br Results br Discussion Here we report on different mutant

Results
Discussion Here we report on 14 different mutant hESC lines, termed DM1 hESCs, that were derived from DM1-affected preimplantation embryos. Previous studies employed DM1 hESCs to explore the causal role of CTG expansion on neurite formation and neuromuscular connections and for characterizing the somatic instability of the CTG repeats in undifferentiated oxytocin receptor (De Temmerman et al., 2008; Marteyn et al., 2011; Seriola et al., 2011). However, neither of these studies addressed the consequences of CTG expansion on DNA methylation and how this change may contribute to disease pathogenesis. In addition, a comprehensive study characterizing aberrant methylation in a wide range of tissues from DM1 aborted fetuses and postmortem samples demonstrates that hypermethylation is largely tissue- and age-specific but not necessarily correlated with expansion size, making it difficult to assign a function to abnormal methylation in disease pathogenesis (López Castel et al., 2011). Taking advantage of a large set of DM1 hESC lines comprised of maternally and paternally inherited expansions bearing from 180 to more than 2,000 CTG repeats, we finely characterized a disease-associated DMR, 650 bp upstream to the CTG repeats, that abnormally gains methylation in a way that strongly correlates with expansion size (ρ = 0.94114; Spearman’s rank correlation, p < 0.01). We show that this association, which is restricted to undifferentiated cells, is triggered by expansions of more than 300 CTG repeats and is tightly linked with allele-specific reduction in SIX5 (but not DMPK) transcription (ρ = 0.84; Spearman’s rank correlation coefficient, p = 0.00134), as determined by allele expression imbalances. Furthermore, we show that the relationship between expansion size, methylation extent, and allele reduction in SIX5 is reproduced in patient-derived iPSCs following cell reprogramming and is maintained in in vitro-differentiated cardiomyocytes, a disease-relevant cell type. Together, our findings in DM1 hESCs and patient-derived iPSCs correspond with the decline in SIX5 expression in DM1 patients and with the expression of various aspects of DM1 pathology in SIX5 heterozygote mice (Klesert et al., 2000; Sarkar et al., 2000, 2004; Wakimoto et al., 2002). Moreover, they are consistent with the negative cis effect of large CTG expansions on the induction of hypermethylation in transgenic mice (Brouwer et al., 2013; López Castel et al., 2011), pointing to a role for chromatin modification in DM1 pathogenesis. However, it should be noted that hypermethylation patterns in transgenic mice are similar, but not identical, to those observed in DM1 individuals (López Castel et al., 2011) and that, in contrast to our study, the reduced expression in SIX5 is uncorrelated with expansion size in transgenic mice (Brouwer et al., 2013). The discrepancies between both studies may stem from epigenetic variations that exist between species or may be attributed to transgene integration site effects. Given the aforementioned relationships, we aimed to mechanistically associate DMR hypermethylation with allele-specific reduction in SIX5 expression. Initially, we ruled out the possibility that the CTG expansion leads to the reduction in SIX5 expression by interfering with CTCF binding via hypermethylation, as proposed previously (Filippova et al., 2001). Our findings do not support the proposition that CTCF normally functions as an insulator binding protein at the DM1 locus to guarantee the independent expression of DMPK and SIX5 (Cho et al., 2005). Although this difference may stem from the different cell types employed in each study, our findings are well in line with the report of Brouwer et al. (2013) regarding transgenic mice that showed no effect of CTG expansion size on CTCF binding. In light of these results regarding CTCF, we questioned whether methylation spreading may exert its effect by an alternative mechanism: by hampering the activity of a putative regulatory element for SIX5 that resides within the DMPK gene. For this purpose, we examined the ability of the DMR to drive reporter gene expression in vivo and in vitro. In vivo, we found that the DMR is capable and sufficient to drive GFP expression in the heads of transgenic fish, predominantly in the epidermis, hindbrain, and branchial arches. We speculate that the partial similarity in the tissue specificity of reporter gene activity in zebrafish to that of SIX5 expression in the branchial arches of developing mice (10.5 days post-coitum [d.p.c.]) (Heath et al., 1997; Klesert et al., 2000) may possibly explain the lack of facial expression and the difficulties in speech and swallowing that are typical in patients.