Deciphering the decisive factors driving fate bifurcations in somatic cell reprogramming.

Single-cell studies have demonstrated that somatic cell reprogramming is a continuous process of cell fates transition. Only partial reprogramming intermediates can overcome the molecular bottlenecks to acquire pluripotency. To decipher the underlying decisive factors driving cell fate, we identified induced pluripotent stem cells or stromal-like cells (iPSCs/SLCs) and iPSCs or trophoblast-like cells (iPSCs/TLCs) fate bifurcations by reconstructing cellular trajectory. The mesenchymal-epithelial transition and the activation of pluripotency networks are the main molecular series in successful reprogramming. Correspondingly, intermediates diverge into SLCs accompanied by the inhibition of cell cycle genes and the activation of extracellular matrix genes, whereas the TLCs fate is characterized by the up-regulation of placenta development genes. Combining putative gene regulatory networks, seven (Taf7, Ezh2, Klf2, etc.) and three key factors (Cdc5l, Klf4, and Nanog) were individually identified as drivers of the successful reprogramming by triggering downstream pluripotent networks during iPSCs/SLCs and iPSCs/TLCs fate bifurcation. Conversely, 11 factors (Cebpb, Sox4, Junb, etc.) and four factors (Gata2, Jund, Ctnnb1, etc.) drive SLCs fate and TLCs fate, respectively. Our study sheds new light on the understanding of decisive factors driving cell fate, which is helpful for improving reprogramming efficiency through manipulating cell fates to avoid alternative fates.

Competitive binding of TET1 and DNMT3A/B cooperates the DNA methylation pattern in human embryonic stem cells.

DNMT3A/B and TET1 play indispensable roles in regulating DNA methylation that undergoes extensive reprogramming during mammalian embryogenesis. Yet the competitive and cooperative relationships between TET1 and DNMT3A/B remain largely unknown in the human embryonic stem cells. Here, we revealed that the main DNA-binding domain of TET1 contains more positive charges by using charge reduction of amino acid alphabet, followed by DNMT3A and DNMT3B. The genome-wide binding profiles showed that TET1 prefers binding to the proximal promoters and CpG islands compared with DNMT3A/B. Moreover, the binding regions of these three transcription factors can be divided into specific and co-binding regions. And a stronger inhibitory effect of DNMT3A on TET1 demethylation was observed in co-binding regions. Furthermore, we integrated TET1 knockout data to further discuss the competitive binding patterns of TET1 and DNMT3A/B. The lack of TET1 increased the occupation of DNMT3A/B at the specific binding regions of TET1 causing focal hypermethylation. The knockout of TET1 was also accompanied by a reduction of DNMT3A/B binding in the co-binding regions, further confirming the cooperative binding function between TET1 and DNMT3A/B. In conclusion, our studies found that the competitive binding of TET1 and DNMT3A/B cooperatively shapes the global DNA methylation pattern in human embryonic stem cells.

The Cumulative Formation of R-loop Interacts with Histone Modifications to Shape Cell Reprogramming.

R-loop, a three-stranded RNA/DNA structure, plays important roles in modulating genome stability and gene expression, but the molecular mechanism of R-loops in cell reprogramming remains elusive. Here, we comprehensively profiled the genome-wide landscape of R-loops during cell reprogramming. The results showed that the R-loop formation on most different types of repetitive elements is stage-specific in cell reprogramming. We unveiled that the cumulative deposition of an R-loop subset is positively correlated with gene expression during reprogramming. More importantly, the dynamic turnover of this R-loop subset is accompanied by the activation of the pluripotent transcriptional regulatory network (TRN). Moreover, the large accumulation of the active histone marker H3K4me3 and the reduction in H3K27me3 were also observed in these R-loop regions. Finally, we characterized the dynamic network of R-loops that facilitates cell fate transitions in reprogramming. Together, our study provides a new clue for deciphering the interplay mechanism between R-loops and HMs to control cell reprogramming.