SPYing on triplet repeat expansions: Insights into FAN1-MLH1 interaction and regulation.

The DNA repair proteins FAN1 and MLH1 have opposing effects on triplet repeat expansions. New studies by Goold et al. (2021) and Porro et al. (2021) pinpoint interactions between FAN1 and MLH1 that cross-regulate each other's activities.

New developments in Huntington's disease and other triplet repeat diseases: DNA repair turns to the dark side.

Huntington's disease (HD) is a fatal, inherited neurodegenerative disease that causes neuronal death, particularly in medium spiny neurons. HD leads to serious and progressive motor, cognitive and psychiatric symptoms. Its genetic basis is an expansion of the CAG triplet repeat in the HTT gene, leading to extra glutamines in the huntingtin protein. HD is one of nine genetic diseases in this polyglutamine (polyQ) category, that also includes a number of inherited spinocerebellar ataxias (SCAs). Traditionally it has been assumed that HD age of onset and disease progression were solely the outcome of age-dependent exposure of neurons to toxic effects of the inherited mutant huntingtin protein. However, recent genome-wide association studies (GWAS) have revealed significant effects of genetic variants outside of HTT. Surprisingly, these variants turn out to be mostly in genes encoding DNA repair factors, suggesting that at least some disease modulation occurs at the level of the HTT DNA itself. These DNA repair proteins are known from model systems to promote ongoing somatic CAG repeat expansions in tissues affected by HD. Thus, for triplet repeats, some DNA repair proteins seem to abandon their normal genoprotective roles and, instead, drive expansions and accelerate disease. One attractive hypothesis-still to be proven rigorously-is that somatic HTT expansions augment the disease burden of the inherited allele. If so, therapeutic approaches that lower levels of huntingtin protein may need blending with additional therapies that reduce levels of somatic CAG repeat expansions to achieve maximal effect.

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

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 MutSß (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 MutSß 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 MutSß 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 MutSß is partially dependent on HDAC3 activity. Together, these results indicate that MutSß 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.

Assessing Triplet Repeat Expansions in Human SVG-A Cell Culture.

Determining the molecular mechanisms that contribute to trinucleotide repeat (TNR) expansions is essential to understanding the origin of genetically inherited diseases, such as Huntington's disease, and to inform efforts in developing therapeutic treatments. As one resource to probe the mechanisms of TNR expansions, we describe an expansion assay in human tissue culture cells. The cell line SVG-A, derived from human astrocytes, has the important property of supporting expansions in culture, unlike many cell lines derived from patients. SVG-A cells are also amenable to standard genetic and biochemical techniques such as siRNA, CRISPR-Cas9 and enzymatic inhibitors. This combination of features allows for mechanistic studies of TNR expansions, using the quantitative genetic assay described here as a readout. The SVG-A assay has correctly identified key proteins that drive expansions and it has facilitated testing of enzymatic inhibitors that suppress expansions as potential therapeutics. This chapter describes how repeat expansions are detected, visualized, and quantified.

MutSß abundance and Msh3 ATP hydrolysis activity are important drivers of CTG•CAG repeat expansions.

CTG•CAG repeat expansions cause at least twelve inherited neurological diseases. Expansions require the presence, not the absence, of the mismatch repair protein MutSß (Msh2-Msh3 heterodimer). To evaluate properties of MutSß that drive expansions, previous studies have tested under-expression, ATPase function or polymorphic variants of Msh2 and Msh3, but in disparate experimental systems. Additionally, some variants destabilize MutSß, potentially masking the effects of biochemical alterations of the variations. Here, human Msh3 was mutated to selectively inactivate MutSß. Msh3-/- cells are severely defective for CTG•CAG repeat expansions but show full activity on contractions. Msh3-/- cells provide a single, isogenic system to add back Msh3 and test key biochemical features of MutSß on expansions. Msh3 overexpression led to high expansion activity and elevated levels of MutSß complex, indicating that MutSß abundance drives expansions. An ATPase-defective Msh3 expressed at normal levels was as defective in expansions as Msh3-/- cells, indicating that Msh3 ATPase function is critical for expansions. Expression of two Msh3 polymorphic variants at normal levels showed no detectable change in expansions, suggesting these polymorphisms primarily affect Msh3 protein stability, not activity. In summary, CTG•CAG expansions are limited by the abundance of MutSß and rely heavily on Msh3 ATPase function.

