Figure 3: Hypomethylated regions in Setdb1MD13/+ correspond to ERVK elements.
(a) Detection of Setdb1, Trim28 and H3K9me3 by immunohistochemistry in testes from 3 months old males. For each, a secondary antibody control (2°Ab) is shown to indicate the specificity of the immunohistochemical staining (first panel of each row), followed by specific staining at 20x and 100x magnification. Setdb1 was found to be present in spermatogonia, Trim28 in spermatocytes and spermatids and H3K9me3 in spermatogonia and spermatids. (b) Box plot of the average DNA methylation in 10Kb windows across the genome (n = 246, 281), excluding outliers (1.5× interquartile range). (c) Plot showing the weighted average DNA methylation for sequence elements annotated as ERVK in sperm from Setdb1MD13/+ and Setdb1+/+ mice (n = 16,331). Elements for which the DNA methylation difference was greater than 10 percentage points are colored blue (n = 607). (d) Counts of ERVK subfamilies that were either hypermethylated or hypomethylated in Setdb1MD13/+ relative to Setdb1+/+ and for which DNA methylation differed by more than 10 percentage points. The 20 most frequently represented subfamilies are shown. (e) UCSC genome browser screen capture showing a reduction of mCG at an IAPLTR3 (top left panel) and a RNERVK23-int (top right panel) in Setdb1MD13 heterozygotes. Sanger sequencing of bisulphite converted sperm genomic DNA from independent males confirmed reduced mCG levels of the IAPLTR3 (bottom left panel; T-test P = 0.05) and the RNERVK23-int (bottom right panel; T-test P < 0.05) in Setdb1MD13heterozygotes. Each boxed block of lines comprises clones derived from one bisulphite conversion and each column represents one individual with the percentage of methylated CpGs indicated above each individual. Open circles indicate an unmethylated CpG, and closed circles a methylated CpG.
