Figure 4.

Methyl-DNA-immunoprecipitation sequencing (meDIP-seq) analysis of DNA from 46 h conjugating cells shown in Figure 2. All reads are presented in reads per kb per million (RPKM) to correct for length of sequences and sequencing depth. (a) IgG controls for both vegetative (x-axis) and 46 h DNA (y-axis). Micronuclear sequences are polyploid at the 46 h stage due to polytenization of chromosomes prior to genome rearrangements, and fall along a 5:1 gradient, shown as a dashed line. (b) Heatmap of methylcytosine immunoprecipitation (meCyt) reads with vegetative reads subtracted; IgG on the x-axis and methylcytosine on the y-axis. (c) A scatter plot to highlight the outliers along the y-axis in (b). The dotted line denotes the threshold (100 excess reads in 46 h meCyt) used to define the methylation cohort. These 11 chromosomes were fed into the MEME algorithm, which identified the CC motif on the right, which was highly statistically significant (MEME e-value = 2.8e-236); control cohorts of chromosomes were selected from the un-enriched population (middle) and depleted population (bottom): no motifs were found and the highest scoring motif in these cases was the telomeric sequence, G₄T₄G₄T₄G₄. (d) Hydroxymethylcytosine (x-axis) versus methylcytosine (y-axis) immunoprecipitation data. Nanochromosomes encoding ribosomal proteins or the ribosomal RNA are shown as cyan diamonds; the rest of the nanochromosomes in the genome are plotted as red circles. Note co-enrichment of the methylation cohort with both methyl- and hydroxymethyl- modifications, and that the chromosomes encoding ribosomal RNA and ribosomal proteins are only enriched for hydroxymethylcytosine. (e) The same analysis as (b) and (c), but for micronuclear contigs separated from the genome assembly in (a). Representative satellite repeats (labeled with their repeat unit length) and TBE elements display a complex mixture, suggesting a heterogeneous combination of DNA modifications in the genome. The strong hydroxymethylcytosine signal for the 170 bp satellite repeat (10,953 hmCyt reads, x-axis; 4,166 meCyt reads, y-axis; Additional file 3) placed it outside the bounds of this figure. (f) Venn diagram of the methylation (mC) cohort, hydroxymethylation (hmC) cohort and CC motif cohort. (g) All 69 CC motif-containing chromosomes plotted with their methylation and hydroxymethylation signals from meDIP-seq data. IgG, immunoglobulin G; MEME, Multiple Em for Motif Election; TBE, telomere bearing element; Veg, vegetative.