A selective inhibitor of histone deacetylase 3 prevents cognitive deficits and suppresses striatal CAG repeat expansions in Huntington's disease mice.

Huntington's disease (HD) is a neurodegenerative disorder whose major symptoms include progressive motor and cognitive dysfunction. Cognitive decline is a critical quality of life concern for HD patients and families. The enzyme histone deacetylase 3 (HDAC3) appears to be important in HD pathology by negatively regulating genes involved in cognitive functions. Furthermore, HDAC3 has been implicated in the aberrant transcriptional patterns that help cause disease symptoms in HD mice. HDAC3 also helps fuel CAG repeat expansions in human cells, suggesting that HDAC3 may power striatal expansions in the HTT gene thought to drive disease progression. This multifaceted role suggests that early HDAC3 inhibition offers an attractive mechanism to prevent HD cognitive decline and to suppress striatal expansions. This hypothesis was investigated by treating HdhQ111 knock-in mice with the HDAC3-selective inhibitor RGFP966. Chronic early treatment prevented long-term memory impairments and normalized specific memory-related gene expression in hippocampus. Additionally, RGFP966 prevented corticostriatal-dependent motor learning deficits, significantly suppressed striatal CAG repeat expansions, partially rescued striatal protein marker expression and reduced accumulation of mutant huntingtin oligomeric forms. These novel results highlight RGFP966 as an appealing multiple-benefit therapy in HD that concurrently prevents cognitive decline and suppresses striatal CAG repeat expansions.

The Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex promotes trinucleotide repeat expansions independently of homologous recombination.

Trinucleotide repeats (TNRs) are tandem arrays of three nucleotides that can expand in length to cause at least 17 inherited human diseases. Somatic expansions in patients can occur in differentiated tissues where DNA replication is limited and cannot be a primary source of somatic mutation. Instead, mouse models of TNR diseases have shown that both inherited and somatic expansions can be suppressed by the loss of certain DNA repair factors. It is generally believed that these repair factors cause misprocessing of TNRs, leading to expansions. Here we extend this idea to show that the Mre11-Rad50-Xrs2 (MRX) complex of Saccharomyces cerevisiae is a causative factor in expansions of short TNRs. Mutations that eliminate MRX subunits led to significant suppression of expansions whereas mutations that inactivate Rad51 had only a minor effect. Coupled with previous evidence, this suggests that MRX drives expansions of short TNRs through a process distinct from homologous recombination. The nuclease function of Mre11 was dispensable for expansions, suggesting that expansions do not occur by Mre11-dependent nucleolytic processing of the TNR. Epistasis between MRX and post-replication repair (PRR) was tested. PRR protects against expansions, so a rad5 mutant gave a high expansion rate. In contrast, the mre11 rad5 double mutant gave a suppressed expansion rate, indistinguishable from the mre11 single mutant. This suggests that MRX creates a TNR substrate for PRR. Protein acetylation was also tested as a mechanism regulating MRX activity in expansions. Six acetylation sites were identified in Rad50. Mutation of all six lysine residues to arginine gave partial bypass of a sin3 HDAC mutant, suggesting that Rad50 acetylation is functionally important for Sin3-mediated expansions. Overall we conclude that yeast MRX helps drive expansions of short TNRs by a mechanism distinct from its role in homologous recombination and independent of the nuclease function of Mre11.

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.

Nucleotide excision repair and the 26S proteasome function together to promote trinucleotide repeat expansions.

