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Wednesday, January 27, 2010

The usual excuses why genetics is all messed up

5-methylcytosine (5mC) and 5-hydroxymethylcytosine (hmC) are bases in mammalian DNA. How could no one have noticed? Does anyone care besides me?

Why has hmC been overlooked as a normal constituent of mammalian DNA? With the exception of two papers that reported high levels of hmC in genomic DNA isolated using unconventional but not standard methods, most previous studies describe hmC as a rare base that is a probable oxidation product of 5mC. One reason that hmC may have been missed is that it might be present at detectable levels only in specific cell types 15 (ES cells and not differentiated cells). Another factor may be the relatively low abundance of hmC. In ES cells, hmC is ~4% of all cytosine species in CpG dinucleotides located in MspI cleavage sites (CCGG). CpG is ~0.8% of all dinucleotides in the mouse genome, thus hmC constitutes ~0.032% of all bases or ~1 in every 3000 nucleotides. For comparison, 5mC is 55-60% of all cytosines in CpG dinucleotides in MspI cleavage sites, about 14-fold higher than hmC. A more trivial explanation is that some TLC running buffers do not resolve hmC from C. We were fortunate that our experimental design led us to focus on areas of the genome containing CpG dinucleotides (and hence enriched for 5mC), and that our TLC running conditions could distinguish hmC.

A full appreciation of the biological significance of hmC will depend heavily on the development of tools that allow hmC, 5mC and C to be distinguished unequivocally. We show here that two of the three most commonly used techniques do not meet this criterion. A widely-used mouse monoclonal antibody to 5mC apparently does not recognize hmC by immunocytochemistry, thus it will be important to reevaluate previous reports of DNA demethylation based solely on the use of this antibody. Similarly,the methylation-sensitive restriction enzyme, HpaII, fails to cut hmC as previously reported, raising the possibility that in some instances hmC-modified DNA was incorrectly judged to be methylated. Another methylation-sensitive restriction enzyme, McrBC, is already known to cleave 5mC- and hmC-containing DNA equivalently, and therefore also does not allow these two nucleotides to be distinguished. It is yet to be determined how bisulfite modification analysis interprets the presence of hmC in DNA. Treatment of DNA with sodium bisulfite promotes the spontaneous deamination of cytosine to uracil, while leaving 5mC unaffected; amplification of the sequence of interest followed by sequencing allows the precise methylation patterns at a given sequence to be determined. It is known that bisulfite reacts rapidly with hmC at the C5 to form a stable cytosine 5-methylenesulfonate adduct, which is not readily deaminated. This substituted species, which is expected to form base pairs similar to those formed by cytosine, could be read by polymerases as C during the amplification steps, resulting in the sequence being interpreted as containing 5mC. Alternatively, polymerases may not copy cytosine 5-methylenesulfonate efficiently, in which case the DNA containing this adduct would not be amplified effectively and the sequence containing the original hmC modification would be underrepresented in the amplified DNA. Notably, disruptions of the TET1 and TET2 genetic loci have been reported in association with hematologic malignancies. A fusion of TET1 with the histone methyltransferase, MLL, has been identified in at least two cases of acute myeloid leukemia (AML) associated with t(10;11)(q22;q23) translocation. Homozygous null mutations and chromosomal deletions involving the TET2 locus have been found in AML and myeloproliferative disorders, suggesting a tumor suppressor function for TET2. It will be interesting to test the involvement of TET proteins and hmC in oncogenictransformation and malignant progression.

This excerpt is from doi:10.1126/science.1170116

The DNA of other species are known to contain additional bases such as beta-D-glucosyl-hydroxymethyluracil. Additional DNA bases are widespread in nature. They can be found in eukaryotes, prokaryotes, and bacteriophages. They may completely replace the standard base or replace only a small fraction. Their constituents vary from simple methyl or hydroxy groups to large moieties like amino acids and multiply hexosylated side chains.

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