GSE139060
De novo methylaton of the unmethylated K. phaffii genome by human DNA methyltransferases (DNMTs) [RNA-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: RNA-seq 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, RNA-seq was performed on total RNA 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 RNA-seq was performed on total RNA extracted 1, 2, 3, and 4 days after generating and validating knock-in strains. All experiments were performed as biological triplicates. We used fragments per kilobase of transcript per million mapped reads (FPKM) normalization to obtain a table of library-size normalized expression values for each gene in each sample.