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. 2009 Aug 6;8(8):878-85.
doi: 10.1016/j.dnarep.2009.04.024. Epub 2009 Jun 3.

Diverse effects of individual mismatch repair components on transcription-induced CAG repeat instability in human cells

Yunfu Lin  1 , John H Wilson
Affiliations

Diverse effects of individual mismatch repair components on transcription-induced CAG repeat instability in human cells

Yunfu Lin et al. DNA Repair (Amst). .

Abstract

Several neurodegerative diseases are caused by expansion of a trinucleotide repeat tract in a critical gene. The mechanism of repeat instability is not yet defined, but in mice it requires MutSbeta, a complex of MSH2 and MSH3. We showed previously that transcription through a CAG repeat tract induces repeat instability in human cells via a pathway that requires the mismatch repair (MMR) components, MSH2 and MSH3, and the entire transcription-coupled nucleotide excision repair pathway [Y. Lin, V. Dion, J.H. Wilson, Transcription promotes contraction of CAG repeat tracts in human cells, Nat. Struct. Mol. Biol. 13 (2006) 179-180; Y. Lin, J.H. Wilson, Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair, Mol. Cell Biol. 27 (2007) 6209-6217]. Here, we examine the role of downstream MMR processing components on transcription-induced CAG instability, using our selection assay for repeat contraction. In contrast to knockdowns of MSH2 or MSH3, which reduce repeat contractions, we show that siRNA-mediated depletion of MLH1 or PMS2 increases contraction frequency. Knockdown of DNMT1, which has been identified as an MMR factor in genetic studies, also elevates the frequency of contraction. Simultaneous knockdowns of MLH1 or DNMT1 along with MSH2, XPA, or BRCA1, whose individual knockdowns each decrease CAG contraction, yield intermediate frequencies. In sharp contrast, double knockdown of MLH1 and DNMT1 additively increases the frequency of CAG contraction. These results show that MMR components can alter repeat stability in diverse ways, either enhancing or suppressing CAG contraction, and they provide insight into the influence of MMR components on transcription-induced CAG repeat instability.

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Figures

Fig. 1
Fig. 1
Selection assay for transcription-induced contraction of CAG repeats. When long CAG tracts are present in the intron of the HPRT minigene, they are incorporated into the mRNA, rendering it nonfunctional and giving the cells an HPRT− (HAT-sensitive) phenotype. When contraction of the repeat generates a tract with fewer than 39 repeats, it is not efficiently incorporated into the mRNA, allowing sufficient normal protein to be made to give an HPRT+ (HAT-resistant) phenotype [39]. The HPRT minigene is controlled by the TRE-pCMVmini promoter, which can be turned on by addition of doxycycline. When the reverse tetracycline transcription activator (rtTA) binds doxycycline, it is converted to an active form that binds to the promoter.
Fig. 2
Fig. 2
Effects of siRNA-mediated knockdowns on transcription-induced CAG repeat contraction. Contraction frequencies were calculated as the number of HPRT+ colonies divided by the number of viable cells, averaged over at least five independent experiments. Values obtained in the absence of doxycycline (transcription turned off) are shown in unfilled boxes. Values obtained in the presence of doxycycline are shown in black boxes. Transcription-induced frequencies that are significantly different from the vimentin siRNA control are indicated with an asterisk; specific p values are indicated in the text.
Fig. 3
Fig. 3
PCR analysis of the lengths of CAG repeat tracts. The CAG repeat tracts in HPRT+ colonies were amplified by PCR and the products were separated by electrophoresis. The number of CAG repeats in PCR products from independent HPRT+ colonies (lanes 9–28) was determined by reference to a ladder of length markers (lanes 1–8). Lanes 1 to 8 contain tracts of 15, 18, 20, 24, 27, 30, 33, and 36 CAG repeats, respectively.
Fig. 4
Fig. 4
Transcription-induced CAG repeat contraction after single or double siRNA-mediated knockdowns. In all cases the concentration of total siRNA was 200 nM. For single knockdowns, 100 nM of siRNA was used and the total concentration of siRNA was adjusted to 200 nM with vimentin siRNA. For double knockdowns, 100 nM of each siRNA was used. Contraction frequencies are the average results of at least five experiments.
Fig. 5
Fig. 5
Possible interactions between MLH1, DNMT1, and the transcription-induced pathway for repeat instability. Transcription through a repeat tract is envisioned to generate a slipped-strand duplex structure with CTG hairpins on one strand and CAG loops on the other. Shown here is a single CTG hairpin, bound by MutSβ, serving as a block to progression of a subsequent RNAPII molecule. This intermediate is processed by TC-NER to produce large contractions, which can be detected in our assay system. MutLα may act to divert intermediates to products that we cannot detect. DNMT1 could act to inhibit formation of the intermediate (by inhibiting transcription), promote an alternative pathway for processing the intermediate (like MutLα), or remove products from detection (for example, by silencing).
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