Somatic instability of CGG repeats, which, unlike CAGs and GAAs, can be methylated, is low in patients harboring the full mutation (>200 CGGs)50, 51
. By contrast, FRAXA males, who express FMR1
although they harbor a large expansion, have unmethylated repeats with a high degree of instability in somatic tissues52, 53
. One such individual displayed instability in some tissues and not in others, a pattern that correlated perfectly with methylation status of the repeat52
. Cultured cells from FRAXA patients show the same absolute correlation: methylated CGG repeats are stable, whereas unmethylated repeats are unstable54-56
. Importantly, in cultured primate cells, DNA methylation of plasmids containing CGG repeats stabilized the repeat tract, specifically implicating DNA methylation within or near repeat tracts as a key modifier of triplet repeat instability37
In addition to epigenetic modifiers, transcription has emerged as an important player in triplet repeat instability. Transcription through a repeat tract can stimulate repeat instability in human cells and in fly models of CAG repeat diseases57
. Lin et al
showed that transcription through a repeat of 95 CAGs caused substantial contractions of the repeat tract7
. Moreover, transcription-induced destabilization required MMR and transcription-coupled NER (TC-NER)57
. Thus, heterochromatin might reduce repeat instability by lowering the rate of transcription through the repeat tract, thereby decreasing the potential for TC-NER (). The absence of transcription through the highly expanded CGG repeats at the FMR1
locus could account for their surprising stability in somatic tissues.
Many repeat loci, however, are more unstable than their shorter counterparts even though they are transcribed at reduced levels57
. For these loci, another mechanism must be at work. We suggest that this mechanism might be antisense transcription. Indeed, a surprisingly high fraction of human genes are associated with antisense transcripts, including most wild type alleles of triplet repeat-associated genes59
(). At the FMR1
locus, heterochromatin appears to shut-down both sense and antisense transcription60
. By contrast, at the SCA8 and DM1 loci, the levels of antisense transcription are higher on the expanded alleles21, 61
. At least at the DMPK
locus, high levels of antisense transcription are associated with heterochromatin marks21
Sense and antisense transcripts at disease loci with normal length repeatsa
At the DM1 locus, the expanded CTG repeat tract is associated with methylation of a CCCTC-binding factor (CTCF) binding site, which appears to insulate the repeat from antisense transcription19
. When the CTCF site is methylated, however, CTCF cannot bind, allowing enhanced antisense transcription through the expanded repeat19
. Thus, paradoxically, heterochromatinization allows for higher levels of antisense transcription through the repeat, which could promote instability via TC-NER ().
Libby et al
investigated the role of the CTCF binding site in triplet repeat instability in a mouse model for SCA710
. In these mice, CTCF binding sites flank the 92 CAG repeats in the transgene. Mutation of the downstream CTCF site dramatically increased instability in the germline. Furthermore, the kidney, cortex, brainstem, and liver of mice mutant for the CTCF binding site also displayed a higher level of CAG instability compared to mice with a wild type binding site10
. Intriguingly, one mouse that carried a wild type CTCF binding site showed elevated levels of instability only in its kidney. Further investigation showed an aberrantly methylated CTCF binding site in this tissue. DNA methyaltion prevented CTCF binding its target sequence in vitro
. The authors concluded that CTCF binding, which is influenced by DNA methylation, contributes directly to preventing repeat instability. These results are akin to those for the expanded CTG repeat in DM1 cells, where methylation of the CTCF binding sites correlate with loss of CTCF binding and increased antisense transcription19, 21
. In the SCA7 mice, however, the role of antisense transcription has not been analyzed. Taken together, these data provide a tantalizing link between CAG repeat instability, CG-rich sequences, DNA methylation, and perhaps antisense transcription through the locus Antisense transcription could promote repeat instability in other ways. Simultaneous sense and antisense transcription might lead to head-on collisions between converging RNA polymerases, which could amplify the problems that arise when a repeat is transcribed in a single direction (e.g. enhance formation of secondary structure, persistence of RNA/DNA hybrids, and increased gratuitous TC-NER) thereby elevating instability.
Antisense transcription could also generate double-stranded RNA, which might trigger the RNA interference (RNAi). RNAi is a specialized pathway that allows post transcriptional repression of specific mRNAs62
. Double stranded RNAs, such as microRNAs, are processed by the ribonuclease Dicer into 21nucleotide fragments. Short RNAs are subsequently loaded in the RNA-induced silencing complex (RISC), which targets mRNAs for degradation or inhibits their translation62
. In fission yeast, the RNAi pathway prevents the appearance of centromeric transcripts by facilitating formation and maintenance of centromeric heterochromatin62
. Whether an analogous process operates in mammalian cells is not yet clear. Nonetheless, repeat-containing transcripts can be processed by Dicer, and enter the RNAi pathway22, 63, 64
. Thus, the RNAi pathway could promote heterochromatin formation at disease loci22
, which could impact instability.
RNAs perform a variety of other functions that might be relevant to repeat instability: they can serve as a template for DNA synthesis during DNA repair65
, guide programmed chromosome rearrangements66
, and help remove T-G mismatches generated during reprogramming67
. One of these functions might ultimately be found to play a role in repeat instability.