Figure 4: Alleviation of epigenetic repression at the IPA1 promoter by qWS8/ipa1-2D.

(a) Schematic map showing the sites of two methylation sensitive enzymes (HpaII and MspI) and probe for methylation detection by Southern blotting. The blue and orange double arrows indicate the mapping region and single repeat region respectively. Mn1–Mn4, positions of PCR detection after MNase digestion. (b) Southern blot analysis of DNA methylation differences between NIL^ipa1-2D and NIL^IPA1. The bands reflecting different methylation pattern are denoted by arrows. Note that the smallest band is a direct reflection of different methylation at sites −2,545 and −2,974. M, DNA markers. (c) Distribution of three cytosine contexts and methylation pattern in ∼800-bp promoter region of IPA1. Filled circles, methylated cytosine; empty circles, unmethylated cytosine. Two distinct regions are labelled by blue lines and the junction region is labelled by black frame. Note that the junction region presents obvious methylation difference especially for CHH and overlaps with the DH site labelled by black double arrows. (d–f) Methylation levels of CG (d), CHG (e) and CHH (f) in eight 100 bp-windows of the promoter region shown in c. Blue, NIL^ipa1-2D; Red, NIL^IPA1. Dark yellow shades highlight the junction region. (g) Schematic map of the approach to detect open chromatin. Chromatin with loose nucleosome occupation is more susceptible to mononuclease (MNase) digestion and inefficiently amplified by PCR. (h) Sensitivity of four regions (Mn1–Mn4) to increasing dosage of MNase digestion between NIL^IPA1 and NIL^ipa1-2D. The position of each region is labelled in a. Note that the Mn3 region was more sensitive to the digestion in NIL^ipa1-2D. (i) Ratio of the NIL^ipa1-2D allele in the Mn3 region after MNase digestion of nucleus from heterozygous NIL. Two alleles are distinguished by the SNP at position −419 shown in a.