We devised a moderately high-throughput chemical screen to provide an unbiased search for new pathways that modulate CAG repeat instability. We chose to screen the Prestwick chemical library because it contains a broad diversity of chemical structures with a wide range of pharmacological effects (45
). As with most screens, ours was blind to certain chemicals that alter repeat instability. First, the screen was designed to detect only those chemicals that increased the frequency of contractions. Chemicals that decrease contraction frequency would not have been detected. Second, chemicals that stimulated repeat contraction but were too toxic or were ineffectual at the single concentration tested would also have been missed. Camptothecin, which is in the library but was missed in the initial screen, may be an example of a compound that was tested below its effective concentration, but we have not investigated that possibility further. In addition, our screen may be biased toward modulators that affect transcription, since transcription through the repeat was induced throughout the screen. Even with these limitations, the screen identified 18 chemicals that substantially increased CAG repeat contraction. Five of these—acacetin, vitexin, amikacin, butirosin, and betulinic acid—provided initial clues that led to the identification of the TOP1-TDP1-SSBR repair pathway as a modulator of CAG repeat contraction. These clues were confirmed by camptothecin treatment and siRNA knockdown. The other 13 chemicals identified in the screen may conceal links to repeat instability that are not obvious at present, but may be revealed with additional experiments.
Supercoiling has been shown to play a role in the instability of CGG, GAA, and CAG repeats in bacteria (38
), but not previously in mammalian cells. In bacteria the link has been ascribed to the effects of negative supercoiling on enhancing the formation of repeat-induced non-B DNA structures (38
), which are thought to constitute the key common event leading to changes in repeat-tract length (24
). The negative supercoiling that develops behind a transcribing RNA polymerase (54
) would provide a natural connection between transcription and repeat instability and could explain our results with TOP1 inhibitors, especially with siRNA knockdowns of TOP1, which might be expected to increase supercoiling stress (9
Supercoiling by itself, however, does not readily accommodate the results with inhibitors of TDP1 and SSBR. The TDP1-SSBR connection suggests that formation of irreversible TOP1 cleavage complexes may be the source of the CAG repeat instability observed in our studies. If an RNA polymerase runs into a TOP1-DNA cleavage intermediate on the template strand, the intermediate will be converted to an irreversible cleavage complex that is unable to religate the DNA and release TOP1 (44
). In the normal course of events, these complexes are resolved by the action of TDP1 and the SSBR pathway (44
). Thus, we favor the interpretation that TOP1, TDP1, and SSBR normally act to suppress CAG repeat instability by restricting the consequences of irreversible TOP1-DNA complex formation. In the presence of TOP1 inhibitors, the frequency of irreversible complexes rises, with a concomitant rise in repeat instability. When TDP1 or the SSBR pathway is inhibited, the frequency of irreversible complexes also rises, resulting in an increase in repeat instability. How such irreversible TOP1-DNA intermediates lead to CAG repeat instability is not clear. In any case, the normal action of the TOP1-TDP1-SSBR pathway suppresses repeat instability, and when this pathway is compromised, expanded CAG repeats become more unstable.
In addition to defining a new pathway—the TOP1-TDP1-SSBR pathway—for CAG repeat instability, we have addressed the fundamental question of what causes the increase in repeat instability when this pathway is compromised by chemical or siRNA treatments. By knocking down the TC-NER pathway at the same time we interfered with the TOP1-TDP1-SSBR pathway, we showed that we could block the increase in repeat contractions. We previously demonstrated that the TC-NER pathway acts to increase transcription-induced CAG contractions; that is, when TC-NER components were knocked down, contraction frequencies were substantially reduced (28
). Thus, it appears that TOP1-DNA irreversible cleavage complexes are removed by TC-NER, when the preferred repair pathway is unavailable, as summarized in .
Fig. 6. Proposed relationship between the TOP1-TDP1-SSBR and the TC-NER pathways. The TOP1-DNA irreversible cleavage complexes (TOP1icc) that arise normally in the course of TOP1 action are usually taken care of by TDP1 and SSBR, which prevent the formation of (more ...)
