Systematic mapping of TF-mediated cell fate changes by a pooled induction coupled with scRNA-seq and multi-omics approaches

Transcriptional regulation controls various cellular functions via the interactions between cell-type-specific transcription factors (TFs) and their chromosomal targets. While multiple TFs capable of converting cell fates from one to another have been reported, only a few systematic studies aiming to understand the fate conversion potential of numerous individual TFs have been reported. Here, we propose an iTF-seq method (pooled induction of individual TFs followed by single-cell RNA sequencing) to identify potent TFs capable of converting cell fates and elucidate their lineage specification potential at a single-cell level. Enabling metabolic biotinylation of TFs during the process, the method allows subsequent multi-omics approaches to understand underlying action mechanisms of TFs during cell fate changes. Our pilot iTF-seq of 80 TFs in mouse embryonic stem (ES) cells has identified multiple previously known and unknown TFs triggering transcriptome changes within one day of TF induction. Subsequent tests revealed that Gcm1 and Otx2 function as activators and pioneer factors, while they resulted in distinct cell fates and gene expression programs upon induction. We generated 80 cell lines to induce individual TFs in J1 ES cells. cDNAs for the TFs were individually cloned, and 80 individual stable lines were generated using a Sleeping Beauty (SB) transposon combined with a metabolic biotinylation (SBFB) system. We performed scRNA-seq of pooled cell lines upon doxycycline treatment for 1, 3, and 5 days with wildtype ES cells. We performed RNA-seq on SBFB cells. First, we conducted time-course experiments involving the removal of Dox for Dlx5, Gata3, Gcm1, Otx2, and Pdx1 cases. In addition to maintaining continuous Dox treatment for 7 days as a control, we generated samples by discontinuing Dox after 1 day, 3 days, and 5 days of culture. Second, we conducted global gene expression profiling upon the induction of Otx2, Gcm1, and Dlx5. The samples were collected from Dox-treated cells at various time points: 0h, 3h, 6h, 12h, day 1, day 3, and day 5.