GSE202540
Transcriptome and 3D genome sequencing depict the regulation of K5aSE
Purpose: Super-enhancers (SEs) have been reported to regulate the expression of core transcription factors in embryonic stem cells (ESCs), but they have not been well identified and evaluated in terms of their functional roles in ESC phenotype. Methods and results: Here we identified an ESC-specific Klf5-adjacent SE (K5aSE) by conducting comprehensive analysis of ESCs in different states. We found that K5aSE is required for ESC clonal growth, cell proliferation, embryoid body (EB) differentiation and Klf5 expression. However, restorative expression of Klf5 only partially rescued the phenotype of K5aSE knockout (K5aSE-KO) ESCs. Meanwhile, Klf5-KO ESCs exhibited similar phenotypes to K5aSE-KO cells, but shown different lineage differentiation in EBs, revealing that K5aSE and Klf5 regulate ESCs differently. Further 4C-seq combined with RNA-seq analysis showed that K5aSE significantly promoted the expression of other four target genes in the same chromosome, such as Clybl, Farp1, Nkx3-1 and Tbc1d4, and that CRISPRa-mediated restoration of these four genes also partially rescued the phenotype of K5aSE-KO cells. Finally, we also identified these four genes as novel ESC regulators required for self-renewal and EB differentiation.  transcripts in the retinas of WT and Nrl-/- mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl-/- retina, with a fold change =1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Together, this study provides new insights into understanding ESC properties and highlights the crucial role of SE as 3D chromatin structure-dependent transcriptional driver in ESCs. Overall design: At least 2 biological replicates were analyzed for each experimental condition. WT and knockout cells were RNA-seq in ESCs or EBs-day 5. 4C-seq of K5aSE were done in ESCs.