It will be important to determine the relevance of the TOP1-TDP1-SSBR pathway to the CAG repeat instability that occurs in human patients. We are currently testing the effects of the TOP1-TDP1-SSBR pathway in a SCA1 mouse model (55
), which allows both germ line and somatic instability to be assessed in an organism that displays patterns of repeat instability similar to those in human patients. There is generally good agreement between results in our assay in human cells and those in mouse models (29
). Among the 20 genes shown to affect CAG repeat contraction in human cells (6
), six have been tested in a mouse model and also shown to modulate CAG repeat instability, including MSH2 (22
), MSH3 (52
), PMS2 (12
), DNMT1 (6
), CSB (20
), and XPA (L. Hubert et al., unpublished data). Similarly, among those genes with little effect on CAG contraction in human cells (28
), three have been tested in mouse models and shown to have little effect on CAG repeats, including MSH6 (52
), XPC (8
), and FEN1 (50
). Interestingly, only in the case of the OGG1 glycosylase discussed below do the results differ, with deficiencies having no effect on CAG contraction in human cells (32
), but large effects in mice (21
). Overall, the otherwise good agreement between these two assays suggests that screening candidates in human cells will be a productive way to select genes to test in mice.
The TOP1-TDP1-SSBR pathway is similar to the repair process that operates during base excision repair (BER), except that TOP1-TDP1 are replaced by a variety of glycosylases and apurinic-apyrimidinic endonuclease 1 (APE1), which control the initial steps in BER: removing the damaged base and breaking the single strand (46
). As in the TOP1-TDP1-SSBR pathway, the final stage of BER—repair of the broken strand—involves XRCC1, DNA polymerase β, and DNA ligase 3, among others (46
). The most extensively studied glycosylase is OGG1. It is responsible for excising 8-oxo-guanine, a common base damage caused by reactive oxygen species (53
). In cell extracts and in mice, it has been shown that OGG1 normally acts to promote CAG repeat expansions (21
). Thus, in the absence of OGG1, CAG repeats become more stable. Strikingly, mouse models of Huntington disease that are genetically deficient for OGG1 have much more stable CAG repeats in brain than do OGG1-positive mice (21
). It is curious that the TOP1-TDP1-SSBR pathway acts to suppress CAG instability, while the OGG1-APE1-BER pathway acts to stimulate repeat instability.
There are several possible reasons for these differences. First, it could be a trivial consequence of the selective detection of large contractions in our study versus the analysis of frequent events in the OGG1 studies (21
). Thus, it is possible that defects in OGG1 promote large contractions, even as they reduce expansions, but that contractions occur at frequencies that are too low to detect by small-pool PCR. Experiments in our selective system, however, have shown that siRNA knockdowns of OGG1 and APE1 do not change the frequency of transcription-induced CAG contractions (32
). A second possibility is that the distinct effects on CAG repeat instability are a consequence of the differences in mechanism. The action of TOP1-TDP1 generates clean ends with 3′ hydroxyls and 5′ phosphates, which are conducive to religation (44
); it is only when the process is interrupted that the repeat tracts are affected. In contrast, OGG1-APE1 leaves ends with 3′ hydroxyls and 5′ sugar phosphates (33
). Although DNA polymerase β can remove this obstruction, it can also displace the strand, allowing a CAG hairpin to form, presumably as a precursor to expansion (33
). In the absence of OGG1, the troublesome ends would not be generated and the repeats would, therefore, be more stable. Resolving these differences may prove enlightening for our understanding of CAG repeat instability.
In summary, by using a selectable CAG contraction system in human cells to screen the Prestwick chemical library, we have made two novel findings. First, we have identified the TOP1-TDP1-SSBR pathway as a modulator of transcription-induced CAG repeat instability in human cells. Although supercoiling has been shown to destabilize triplet repeats in bacteria, this report is the first that identifies the repair of transcription-induced DNA damage—presumably, the TOP1 irreversible cleavage complexes—as a critical determinant of repeat stability. Second, we have shown that TC-NER drives CAG repeat instability not only when transcription through the repeat is induced but also when the TOP1-TDP1-SSBR pathway is compromised. These studies substantially enrich our understanding of transcription-induced CAG repeat instability. Also, as a result of this work we have discovered a set of bioactive small molecules that modulate CAG repeat instability. These compounds may serve as tools for dissecting the molecular mechanism of CAG repeat instability and as aids in the development of preventive and therapeutic approaches for repeat-associated diseases.