5hmC levels in cerebellum and heart.

<p>EpiMark^TM 5hmC and 5mC analysis of CpG site 12 in the FXN upstream GAA repeat region of DNA from FRDA and control cerebellum (<b>A</b>) and heart (<b>B</b>) tissues. G = glucosylated DNA. MspI values represent complete digestion control levels of DNA; MspI + G represents 5hmC levels, HpaII and HpaII + G represent combined 5mC and 5hmC levels; Mock and Mock +G represent undigested control levels of DNA. Error bars = s.e.m. n=4.</p

DNA methylation levels in heart.

<p>MethylScreen analysis of four CpG sites in the FXN upstream GAA repeat region of DNA from FRDA and control heart tissues. UM = unmethylated, IM = intermediately methylated, DM = densely methylated. Error bars = s.e.m. n=4.</p

CTCF analysis.

<p>(<b>A</b>) EMSA showing a mobility shift for the <i>FXN</i> 5’ UTR probe and CTCF-positive DM1 (1) probe [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074956#B35" target="_blank">35</a>], while there is no such shift detected for the FXN upstream GAA (Up) probe, <i>FXN</i> downstream GAA (Down) probe or CTCF-negative DM1 (3) probe [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074956#B35" target="_blank">35</a>]. (<b>B</b>) ChIP analysis showing the relative CTCF occupancy in the <i>FXN</i> 5’ UTR of DNA from FRDA and control cerebellum tissues. Error bars = s.e.m. n=3. * = P<0.05.</p

Absence of Pms2 does not affect somatic instability of the (GAA·TTC)_n sequence in non-neuronal tissues.

<p>Panels show the distribution of repeat lengths measured in (A,B) heart and (C,D) kidney from (A,C) <i>Pms2^+/+</i> and (B,D) <i>Pms2^−/−</i> littermates. The number of products measured at each repeat length is on the Y-axis. Gray bars represent the length of the progenitor alleles, +/−5%. Products that fell outside of these ranges were considered mutations.</p

Effect of Pms2 on large expansions in cerebellum and DRG.

*<p><i>P</i> values indicate statistical significance between +/+ and −/−.</p><p><i>P</i> values are not indicated when not significant.</p

Effect of Pms2 on instability in somatic tissues.

*<p><i>P</i> values indicate statistical significance between +/+ and −/−. <i>P</i> values are not indicated when not significant.</p>∧<p>Because multiple progenitor alleles were present, changes between progenitors could not be definitively identified as contractions or expansions. Contractions and expansions were conservatively defined as molecules that were at least 5% shorter than the smallest progenitor or 5% longer than the largest progenitor, respectively. Contractions, expansions, and changes between progenitors were included in “Total Mutations”.</p

Absence of MutSα reduces somatic mutation load of the (GAA·TTC)_n sequence.

<p>Panels A, B, E, and F show representative autoradiographs from SP-PCR analysis of DNA extracted from (A) <i>Msh2^+/+</i>, (B) <i>Msh2^−/−</i>, (E) <i>Msh6^+/+</i>, and (F) <i>Msh6^−/−</i> cerebellum. Progenitor allele lengths determined by conventional PCR are 192, 130, and 89 repeats for <i>Msh2^+/+</i>; 188, 128, and 88 repeats for <i>Msh2^−/−</i>; 186, 133, and 98 repeats for <i>Msh6^+/+</i>; and 187, 129, and 90 repeats for <i>Msh6^−/−</i>, as indicated by arrowheads at the right of each panel. Results from the total analysis of 150 to 282 molecules per genotype are quantified in C and D for <i>Msh2</i>, and G and H for <i>Msh6</i>.</p

Effect of Msh2 and Msh6 on instability in somatic tissues.

