Figure 5. DNA methylation at the Gtl2 secondary DMR correlates with biallelic expression in Trim28^chatwo mutants.

DNA methylation at the Gtl2 secondary DMR was measured in single E8.5 wild type and Trim28^chatwo embryos by bisulfite sequencing (A) and pyrosequencing (B-D). (A) Representative bisulfite sequencing results for wild type and Trim28^chatwo embryos, additional results are shown in Figure S3. Bisulfite sequencing in A and Figure S3 shows results from Trim28^chatwo embryos that biallelically expressed Gtl2. Filled circles, methylated DNA. Empty circles, unmethylated DNA. Maternal (mat) and paternal (pat) chromosomes were identified by allele-specific SNPs. (B) DNA methylation at the germline IG-DMR versus the Gtl2 secondary DMR as measured by pyrosequencing in single wild type and Trim28^chatwo embryos. (C) Average DMR methylation for all wild type and Trim28^chatwo embryos analyzed. (D) Gtl2 allele expression ratios versus Gtl2 secondary DMR methylation as measured by pyrosequencing in single wild type and Trim28^chatwo embryos. The red line in D shows the linear regression model for Trim28^chatwo embryos. The P-value (red) indicates the correlation between biallelic expression and DNA methylation. The data represented in B-D includes the same E8.5 wild type and Trim28^chatwo embryos as shown in Figure 4C. n = number of embryos analyzed. Error bars represent standard deviation. Statistical significance was measured using a paired student’s t-test, ns-not significant, ****p<0.0001.