HDAC3 deacetylates the DNA mismatch repair factor MutS beta to stimulate triplet repeat expansions

Trinucleotide repeat (TNR) expansions cause nearly 20 severe human neurological diseases which are currently untreatable. For some of these diseases, ongoing somatic expansions accelerate disease progression and may influence age of onset. This new knowledge emphasizes the importance of understanding the protein factors that drive expansions. Recent genetic evidence indicates that the mismatch repair factor MutSβ (Msh2-Msh3 complex) and the histone deacetylase HDAC3 function in the same pathway to drive triplet repeat expansions. Here we tested the hypothesis that HDAC3 deacetylates MutSβ and thereby activates it to drive expansions. The HDAC3-selective inhibitor RGFP966 was used to examine its biological and biochemical consequences in human tissue culture cells. HDAC3 inhibition efficiently suppresses repeat expansion without impeding canonical mismatch repair activity. Five key lysine residues in Msh3 are direct targets of HDAC3 deacetylation. In cells expressing Msh3 in which these lysine residues are mutated to arginine, the inhibitory effect of RGFP966 on expansions is largely bypassed, consistent with the direct deacetylation hypothesis. RGFP966 treatment does not alter MutSβ subunit abundance or complex formation but does partially control its subcellular localization. Deacetylation sites in Msh3 overlap a nuclear localization signal, and we show that localization of MutSβ is partially dependent on HDAC3 activity. Together, these results indicate that MutSβ is a key target of HDAC3 deacetylation and provide insights into an innovative regulatory mechanism for triplet repeat expansions. The results suggest expansion activity may be druggable and support HDAC3-selective inhibition as an attractive therapy in some triplet repeat expansion diseases

The 8-oxoG glycosylase activity of nuclear extracts derived from brain of AD patients

<p><b>Copyright information:</b></p><p>Taken from "Identification and characterization of mutations in patients with Alzheimer's disease"</p><p></p><p>Nucleic Acids Research 2007;35(8):2759-2766.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885677.</p><p>© 2007 The Author(s)</p> Nuclear extracts (40 μg) from AD patients (AD1, AD2 and AD3) or age-matched controls (C1, C2 and C3) were incubated with 50 fmol of P-labeled 36-mer duplex containing a centrally located 8-oxoG residue. 8-oxoG cleavage activity in AD patients, except AD1, was lower than that in age-matched controls

Mismatch Repair Genes <i>Mlh1</i> and <i>Mlh3</i> Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

<div><p>The Huntington's disease gene (<i>HTT</i>) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease <i>Hdh^Q111</i> mice exhibit higher levels of somatic <i>HTT</i> CAG expansion on a C57BL/6 genetic background (B6.<i>Hdh^Q111</i>) than on a 129 background (129.<i>Hdh^Q111</i>). Linkage mapping in (B6x129).<i>Hdh^Q111</i> F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene <i>Mlh1</i> as the most likely candidate modifier. Crossing B6.<i>Hdh^Q111</i> mice onto an <i>Mlh1</i> null background demonstrated that <i>Mlh1</i> is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. <i>Hdh^Q111</i> somatic expansion was also abolished in mice deficient in the <i>Mlh3</i> gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, <i>Mlh1</i> and <i>Mlh3</i> genes encoding MMR effector proteins were as critical to somatic expansion as <i>Msh2</i> and <i>Msh3</i> genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The <i>Mlh1</i> locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify <i>Mlh1</i> and <i>Mlh3</i> as novel critical genetic modifiers of <i>HTT</i> CAG instability, point to <i>Mlh1</i> genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.</p></div

The <i>Mlh1</i> locus is highly polymorphic between B6 and 129 strains.

<p>The <i>Mlh1</i> locus is highly polymorphic between B6 and 129 strains.</p

Reduced MLH1 expression in 129 versus B6 mice.

<p>Quantification of MLH1 (A) mRNA and (B, C) protein levels in the striatum of B6.<i>Mlh1^+/+</i>, 129.<i>Mlh1^+/+</i> and B6.<i>Mlh1^+/−</i> 10-week-old mice (<i>n</i> = 3). (A) Striatal <i>Mlh1</i> mRNA levels (TaqMan Mm00503449_m1, exons 11–12) in 129.<i>Mlh1^+/+</i> mice were significantly reduced by approximately 50% when compared to B6.<i>Mlh1^+/+</i> (<i>p</i><0.05), and were comparable to levels in B6.<i>Mlh1^+/−</i> mice. (B, C) Western blot analysis of MLH1 protein revealed significantly reduced levels in 129.<i>Mlh1^+/+</i> striata compared to B6.<i>Mlh1^+/+</i> striata. Bar graphs represent mean ±SD. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

<i>Mlh1</i> is required for striatal and liver <i>HTT</i> CAG repeat instability in B6.<i>Hdh^Q111/+</i> mice.

