De novo methylaton of the unmethylated K. phaffii genome by human DNA methyltransferases (DNMTs) [BiSulfite-seq]

We engineered combinatorial knock-in of human DNMTs in Komagataella phaffii, a yeast species lacking endogenous DNA methylation, to characterize methylation preferences and functional consequences of individual de-novo DNMTs as well as their combinations. Methylation preferences were determined through whole-genome bisulfite sequencing (WGBS) experiments. Effects of knock-in on gene expression were determined by RNA-seq. Measurements of genome-wide methylation produced by distinct de-novo DNMTs permitted determination of methylation preferences and aversions specific to individual DNMTs. Combining methylation and gene expression data provided insight about cellular response to the stress of DNA methylation and about the association between gene methylation patterns and changes in transcriptional output. Overall design: Whole-genome bisulfite sequencing (WGBS) for 13 experimental conditions. Five of these conditions were single gene knock-ins of DNMT1A, DNMT3A1, DNMT3A2, DNMT3B1,or DNMT3L. Three conditions were double knock-ins of DNMT1A and DNMT3L, DNMT3A1 and DNMT3L, or DNMT3A2 and DNMTL. For each of these conditions as well as for the control condition, WGBS was performed on DNA extracted from K. phaffii cells 5 days after generating and validating knock-in strains. The remaining conditions composed a time-course experiment for the double knock-in of DNMT3B1 and DNMT3L in which WGBS was performed on DNA extracted 1, 2, 3, and 4 days after generating and validating knock-in strains. All experiments were performed as biological duplicates. Sequencing data from each replicate was analyzed separately, and final methylation rates of CpG context cytosines were determined from the sum of methylated and unmethylated observations across replicates corresponding the same condition.