5-methylcytosine (5mC) is a major biological DNA modification that is involved in mammalian development and disease. DNA methylation in cells is catalyzed by a collective activity of DNA methyltransferase (Dnmt) proteins, explicitly Dnmt1, Dnmt3a, and Dnmt3b, which transfer the methyl group from S-adenosyl-L-methionine (SAM or AdoMet) to the cytosine base in the DNA CpG sequence, while levels of DNA modification are regulated by the modulation of the amount of the individual enzyme or its catalytic efficiency. Despite recent advances, the precise role of each methyltransferase in the maintenance of cellular epigenomic status remains unclear.
Classically, mouse embryonic stem cells (mESCs) are cultured in the presence of leukemia inhibitory factor (LIF), which sustains the naive state of pluripotency but exhibit a significant heterogeneity. When mESCs are cultured in the presence of LIF and two additional kinase inhibitors (2i; MEK/ERK pathway inhibitor PD0325091 and Gsk-3 inhibitor CHIR99021), they can maintain a more homogeneous ground state of pluripotency. Furthermore, mESCs grown in LIF medium maintain a relatively high level of DNA 5mC, whereas 2i maintains mESC genomic DNA hypomethylation by promoting Dnmt3a and Dnmt3b protein degradation [1]. However, the regulation of specific Dnmt1 activity by individual MEK/ERK or Gsk-3 pathways is unknown.
In this work we aimed to evaluate a specific Dnmt1 activity in mouse embryonic stem E14tg2a cells after the treatment with 2i or each kinase inhibitor individually. For this purpose, we used a novel Dnmt1-specific genome tagging platform [2] to assess 5mC, 5azidoC and Dnmt gene expression levels in cells exposed to corresponding inhibitor. HPLC-MS analysis revealed that the levels of genomic DNA 5mC significantly decreased when E14tg2a cells were cultured with both or individual kinase inhibitors. Similarly, 5azidoC levels were significantly decreased in cells cultured with 2i, whereas the treatment with single inhibitor yielded different outcome: the MEK/ERK pathway inhibitor PD0325091 significantly reduced Dnmt1-specific genome tagging in cells, while CHIR99021 had no effect on 5azidoC levels. Real-time qPCR analysis unveiled that Dnmt1 and Uhrf1 mRNA expression remained stable after treatment with a single kinase inhibitor or their combination.
In summary, MEK/ERK signaling pathway is important in the regulation of Dnmt1 activity in mouse embryonic cells. As a result, the application of Dnmt1-specific genome tagging platform could be further expanded to investigate the regulation of catalytic Dnmt1 activity in cellulo by a variety of signaling pathways or particular "druggable" proteins.