MutSβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

Abstract

Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington's disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutSβ (MSH2-MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (∼30-40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG•CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutSβ subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutSα subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutSβ, but not MutSα, was enriched at the TNR. These findings imply a direct role for MutSβ in promoting expansion of threshold-length CTG•CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutSβ, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs.

Figure 1.

Expansions are suppressed following siRNA knockdown of MSH2 and MSH3, but not by knockdown of MSH6, CtIP or Mre11. (A) Expansion frequencies subsequent to treatment with MSH2 siRNA, MSH3 siRNA, MSH6 siRNA, CtIP individual siRNAs (denoted #1 and #2) and Mre11 siRNA. All frequencies are normalized to scrambled siRNA, denoted as ‘Scr’ (white bars). Error bars denote ±1 SEM; *P < 0.05 compared to scrambled siRNA control; n = 4 for MSH2 and MSH6 siRNA, n = 3 for MSH3, CtIP and Mre11 siRNA. (B) Quantification of protein levels following knockdown, normalized to actin and to the scrambled siRNA control. Error bars denote ±1 SEM; n = 3.

Figure 2.

(A) MSH2 and MSH3, but not MSH6, are enriched at the (CTG)₂₂ repeat tract in human SVG-A cells. Cells were transfected with a shuttle vector containing either a (CTG)₂₂ repeat tract (red bars) or a corresponding randomized (C,T,G)₂₂ sequence as a control (white bars). ChIP reactions were performed subsequently with antibodies specific for MSH2, MSH3 or MSH6, while background signals were assessed using a FLAG tag antibody. Real-time PCR signals for occupancy of MSH2, MSH3 and MSH6 are presented as fold enrichment over background signals. Error bars, ±SEM; *P < 0.05 compared to randomized (C,T,G)₂₂ control; n = 5. (B) Schematic showing position of real-time PCR primers for ChIP experiments, relative to the (CTG)₂₂ tract or randomized (C,T,G)₂₂ sequence. The ChIP amplicon is 89 bp, with 69 bp of flanking sequence between the amplicon and the repeat tract or control sequence.

Figure 3.

Double knockdown of HDAC3 and MSH2 leads to an expansion phenotype that is indistinguishable from knockdown of either protein alone. (A) Expansion frequencies following separate siRNA treatments against HDAC3 and MSH2 and a simultaneous HDAC3+MSH2 siRNA treatment. All frequencies were normalized to scrambled siRNA (‘Scr’, white bar). Error bars denote ±1 SEM; *P < 0.05 compared to scrambled control; n = 3. (B) Representative western blot confirming knockdown of HDAC3 and MSH2 expression following siRNA treatments. Actin was used as a loading control.

Figure 4.

RNAi knockdown of HDAC3 does not affect occupancy of MSH2, MSH3 or MSH6 at (CTG)₂₂ repeat tracts, or protein expression of MSH2 or MSH3, in SVG-A cells. (A) SVG-A cells were transiently transfected with a (CTG)₂₂-repeat containing shuttle vector, in the presence of scrambled siRNA (white bars) or HDAC3 siRNA (navy bars). ChIP reactions were performed subsequently with antibodies specific for MSH2, MSH3 or MSH6, while background signals were assessed using a FLAG tag antibody. Signals for occupancy of MSH2, MSH3 and MSH6 are presented as fold enrichment over background signals. Error bars, ±SEM; *P < 0.05 compared to control; n = 5. (B) Representative immunoblots for MSH2 and MSH3 protein expression in SVG-A cells following transfection with scrambled siRNA or HDAC3 siRNA. For assessment of MSH2 and MSH3 protein expression, α-tubulin was used as a loading control. For assessment of HDAC3 protein expression, actin was used as a loading control.

Figure 5.

Single and double knockdowns of HDAC3 and HDAC5 lead to similar reductions in expansions. (A) Expansion frequencies subsequent to treatment with HDAC5 SMARTpool siRNA (labelled HDAC5 in the graph; n = 6), individual HDAC5 siRNAs (labelled #5 and #7; n = 3), HDAC3 siRNA (n = 3), HDAC5 SMARTpool siRNA (n = 3) and simultaneous HDAC3 and HDAC5 siRNA treatments (n = 3). Error bars denote ± 1 SEM; *P < 0.05 compared to scrambled siRNA control. (B) Expression levels of HDAC5 and HDAC3 determined by real-time RT–PCR normalized to scrambled siRNA and HPRT levels following siRNA treatment with HDAC5 SMARTpool (n = 3), HDAC5 siRNAs #5 and HDAC #7 (n = 2), HDAC3 SMARTpool (n = 2), separate siRNA treatments against HDAC3 and HDAC5 (n = 2) and a simultaneous HDAC3+HDAC5 siRNA treatment (n = 2). Error bars denote range.

Figure 6.

Double knockdown of HDAC5 and MSH2 leads to an expansion phenotype that is indistinguishable from knockdown of either protein alone. (A) Expansion frequencies following separate siRNA treatments against HDAC5 or MSH2 or a simultaneous HDAC5+MSH2 siRNA treatment. All frequencies were normalized to scrambled siRNA. Error bars denote ±1 SEM; *P < 0.05 compared to scrambled control; n = 3–4. (B) Expression levels of HDAC5 and MSH2 determined by real-time RT–PCR. Expression was normalized to scrambled siRNA and HPRT levels following siRNA treatment with individual HDAC5 or MSH2 SMARTpool siRNAs or a simultaneous HDAC5+MSH2 siRNA treatment (n = 2). Error bars denote range. (C) Schematic diagram of the genetic relationship between HDAC3, HDAC5 and MutSβ in regard to TNR expansions. Double-headed arrows signify genetic epistasis, as inferred from siRNA double knockdowns.