Figure 1.
Somatic HTT CAG instability differs between B6.HdhQ111/+ and 129.HdhQ111/+ mice.
(A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 10-week-old B6.HdhQ111/+ and 129.HdhQ111/+ mice, highlighting the altered contribution of B6 and 129 genetic background to somatic HTT CAG repeat expansion, as previously described [17]. Tail and striatum: B6.HdhQ111/+, CAG116; 129.HdhQ111/+, CAG112. Liver: B6.HdhQ111/+, CAG113; 129.HdhQ111/+, CAG111 (B) Quantification of CAG instability index reveals a statistically significant decrease in somatic HTT CAG instability in the striatum and liver of 129.HdhQ111/+ mice compared to B6.HdhQ111/+ mice. B6.HdhQ111/+ striatum, n = 10, CAG116.9±1.2SD; B6.HdhQ111/+ liver, n = 10, CAG114.3±1.2SD; 129.HdhQ111/+ striatum, n = 12, CAG110.9±1.2SD; 129.HdhQ111/+ liver, n = 9, CAG109.5±1.4SD; Bar graphs represent mean ±SD; ****, p<0.0001.
Figure 2.
Striatal HTT CAG instability in 10-week-old HdhQ111/+ mice on different genetic backgrounds.
Graphical representation of striatal CAG instability indices from individual (A) B6, 129, (B6x129).F1 and (B6x129).F2 mice, color-coded based on strain genetic background; and from (B) (B6x129).F2 mice color-coded by genotype at the Mlh1, Msh3 and Msh2 genes (“undetermined” indicates failed genotype). F2 mice homozygous or heterozygous for B6 Mlh1 alleles display significantly higher levels of striatal somatic CAG instability than F2 mice homozygous for 129 Mlh1 alleles (p<0.0001 for both). No relationship could be established between Msh3 or Msh2 genotype and striatal CAG instability. B6.HdhQ111/+, n = 10, CAG116.9±1.2SD; 129.HdhQ111/+, n = 12, CAG110.9±1.2SD; (B6x129).HdhQ111/+ F1, n = 11, CAG114.7±6.4SD; (B6x129).HdhQ111/+ F2, n = 69, CAG107.7±3.2SD. dbSNP markers located within MMR genes: Mlh1, rs30131926 and rs30174694 (concordant genotypes detected with both markers); Msh3, rs29551174; Msh2, rs33609112 and rs49012398 (concordant genotypes detected with both markers). Horizontal bars represent the mean CAG instability indices of the respective groups.
Figure 3.
Identification of a quantitative trait locus (QTL) associated with striatal HTT CAG instability.
Linkage analysis in 10-week-old (B6x129).HdhQ111/+ F2 mice (n = 69) identified a single QTL on chromosome 9, with a maximum LOD score of approximately 14 and a 2-LOD-dropoff interval of 5 Mb (chr9:107,982,655–113,057,967; GRCm38/mm10) (Figure S6). Note that the 2 markers positioned within the Mlh1 gene (dbSNP rs30131926 and rs30174694) define the QTL peak. The red dashed line represents the threshold (LOD = 4.3) considered for the identification of significant QTLs [85]. The coordinates (cM) of the 147 genetic markers used are represented by open triangles.
Figure 4.
Mlh1 is required for striatal and liver HTT CAG repeat instability in B6.HdhQ111/+ mice.
(A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 22-week-old B6.HdhQ111/+ mice on different Mlh1 genetic backgrounds. Mlh1+/+, CAG113; Mlh1+/−, CAG113; Mlh1−/−, CAG110. (B) Quantification of striatal and liver HTT CAG instability indices in these mice reveals a statistically significant decrease in HTT CAG instability in the absence of Mlh1. Mlh1+/+, CAG115.3±4.9SD, n = 6; Mlh1+/−, CAG112.0±2.1SD, n = 6; Mlh1−/−, CAG109.3±2.6SD, n = 6. Bar graphs represent mean ±SD. ****, p<0.0001.
Figure 5.
Mlh1 is an enhancer of nuclear mutant huntingtin immunostaining in B6.HdhQ111/+ mice.
(A) Representative EM48 immunostained histological sections from striata of 22-week-old B6.HdhQ111/+ mice on different Mlh1 genetic backgrounds. Mlh1+/+, CAG113; Mlh1+/−, CAG108; Mlh1−/−, CAG110. (B) Quantification of diffuse nuclear EM48 staining demonstrates a statistically significant reduction in the absence of Mlh1. Mlh1+/+, CAG115.3±4.9SD, n = 6; Mlh1+/−, CAG112.0±2.1SD, n = 6; Mlh1−/−, CAG109.2±2.9SD, n = 5. Bar graphs represent mean ±SD. **, p<0.01.
Figure 6.
Mlh3 is required for striatal and liver HTT CAG repeat instability in B6.HdhQ111/+ mice.
(A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6.HdhQ111/+ mice on different Mlh3 genetic backgrounds. Mlh3+/+, CAG103; Mlh3+/−, CAG101; Mlh3−/−, CAG102. (B) Quantification of striatal and liver HTT CAG instability indices in these animals reveals a statistically significant suppression of HTT CAG instability in the absence of Mlh3. Mlh3+/+, CAG103.3±1.5SD, n = 3; Mlh3+/−, CAG101.3±0.5SD, n = 4; Mlh3−/−, CAG101.3±0.6SD, n = 3. Bar graphs represent mean ±SD. *, p<0.05; ***, p<0.001; ****, p<0.0001.
