The Zygotic Methylation Landscape
(A) Global methylation levels in gametes and zygotes. The graph shows the mean and SD of the methylation values of 20 kb tiles across the genome.
(B) Frequency distribution of 20 kb tile methylation values. Tiles were allocated to bins centered every 5%.
(C) Mean methylation levels in repetitive-element classes. “Zygotes” refers to overall levels, as natural sequence variability prohibits assignment of parental alleles using SNPs. LINE, long interspersed nuclear element; SINE, short interspersed nuclear element; MuLV, murine leukemia virus; IAP, intracisternal A particle; Etn, early transposon; MERV-L, murine endogenous retrovirus with leucine tRNA primer; ERV, endogenous retrovirus.
See also .

Methylation Trajectories in Different Genomic Contexts
(A) Distribution of methylation values at sequence features in sperm and the paternal component of control zygotes. The plot displays the median (bar), interquartile range (box), and maximum and minimum (whiskers). The p values shown are the result of a paired ANOVA multiple-comparison test with Sidak correction; “n” denotes the number of sequences that met quantification criteria.
(B) Absolute methylation change between sperm and the paternal component of control zygotes. Only changes ≥ 10% and significant according to a chi-square test (corrected p value < 0.05) are shown; all others are recorded as “no change.”
(C) RDL at identified demethylated loci, calculated by dividing the absolute paternal methylation change (as in B) by the sperm methylation level. Demethylated loci are defined as those that had an absolute paternal methylation loss of ≥10% and were significant according to a chi-square test (corrected p value < 0.05). n = 5,393 intergenic sequences, 7,405 gene body sequences, 4,213 CGI promoters, 1,366 non-CGI promoters, 526 promoter CGIs, and 257 nonpromoter CGIs.
(D) Distribution of early embryo paternal transcriptional noise levels at genes allocated into quartiles according to average intragenic methylation levels in the paternal pronucleus. Normalized transcriptional noise refers to expression-corrected variation in transcription from the paternal allele between single cells as calculated in . The p values shown are the result of an ANOVA multiple-comparison test between indicated data sets with Sidak correction. The differences between second and third quartiles, and third and fourth quartiles are not significant at any stage according to this test. The total number of genes that fell into each quartile was as follows: 0%–25%, 1,379; 25%–50%, 2,468; 50%–75%, 2,605; and 75%–100%, 270. Genes that were not expressed at a particular stage were excluded from analysis of that stage. The number of genes analyzed at each stage is given in .
(E) Relationship between the average paternal pronuclear gene body methylation and paternal transcriptional noise at the late 2-cell stage. A quadratic equation is a significantly better fit to the data than either a linear or no relationship (p < 0.0001 in both cases, extra sum-of-squares F test).
See also and .

Impact of TET3 Deletion on Methylation Dynamics
(A) Distribution of methylation values at demethylated loci, defined as in C. The p values shown are the result of a paired ANOVA multiple-comparison test with Sidak correction.
(B) Impact of TET3 deletion on demethylated loci. The ranges indicate the absolute change in methylation level between the paternal component of control and TET3 zygotes. Only changes ≥10% and significant according to a chi-square test (corrected p value < 0.05) are shown; all others are recorded as “no change.”
(C) Dendrogram derived from hierarchical clustering using the Pearson correlation distance for the indicated sequence features. The number above the branch denotes the approximately unbiased p values computed by multiscale bootstrap resampling (n = 10,000) using the pvclust package for R (). This was identical for both features.
(D) Proportion of normal demethylation affected by the loss of TET3 at TET3-demethylated loci, calculated by dividing the absolute paternal methylation change between control and TET3 zygotes (as in B) by the normal absolute demethylation in control zygotes (as in B). TET3-demethylated loci are defined as demethylated loci that underwent an absolute paternal methylation gain of ≥10% in the absence of TET3 and were significant according to a chi-square test (corrected p value < 0.05). n = 1,310 intergenic sequences, 2,487 gene body sequences, 259 CGI promoters, and 398 non-CGI promoters. The number of promoter and nonpromoter CGIs that met these criteria was insufficient for meaningful analysis. ^∗Proportional change > 1 indicates that paternal methylation in TET3 zygotes increased to higher levels than in sperm.
(E) Examples of complete (Col14a1 intragenic sequence and Plbd1 CGI promoter) and partial (Zfp356 intragenic sequence and Coro1c non-CGI promoter) impairment of demethylation due to loss of TET3 oxidation.
See also and .

A Protective Role for TET3
(A) Distribution of methylation values for TET3 targets at CGI-associated sequences. TET3 targets are defined as loci where the absence of TET3 activity resulted in a paternal methylation gain of ≥10%, which is significant according to a chi-square test (corrected p value < 0.05).
(B) Function of TET3 at its targets and their methylation behavior. The area of each circle is proportional to the percentage of loci in that category. TET3 targets are defined as in (A). Protected, paternal methylation ≥10% higher in TET3 zygotes than in sperm and significant according to a chi-square test (corrected p value < 0.05); demethylated, criteria as in C; normal gain methylation, paternal methylation ≥10% higher in control zygotes than in sperm and significant according to a chi-square test (corrected p value < 0.05). Percentages do not necessarily add to 100 due to the application of statistical criteria.
(C) Examples of loci protected by TET3 classified according to whether they are normally demethylated, stable, or gain methylation. Agrn and Gm106 are CGI promoters, Lmtk3 is an intragenic CGI, Kif6 is a non-CGI promoter, and Tbc1d2b is a promoter CGI. Chr8 Orphan CGI is located at chromosome 8 29088955-29089878 (NCBI37 assembly).
See also .