird and colleagues successfully obtained methylation information for the two complementary DNA strands simultaneously (18). In this study, genomic DNA isolated from subcloned normal fibroblast cells was first digested with the methylation-insensitive TaqI restriction endonuclease, and then ligated to hairpin linkers. After bisulfite conversion and PCR cloning, sequencing reactions were conducted to generate methylation profiles for two genomic loci that exhibited high methylation entropies in normal cerebellum and in ependymomas (Supplementary Figure S3). More detailed methylation patterns of these two genomic loci can be visualized at http://cmbteg.childrensmemorial.org/cgi-bin/gbrowse/btech (32). To ensure an accurate calculation of the fidelity of inheritance of DNA methylation, the sequence reads with incomplete bisulfite conversion (containing unconverted cytosines at non-CpG dinucleotides) were discarded. A total of 27 sequence-reads comprising 12 distinct methylation patterns and 30 sequence-reads comprising 10 distinct methylation patterns were obtained for genomic locus 1 (chr9:139174924-139175041) and genomic locus 2 (chr10:134480046-134480230), respectively. Both genomic loci exhibited high methylation variations, similarly to those observed in normal cerebellum and in ependymomas (Figure 5).Figure 5.

Bottom Line: However, little is known about genome-wide variation of DNA methylation patterns.We further identified 12 putative allelic-specific methylated genomic loci, including four Alu elements and eight promoters.Lastly, using subcloned normal fibroblast cells, we demonstrated the highly variable methylation patterns are resulted from low fidelity of DNA methylation inheritance.

View Article: PubMed Central - PubMed

Affiliation: Falk Brain Tumor Center, Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago IL 60614-3394, USA. hxie@childrensmemorial.org

ABSTRACT
Genomic DNA methylation contributes substantively to transcriptional regulations that underlie mammalian development and cellular differentiation. Much effort has been made to decipher the molecular mechanisms governing the establishment and maintenance of DNA methylation patterns. However, little is known about genome-wide variation of DNA methylation patterns. In this study, we introduced the concept of methylation entropy, a measure of the randomness of DNA methylation patterns in a cell population, and exploited it to assess the variability in DNA methylation patterns of Alu repeats and promoters. A few interesting observations were made: (i) within a cell population, methylation entropy varies among genomic loci; (ii) among cell populations, the methylation entropies of most genomic loci remain constant; (iii) compared to normal tissue controls, some tumors exhibit greater methylation entropies; (iv) Alu elements with high methylation entropy are associated with high GC content but depletion of CpG dinucleotides and (v) Alu elements in the intronic regions or far from CpG islands are associated with low methylation entropy. We further identified 12 putative allelic-specific methylated genomic loci, including four Alu elements and eight promoters. Lastly, using subcloned normal fibroblast cells, we demonstrated the highly variable methylation patterns are resulted from low fidelity of DNA methylation inheritance.

Show MeSH
Related in: MedlinePlus