Dynamic network-guided CRISPRi screen identifies CTCF-loop-constrained nonlinear enhancer gene regulatory activity during cell state transitions

Renhe Luo, Jielin Yan, Jin Woo Oh, Wang Xi, Dustin Shigaki, Wilfred Wong, Hyein S Cho, Dylan Murphy, Ronald Cutler, Bess P Rosen, Julian Pulecio, Dapeng Yang, Rachel A Glenn, Tingxu Chen, Qing V Li, Thomas Vierbuchen, Simone Sidoli, Effie Apostolou, Danwei Huangfu, Michael A Beer.
at Genet. 2023-07-24;55(8):1336-1346.
Comprehensive enhancer discovery is challenging because most enhancers, especially those contributing to complex diseases, have weak effects on gene expression. Our gene regulatory network modeling identified that nonlinear enhancer gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Using human embryonic stem cell definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. We discovered a comprehensive set of enhancers for each of the core endoderm-specifying transcription factors. Many enhancers had strong effects mid-transition but weak effects post-transition, consistent with the nonlinear temporal responses to enhancer perturbation predicted by the modeling. Integrating three-dimensional genomic information, we were able to develop a CTCF-loop-constrained Interaction Activity model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and systematic enhancer discovery in both normal and pathological cell state transitions.

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Data summary
The parental HUES8 hESC line was obtained from Harvard University under a material transfer agreement. Sequencing data are available at GEO under accession GSE213394 (new data from this study) and GSE114102 (published DE-72 h H3K4me1 ChIP–seq data), GSE63525 (published K562 Hi-C data) and GSE72816, GSE177081, GSE177471 (published ChIA–PET data). The Hi-C data are available in the 4D Nucleome Data Portal (https://data.4dnucleome.org/) under accession numbers 4DNESDO2ZYBM, 4DNESQMUTYXH, 4DNESFL8KDMT, 4DNESW8SIXN7, 4DNESW9GVC97, and 4DNESI1DNSGF. Mass spectrometry data are available in the PRIDE database under ProteomeXchange accession PXD043070. Source data are provided with this paper.