Ten-eleven translocation 1 mediating DNA demethylation regulates the proliferation of chicken primordial germ cells through the activation of Wnt4/β-catenin signaling pathway

Abstract

Objective: The objective of this study was to investigate the regulation relationship of Teneleven translocation 1 (<i>Tet1</i>) in DNA demethylation and the proliferation of primordial germ cells (PGCs) in chickens.Methods: siRNA targeting <i>Tet1</i> was used to transiently knockdown the expression of <i>Tet1</i> in chicken PGCs, and the genomic DNA methylation status was measured. The proliferation of chicken PGCs was detected by flow cytometry analysis and cell counting kit-8 assay when activation or inhibition of Wnt4/β-catenin signaling pathway. And the level of DNA methylation and hisotne methylation was also tested.Results: Results revealed that knockdown of <i>Tet1</i> inhibited the proliferation of chicken PGCs and downregulated the mRNA expression of <i>Cyclin D1</i> and cyclin-dependent kinase 6 (<i>CDK6</i>), as well as pluripotency-associated genes (<i>Nanog, PouV</i>, and <i>Sox2</i>). Flow cytometry analysis confirmed that the population of PGCs in <i>Tet1</i> knockdown group displayed a significant decrease in the proportion of S and G2 phase cells, which meant that there were less PGCs entered the mitosis process than that of control. Furthermore, <i>Tet1</i> knockdown delayed the entrance to G1/S phase and this inhibition was rescued by treated with BIO. Consistent with these findings, Wnt/β-catenin signaling was inactivated in <i>Tet1</i> knockdown PGCs, leading to aberrant proliferation. Further analysis showed that the methylation of the whole genome increased significantly after <i>Tet1</i> downregulation, while hydroxymethylation obviously declined. Meanwhile, the level of H3K27me3 was upregulated and H3K9me2 was downregulated in <i>Tet1</i> knockdown PGCs, which was achieved by regulating Wnt/β-catenin signaling pathway.Conclusion: These results suggested that the self-renewal of chicken PGCs and the maintenance of their characteristics were regulated by <i>Tet1</i> mediating DNA demethylation through the activation of Wnt4/β-catenin signaling pathway.