TEAD2 mediates the ground-state pluripotency by chromatin looping

Mouse embryonic stem cells (mESCs) cultured in 2i (MEK and GSK3 kinase inhibitor)/LIF and serum/LIF that we called 2i-ESCs and serum-ESCs represent ground and confused pluripotent states, respectively. However, the transcription factors that regulate ground pluripotency through chromatin-associated characteristics are not yet fully understood. By mapping chromatin accessibility and transcription factor regulatory networks during the interconversion of 2i-ESCs and serum-ESCs, we have identified TEAD2 as highly enriched in 2i-specific peaks. While Tead2 knockout did not affect the pluripotency or differentiation ability of either 2i-ESCs or serum-ESCs, it did prevent the establishment of the 2i-specific state and the exit from the serum-specific state. TEAD2 binds to active regions in 2i-specific genes and activates their expression by regulating enhancer-promoter (EP) interactions during serum-to-2i transition. Remarkably, TEAD2-mediated EP interactions were independent of chromatin architecture proteins YY1 and CTCF, but instead appear to be facilitated by TEAD2 homodimer formation. Overall design: Dynamic gene expression and chromatin accessibility profiling of interconversion of serum-ESCs and 2i-ESCs. Examination of TEAD2, H3K27ac, YY1, SMC1 binding sites in 2i-ESCs or cells on day 6 of serum-to2i transition. Examination of chromatin interactions in cells on day 6 of serum-to2i transition with or without Tead2-knockout. Tead2-knockout SL-ESC lines and 2iL-ESCs with mutated TEAD2 motifs at B4galt6 promoter were generated by using CRISPR/Cas9. Cells from the sixth day of SL-to-2iL transition after Tead2-knockout, as well as mutated 2iL-ESCs, were collected and 4C-seq analysis was performed. Tead2-FLAG-AviTag knock-in 2iL- and SL-ESC lines were genetically generated by using the CRISPR/Cas9 system. Cells were then collected for Biotin ChIP-seq. Tead2-knockout SL-ESC lines were also generated by using CRISPR/Cas9. Wild-type and Tead2-knockout cells were collected from the sixth day of SL-to-2iL transition and H3K27ac and H3K4me1 CUT&Tag experiments were performed. 2iL-ESC lines with mutation of TEAD2 motifs in the B4galt6 promoter region were generated by using CRISPR/Cas9, and wild-type and mutated 2iL-ESCs were then collected for H3K27ac CUT&Tag experiments. Tead2-knockout SL-ESC lines were generated by using the CRISPR/Cas9 system. Wild-type and Tead2-knockout cells were collected from the day 0 and day 21 of SL-to-2iL transition and BL-HiC analysis was performed. Tead2-knockout SL-ESC lines were generated by using the CRISPR/Cas9 system. Cells from the day 15 and 21 of SL-to-2iL transition after Tead2 knockout were collected and RNA-seq was performed. During the SL-to-2iL transition, siRNA targeting Tead4 gene and negative control were transfected, and siNC and siTead4 cells from the day 0 and 6 of transition were collected and RNA-seq was performed.