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

Abstract

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.

Keywords: histone deacetylase 3; mismatch repair; triplet repeat expansion.

Fig. 1.

Putative HDAC3 sites in MutSβ affect triplet repeat expansions. (A) Previous proteomic studies (https://www.phosphosite.org//homeAction.action) identified five acetylated lysine residues in Msh3. The Msh3-5KR mutant changes these lysine residues to arginines, mimicking a permanently deacetylated Msh3 protein. (B) Representative Western blot after HDAC3 inhibition by RGFP966 treatment. (C) Quantification of Western blot signals as a function of RGFP966 dose. **P < 0.001, n = 4. (D) TNR expansions as a function of RGF966 dose in cells expressing Msh3 or Msh3-5KR. *P < 0.05, n = 4. See SI Appendix, Fig. S1C for statistical details.

Fig. 2.

Deacetylation assays of acetylated peptides derived from Msh3 in the presence of SMRT(389-480)/HDAC3 complex. (A) KAc and CH₃COO⁻ peak integral quantification. Dynamics Center (Bruker) interface showing the alignment of peaks corresponding to the acetyl group of the acetylated lysines (substrate, ∼1.83 ppm) and the free acetic acid (product, ∼1.78 ppm) after incubation with SMRT(389-480)/HDAC3 at 20 °C for 60 min. (B) SMRT(389-480)/HDAC3 complex preferentially deacetylates Msh3 K122 peptide over the rest of the substrates. The ratio of the integrals of the products (free acetic acid) over the integrals of the substrates (acetyl group of lysines) was plotted against reaction time.

Fig. 3.

DNA mismatch repair and DNA hairpin repair assays after RGFP966 treatment. (A) Western blots showing expression of MMR proteins and related histone marks. (B) In vitro MMR assay using a 5′ nicked heteroduplex containing a G-T mismatch or a 2-nt insertion/deletion mispair. The % of repair represents the average repair of three independent experiments. (C) Diagram of DNA substrate containing a (CAG)₅ or (CAG)₁₅ hairpin structure in the continuous strand. (D) Southern blot analysis showing nick-directed hairpin repair. (E) Quantification of hairpin repair. P < 0.04, n = 3. n.s., not significant. See SI Appendix, Fig. S1C for statistical details.

Fig. 4.

Subcellular fractionation of MutSβ following treatment with RGFP966. (A) Sequence alignment of a known NLS in Msh6 (67, 68) with Msh3 and Msh3-5KR. Msh3 residues 98, 99, and 103 are shown in boldface. (B) Representative Western blot of subcellular fractionation of MutSβ. (C) Representative Western blot of subcellular fractionation of MutSβ containing Msh3-5KR. (D) Quantification of subcellular fractionation experiments performed in Msh3-expressing cells. Graph depicts percentage of total Msh3 or Msh2 protein that is localized to the cytoplasm. *P = 0.0183, n = 3, SI Appendix, Fig. S1C. (E) Quantification of subcellular fractionation experiments performed in Msh3-5KR-expressing cells. Graph depicts percentage of total Msh3 or Msh2 protein that is localized to the cytoplasm, n = 3.

Fig. 5.

Immunofluorescence microscopy using EGFP tags. In all panels, DAPI + GFP indicates overlay of separate imaging for DAPI and EGFP. When present, RGFP966 dose was 10 μM. Scale bars are indicated in each image. (A) Transient transfections of EGFP fusion peptides. Top Left, EGFP alone; Top Right, SV40-NLS^3x-EGFP positive control; Bottom Left, Msh3^91-130-EGFP; Bottom Right, Msh3-5KR^91-130-EGFP. FITC channel for EGFP signal. (B) Stable transfectants expressing full-length Msh3-EGFP. White arrows indicate cytoplasmic accumulation of fluorescent protein. (C) Stable transfectants expressing full-length Msh3-5KR-EGFP.

Fig. 6.

Model for SMRT/HDAC3 control of MutSβ subcellular localization. MutSβ is able to translocate into the nucleus due to the nuclear localization sequence in Msh3. Inside the nucleus, MutSβ is subject to acetylation by p300/CBP and deacetylation by SMRT/HDAC3. The deacetylated form of MutSβ can translocate in and out of the nucleus (Left). MutSβ containing Msh3-5KR simulates this situation. In contrast, the acetylated form of MutSβ can exit the nucleus (Right) but its reentry occurs with reduced efficiency (red vertical bar). Treatment with RGFP966 favors acetylated MutSβ, hinders its nuclear reentry, and thereby suppresses triplet repeat expansions.