Figure 7.
B6-129 MLH1 protein polymorphisms.
(A) Schematic representation of the murine MLH1 protein showing the location of B6-129 nonsynonymous polymorphisms identified (purple triangles) and their positions relative to conserved ATP binding motifs and ATP binding domain (dark and light red boxes, respectively) [46], as well as proposed MMR protein interaction domains (blue boxes) [41]. (B) Cross-species alignment of B6 and 129 MLH1 proteins in regions encompassing the polymorphic sites between the two strains. Protein sequence alignment was performed using Clustal Omega [96] and visualized in Jalview [97] with BLOSUM62 color scheme: white, residue does not match the consensus residue at that position; light blue, residue does not match the consensus residue but the two residues have a positive BLOSUM62 score; dark blue, residue matches consensus sequence.
Table 1.
The Mlh1 locus is highly polymorphic between B6 and 129 strains.
Figure 8.
Repair of a single CTG slip-out in a cell-free MMR assay is MLH1 dose-dependent.
(A) Short slipped-DNA repair using HeLa or HEK293T (MutLα-deficient) whole cell extracts complemented with equal amounts (100 ng) of purified MutLα protein complexes: hMLH1-hPMS2, mMLH1.B6-hPMS2 or mMLH1.129-hPMS2. Both B6 and 129 MLH1 proteins show ability to repair the mismatch when in a complex with hPMS2. The individual lanes represented are from the same blot. (B) Repair using MutLα-deficient HEK293T cell extracts complemented with increasing concentrations (5, 25 and 100 ng) of either mMLH1.B6-hPMS2 or mMLH1.129-hPMS2 protein complexes. Quantification of repair suggests that both B6 and 129 MLH1 proteins are comparably efficient at repairing CTG slip-outs. In addition, it suggests a MutLα dose-dependency, with higher concentrations of mMLH1-hPMS2 resulting in higher levels of MMR activity (p = 0.0013). The individual lanes represented are from the same blot and the experiment was reproduced three times. Bars graphs represent mean ±SD.
Figure 9.
Reduced MLH1 expression in 129 versus B6 mice.
Quantification of MLH1 (A) mRNA and (B, C) protein levels in the striatum of B6.Mlh1+/+, 129.Mlh1+/+ and B6.Mlh1+/− 10-week-old mice (n = 3). (A) Striatal Mlh1 mRNA levels (TaqMan Mm00503449_m1, exons 11–12) in 129.Mlh1+/+ mice were significantly reduced by approximately 50% when compared to B6.Mlh1+/+ (p<0.05), and were comparable to levels in B6.Mlh1+/− mice. (B, C) Western blot analysis of MLH1 protein revealed significantly reduced levels in 129.Mlh1+/+ striata compared to B6.Mlh1+/+ striata. Bar graphs represent mean ±SD. *, p<0.05; **, p<0.01.
Figure 10.
The 129 and B6 3′-flanking regions of Mlh1 confer differential mRNA regulation.
Investigation of the regulatory potential of B6 and 129 immediate (A) 5′- and (B) 3′-flanking regions of Mlh1 using dual luciferase reporter assays. (A) The immediate 5′-flanking region of Mlh1 containing 17 B6-129 polymorphisms (2,441 bp) was used to drive firefly luciferase expression. (B) The immediate 3′-flanking region of Mlh1 (i–iv) containing either 19, 15, 4 or 1 B6-129 polymorphism(s) (1,676 bp, 1,280 bp, 591 bp and 205 bp, respectively) was cloned downstream of a firefly luciferase gene. “Swap” constructs (v) of the immediate 3′-flanking region of Mlh1 containing either 4, 5 or 10 129 polymorphisms (530 bp, 438 bp and 708 bp, respectively; total 1676 bp) were cloned downstream of a firefly luciferase gene. Relative luciferase activity was determined by normalization to internal Renilla luminescence and determined relative to the analogous B6 construct. B6-129 polymorphisms are represented by open triangles. Bar graphs represent mean ±SD. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 11.
Proposed model of MutS and MutL-dependent events leading to CAG•CTG somatic instability.
CAG•CTG repeat structures are initially recognized by the MutSβ (MSH2-MSH3) complex [25], [98]. The loop in the CAG•CTG repeat tract represents a short slip-out, previously identified as the main substrate for MMR protein-dependent repair of CAG•CTG structures in cell free systems [50], [51]. However, the nature of the putative CAG•CTG structure(s) that leads to MutS and MutL-dependent somatic instability in vivo is unknown. Following ATP hydrolysis by DNA-bound MutSβ [27], a MutLγ (MLH1–MLH3) heterodimer is preferentially recruited to the complex (thick arrow) over the MutLα (MLH1-PMS2) heterodimer (thin arrow). The total absence of HTT CAG expansion in Mlh3−/− mice suggests that PMS2 plays no role at all in this process. However, PMS2 has been shown to play a role in the expansion of CTG repeats in a DM1 mouse model [24], suggesting that these events may be genetic locus and/or mouse strain dependent. Following MutLγ binding, various pathways, e.g. canonical mismatch repair (MMR), noncanonical mismatch repair (ncMMR) and/or other DNA repair processes may be engaged and process the repeats such that they ultimately undergo expansion. Other members of alternative DNA repair pathways, namely OGG1, XPA and NEIL1 have been directly implicated in CAG/CTG somatic instability in mice [76]–[78], however, how these proteins intersect with MMR protein-dependent pathways has yet to be demonstrated.