Restarted replication forks are error-prone and cause CAG repeat expansions and contractions

Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2–7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.

Expanded CAG/CTG repeats resist gene silencing mediated by targeted Epigenome editing

Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Previous studies have implicated the histone deacetylase HDAC5 and the DNA methyltransferase DNMT1 as modulators of repeat instability via mechanisms that are not fully understood. Using PInT, we found no evidence that HDAC5 or DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by targeting HDAC5 and DNMT1. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

Rad9-mediated checkpoint activation is responsible for elevated expansions of GAA repeats in CST-deficient yeast

Large-scale expansion of (GAA)n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich’s ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system (Zhang et al. 2012). The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that, in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)100 repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53 and EXO1 belonging to this pathway rescued the increased GAA expansions. Further, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA Polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.

Brain Regional Differences in Hexanucleotide Repeat Length in X-Linked Dystonia-Parkinsonism Using Nanopore Sequencing

ObjectiveOur study investigated the presence of regional differences in hexanucleotide repeat number in postmortem brain tissues of 2 patients with X-linked dystonia-parkinsonism (XDP), a combined dystonia-parkinsonism syndrome modified by a (CCCTCT)n repeat within the causal SINE-VNTR-Alu retrotransposon insertion in the TAF1 gene.MethodsGenomic DNA was extracted from blood and postmortem brain samples, including the basal ganglia and cortex from both patients and from the cerebellum, midbrain, and pituitary gland from 1 patient. Repeat sizing was performed using fragment analysis, small-pool PCR-based Southern blotting, and Oxford nanopore sequencing.ResultsThe basal ganglia (p < 0.001) and cerebellum (p < 0.001) showed higher median repeat numbers and higher degrees of repeat instability compared with blood.ConclusionsSomatic repeat instability may predominate in brain regions selectively affected in XDP, thereby hinting at its potential role in disease manifestation and modification.

Association Analysis of Chromosome X to Identify Genetic Modifiers of Huntington’s Disease

Background: Huntington’s disease (HD) is caused by an expanded (>35) CAG trinucleotide repeat in huntingtin (HTT). Age-at-onset of motor symptoms is inversely correlated with the size of the inherited CAG repeat, which expands further in brain regions due to somatic repeat instability. Our recent genetic investigation focusing on autosomal SNPs revealed that age-at-onset is also influenced by genetic variation at many loci, the majority of which encode genes involved in DNA maintenance/repair processes and repeat instability. Objective: We performed a complementary association analysis to determine whether variants in the X chromosome modify HD. Methods: We imputed SNPs on chromosome X for ∼9,000 HD subjects of European ancestry and performed an X chromosome-wide association study (XWAS) to test for association with age-at-onset corrected for inherited CAG repeat length. Results: In a mixed effects model XWAS analysis of all subjects (males and females), assuming random X-inactivation in females, no genome-wide significant onset modification signal was found. However, suggestive significant association signals were detected at Xq12 (top SNP, rs59098970; p-value, 1.4E-6), near moesin (MSN), in a region devoid of DNA maintenance genes. Additional suggestive signals not involving DNA repair genes were observed in male- and female-only analyses at other locations. Conclusion: Although not genome-wide significant, potentially due to small effect size compared to the power of the current study, our data leave open the possibility of modification of HD by a non-DNA repair process. Our XWAS results are publicly available at the updated GEM EURO 9K website hosted at https://www.hdinhd.org/ for browsing, pathway analysis, and data download.

A transgenic cell line with inducible transcription for studying (CGG)n repeat expansion mechanisms

There are more than 30 inherited human disorders connected with repeat expansion (myotonic dystrophy type I, Huntington’s disease, Fragile X syndrome). Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear. An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures. There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structures can block various stages of DNA metabolism processes, such as replication, repair and recombination and it is considered as the source of repeat instability. However, none of the hypotheses is fully conf irmed or is the only valid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription and TCR­-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of (CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNA metabolism and, therefore, the developed systems are universal and applicable for various studies. Transgenic cell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantif ication was developed. One type of the cell lines contains an exogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vector is maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for f inding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed for the quantif ication of exogenous (CGG)n repeat instability in the transgenic cell lines’ genome.