Inhibition of DNA Methylation in Picochlorum soloecismus Alters Algae Productivity

Abstract

Eukaryotic organisms regulate the organization, structure, and accessibility of their genomes through chromatin remodeling that can be inherited as epigenetic modifications. These DNA and histone protein modifications are ultimately responsible for an organism's molecular adaptation to the environment, resulting in distinctive phenotypes. Epigenetic manipulation of algae holds yet untapped potential for the optimization of biofuel production and bioproduct formation; however, epigenetic machinery and modes-of-action have not been well characterized in algae. We sought to determine the extent to which the biofuel platform species Picochlorum soloecismus utilizes DNA methylation to regulate its genome. We found candidate genes with domains for DNA methylation in the P. soloecismus genome. Whole-genome bisulfite sequencing revealed DNA methylation in all three cytosine contexts (CpG, CHH, and CHG). While global DNA methylation is low overall (∼1.15%), it occurs in appreciable quantities (12.1%) in CpG dinucleotides in a bimodal distribution in all genomic contexts, though terminators contain the greatest number of CpG sites per kilobase. The P. soloecismus genome becomes hypomethylated during the growth cycle in response to nitrogen starvation. Algae cultures were treated daily across the growth cycle with 20 µM 5-aza-2-deoxycytidine (5AZA) to inhibit propagation of DNA methylation in daughter cells. 5AZA treatment significantly increased optical density and forward and side scatter of cells across the growth cycle (16 days). This increase in cell size and complexity correlated with a significant increase (∼66%) in lipid accumulation. Site specific CpG DNA methylation was significantly altered with 5AZA treatment over the time course, though nitrogen starvation itself induced significant hypomethylation in CpG contexts. Genes involved in several biological processes, including fatty acid synthesis, had altered methylation ratios in response to 5AZA; we hypothesize that these changes are potentially responsible for the phenotype of early induction of carbon storage as lipids. This is the first report to utilize epigenetic manipulation strategies to alter algal physiology and phenotype. Collectively, these data suggest these strategies can be utilized to fine-tune metabolic responses, alter growth, and enhance environmental adaption of microalgae for desired outcomes.

DNA Methylation in Algae and Its Impact on Abiotic Stress Responses

“…Nuclear genome 5-mC patterns have been recently profiled in several Archaeplastida microalgae, as well as in a few stramenopiles, showing low levels of DNA methylation in algae [ 50 , 72 , 75 , 76 , 77 , 79 , 80 , 81 , 82 ]. The C .…”

“…The Picochlorum soloecismus genome contains approximately 1.15% of cytosine methylated. Contextually, this methylation occurs in a bimodal distribution, predominately in CG sites (in ~12.1% of them) and a few in CHH and CHG sites (<1%) [ 81 ]. In Scenedesmus acutus , 14% of the genome is methylated and cytosine methylation occurred mainly in the symmetric CG rather than in CHG and CHH contexts (76.9% of CG, 2.6% of CHG and 1.6% of CHH context, respectively) [ 82 ].…”

“…In C. variabilis , genes are universally CG methylated within their bodies [ 50 ]; CHG methylation is also substantial but, similarly to what occurs in land plants, is concentrated in repetitive (presumably transposon) sequences and excluded from genes [ 50 ]. In P. soloecismus, DNA methylation occurs globally in all genome feature regions, with greater methylation in the CG context [ 81 ]. The V .…”

“…DNA methylation plays a role in N responses in the Chlorophyta Picochlorum soloecismus and potentially regulates genes that are involved in stress responses and lipid accumulation [ 81 ]. Indeed, the genome of this alga becomes hypomethylated during the growth cycle in response to N starvation.…”

“…The greatest DNA hypomethylation occurs after 10 days in culture under severe N depletion conditions. During N starvation, several of the hypomethylated CG sites of P. soloecismus genome are annotated as genes in pathways involved in lipid biosynthesis, including diacylglycerol acyltransferase 2 ( DGAT2 ) [ 81 ]. Fei et al (2017) [ 28 ] explored the relationship between DNA methylation and transgenic silence upon N deprivation in C. reinhardtii .…”