Dnmt1 deficiency promotes CAG repeat expansion in the mouse germline

Abstract

Expanded CAG repeat tracts are the cause of at least a dozen neurodegenerative disorders. In humans, long CAG repeats tend to expand during transmissions from parent to offspring, leading to an earlier age of disease onset and more severe symptoms in subsequent generations. Here, we show that the maintenance DNA methyltransferase Dnmt1, which preserves the patterns of CpG methylation, plays a key role in CAG repeat instability in human cells and in the male and female mouse germlines. SiRNA knockdown of Dnmt1 in human cells destabilized CAG triplet repeats, and Dnmt1 deficiency in mice promoted intergenerational expansion of CAG repeats at the murine spinocerebellar ataxia type 1 (Sca1) locus. Importantly, Dnmt1(+/-) SCA1 mice, unlike their Dnmt1(+/+) SCA1 counterparts, closely reproduced the intergenerational instability patterns observed in human SCA1 patients. In addition, we found aberrant DNA and histone methylation at sites within the CpG island that abuts the expanded repeat tract in Dnmt1-deficient mice. These studies suggest that local chromatin structure may play a role in triplet repeat instability. These results are consistent with normal epigenetic changes during germline development contributing to intergenerational instability of CAG repeats in mice and in humans.

Figure 1

Intergenerational changes in CAG repeat tract lengths in SCA1 progeny mice. Percent transmission is the number of alleles with a given change in repeat length divided by the total number of alleles (n) multiplied by 100%. Change in repeat length is defined as the number of repeats in a progeny mouse minus the number of repeats in its donor parent. A) Dnmt1^+/+ SCA1 mouse donors. B) Dnmt1^+/− SCA1 mouse donors. The progeny from the male and female jDnmt1^+/− SCA1 donors includes Dnmt1^+/+ SCA1 and Dnmt1^+/− SCA1 mice.

Figure 2

Age association of track-length changes in progeny of Dnmt1^+/+ SCA1 and Dnmt1^+/− SAC1 donor mice. The number of transmissions taken from figure 1A&B were plotted as a function of age of the donor parent at conception. Top row: 5–15 week-old mouse donors. Middle row: 15 week and 1 day to 25 week-old donors. Bottom row: donors older than 25 weeks.

Figure 3

Progeny genotype does not alter CAG tract-length changes in Dnmt1^+/− SCA1 mouse donors. The breeding schemes are shown at the top. The Dnmt1^+/+ SCA1 and Dnmt1^+/− SCA1 progeny are labelled in green and orange, respectively. A) Progeny from female Dnmt1^+/− SCA1 donors. Only the progeny from donors that were 25 weeks of age or younger are shown. B) Progeny from male Dnmt1^+/− SCA1 donors.

Figure 4

DNA methylation analysis of the Sca1 repeat region. A) Map of the CpG sites for the expanded allele at the Sca1 locus. The two upstream CpG sites are 17 bp and 20 bp in front of the CAG repeat tract, the third site is 32 bp downstream of the repeat. B) Representative bisulfite sequencing gel of the expanded allele of the Sca1 locus. The ovary and the testis are from Dnmt1^+/− SCA1 mice. Arrows indicate the CpG sites analyzed. C) Box plot of DNA methylation for each CpG site shown in A in ovaries and testes from Dnmt1^+/+ SCA1 (green) and Dnmt1^+/− SCA1 (orange) mice. Boxes represent the middle 50% of all data points. The median is shown as a horizontal bar. Vertical lines represent the 95^th percentile.

Figure 5

Chromatin immunoprecipitation of the Sca1 repeat region. A) Map of the Sca1 locus around the repeat tract showing the regions analyzed by ChIP. H3Ac and H3K9me2 levels upstream (B) and downstream (C) of the Sca1 repeat tracts in 40-week old testes. Error bars represent one standard error.