I searched via Google Scholar to see if it is currently possible to map the human genome or that of any other mammal. The human genome was "mapped" in terms of four bases before the discovery of another two nucleotides, viz., 5-hydroxymethylcytosine and 5-methylcytosine, which are crucial to mammalian development. I found this paper:
Nucleic Acids Research Advance Access published online on April 5, 2010
Nucleic Acids Research, doi:10.1093/nar/gkq223
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Methods Online
Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine
Seung-Gi Jin, Swati Kadam and Gerd P. Pfeifer*
Department of Cancer Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA
*To whom correspondence should be addressed. Tel: +1 626 301 8853 begin_of_the_skype_highlighting +1 626 301 8853 end_of_the_skype_highlighting; Fax: +1 626 358 7703; Email: gpfeifer@coh.org
Received November 5, 2009. Revised March 12, 2010. Accepted March 17, 2010.
DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base.
Showing posts with label 5-hydroxymethylcytosine. Show all posts
Showing posts with label 5-hydroxymethylcytosine. Show all posts
Thursday, July 29, 2010
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.
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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