Epigenetic Response of Yarrowia lipolytica to Stress: Tracking Methylation Level and Search for Methylation Patterns via Whole-Genome Sequencing

Abstract

DNA methylation is a common, but not universal, epigenetic modification that plays an important role in multiple cellular processes. While definitely settled for numerous plant, mammalian, and bacterial species, the genome methylation in different fungal species, including widely studied and industrially-relevant yeast species, Yarrowia lipolytica, is still a matter of debate. In this paper, we report a differential DNA methylation level in the genome of Y. lipolytica subjected to sequential subculturing and to heat stress conditions. To this end, we adopted repeated batch bioreactor cultivations of Y. lipolytica subjected to thermal stress in specific time intervals. To analyze the variation in DNA methylation between stressed and control cultures, we (a) quantified the global DNA methylation status using an immuno-assay, and (b) studied DNA methylation patterns through whole-genome sequencing. Primarily, we demonstrated that 5 mC modification can be detected using a commercial immuno-assay, and that the modifications are present in Y. lipolytica's genome at ~0.5% 5 mC frequency. On the other hand, we did not observe any changes in the epigenetic response of Y. lipolytica to heat shock (HS) treatment. Interestingly, we identified a general phenomenon of decreased 5 mC level in Y. lipolytica's genome in the stationary phase of growth, when compared to a late-exponential epigenome. While this study provides an insight into the subculturing stress response and adaptation to the stress at epigenetic level by Y. lipolytica, it also leaves an open question of inability to detect any genomic DNA methylation level (either in CpG context or context-less) through whole-genome sequencing. The results of ONT sequencing, suggesting that 5 mC modification is either rare or non-existent in Y. lipolytica genome, are contradicted with the results of the immunoassay.

Keywords: Yarrowia lipolytica; epigenome; genomics; methylation level; next generation sequencing; stress response; yeast.

Figure 1

Frequency of 5 mC [%] in (A) nucleic acid isolates from Y. lipolytica W29 strain and in PCR-amplified DNA template prior (grey) and after (black) RNAse A treatment; upper panel shows electrophoretic separation of the genomic DNA (gDNA) preparation in 1% agarose gel (M: Gene Ruler Express; Thermo Fisher Scientific); (B) positive and negative control samples provided by the immunoassay manufacturer; upper panel–standard curve for the assay showing its linearity and detection ranges; Negative control was an unmethylated polynucleotide containing 50% of cytosine. Important: the value of the 5 mC (A450 nm) read for the negative control sample (C-) was subtracted from all the analyzed samples in (A). Error bars indicate the ±SD from biological triplicate, each analyzed in technical triplicate.

Figure 2

Level of global DNA methylation (5 mC) in Y. lipolytica W29 strain cultivated in repeated-batch cultures, under HS (grey) and control (black) conditions. Sample names are encoded: hours after HS/number of cycles. Error bars indicate the ±SD from biological triplicate, each analyzed in technical triplicate.

Figure 3

Representative images from light microscopy observations of Y. lipolytica cells in repeated-batch cultures at different stages. Magnification 1000× (PrimoStar, Carl Zeiss light microscope). Mean percentage of filamentous (F) and ovoid forms (O) ± SD from five microscopic preparations.

Figure 4

The level of global DNA methylation (5 mC and N6mA) using dam/dcm and CpG model (ONT Guppy basecalling software version 3.6.1). Sample names are encoded: hours after HS/number of cycles.

Figure 5

Level of global DNA methylation (5-methylcytosine and N6-methyladosine content) in all contexts (ONT Guppy basecalling software version 3.6.1). Sample names are encoded: hours after HS/number of cycles.