Investigating The Role Of Epigenetic Regulation Mediated By DNMT1 In Early Cell Fate Differentiation In Embryoid Body And Zebrafish Embryo Models

Abstract

Abstract Introduction: Epigenetics refers to changes in genomic functions that are not caused by alterations in the DNA sequence but may be heritable through cell lineages. DNA methylation is an epigenetic DNA base modification that has been linked to gene repression. DNA methyltransferase 1 (DNMT1) is an enzyme that copies the patterns of DNA methylation onto newly replicated DNA strands prior to cell division. During the early cleavage stages of embryogenesis, the genome undergoes a demethylation process. Around the time of blastocyst implantation, DNA methylation increases concurrently with the differentiation of the three germ layers. In parallel, various metabolic changes occur during embryo development to provide necessary metabolites at each developmental stage. The presence of DNMT1 is crucial during embryogenesis, as knockout embryos fail to progress beyond 9.5 days post fertilisation. The importance of DNMT1 is highlighted during gastrulation, which is the period of germ layer formation and tissue specification. The aim of this study was to investigate the requirement for DNA methylation maintenance for correct cellular differentiation. Additionally, it was investigated whether DNA methylation maintenance could play an alternative role, such as in regulating the pathways in metabolism that ensure proper progression of embryo development. Method: Mouse Embryonic Stem Cells (mESC) were used to generate Embryoid Bodies (EBs) as a 3D in vitro model of embryo development. To induce DNA hypomethylation, DNMT1 was targeted either by utilising a recently developed non-covalent chemical inhibitor GSK-3484862 (DNMT1i) or by employing established Dnmt1 knockout (D1KO) mESC lines. Through random and targeted differentiation of EBs, cellular differentiation was verified by visualising cell-specific markers typical of progenitors or terminally differentiated cells originating from different germ layers. Additionally, fertilised zebrafish eggs were subjected to various DNMT1i treatment protocols to visualise the inhibitor effect on cellular differentiation and tissue patterning in an in vivo model. In addition, cell cycle and various metabolites were assayed in mouse embryonic stem cells and in vitro embryoid models using flow cytometry and chemiluminescence assays, to test if failure in DNA methylation maintenance affects cell cycle profile and cellular metabolism. Results/Discussion: Inhibition studies using DNMT1i indicated that early cellular differentiation of mesoderm, endoderm, and ectoderm can occur under DNA hypomethylation conditions induced by DNMT1i. However, late differentiation of cardiomyocytes was delayed. Treating zebrafish embryos with DNMT1i showed a dose-dependent phenotype. The main effects observed were pericardial oedema, embryonic short axis and stochastic patterning of intersegmental vessels, and delayed/absence of differentiation of downstream cell fates, such as T cells. Although DNMT1i treatment of mESC reduces DNA methylation levels to comparable levels observed in D1KO mESC, a few differences were observed with respect to mESC proliferation, and the cell distribution across the S and G2 phases. In addition, the late differentiation towards hepatocytes was detected in DNMT1i-treated EBs but absent in D1KO EBs. Investigation of various metabolites under DNA hypomethylation conditions showed an elevation of lactate levels, with mitochondrial elongation in D1KO and DNMT1i treated mESC. Finally, an increase in γH2AX as an early marker of the DNA damage response was observed in association with DNMT1 absence. Conclusion: DNA hypomethylation did not prevent cellular differentiation of progenitors and many terminal fates derived from mesoderm, endoderm, and ectoderm. Although both DNMT1i treatment and D1KO caused hypomethylation, the differences between these models indicated some differences in the potential pathways implicated and their effects on cell cycle profile. Conversely, both hypomethylated models demonstrated that cellular metabolism was affected with raised lactate levels, the full implications of which require future investigation. Finally, genomic stress was inferred by the increased activity of the DNA damage response.