*<p><i>P</i> values indicate statistical significance between +/+ and −/−. <i>P</i> values are not indicated when not significant.</p>∧<p>Because multiple progenitor alleles were present, changes between progenitors could not be definitively identified as contractions or expansions. Contractions and expansions were conservatively defined as molecules that were at least 5% shorter than the smallest progenitor or 5% longer than the largest progenitor, respectively. Contractions, expansions, and changes between progenitors were included in “Total Mutations”.</p

Absence of Pms2 increases somatic expansion load of the (GAA·TTC)_n sequence in cerebrum.

<p>Analysis of instability in cerebrum of <i>Pms2^+/+</i> and <i>Pms2^−/−</i> mice. Representative autoradiographs from (A) <i>Pms2^+/+</i> and (B) <i>Pms2^−/−</i> cerebrum are shown. Progenitor allele lengths of 229, 194, 135, and 94 repeats for <i>Pms2^+/+</i> and 229, 185, 130, and 86 repeats for <i>Pms2^−/−</i> are indicated by arrowheads at the right of each panel. Mutation load and expansion load, quantified from the analysis of ∼550 molecules per genotype, are shown in panels C and D, respectively. Panels E and F indicate the distribution of repeat lengths for all SP-PCR products of (E) <i>Pms2^+/+</i> and (F) <i>Pms2^−/−</i>. These graphs are therefore an exact, combined representation of all blots analyzed for <i>Pms2^+/+</i> and <i>Pms2^−/−</i> cerebrum. The number of products measured at each repeat length is on the Y-axis. Note that the actual frequency of products at the progenitor lengths may be slightly higher than the counted frequency, because a single band may represent PCR products amplified from multiple progenitor molecules. The gray bands represent the progenitor allele lengths +/−5%. Lines within the gray bands represent unchanged bands. Lines outside of these ranges represent mutations. Those to the right of the rightmost band represent expansions, those to the left of the leftmost band represent contractions, and those in the middle represent unclassified mutations. Panel G shows the cumulative number of products observed with increasing expansion size. Expansion size was calculated as the percent increase in repeat length above the largest progenitor. An incremental increase is seen in <i>Pms2^−/−</i> versus <i>Pms2^+/+</i> mice at all expansion sizes, but the increase in magnitude of the difference at higher expansion sizes points to the role of Pms2 in preferentially suppressing large expansions in the cerebrum.</p

Absence of Pms2 increases somatic expansion load of the (GAA·TTC)_n sequence in cerebellum.

<p>Analysis of instability in cerebellum of <i>Pms2^+/+</i> and <i>Pms2^−/−</i> mice. Progenitor allele lengths of 232, 199, 143, and 99 repeats for (A) <i>Pms2^+/+</i>, and 224, 195, 152, and 101 repeats for (B) <i>Pms2^−/−</i> are indicated by arrowheads. Mutation load and expansion load, calculated from analysis of ∼400 molecules per genotype, are shown in C and D, respectively. Panels E and F indicate the distribution of repeat lengths for all SP-PCR products of (E) <i>Pms2^+/+</i> and (F) <i>Pms2^−/−</i>. These graphs are therefore an exact, combined representation of all blots analyzed for <i>Pms2^+/+</i> and <i>Pms2^−/−</i> cerebrum. The number of products measured at each repeat length is on the Y-axis. Note that the actual frequency of products at the progenitor lengths may be slightly higher than the counted frequency, because a single band may represent PCR products amplified from multiple progenitor molecules. The gray bars represent the progenitor allele lengths +/−5%. Lines within the gray bands represent unchanged bands. Lines outside of these ranges represent mutations. Those to the right of the rightmost band represent expansions, those to the left of the leftmost band represent contractions, and those in the middle represent unclassified mutations. Products outside of these ranges were counted as mutations. Panel G shows the cumulative number of products observed with increasing expansion size. Expansion size was calculated as the percent increase in repeat length above the largest progenitor. An incremental increase is seen in <i>Pms2^−/−</i> versus <i>Pms2^+/+</i> mice at all expansion sizes, but the increase in magnitude of the difference at higher expansion sizes points to the role of Pms2 in preferentially suppressing large expansions in the cerebellum.</p