Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Other
Summary
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.
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