Histone Deacetylase Complexes Promote Trinucleotide Repeat Expansions

et al. (2012) Histone Deacetylase Complexes Promote Trinucleotide Repeat Expansions. PLoS Biol 10(2): e1001257. doi:10.1371/journal.pbio.1001257 Histone Deacetylase Complexes Promote Trinucleotide Repeat Expansions Kim Debacker. 0 Aisling Frizzell. 0 Olive Gleeson 0 Lucy Kirkham-McCarthy 0 Tony Mertz 0 Robert S. Lahue 0 Nancy Maizels, University of Washington, United States of America 0 Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway , Galway , Ireland Expansions of DNA trinucleotide repeats cause at least 17 inherited neurodegenerative diseases, such as Huntington's disease. Expansions can occur at frequencies approaching 100% in affected families and in transgenic mice, suggesting that specific cellular proteins actively promote (favor) expansions. The inference is that expansions arise due to the presence of these promoting proteins, not their absence, and that interfering with these proteins can suppress expansions. The goal of this study was to identify novel factors that promote expansions. We discovered that specific histone deacetylase complexes (HDACs) promote CTGNCAG repeat expansions in budding yeast and human cells. Mutation or inhibition of yeast Rpd3L or Hda1 suppressed up to 90% of expansions. In cultured human astrocytes, expansions were suppressed by 75% upon inhibition or knockdown of HDAC3, whereas siRNA against the histone acetyltransferases CBP/p300 stimulated expansions. Genetic and molecular analysis both indicated that HDACs act at a distance from the triplet repeat to promote expansions. Expansion assays with nuclease mutants indicated that Sae2 is one of the relevant factors regulated by Rpd3L and Hda1. The causal relationship between HDACs and expansions indicates that HDACs can promote mutagenesis at some DNA sequences. This relationship further implies that HDAC3 inhibitors being tested for relief of expansion-associated gene silencing may also suppress somatic expansions that contribute to disease progression. - Funding: This work was supported by Science Foundation Ireland (SFI; www.sfi.ie) grants 06/IN.1/B73 and 10/IN.1/B2973, by an SFI equipment award, and by the Millenium Fund of National University of Ireland, Galway (www.nuigalway.ie) (all to R.S.L.); by a postdoctoral fellowship from the Irish Research Council for Science Engineering and Technology (IRCSET, www.ircset.ie; to K.D.); and by an IRCSET postgraduate scholarship and by the Thomas Crawford Hayes Fund www. nuigalway.ie (both to A.F.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: DM1, myotonic dystrophy type 1; HAT, histone acetyltransferase; HD, Huntington9s disease; HDAC, histone deacetylase complex; TNR, trinucleotide repeat; TSA, trichostatin A . These authors contributed equally to this work. The relentless expansion of trinucleotide repeats (TNRs) causes Huntingtons disease (HD), myotonic dystrophy type 1 (DM1), and at least 15 other inherited neurological disorders [1]. It is thought that expansions are actively promoted by the presence of key proteins, not their absence, probably due to the corruption of their normal biochemical activities by TNR DNA [24]. Evidence for promoting factors includes the fact that disease alleles expand at high frequencies, sometimes approaching 100% [5], in otherwise normal individuals and in a number of transgenic and knockin mouse models of HD and DM1 [612]. Using candidate gene approaches, the DNA repair factors Msh2, Msh3, Pms2, Ogg1, and Xpa were identified as promoting proteins in mice, based on the fact that somatic expansions are suppressed ,50% 90% by homozygous knockout of Msh2, Msh3, Pms2, Ogg1, or Xpa [613]. Knockout of Msh2 or Msh3 also largely eliminates intergenerational expansions [7,9,10,14]. Thus, key DNA repair components promote expansions in certain mouse models. The transgenic mice studies described above monitor long, diseasecausing TNRs becoming even longer. For example, commonly used HD mouse models carry CAG tracts of 110120 repeats [10,12]. A human inheriting an HD allele in this length range would develop the disease as a young child [15]. As an alternative approach, we focus on expansions near the crucial threshold, a narrow range of allele lengths (,3040 uninterrupted repeats in humans [2,4,16]) that demarcates stable shorter repeats from unstable longer tracts. Expansion risk in humans and in yeast increases sharply once the threshold is crossed [17,18]. Expansions crossing the threshold are critical initiating mutations leading to enhanced instability and disease [24]. It is not known whether the mechanism of expansion is the same for threshold-length alleles and long, disease-causing tracts. In this study, we find that yeast mutants lacking the nucleases Sae2 or Mre11 reduce expansion rates for (CTG)20 alleles, whereas sae2 or mre11 mutants show increased expansion frequencies for long (CAG)70 repeats [19]. This new evidence suggests that triplet repeat length helps determine expansion mechanism. The goal of this study was to identify novel factors in yeast and human cells that promote expansions of TNR alleles near the threshold. We found specific histone deacetylase complexes (HDACs) that promote expansions, plus one human histone acetyltransferase (HAT) that inhibits expansions, and we suggest a mechanistic link between HDACs and DNA repair. These results indicate a causal relationship between HDACs and expansions, and they show that protein acetylation and deacetylation are key modulators of TNR instability. The human genome contains numerous DNA trinucleotide repeats, which mutate infrequently in most situations. However, in families affected by certain inherited neurological diseases such as Huntingtons, a trinucleotide repeat has undergone an expansion mutation that lengthens the repeat tract. This expansion is generally sufficient to cause disease. Further germline and somatic expansions in affected families occur at very high frequenciesapproaching 100% in some casessuggesting that mutation of the trinucleotide repeat becomes the norm rather than the exception, while the rest of the genome remains genetically stable. These observations indicate that trinucleotide repeat expansions are localized in the genome and occur by novel mutational mechanisms. We searched for proteins that favor expansions and identified specific histone deacetylase complexes (HDACs)comprising enzymes that remove acetyl groups from histonesin budding yeast and in human astrocytes. Interfering with these HDACs by mutation, RNA interference, or small molecule inhibitors blocked 50%90% of expansion events. We also found that yeast HDACs promote expansions via a downstream deacetylation target, the nuclease Sae2. These results indicate that HDACs promote trinucleotide repeat expansions by modu (...truncated)