Genetic analysis in budding yeast and in cultured human astrocytes reveals that specific histone deacetylase complexes accelerate expansion mutations in DNA triplet repeats.

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.

SUMMARY Human RTEL1 is an essential, multifunctional helicase that maintains telomeres, regulates homologous recombination, and helps prevent bone marrow failure. Here, we show that RTEL1 also blocks trinucleotide repeat expansions, the causal mutation for 17 neurological diseases. Increased expansion frequencies of (CTG·CAG) repeats occurred in human cells following knockdown of RTEL1, but not the alternative helicase Fbh1, and purified RTEL1 efficiently unwound triplet repeat hairpins in vitro. The expansion-blocking activity of RTEL1 also required Rad18 and HLTF, homologs of yeast Rad18 and Rad5. These findings are reminiscent of budding yeast Srs2, which inhibits expansions, unwinds hairpins, and prevents triplet-repeat-induced chromosome fragility. Accordingly, we found expansions and fragility were suppressed in yeast srs2 mutants expressing RTEL1, but not Fbh1. We propose that RTEL1 serves as a human analog of Srs2 to inhibit (CTG·CAG) repeat expansions and fragility, likely by unwinding problematic hairpins.

Histone deacetylase complexes (HDACs) are powerful regulators of the epigenome. It is now clear that a subset of HDACs also regulate the stability of the genome itself, but not primarily through transcription. Instead, these key HDACs control genome stability more directly by stabilizing enzymes important for DNA mutagenesis and repair, or by modifying histones at sites of DNA damage. Surprisingly, certain HDACs in budding yeast and human cells accelerate the pace of genetic expansions in trinucleotide repeats, the type of mutation that causes Huntington disease. In other words, HDACs promote mutagenesis in some settings. At double-strand breaks, however, the same HDACs in budding yeast help stabilize the genome by facilitating homology-dependent repair. Double-strand breaks can also be repaired without the requirement for homology, and two specific human HDACs are now known to promote this event. These new findings highlight certain HDACs as caretakers of genome stability, and also underscore the potential medical complexities in using HDAC inhibitors for treatment of disease.

No data exist on the ability of thiolation domains from fungal non-ribosomal peptide synthetases to undergo 4′-phosphopantetheinylation, using either biotinylated or fluorescently labeled coenzyme A analogues, mediated by 4′-phosphopantetheinyl transferases (PPTase). Yet, this is a key requirement to confirm the amino acid recognition function, and coding potential, of either non-ribosomal peptide synthetases or recombinantly expressed regions of these enzymes (e.g., didomains or modules). Moreover, determination of 4′-phosphopantetheinylation activity remains cumbersome. Here, we demonstrate that a recombinant fungal PPTase catalyzes the solutionphase transfer of either biotin-or fluorescein-labeled 4′-phosphopantetheine region of coenzyme A to a fungal thiolation domain, which is either part of a non-ribosomal peptide synthetase didomain (72 kDa), derived from Aspergillus fumigatus, or fused to a non-native protein (glutathione s-transferase). Significantly, we demonstrate that this reaction can unexpectedly occur when the target protein (4.4 pmol) is immobilized on a solid surface. These findings (i) confirm that thiolation domains of fungal origin, in native or non-native configuration, can accept modified 4′-phosphopantetheine residues via PPTase-mediated labeling and (ii) illustrate a novel, highthroughput method to determine PPTase activity.