The Epigenetic Regulation Of Tbx3 Expression In Mouse Embryonic Stem Cell

Objective:To investigate the epigenetic regulation of Tbx3 expression in mouse embryonic stem cells.Methods: 1.RT-q PCR results of DPF2-deletion ESCs imply that DPF2 acts on Tbx3 locus in mouse embryonic stem cells.First,the potential enhancer site of Tbx3 was found by CHIP-seq and luciferase Assay in m ESCs.Second,we knocked out these genetic sites--Enhancer-2,Enhancer-3-6,and Enhancer-7 to identify the functional enhancer by CRISPR/Cas9 system.Finally,CHIP-seq analysis of DPF2 knockout cells was performed to verify the role of DPF2 through acting on Tbx3 genius loci.2.CHIP-seq analysis of DPF2 knockout cells revealed the presence of PRC2 interacting with Enhancer-2 genius loci of Tbx3.Then,conditional knockout experiment of EED in m ESCs was performed to verify the interaction by CHIP-seq in EED-induced knockout cells.Results: 1.The expression of Tbx3 was significantly reduced in DPF2-deletion ESCs.2.Knockout of Enhancer-7 remarkably impaired the expression of Tbx3 in m ESCs.3.H3K27Ac3 chromatin marker of Enhancer-7 and its ability of binding to the ESC-specific transcription factor OCT4 are weakened on account of the deletion of Dpf2.4.The expression of Tbx3 was increased significantly in EED conditional knockout cells.5.Knocking out EED weakened H3K27me3 modification of Enhancer-2 and improved its ability of binding to Oct4 and Sox2.Conclusion: Above all,the expression of Tbx3 is regulated by DPF2,a subunit of BAF complex and PRC2 in mouse embryonic stem cells.There are two stem cell-specific enhancers of Tbx3-Enhancer-7 and Enhancer-2.DPF2 promotes the expression of Tbx3 through affecting the H3K27 Ac chromatin marker and the interaction of OCT4 with enhancer-7 while the PRC2 inhibits the expression of Tbx3 by influencing the H3K27me3 marker of Enhancer-2 which furthermore controls the binding of Oct4 and Oct2 on Enhancer-2.