Maternal germline-specific effect of DNA ligase I on CTG/CAG instability.

The instability of (CTG)•(CAG) repeats can cause >15 diseases including myotonic dystrophy, DM1. Instability can arise during DNA replication, repair or recombination, where sealing of nicks by DNA ligase I (LIGI) is a final step. The role of LIGI in CTG/CAG instability was determined using in vitro and in vivo approaches. Cell extracts from a human (46BR) harbouring a deficient LIGI (∼3% normal activity) were used to replicate CTG/CAG repeats; and DM1 mice with >300 CTG repeats were crossed with mice harbouring the 46BR LigI. In mice, the defective LigI reduced the frequency of CTG expansions and increased CTG contraction frequencies on female transmissions. Neither male transmissions nor somatic CTG instability was affected by the 46BR LigI - indicating a post-female germline segregation event. Replication-mediated instability was affected by the 46BR LIGI in a manner that depended upon the location of Okazaki fragment initiation relative to the repeat tract; on certain templates, the expansion bias was unaltered by the mutant LIGI, similar to paternal transmissions and somatic tissues; however, a replication fork-shift reduced expansions and increased contractions, similar to maternal transmissions. The presence of contractions in oocytes suggests that the DM1 replication profile specific to pre-meiotic oogenesis replication of maternal alleles is distinct from that occurring in other tissues and, when mediated by the mutant LigI, is predisposed to CTG contractions. Thus, unlike other DNA metabolizing enzymes studied to date, LigI has a highly specific role in CTG repeat maintenance in the maternal germline, involved in mediating CTG expansions and in the avoidance of maternal CTG contractions.

CTG trinucleotide repeat "big jumps": large expansions, small mice.

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions ("big jumps"), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG*CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis.

Cell recovery from DM1 transgenic mouse tissue to study (CTG) n instability and DM1 pathogenesis.

Myotonic dystrophy type 1 results from an unstable expanded CTG repeat ((CTG) n ) in the 3' UTR of the DMPK gene. Transgenic mouse models have been developed to reproduce the (CTG) n instability seen in DM1 patients. These transgenic mice provide an excellent tool to study the disease mechanism as well as the molecular mechanisms underlying trinucleotide repeat instability. The propensity for somatic instability differs per tissue and cell type. Expansion of the (CTG) n over time in certain tissues is thought to underlie progression of the clinical picture. It is therefore crucial to understand what causes the (CTG) n to expand in certain cells and not in others, as well as to see possibly distinct downstream cellular effects of different (CTG) n lengths in different cell populations. We describe here an updated method to determine the genotype (homozygous, hemizygous, or non-transgenic) of the transgene, as well as length of the very long (CTG) n tracts now commonly obtained in our mouse model. Furthermore, in order to facilitate research into cell populations that show different degrees of instability, we present here a fast technique to recover cells from mouse tissues, which can serve as a basis for multiple downstream applications, including cell culture and biochemical or molecular studies.

Tissue- and age-specific DNA replication patterns at the CTG/CAG-expanded human myotonic dystrophy type 1 locus.

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.

A simple and fast method for cell recovery and DNA content analysis from various mouse tissues by flow cytometry.

Cell division in tissues can be investigated in various ways. We present here a method for improving cell recovery and cell cycle analysis for a wide range of mouse tissues. This strategy combines a cell isolation procedure for various mouse tissues based on intracardiac perfusion and subsequent treatment followed by flow cytometry. This easy and reproducible method allows a rapid analysis of nuclear DNA content, providing an estimate of the cell number at different phases of the cell cycle. This combined procedure could also be used for the isolation of specific cell subpopulations from different mouse tissues by fluorescence activated cell sorting.

Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice.

The CTG repeat involved in myotonic dystrophy is one of the most unstable trinucleotide repeats. However, the molecular mechanisms underlying this particular form of genetic instability-biased towards expansions-have not yet been completely elucidated. We previously showed, with highly unstable CTG repeat arrays in DM1 transgenic mice, that Msh2 is required for the formation of intergenerational and somatic expansions. To identify the partners of Msh2 in the formation of intergenerational CTG repeat expansions, we investigated the involvement of Msh3 and Msh6, partners of Msh2 in mismatch repair. Transgenic mice with CTG expansions were crossed with Msh3- or Msh6-deficient mice and CTG repeats were analysed after maternal and paternal transmissions. We demonstrated that Msh3 but not Msh6 plays also a key role in the formation of expansions over successive generation. Furthermore, the absence of one Msh3 allele was sufficient to decrease the formation of expansions, indicating that Msh3 is rate-limiting in this process. In the absence of Msh6, the frequency of expansions decreased only in maternal transmissions. However, the significantly lower levels of Msh2 and Msh3 proteins in Msh6 -/- ovaries suggest that the absence of Msh6 may have an indirect effect.