Trinucleotide repeat (TNR) expansion underpins a number of inheritable neurological human disorders. Multiple mechanisms are thought to contribute to the expansion process. The incorrect processing of the repeat tract by DNA repair proteins can drive this mutation process forward, as expansions are suppressed following ablation of certain repair factors in mouse models and cell models of disease. Nucleotide excision repair (NER) is one repair pathway implicated in TNR instability, although most previous work focussed on TNR contractions, not expansions. Here we investigated the role of NER in modulating expansions of threshold-length (CTG·CAG) repeats in yeast. We show that both the global genome and transcription-coupled repair subpathways promote expansions of threshold-length TNRs. Furthermore, NER works with the 26S proteasome to drive expansions, based on analysis of double mutants defective in both pathways, and of Rad23, a protein involved in both NER and the shuttling of ubiquitinated proteins to the proteasome. This work provides the first evidence that both subpathways of NER can promote threshold-length TNR expansions and that NER interacts with the proteasome to drive expansions.

The 26S proteasome drives trinucleotide repeat expansions.

Trinucleotide repeat (TNR) expansion is the causative mutation for at least 17 inherited neurological diseases. An important question in the field is which proteins drive the expansion process. This study reports that the multi-functional protein Sem1 is a novel driver of TNR expansions in budding yeast. Mutants of SEM1 suppress up to 90% of expansions. Subsequent analysis showed that Sem1 facilitates expansions via its function in the 26S proteasome, a highly conserved multi-subunit complex with both proteolytic and non-proteolytic functions. The proteolytic function of the 26S proteasome is relevant to expansions, as mutation of additional proteasome components or treatment of yeast with a proteasome inhibitor suppressed CTG•CAG expansions. The 26S proteasome also drives expansions in human cells. In a human astrocytic cell line, siRNA-mediated knockdown of 26S proteasome subunits PSMC5 or PSMB3 reduced expansions. This expansion phenotype, both in yeast and human cells, is dependent on the proteolytic activity of the proteasome rather than a stress response owing to depletion of free ubiquitin. Thus, the 26S proteasome is a novel factor that drives expansions in both yeast and human cells by a mechanism involving protein degradation.

Trinucleotide repeat expansions catalyzed by human cell-free extracts.

Trinucleotide repeat expansions cause 17 heritable human neurological disorders. In some diseases, somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited. This finding stimulated significant interest in replication-independent expansion mechanisms. Aberrant DNA repair is a likely source, based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutSß (Msh2-Msh3 complex). Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats. The findings included expansions on one strand but not the other, or processing of DNA hairpin structures thought to be important intermediates in the expansion process. However, it has been difficult to recapitulate complete expansions in vitro, and the biochemical role of MutSß remains controversial. Here, we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication. The extract promotes a size range of expansions that is similar to certain diseases, and triplet repeat length and sequence govern expansions in vitro as in vivo. MutSß stimulates expansions in the extract, consistent with aberrant repair of endogenous DNA damage as a source of expansions. Overall, this biochemical system retains the key characteristics of somatic expansions in humans and mice, suggesting that this important mutagenic process can be restored in the test tube.

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.

New functions of Ctf18-RFC in preserving genome stability outside its role in sister chromatid cohesion.

Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability--expansions, contractions, and fragility--with effect over a wide range of allele lengths from 20-155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair.

Anc1, a protein associated with multiple transcription complexes, is involved in postreplication repair pathway in S. cerevisiae.

Yeast strains lacking Anc1, a member of the YEATS protein family, are sensitive to several DNA damaging agents. The YEATS family includes two human genes that are common fusion partners with MLL in human acute leukemias. Anc1 is a member of seven multi-protein complexes involved in transcription, and the damage sensitivity observed in anc1Delta cells is mirrored in strains deleted for some other non-essential members of several of these complexes. Here we show that ANC1 is in the same epistasis group as SRS2 and RAD5, members of the postreplication repair (PRR) pathway, but has additive or synergistic interactions with several other members of this pathway. Although PRR is traditionally divided into an "error-prone" and an "error-free" branch, ANC1 is not epistatic with all members of either established branch, and instead defines a new error-free branch of the PRR pathway. Like several genes involved in PRR, an intact ANC1 gene significantly suppresses spontaneous mutation rates, including the expansion of (CAG)(25) repeats. Anc1's role in the PRR pathway, as well as its role in suppressing triplet repeats, point to a possible mechanism for a protein of potential medical interest.

Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae.

Small looped mispairs are corrected by DNA mismatch repair. In addition, a distinct process called large loop repair (LLR) corrects heteroduplexes up to several hundred nucleotides in bacteria, yeast and human cells, and in cell-free extracts. Only some LLR protein components are known, however. Previous studies with neutralizing antibodies suggested a role for yeast DNA polymerase delta (Pol delta), RFC and PCNA in LLR repair synthesis. In the current study, biochemical fractionation studies identified FEN1 (Rad27) as another required LLR component. In the presence of purified FEN1, Pol delta, RFC and PCNA, repair occurred on heteroduplexes with loops ranging from 8 to 216 nt. Repair utilized a 5' nick, with correction directed to the nicked strand, irrespective of which strand contained the loop. In contrast, repair of a G/T mismatch occurred at low levels, suggesting specificity of the reconstituted system for looped mispairs. The presence of RPA enhanced reactivity on some looped substrates, but RPA was not required for activity. Although additional LLR factors remain to be identified, the excision and resynthesis steps of LLR from a 5' nick can be reconstituted in a purified system with FEN1 and Pol delta, together with PCNA and its loader RFC.

Rapid unwinding of triplet repeat hairpins by Srs2 helicase of Saccharomyces cerevisiae.

Expansions of trinucleotide repeats cause at least 15 heritable human diseases. Single-stranded triplet repeat DNA in vitro forms stable hairpins in a sequence-dependent manner that correlates with expansion risk in vivo. Hairpins are therefore considered likely intermediates during the expansion process. Unwinding of a hairpin by a DNA helicase would help protect against expansions. Yeast Srs2, but not the RecQ homolog Sgs1, blocks expansions in vivo in a manner largely dependent on its helicase function. The current study tested the idea that Srs2 would be faster at unwinding DNA substrates with an extrahelical triplet repeat hairpin embedded in a duplex context. These substrates should mimic the relevant intermediate structure thought to occur in vivo. Srs2 was faster than Sgs1 at unwinding several substrates containing triplet repeat hairpins or another structured loop. In contrast, control substrates with an unstructured loop or a Watson-Crick duplex were unwound equally well by both enzymes. Results with a fluorescently labeled, three-way junction showed that Srs2 unwinding proceeds unabated through extrahelical triplet repeats. In summary, Srs2 maintains its facile unwinding of triplet repeat hairpins embedded within duplex DNA, supporting the genetic evidence that Srs2 is a key helicase in Saccharomyces cerevisiae for preventing expansions.

Mrc1, Tof1 and Csm3 inhibit CAG.CTG repeat instability by at least two mechanisms.

Trinucleotide repeats frequently expand and contract in humans and model organisms. Protein factors that modulate this process have been found by candidate gene approaches or mutant screens for increased expansion rates. To extend this effort, Saccharomyces cerevisiae mutants with higher CAG.CTG repeat contraction rates were sought using a disruption library. This screen identified Mrc1, the homolog of human Claspin, which mediates the replication and DNA damage checkpoints, and also couples the replicative helicase and polymerase. Genetic analysis showed that Mrc1, along with Tof1 and Csm3, inhibits instability in two distinct ways. Contraction rates of (CAG)(20) tracts are elevated by loss of Mrc1, Tof1 or Csm3, but not by defects in most replication checkpoint or DNA damage checkpoint proteins. The three proteins likely inhibit contractions primarily through their coupling activity, which would prevent accumulation of single-strand template DNA prior to the formation of aberrant secondary structure. In contrast, expansion rates of (CTG)(13) are elevated in strains defective for Mrc1, Tof1, Csm3, Mec1, Ddc2, Rad24, Ddc1, Mec3, Rad17, Rad9, Rad53 or Chk1, suggesting that the DNA damage checkpoint inhibits expansions after formation of repeat-dependent structures. Together, these results indicate that at least two Mrc1-dependent mechanisms function to reduce CAG.CTG repeat instability.