RA-dependent proximal and distal enhancers' architecture promotes RA-induced early ESC differentiation

Retinoic acid (RA) plays a pivotal role in different biological processes including inducing differentiation of embryonic stem cells (ESCs). We first knocked out one or both of RA receptors- Rara and Rxra to partially abolish the RA signaling, and then overexpressed one of them separately to hyper activation of RA signaling pathway. By transcriptome analysis, we show that RA signaling effects multi-lineage differentiation, particularly the endoderm, and identify RA signaling target genes, interesting, there are two family cluster genes- Hoxb and Cyp26. Chromosome immunoprecipitation (ChIP-seq) shows that RA induces novel proximal and distal enhancers around the cluster target genes and these enhancers are dependent of RA receptors(Rara/Rxra). 4C-seq reveals enhancer-enhancer and enhancer-promoter interactions in their regions and shows a decrease in the relative frequency of contact relative to WT after Rara/Rxra-knockout, suggesting Rara/Rxra participate in mediating chromatin interaction. We also identify that enhancers significantly maintain expression of RA-induced Hoxb and Cyp26 family genes and regulate multi-lineage marker genes. At the same time, we reveal that disrupting RA-response elements (RAREs) in the enhancer affects the expression of the target gene. Our study provides mechanistic insight into RA-induced early ESC differentiation to endoderm and potential regular regulation of downstream target genes. Overall design: 2 biological replicates were analyzed for WT (RARX), Rara-KO, Rxra-KO and RARX-DKO by RNA-seq in RA-treated ESCs for 24 h, 2 biological replicates were analyzed for V-OE, Rara-OE and Rxra-OE by RNA-seq in RA-treated ESCs for 24 h, 2 biological replicates of H3K27Ac ChIP-seq for WT in normal growth condition,2 biological replicates of H3K27Ac ChIP-seq for WT (RARX), Rara-KO, Rxra-KO and RARX-DKO in RA-treated ESCs for 24 h, 3 biological replicates were analyzed for WT(Cyp26), Cyp26-E1-KO and Cyp26-E2-KO by RNA-seq in RA-treated ESCs for 24 h, 2 biological replicates were analyzed for WT (RARX), Rara-KO, Rxra-KO and RARX-DKO at Hoxb-E1, Hoxb-E2, Cyp26-E1 and Cyp26-E2 by 4C-seq in RA-treated ESCs for 24 h