Project description:Enhancers play key roles in gene regulation. However, comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Through gene regulatory network modeling, we identified that dynamic cell state transitions, a critical missing component in prevalent enhancer discovery strategies, can be utilized to improve the cells’ sensitivity to enhancer perturbation. Guided by the modeling results, we performed a mid-transition CRISPRi-based enhancer screen utilizing human embryonic stem cell definitive endoderm differentiation as a dynamic transition system. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core lineage-specifying transcription factors (TFs), including many enhancers with weak to moderate effects. Integrating the screening results with enhancer activity measurements (ATAC-seq, H3K27ac ChIP-seq) and three-dimensional enhancer-promoter interaction information (CTCF looping, Hi-C), we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Together, our dynamic network-guided enhancer screen and the CIA enhancer prediction model provide generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.
Project description:The evidence that several signal and ligand-dependent pathways function by activating regulatory enhancer programs suggests that a “checkpoint” strategy may underline activation of some or even many diversely-regulated enhancers. Here, we report a molecular mechanism common to several acute signal- and ligand-dependent enhancer activation programs based on release of a shared eRNA transcription checkpoint. This requires recruitment of a DNA-PKcs-phosphorylated RING finger repressor, KAP1, functioning as a modulator, inhibiting its association with 7SK and E3 SUMO ligase activity on the CDK9 subunit of P-TEFb, facilitating formation of an activated P-TEFb complex, licensing eRNA elongation. Overcoming this checkpoint for signal-dependent enhancer activation occurs in diverse pathways including estrogen receptor α, NF-κB-regulated proinflammatory, androgen receptor and neuronal depolarization. Therefore, a specific strategy required to convert a basal state enhancer P-TEFb complex to an active state to release a conserved checkpoint is apparently employed by several functionally-important signal/ligand-regulated regulatory enhancers to implement the instructions of endocrine/paracrine system