Genome binding/occupancy profiling by high throughput sequencing
Summary
The molecular components governing neural induction remain largely unknown. Here, we used a suite of genomic and computational tools to comprehensively identify these components. We performed RNA-seq, ChIP-seq (H3K27ac, H3K27me3) and ATAC-seq on human embryonic stem cells (hESCs) at seven early neural differentiation time points (0-72 hours) and identified thousands of induced genes and their putative regulatory regions. We analyzed the function of ~2,500 selected regions using massively parallel reporter assays at all time points. We found numerous temporal enhancers that correlated with similarly timed epigenetic marks and gene expression. Development of a prioritization method that incorporated all genomic data identified key transcription factors (TFs) involved in neural induction. Individual overexpression of eleven TFs and several combinations in hESCs found novel neural induction regulators. Combined, our results provide a comprehensive map of genes and functional regulatory elements involved in neural induction and identify master regulator TFs that are instrumental for this process.
Overall design
RNA-seq (3 replicates), ChIP-seq (H3K27ac, H3K27me3 - 2 replicates) and ATAC-seq (2 replicates) were preformed on human embryonic stem cells (hESCs) at seven early neural differentiation time points (0-72 hours). Additionally, the function of ~2,500 selected regions was analyzed using massively parallel reporter assays at all time points (3 replicates).
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