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. At least 2 biological replicates were analyzed for each experimental condition. WT and knockout cells were RNA-seq after RA treatment for 24h or untreatment. Enhancer or Hoxa1 Capture-C were done in WT and Halr1-KO cells, in triplicate, under RA induction or untreatmnet for 24 h.

信号通路介导的靶基因转录对于胚胎干细胞（ESC）的命运决定至关重要，但该过程中依赖增强子架构的转录调控机制仍未得到充分阐明。本研究针对视黄酸（RA）信号诱导的胚胎干细胞早期分化过程，阐明了一套复杂的依赖增强子架构的多层转录调控网络。具体而言，本研究鉴定出Hoxa1与长链非编码RNA（lncRNA）Halr1为RA信号的直接下游靶基因。基于染色体构象捕获的筛选实验显示，在胚胎干细胞早期分化进程中，RA信号介导的增强子相互作用增强，对于Hoxa1与Halr1的表达至关重要。进一步研究发现，HOXA1可通过直接结合增强子促进Halr1的表达；反之，缺失Halr1 RNA会增强Hoxa1染色质与多个增强子之间的相互作用，但削弱其与HoxA簇内部染色质的相互作用，进而促进RA信号诱导的Hoxa1过度激活与胚胎干细胞早期分化。上述结果表明，结合于染色质的Halr1不仅可作为“刹车分子”调控增强子与Hoxa1染色质之间的相互作用，还可作为结合因子维持HoxA簇内部的染色质相互作用。综上，本研究揭示了一套涉及增强子、转录因子与长链非编码RNA协同调控的复杂多层转录调控机制，加深了我们对RA信号诱导胚胎干细胞分化内在分子机制的认知。本研究中每个实验条件均至少设置2次生物学重复。分别在RA处理24小时与未处理的条件下，对野生型（WT）与敲除细胞进行RNA测序（RNA-seq）；在RA诱导24小时与未处理的条件下，对野生型与Halr1敲除（Halr1-KO）细胞进行增强子或Hoxa1 Capture-C实验，每组设置3次生物学重复。