Chromosomal maps comparing cytosine methylation in WT and Lsh^−/− MEFs. (A) HPLC analysis for detection of methylated cytosine comparing genomic DNA from diverse WT (blue) and Lsh^−/− (red) tissues derived from day 13.5 embryos. (B) Comparison of methylated cytosine enrichment (−log₁₀ P value) after hybridization on a murine genome tiling array comparing genomic DNA from WT and Lsh^−/− MEF cell lines derived from day 13.5 embryos. WT: median 0.04, mean 0.036, third quartile 0.45. Lsh^−/−: median 0.04, mean 0.37, third quartile 0.48. (C–E) Chromosomal maps at 2 Mb resolution comparing WT (blue) to Lsh^−/− samples (red). (F and G) Chromosomal maps that illustrate differential methylation at 50 kb resolution. The graph shows the difference between WT mean methylation on values at 50 kb bins and Lsh^−/− samples; thus, chromosomal regions above zero represent areas hypermethlated in WT and those below zero are regions hypermethylated in Lsh^−/− MEFs. (H) Representation of differential methylated regions in the University of California Santa Cruz genome browser. The region indicated in the graph (G) on chromosome 8:81,003,000 to 95,738,000 is shown. Gene density is presented at the bottom of the graph. (I) Percentage of differentially methylated regions. The pie graph represents the fraction of the genome with a ratio of Lsh^−/− over WT cytosine methylation larger than 10- or 5-fold (hypermethylated in Lsh^−/−) or less than 10- or 5-fold (hypomethylated in Lsh^−/−).

Cytosine methylation pattern at the 5′ and 3′ ends in Lsh^−/− MEFs. (A) Cytosine methylation profiles comparing WT (blue) and Lsh^−/− MEFs (red) at 100 bp resolution around 5′ ends (TSS) (n = 44,742) and flanking regions [5 kb upstream and 5 kb downstream (gene body)]. (B) Cytosine methylation distribution around the 3′ end of genes (n = 48,680) with flanking regions [5 kb upstream (gene body) and 5 kb downstream] comparing WT (blue) and Lsh^−/− (red) MEFs at 100 bp resolution. (C and D) Cytosine methylation distribution around the 5′ ends and flanking regions comparing WT (blue) and Lsh^−/− (red) at genes with CpG island (C) or without CpG island (D). (E) Cytosine methylation distribution in Lsh^−/− MEFs at the 5′ end of genes classified into four groups (first to fourth) based on their density of CpG sites, with first being the lowest. (F) Cytosine methylation distribution in Lsh^−/− MEFs at the 5′ end of CpG island genes that are classified into six groups based on the position of the center of the CpG islands: −300 bp (blue), −200 bp (pale blue), −100 bp (purple), 0 (green), 100 bp (pink), 200 bp (pale green), 300 bp (red).

H3K4me3 changes in dependence of Lsh. (A) H3K4me3 methylation distribution at the 5′ ends (TSS) (n = 23,365) and flanking regions (5 kb upstream and 5 kb downstream) comparing WT (blue) and Lsh^−/− MEFs (red) at 100 bp resolution. (B and C) H3K4me3 methylation distribution at the 5′ ends and flanking regions comparing WT (blue) and Lsh^−/− MEFs (red) at genes with CpG island (B) or without CpG island (C). (D) Link between increases in DNA methylation and H3K4me3 gains at the 5′ ends. All genes (unique genes n = 23,365) were classified into three groups based on their gain of H3K4me3 in Lsh^−/− cells (Fig. S5B) into high (green), medium (blue), or no gain (brown). The graph depicts the change of DNA methylation (the difference between Lsh^−/− cytosine methylation and WT methylation) at the 5′ ends. (E) Distribution of H3K4me3 changes and DNA methylation changes at the 5′ ends. The gain of H3K4me3 in Lsh^−/− MEFs (difference of Lsh^−/− values minus WT values) and the gain of cytosine methylation in Lsh^−/− MEFs (difference of Lsh^−/− values minus WT values) were plotted around the 5′ end to illustrate the position of each modification. (F) Histogram illustrating the distance between H3K4me3 peaks and cytosine methylation peaks for single genes and the frequency with which they occur.

Relation of cytosine methylation changes and gene expression. (A) Box plot to demonstrate changes of gene expression changes for all genes (All) and those that were subgrouped based on changes in H3K4me3 and cytosine methylation. Genes were ranked according to their H3K4me3 changes in Lsh^−/− MEFs (Fig 3D and Fig. S5B) from highest (H3K4me3 high) to lowest gain (H3K4me3 low, including those genes with H3K4me3 losses). (Left) Gene-expression level in WT MEFs according to the classification. (Right) Ratio of gene expression (Lsh^−/− expression over WT expression), with values above 1 indicating up-regulation and below 1 indicating down-regulation. *P < 0.005. (B) Distribution of cytosine methylation changes in Lsh^−/− MEFs at the 5′ end of genes that are twofold down-regulated in Lsh^−/− MEFs (red, n = 581), twofold up-regulated in Lsh^−/− MEFs (green, n = 320), or a control group without any change (purple, n = 543). (C) Distribution of cytosine methylation changes (green) and H3K4me3 changes (blue) in Lsh^−/− MEFs at the 5′ end of genes that are twofold up-regulated in Lsh^−/− MEFs. (D) Distribution of cytosine methylation changes (red) and H3K4me3 (blue) changes in Lsh^−/− MEFs at the 5′ end of genes that are twofold down-regulated in Lsh^−/− MEFs.