Discovery of Competent Chromatin Regions in Human Embryonic Stem Cells [4C]

It remains unclear how ESCs rapidly establish enhancer regulatory elements to activate lineage-specific genes at the earliest differentiation stages. Previous studies suggest that some tissue-specific enhancers are pre-marked in ESCs with permissive features that support their competence to induce gene expression in response to the binding of lineage transcription factors (TFs). However, the absence of functional assays to identify chromatin regions with the competence to activate gene expression upon TF binding has impeded to better define any pre-marking features and their role on the transcriptional competence of the chromatin. To address this, we leveraged a CRISPR-activation(a) system to systematically interrogate the presence of chromatin regions able to induce the expression of 5 lineage-specific genes that are inactive in human ESCs (hESCs). Multiple CRISPRa screens discovered 42 regions, which we termed Competent Chromatin Regions (CCRs), that can induce the expression of the 5 lineage genes upon CRISPRa targeting. ChIP seq analyses showed that CCRs do not represent primed or active enhancers, since they are not enriched in H3K4me1 or H3K27ac histone modifications. Instead, CCRs exhibit a significant enrichment in POU sequence motifs, along with higher levels of binding by OCT4 and other pluripotent TFs. CCRs have an enriched presence of several repressive and activating chromatin-associated factors, including the DNA demethylation factors TET1 and QSER1 and various HDAC enzymes, which regulate their transcriptional competence. Although CCRs are exclusively found at the topologically associated domains shared with their related genes, we found they do not require an enrichment in pre-established chromatin contacts to exert their function. To assess the extent of our findings, we simulated the earliest stages of lineage differentiation by inducing the expression of the lineage TF FOXA2 in undifferentiated hESCs and we found it preferentially binds to the regions predicted as CCRs. Our results suggest that the factors enriched at CCRs facilitate the binding of lineage TFs and enable ESCs to promptly activate the expression of lineage genes. This study advances our understanding of pluripotency regulation in ESCs by identifying a novel type of regulatory regions that supports their developmental plasticity. Overall design: 4C using as viewpoints the GATA6, EOMES, PDX1 and SOX17 loci in hESCs. Each 4C experiment had n=2 biological replicates.