Functional chromatin signatures premark future lineage-specific enhancers [scRNA-seq]. Functional chromatin signatures premark future lineage-specific enhancers [scRNA-seq]

It remains unknown whether early embryonic cells harbor a blueprint for future enhancers that regulate the expression of lineage-specific genes in adult tissues. Here, we demonstrate that embryonic stem cells (ESCs) have transcriptionally competent chromatin regions (CCRs) prepared to induce the expression of lineage genes prior to differentiation. CCRs represent activatable pre-enhancers within the topological chromatin domains of lineage genes, marked by chromatin signatures distinguishable from primed/poised enhancers, enabling their genome-wide identification. The pioneer transcription factor (TF) FOXA2 preferentially binds CCRs during early lineage specification, promoting their conversion into active enhancers. CCRs can be harnessed to boost the expression of master TFs and promote the direct reprogramming of ESCs into differentiated cells, showcasing their potential for practical applications. Our findings identify a mechanism by which ESCs rapidly establish enhancer activity during early lineage differentiation and expand our understanding of the epigenetic features supporting transcriptional regulation and cellular plasticity. Overall design: Single-cell 10x genomics experiments were performed using hESCs after interrogating candidate CCRs through CRISPRa screens. We employed a gRNA backbone inspired on the CROP-Seq system that allows the dual identification of gRNAs and single-cell transcriptomics. gRNA and transcriptome libraries were generated and sequenced. Final analyses were done using CellRanger and the SCEPTRE algorithm.