The CpG Paradox

Preamble :

It has been previously reported that the Cpg sequences in Big Blue (TM) mice are a dominant feature in spontaneous mutagenesis. This has been a techncial problem since it must tend to obscure important but less frequent changes due to specific mutagens. It has also been reported that the CpG sequences in the lacI gene in the Big Blue (TM) mouse are essentially fully methylated. Monroe et al constructed a novel mouse (the "Merck mouse") with 80% of the lacI CpG sequences removed, in an attempt to reduce the CpG-derived background. What happened however, is that the mutant frequency did not decline, and the remaining CpG sequences became much more frequently mutated, still accounting for most of the background mutations. This is is difficult to explain under current models.

Gerald P. Holmquist : The possibility of RNA interference-associated CG depletion to explain the high spontaneous CpG transition frequency in Big Blue mice. Biology Department Beckman Research Institute of the City of Hope Duarte, CA 91010 626-301-8350 gholm@coh.org 10 January 2005

In transgenic Big Blue mice, forty tandem duplications of the lambda phage genome each copy containing a selectable lacI sequence methylated at CpG dinucleotides show a high spontaneous CpG transition frequency (You et al., 1998). However, when tandem lambda inserts with an 80% decreased lacI CpG dinucleotide content were constructed and tandemly inserted into Merck mice, the CpG transition frequency remained unchanged due to an increased transition frequency at each of the remaining CpG sequences (Monroe et al., 2001). The spontaneous mutational spectrum in this common mammalian model for mutagenesis behaves anomalously (Skopek, 1998). Tandem duplications in vascular plants show evidence for a mutational asymmetry called CG depletion that appears limited to tandem repeats (Lund et al., 2003).

Tandem gene duplication in maize results in increased turnover of cytosine at CG and CNG (plants methylate the cytosine of CNG as well as CG sequences) via polarized C:T and G:A transitions in tandemly duplicated zein genes, long terminal repeats (LTRs), and miniature inverted-repeat transposable elements (MITEs). The extent of CG depletion increases with the number of duplications in the tandem cluster. While LTRs and miniature inverted-repeat transposable elements (MITEs) are usually methylated wherever they occur, they show CG depletion only when in tandem or located within a tandemly duplicated sequence; thus, methylation is a necessary but insufficient while being methylated and tandemly duplicated is sufficient for CG suppression (Lund et al., 2003). GC depletion appears related to two other phenomena. In Neurospora crassa, duplicated sequences are targeted for methylation and C:T transitions, a process called repeat-induced point mutation (Selker 1990). Methylation induced premeiotically (MIP) of tandem repeats is observed in Ascobolus (Goyon and Faugeron, 1989).

RNA interference is a likely mechanism acting to produce CG depletion in tandem repeats. Small interfering RNAs are processed from long RNA duplexes often from viruses, transposons, and tandem repeats leading to many different siRNA molecules accumulating from both strands of extended dsRNA. These guide the RNAi machinery to heterochromatinize and suppress tandemly repetitive genes and integrated transposons as a general defense against genomic intruders (Grewal and Moazed, 2003); (Schramke and Allshire, 2003) (Pal-Bhadra et al., 2004) (Verdel et al., 2004). It is quite possible that the tandem lambda repeats in Big Blue mice are sequestered in a heterochromatic state that is targeted for elimination by unusual mutagenic mechanisms loosely classified as methylation-dependent CG depletion.

Reference List

Grewal,S.I. and Moazed,D. (2003). Heterochromatin and epigenetic control of gene expression. Science 301, 798-802.
Lund,G., Lauria,M., Guldberg,P., and Zaina,S. (2003). Duplication-dependent CG suppression of the seed storage protein genes of maize. Genetics 165, 835-848.
Monroe,J.J., Manjanatha,M.G., and Skopek,T.R. (2001). Extent of CpG methylation is not proportional to the in vivo spontaneous mutation frequency at transgenic loci in Big Blue rodents. Mutat. Res. 476, 1-11.
Pal-Bhadra,M., Leibovitch,B.A., Gandhi,S.G., Rao,M., Bhadra,U., Birchler,J.A., and Elgin,S.C. (2004). Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303, 669-672.
Schramke,V. and Allshire,R. (2003). Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing. Science 301, 1069-1074.
Skopek,T.R. (1998). Transgenic mutation models: research, testing, and reality checks. Environ. Mol. Mutagen. 32, 104-105.
Verdel,A., Jia,S., Gerber,S., Sugiyama,T., Gygi,S., Grewal,S.I., and Moazed,D. (2004). RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672-676.
You,Y.H., Halangoda,A., Buettner,V., Hill,K., Sommer,S., and Pfeifer,G. (1998). Methylation of CpG dinucleotides in the lacI gene of the Big Blue transgenic mouse. Mutat. Res. 420, 55-65.