RTEL1 inhibits trinucleotide repeat expansions and fragility.

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.

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

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 MutSß (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 MutSß 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 MutSß, but not MutSα, was enriched at the TNR. These findings imply a direct role for MutSß 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 MutSß, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs.

Histone deacetylase complexes as caretakers of genome stability.

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.

Histone deacetylase complexes promote trinucleotide repeat expansions.

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 CTG•CAG 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.

Solid phase 4'-phosphopantetheinylation: fungal thiolation domains are targets for chemoenzymatic modification.

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 solution-phase 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, high-throughput method to determine PPTase activity.