CpG dinucleotides

The fact that CpG dinucleotides exist in an unmethylated state in CpG islands is amazing, given that the frequency of CpGs in such islands is six to ten times higher than in bulk DNA [17] (Table 1). A great deal of investigation has been and is being... 

Fig. 2. Distribution of methylated CpGs in genomic DNA and their effect on transcription. 70-80% of all CpG dinucleotides are methylated in vertebrate genomes. mCpGs (filled lollipops) are randomly distributed throughout the genome but are excluded from regions with unusually high CpG density—CpG islands [16,17]. Most of the CpG islands are associated with gene promoters and maintained unmethylated (white lollipops) in all types of somatic cells. Aberrant methylation of CpG islands occurs in cancer cells and leads to silencing of tumor-suppressor and other essential genes [19-21].

DNMTs catalyze the methylation of cytosines located 5 to a guanosine as part of a CpG dinucleotide (CpG) in DNA to 5-methylcytosines using S-adenosyl methionine... 

Figure 8.1 CpG dinucleotide as part of the DNA. The arrow indicates the methylation site.

This massive amount of information should not be considered as insurmountable or only material to be marveled at but not understood. Much of the chemistry is already available to mine this information successfully. Much of it is understandable in somewhat simple terms, generally only after we have discovered the key to this simplicity. For instance, there is a marked decrease in the frequency of the dinucleotide CpG in some areas of the genome. The deficiency is believed to be due to the fact that most CpG nucleotides are methylated on the cytosine base, and spontaneous deamination of the methyl-cytosine residue creates T residues. Thus, CpG dinucleotide sequences mutate to TpG dinucleotides. But there still remain some questions. There are certain regions or islands where the CpG sequences exist in a nonmethylated form and where the frequency of CpG occurs within the expected or normal rate. Why These CpG islands are of particular interest because they are associated with the 5 ends of genes. 

It has recently been established that bacterial DNA, but not vertebrate DNA, has direct immunostimulatory effects on leukocytes in vitro. The immunostimulatory effect is due to unmethylated CpG dinucleotides, which are underrepresented and methylated in vertebrate DNA. CpG DNA and synthetic oligonucleotides from a variety of sources have shown significant promise as new adjuvants (10-12). CpG induces a strong Thl response, mainly by stimulating cytokine induction and through the expression of costimulatory molecules on antigen-presenting cells. CpG is currently in clinical trials and may become part of a licensed product in the future. 

Figure 22.1 Optimal CpG motifs. The stimulatory effects of a particular sequence of DNA are determined by the base context in which the CpG dinucleotides appear. The most immune stimulatory CpG motifs are preceded by any base except a C, and followed by any base except a G. The positions of two bases further from the CpG are less important, although a purine on the 5 side and pyrimidines on the 3 side generally lead to a more immune stimulatory CpG motif. Although there is some cross-stimula-tion, the preferred human and mouse motifs are slightly different, as shown above.

In parallel to these initial descriptions, observations in the antisense field of study revealed that some antisense ODNs displayed sequence-specific immunostimulatory properties (Branda et al., 1993 Krieg et al., 1989, 1993 McIntyre et al., 1993 Pisetsky and Reich 1994 Tanaka et al., 1992). Exhaustive study revealed that immunostimulation by these ODNs could be attributed to the presence of CpG dinucleotides, in particular to base contexts (most importantly a hexamer) that were not restricted to a palindrome (Krieg et al., 1995) (Figure 22.1). Further, methylation of the cytosine in the CpG dinucleotide completely abrogated any immunostimulatory properties, confirming that the immunostimulatory properties of these ODNs were dependent on the presence of the unmethylated CpG dinucleotides (Krieg et al., 1995). Such immunostimulatory sequences based on unmethylated CpG dinucleotides are now known as CpG motifs. 

Cooper DN, Youssoufian H. The CpG dinucleotide and human genetic disease. Hum Genet 1988 78 151-155. 

DNA methylation is accomplished by DNA methyltransferases (DNMTs), which catalyze the transfer of a methyl group from S-adcnosyl-meth ionine to the 5-position of cytosine in the CpG dinucleotide. DNA hypermethylation within the promoter region of tumor suppressor genes involved in cell proliferation and differentiation is a common phenomenon in numerous solid tumors.4-5... 

Once a sequence amplification event occurs, the nature of any selection on the copies is important. In many (or even most) cases, it appears that the majority of repeated DNA sequences represent pseudogenes, which mutate at a neutral rate of evolution.8 Along with amplification dynamics, the possible removal of repeated sequences must also be considered. Removal does not seem to play a major role with the interspersed repeated DNA elements,8,29 30 but it is likely to be important in tandemly repeated satellite elements. Other mechanisms might also alter evolution of parts of a repeated DNA sequence. For instance, human Alu family copies are initially rich in CpG dinucleotides. These sites appear to be approximately 10-fold more subject to mutation than other sites in the genome,19,31... 

Methylation of cytosine residues within CpG dinucleotides is important in the regulation of genes. The interest in 5-MeCytosine results from its low ionization potential. It would be very interesting to continue the work on GG stacks (Section 18.6.4) by including 5-MeCytosine in the ionization potential calculations. 

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