DNA methylation during differentiation impacts mouse L1 TSS distribution. (A) Gel image of 5′ RACE products. Total RNA was extracted from two independent replicate cultures at d0 (pluripotent mESCs), d3 of differentiation, and d21 of differentiation and subjected to 5′ RACE NTC; no template control. mw; molecular weight marker. (B) Position of TSSs within the L1 TF monomer sequence. The TSS count is shown on the y-axis. The position within the 212 bp L1 TF monomer consensus sequence is displayed on the x-axis, and a schematic of the L1 TF monomer consensus with the YY1 binding site highlighted in red is shown at the top. Blue, d0; orange, d3; green, d21. (C) Percentage of TSSs upstream of L1 TF (orange), within L1 TF monomers (blue) and downstream of monomeric region (green) for d0, d3, and d21. The number of loci represented with upstream TSSs at each time point is indicated. (D) Above, schematic of the first 2000 bp of an L1 TF element containing five monomer units. Alternating green shading represents monomer units. Light gray shading represents the nonmonomeric region of the L1 TF promoter. Dark gray represents ORF1. Orange lines show the position of CpG dinucleotides. The positions of the YY1 binding sites are labeled and represented as vertical light gray lines extending down the figure panel. The position of the L1-specific 5′ RACE primer is indicated. Below, TSS counts and mean CpG methylation are shown for time points d0 (blue), d3 (orange), and d21 (green). The histograms in the upper plots show TSS count for 137 L1 TF elements with 5 monomer units, with each bar representing a 10 bp bin. The lower plots display composite DNA methylation profiles with mean (thick line) and standard deviation (shaded region) indicated.