<p>(A) Representative GeneMapper profiles of <i>HTT</i> CAG repeat size distributions in the tail, striatum and liver of 22-week-old B6.<i>Hdh^Q111/+</i> mice on different <i>Mlh1</i> genetic backgrounds. <i>Mlh1^+/+</i>, CAG113; <i>Mlh1^+/−</i>, CAG113; <i>Mlh1^−/−</i>, CAG110. (B) Quantification of striatal and liver <i>HTT</i> CAG instability indices in these mice reveals a statistically significant decrease in <i>HTT</i> CAG instability in the absence of <i>Mlh1</i>. <i>Mlh1^+/+</i>, CAG115.3±4.9SD, <i>n</i> = 6; <i>Mlh1^+/−</i>, CAG112.0±2.1SD, <i>n</i> = 6; <i>Mlh1^−/−</i>, CAG109.3±2.6SD, <i>n</i> = 6. Bar graphs represent mean ±SD. ****, <i>p</i><0.0001.</p

Repair of a single CTG slip-out in a cell-free MMR assay is MLH1 dose-dependent.

<p>(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 (<i>p</i> = 0.0013). The individual lanes represented are from the same blot and the experiment was reproduced three times. Bars graphs represent mean ±SD.</p

The 129 and B6 3′-flanking regions of <i>Mlh1</i> confer differential mRNA regulation.

<p>Investigation of the regulatory potential of B6 and 129 immediate (A) 5′- and (B) 3′-flanking regions of <i>Mlh1</i> using dual luciferase reporter assays. (A) The immediate 5′-flanking region of <i>Mlh1</i> containing 17 B6-129 polymorphisms (2,441 bp) was used to drive firefly luciferase expression. (B) The immediate 3′-flanking region of <i>Mlh1</i> (<i>i–iv</i>) 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 (<i>v</i>) of the immediate 3′-flanking region of <i>Mlh1</i> 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 <i>Renilla</i> luminescence and determined relative to the analogous B6 construct. B6-129 polymorphisms are represented by open triangles. Bar graphs represent mean ±SD. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Somatic <i>HTT</i> CAG instability differs between B6.<i>Hdh^Q111</i>^/+ and 129.<i>Hdh^Q111</i>^/+ mice.

<p>(A) Representative GeneMapper profiles of <i>HTT</i> CAG repeat size distributions in the tail, striatum and liver of 10-week-old B6.<i>Hdh^Q111/+</i> and 129.<i>Hdh^Q111/+</i> mice, highlighting the altered contribution of B6 and 129 genetic background to somatic <i>HTT</i> CAG repeat expansion, as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003930#pgen.1003930-Lloret1" target="_blank">[17]</a>. Tail and striatum: B6.<i>Hdh^Q111/+</i>, CAG116; 129.<i>Hdh^Q111/+</i>, CAG112. Liver: B6.<i>Hdh^Q111/+</i>, CAG113; 129.<i>Hdh^Q111/+</i>, CAG111 (B) Quantification of CAG instability index reveals a statistically significant decrease in somatic <i>HTT</i> CAG instability in the striatum and liver of 129.<i>Hdh^Q111</i>^/+ mice compared to B6.<i>Hdh^Q111</i>^/+ mice. B6.<i>Hdh^Q111/+</i> striatum, <i>n</i> = 10, CAG116.9±1.2SD; B6.<i>Hdh^Q111/+</i> liver, <i>n</i> = 10, CAG114.3±1.2SD; 129.<i>Hdh^Q111/+</i> striatum, <i>n</i> = 12, CAG110.9±1.2SD; 129.<i>Hdh^Q111/+</i> liver, <i>n</i> = 9, CAG109.5±1.4SD; Bar graphs represent mean ±SD; ****, <i>p</i><0.0001.</p

Striatal <i>HTT</i> CAG instability in 10-week-old <i>Hdh^Q111/+</i> mice on different genetic backgrounds.

<p>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 <i>Mlh1</i>, <i>Msh3</i> and <i>Msh2</i> genes (“undetermined” indicates failed genotype). F2 mice homozygous or heterozygous for B6 <i>Mlh1</i> alleles display significantly higher levels of striatal somatic CAG instability than F2 mice homozygous for 129 <i>Mlh1</i> alleles (<i>p</i><0.0001 for both). No relationship could be established between <i>Msh3</i> or <i>Msh2</i> genotype and striatal CAG instability. B6.<i>Hdh^Q111/+</i>, <i>n</i> = 10, CAG116.9±1.2SD; 129.<i>Hdh^Q111/+</i>, <i>n</i> = 12, CAG110.9±1.2SD; (B6x129).<i>Hdh^Q111/+</i> F1, <i>n</i> = 11, CAG114.7±6.4SD; (B6x129).<i>Hdh^Q111/+</i> F2, <i>n</i> = 69, CAG107.7±3.2SD. dbSNP markers located within MMR genes: <i>Mlh1</i>, rs30131926 and rs30174694 (concordant genotypes detected with both markers); <i>Msh3</i>, rs29551174; <i>Msh2</i>, rs33609112 and rs49012398 (concordant genotypes detected with both markers). Horizontal bars represent the mean CAG instability indices of the respective groups.</p