Enhancer architecture-dependent multilayered transcriptional regulation orchestrates RA signal-induced early differentiation of ESC

Signaling pathway driven target gene transcriptions are critical for fate determination of embryonic stem cells (ESC), but enhancer architecture-dependent transcriptional regulation remains largely unclear in this process. Here, we described a complex enhancer architecture-dependent multilayered transcriptional regulation that orchestrates retinoic acid (RA) signal-induced early differentiation of ESC. Specifically, we identified Hoxa1 and lncRNA Halr1 as the direct downstream target genes of RA signal. Chromosome conformation capture based screens show that increased enhancer interactions promoted by RA signal are essential for Hoxa1 and Halr1 expressions during early ESC differentiation. Furthermore, we find that HOXA1 promotes Halr1 expression through direct binding to enhancers; conversely, absence of Halr1 RNA enhances interaction between Hoxa1 chromatin and multiple enhancers, but weakens interaction with HoxA cluster internal chromatin, thereby promoting RA signal-induced Hoxa1 overactivation and early differentiation of ESC. These results indicate that Halr1 binds to chromatin not only acts as a brake to orchestrate interaction between enhancers and Hoxa1 chromatin, but also acts as a binder to maintain chromatin interaction within HoxA cluster. In summary, these findings reveal a complex multilayered transcriptional regulation involving the synergistic regulation of enhancer, transcription factor and lncRNA, and that increases our understanding of the intrinsic molecular mechanisms of RA signal-induced ESC differentiation.