Antisense transcripts are common in human genes 
, suggesting that head-to-head, convergent transcription may be a frequent occurrence on human chromosomes. Antisense transcripts have been found in several trinucleotide repeats (TNR) disease genes, with 8 identified in vivo 
and at least 10 others in human cell lines 
. Previously, we examined the biological consequences of convergent transcription through a CAG tract, showing that it promotes repeat instability and causes massive cell death 
. Here, we have examined the influences of three DNA metabolic processes on convergent transcription-induced cell death and repeat instability. The TC-NER pathway of DNA repair, the mismatch repair recognition component MSH2, and the RNase H species involved in R-loop resolution, which were first identified as playing critical roles in repeat instability induced by sense transcription 
, all affect the repeat instability and cell death induced by convergent transcription. These results suggest that a common structure, generated by convergent transcription through a CAG repeat tract, is likely to be ultimately responsible for both repeat instability and cell death.
For sense transcription-induced repeat instability, we suggested that transcription allowed slipped duplexes to form with looped out CAG and CTG segments 
, and that R-loops enhanced the formation of these aberrant structures 
. Stabilization of CAG and CTG loops by MSH2/MSH3 (MutSβ) binding can block the progress of RNAPII 
, thereby creating a signal that called TC-NER into play to resolve the block 
. This working model was created to be consistent with the results from siRNA knockdowns. Depletion of RNase H, which would increase the lifetime of R-loops, would be expected to increase the formation of slipped duplexes, leading to more repeat instability, as observed 
. Knockdown of MSH2, which would decrease binding to and stabilization of CAG and CTG loops, would reduce stalling of RNAPII, leading to the observed decrease in repeat instability 
. Knocking down of components of TC-NER prevent the resolution of the block, which is the mechanism by which the repeat is rendered unstable, and thus decrease repeat instability 
. Here we have shown that this same reasoning applies to convergent transcription-induced repeat instability.
We have speculated elsewhere 
that convergent transcription through a repeat tract can generate aberrant structures with stalled RNAPII complexes on both strands, creating what we have termed a double bubble 
. Because the structures on each strand are analogous to the one described above for sense transcription, it was our expectation that knockdown of RNase H, MSH2, and TC-NER would produce the same effects on repeat instability induced by convergent transcription as they do on repeat instability induced by sense transcription. Our results match these expectations: depletion of RNase H increases instability, while depletion of MSH2 and TC-NER decrease repeat instability.
The more surprising result of convergent transcription through a CAG repeat tract–massive cell death–depends on simultaneous induction of both sense and antisense transcription on either side of a CAG repeat tract, so that converging RNAPII complexes encounter the same tract 
. The resulting double bubble, produced by stalled RNAPII complexes on both strands, must present some significant complication for the cell, which induces an ATR response and triggers cell death, two consequences that are not associated with sense transcription alone 
. At the outset, it was unclear whether the processes involved in convergent transcription-induced repeat instability would also be involved in the associated cell death. Our knockdown experiments show clearly, however, that RNase H, MSH2, and TC-NER are all involved in both repeat instability and cell death. We can interpret our results in terms of the likely effects on the formation or persistence of the convergent transcription-induced double bubble. Knockdown of RNase H increases R-loops, which favors formation of the slipped duplexes that are key to formation of the double bubbles, thereby increasing the structure formation and increasing cell death. Knockdown of MSH2 prevents stabilization of the CAG and CTG loops, thereby decreasing structure formation and cell death. Similarly, knockdown of MSH3 also reduces cell death, while double knockdown of MSH2 and MSH3 reduces cell death to the same level as either single knockdown (data not shown), consistent with MutSβ playing a role in the stabilization of CAG and CTG loops 
. Finally, depletion of TC-NER components prevents resolution of the block to RNAPII, prolonging the aberrant structure and increasing cell death.
One striking feature of the effects of siRNA knockdowns on cell death is that DIT7-R103 cells, which carry a short repeat (CAG15
), are more strongly affected than DIT7 cells, which carry a long repeat (CAG95
). This counterintuitive result cannot be due to different locations of the repeat in the genome, for example, because DIT7-R103 cells were derived from DIT7 cells by contraction of the repeat. Although we do not know the basis for the difference, we speculate that it reflects the different numbers of CAG and CTG loops that can form in the two repeats. The long CAG tract can potentially form multiple loops, consistent with our measurements of single-stranded regions within the tract 
, while the short tract is unlikely to form more than one. Reduction of MSH2, for example, would reduce the number of stabilized loops in a tract. If the tract has multiple loops, however, some may still be stabilized, resulting in a small effect on cell death. By contrast, in a tract with a single loop, reduction of MSH2 would decrease the number of cells in which the loop is stabilized, thereby reducing cell death. Similar arguments can be made for the effects of knockdowns of RNase H and TC-NER, both of which would be expected to increase the number of stabilized loops. If cells with long repeats already have multiple stabilized loops, an increase may have little effect on cell death, whereas in cells with a single repeat, knockdowns may increase the proportion of cells with a stabilized loop, resulting in more substantial increases in cell death.
Our results are consistent with the idea that the stalled RNAPII is the original signal triggering cell death during convergent transcription 
. Previous studies showed that agents such as UV light, actinomycin D, psoralen, or antibodies against the RNAPII elongation complex–all of which interfere with transcription by blocking RNAPII genome wide–can stimulate apoptosis 
. Both genome-wide arrest of RNAPII and its stalling at CAG tracts stimulate a cellular response via the ATR signaling pathway 
. It is remarkable that RNAPII arrested at a single locus in the genome has such a similar effect on cells as genome-wide transcriptional interference, which occurs at thousands of actively transcribed genes. The critical feature of this locus appears to be the ability of CAG repeats to form abnormal secondary structures capable of blocking transcription on both template strands. It is not yet clear whether convergent transcription-induced cell death is unique to CAG repeats, or is a more general attribute of other structure-forming repeats, as well. Supporting this possibility is the observation that transcription stalls at other types of repeat tracts and at DNA sequences that can form secondary structures in vitro 
; thus, noncanonical DNA structures can cause problems for RNAP.
The pathogenic mechanisms of CAG diseases are complicated and appear to include toxic proteins and RNA molecules 
. Convergent transcription-induced cell death raises the possibility that DNA toxicity may also contribute to pathogenesis of these diseases. We showed previously that convergent transcription through CAG repeats can trigger cell death in both proliferating and nonproliferating cells 
, indicating that it is a potential mechanism of cell death in the terminally differentiated cells that are affected in repeat diseases. In addition, antisense transcripts have been found for several TNR disease genes, supporting the idea that convergent transcription occurs in vivo and could potentially affect cell health. The contribution of convergent transcription to the pathogenesis of repeat diseases, however, remains to be tested.
In summary, we have shown that TC-NER pathway, MSH2, and R-loops modulate convergent transcription-induced repeat instability and cell death in human cells. These observations link the mechanisms of convergent transcription-induced repeat instability and convergent transcription-induced cell death, suggesting that a common structure may trigger both outcomes.