Expansions of repetitive DNA sequences cause numerous human neurological and neuromuscular diseases. Ongoing repeat expansions in patients can exacerbate disease progression and severity. As pathogenesis is connected to repeat length, a potential therapeutic avenue is to modulate disease by manipulating repeat expansion size--targeting DNA, the root-cause of symptoms. How repeat instability is mediated by DNA replication, repair, recombination, transcription and epigenetics may explain its contribution to pathogenesis and give insights into therapeutic strategies to block expansions or induce contractions.

Myotonic dystrophy (DM1) affects multiple organs, shows age-dependent progression and is caused by CTG expansions at the DM1 locus. We determined the DM1 CpG methylation profile and CTG length in tissues from DM1 foetuses, DM1 adults, non-affected individuals and transgenic DM1 mice. Analysis included CTCF binding sites upstream and downstream of the CTG tract, as methylation-sensitive CTCF binding affects chromatinization and transcription of the DM1 locus. In humans, in a given foetus, expansions were largest in heart and smallest in liver, differing by 40-400 repeats; in adults, the largest expansions were in heart and cerebral cortex and smallest in cerebellum, differing by up to 5770 repeats in the same individual. Abnormal methylation was specific to the mutant allele. In DM1 adults, heart, liver and cortex showed high-to-moderate methylation levels, whereas cerebellum, kidney and skeletal muscle were devoid of methylation. Methylation decreased between foetuses and adults. Contrary to previous findings, methylation was not restricted to individuals with congenital DM1. The expanded repeat demarcates an abrupt boundary of methylation. Upstream sequences, including the CTCF site, were methylated, whereas the repeat itself and downstream sequences were not. In DM1 mice, expansion-, tissue- and age-specific methylation patterns were similar but not identical to those in DM1 individuals; notably in mice, methylation was present up- and downstream of the repeat, but greater upstream. Thus, in humans, the CpG-free expanded CTG repeat appears to maintain a highly polarized pattern of CpG methylation at the DM1 locus, which varies markedly with age and tissues.

Myotonic dystrophy, caused by DM1 CTG/CAG repeat expansions, shows varying instability levels between tissues and across ages within patients. We determined DNA replication profiles at the DM1 locus in patient fibroblasts and tissues from DM1 transgenic mice of various ages showing different instability. In patient cells, the repeat is flanked by two replication origins demarcated by CTCF sites, with replication diminished at the expansion. In mice, the expansion replicated from only the downstream origin (CAG as lagging template). In testes from mice of three different ages, replication toward the repeat paused at the earliest age and was relieved at later ages-coinciding with increased instability. Brain, pancreas and thymus replication varied with CpG methylation at DM1 CTCF sites. CTCF sites between progressing forks and repeats reduced replication depending on chromatin. Thus, varying replication progression may affect tissue- and age-specific repeat instability.

Mechanisms contributing to disease-associated trinucleotide repeat instability are poorly understood. DNA ligation is an essential step common to replication and repair, both potential sources of repeat instability. Using derivatives of DNA ligase I (hLigI)-deficient human cells (46BR.1G1), we assessed the effect of hLigI activity, overexpression, and its interaction with proliferating cell nuclear antigen (PCNA) upon the ability to replicate and repair trinucleotide repeats. Compared with LigI ؉/؉ , replication progression through repeats was poor, and repair tracts were broadened beyond the slipped-repeat for all mutant extracts. Increased repeat instability was linked only to hLigI overexpression and expression of a mutant hLigI incapable of interacting with PCNA. The endogenous mutant version of hLigI with reduced ligation activity did not alter instability. We distinguished the DNA processes through which hLigI contributes to trinucleotide instability. The highest levels of repeat instability were observed under the hLigI overexpression and were linked to reduced slipped-DNAs repair efficiencies. Therefore, the replication-mediated instability can partly be attributed to errors during replication but also to the poor repair of slipped-DNAs formed during this process. However, repair efficiencies were unaffected by expression of a PCNA interaction mutant of hLigI, limiting this instability to the replication process. The addition of purified proteins suggests that disruption of LigI and PCNA interactions influences trinucleotide repeat instability. The variable levels of age-and tissue-specific trinucleotide repeat instability observed in myotonic dystrophy patients and transgenic mice may be influenced by varying steady state levels of DNA ligase I in these tissues and during different developmental windows.More than 40 hereditary diseases are caused by gene-specific repeat instability (1). Changes at trinucleotide repeats (TNRs) 3 constitute the largest component of this group, causing at least 15 different human diseases, including myotonic dystrophy (DM1), Huntington disease, and fragile X syndrome (FRAXA). Repeat changes in humans are expansion-biased and occur both in parent-to-offspring transmissions and in somatic tissues. The formation of unusual DNA structures during DNA replication and/or aberrant repair of these intermediates has been postulated as the likely source for the development of repeat tract changes (1-3), although the exact molecular mechanisms are unclear. Various proteins have been identified as players in the mutagenic process of TNR instability, including FEN1 (4 -6), OGG1 (7), and some mismatch repair factors, such as MSH2, MSH3, and PMS2 (8 -13). All processes suggested to be involved in repeat instability require a nick located within or proximal to the repeat tract, which ultimately must be ligated. Importantly, many proposed mechanisms of repeat instability involve slippage at the nick (1, 2, 7).Ligation is an essential step in DNA replication, repair, and recombination (14, 1...