L1 donor/daughter elements are methylated in somatic tissues of adult mice. (A) Schematic of CpG dinucleotides in a mouse L1 TF element. Triangles in 5′ UTR represent monomer units. A magnification of the 5′ UTR is shown below. Black boxes represent monomer units. Dark gray box represents unique (nonmonomeric) region within 5′ UTR. Orange strokes represent CpG dinucleotides. Red boxes represent YY1 binding sites. (B) Experimental design of mouse L1 locus-specific bisulfite sequencing. Genomic DNA was extracted from tissues of C57BL6/J mice harboring previously identified donor and daughter L1 insertions (Richardson et al. 2017). The parental generation “P” (square = male, circle = female) is mosaic for the de novo daughter L1 insertion (represented by stripes). F1-F3 generations are heterozygous for L1 insertions (filled diamond = male or female). F2 and F3 generation animals were only available for Insertion 5 and polyL1Tf_4. Tissues for analysis of donor elements were only collected from the P generation. DNA was isolated from brain (green), heart (red), liver (orange), and gonads (gray) if available. After bisulfite conversion, the 5′ monomeric region of each L1 was PCR amplified. Amplicons were pooled and sequenced as 2 × 300-mer Illumina reads. Circles represent methylated (black circles) and unmethylated (white circles) CpG dinucleotides in L1 5′ UTR. (C) Methylation of a de novo L1 promoter sequence (Insertion 2) shown in the germline mosaic parental testis (animal B) and in the following F1 generation embryonic tissue (animal AB15). Displayed are 50 nonidentical sequences extracted at random from a much larger pool of available Illumina reads. Each cartoon panel corresponds to an amplicon (black circle, methylated CpG; white circle, unmethylated CpG; ×, mutated CpG). Colored line above the cartoon represents amplicon (gray = genomic sequence, colored = L1 sequence). The overall percentage of methylated CpG dinucleotides is indicated below each cartoon. Gray letters indicate methylation of CpG dinucleotides in genomic sequence. Colored letters indicate methylation of CpG dinucleotides in L1 sequence. (D) Locus-specific methylation analysis schematic representation for L1 TF monomer, 3 full-length de novo L1 insertions (Insertion 2, 5, 7), 2 polymorphic L1 insertions (polyL1Tf_3 and polyL1Tf_4) and their 5 respective donor elements (Donor 2, 5, 7, 3, 4). 5′ monomeric sequences of each L1 were PCR amplified using primer pairs (green arrows) specific to that locus. Orange strokes indicate L1 CpG dinucleotides covered by the assay. Blue strokes represent covered genomic CpG dinucleotides. Gray strokes in the gray shaded area represent CpG dinucleotides not reached by Illumina sequencing. Red boxes indicate YY1 binding sites. Colored boxes represent L1 monomer units. (E) Genome-wide methylation of L1 TFI/TFII promoter sequence shown in all animals. Animals are labeled E, EF19, 137, 138, 235, B, AB5, AB15, CD14 and 55 in the bottom part of the x-axis label, with the generation (P, F1, F2 or F3) indicated in brackets. Each graph contains animals from the same family. The different tissues used for DNA extraction and bisulfite sequencing in each animal is indicated in the top part of the x-axis labeled as: brain, B; heart, H; liver, L; testis, T; ovaries, O; and embryonic tissues, E. Displayed are 1000 nonidentical sequences extracted at random from a much larger pool of available Illumina reads. The violin plots represent the methylation distribution as per Supplemental Figure S2. The black line and dashed lines show the distribution median and quartiles, respectively. The percentage of methylated CpGs per read is indicated on the y-axis. (F) As for (E) but for de novo L1 promoter sequences shown in the mosaic P generation in which each de novo L1 insertion was identified (animals E and CD14) and in following F1–F3 generations if available (animals EF19, 138, 235 and 55). Displayed are 1000 nonidentical sequences (if available) extracted at random from a much larger pool of available Illumina reads (exceptions: Insertion 5: 138 H, 235 B, H, L, O, EF19 T [368, 328, 337, 352, 958, 282 reads, respectively]; Insertion 7: 55 B, H, L, CD14 T [598, 885, 890, 662 reads, respectively]). (G) As for (E,F) but for polymorphic L1 insertions. (H) As for (E,F) but showing methylation of 5 donor L1 elements in the animal they mobilized in (P generation).
