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Updated: Dec 3, 2025
A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Published on: May 12, 2017
Defining Essential Enhancers for Pluripotent Stem Cells Using a Features-Oriented CRISPR-Cas9 Screen
Hao Fei Wang1, Tushar Warrier1, Chadi A Farran2
1Laboratory for Epigenetics, Stem Cells and Cell Therapy, Programme in Stem Cell, Regenerative Medicine and Aging, A(∗)STAR Institute of Molecular and Cell Biology, Singapore 138673, Singapore; Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.
Researchers identified essential stem cell enhancers using a CRISPR-Cas9 screen. These cis-regulatory elements (CREs) maintain pluripotency and regulate gene networks, with CRE111 modulating the LIF-JAK1-STAT3 pathway.
Area of Science:
- Genomics
- Molecular Biology
- Stem Cell Biology
Background:
- Cis-regulatory elements (CREs) control gene expression and cellular functions like differentiation.
- Significant gaps exist in understanding CRE function and their target genes in vivo.
- Pluripotency maintenance in mouse embryonic stem cells (mESCs) relies on precise gene regulation.
Purpose of the Study:
- To systematically identify functional CREs critical for maintaining pluripotency in mESCs.
- To characterize the regulatory networks governed by these CREs.
- To elucidate the role of specific CREs in key signaling pathways.
Main Methods:
- Features-oriented CRISPR-utilized systematic (FOCUS) screen utilizing CRISPR-Cas9.
- RNA sequencing (RNA-seq) to analyze gene expression changes.
- Genomic 4C-seq to map CRE-target gene interactions.
Main Results:
- A FOCUS screen identified 16 essential stem cell enhancers from 235 OCT4-bound CRE candidates.
- RNA-seq and 4C-seq revealed complex CRE networks regulating mESC growth and self-renewal.
- CRE111 was identified as a key enhancer regulating the LIF-JAK1-STAT3 pathway via its target gene, Lrrc31.
Conclusions:
- The study successfully identified functional CREs crucial for mESC pluripotency.
- A comprehensive map of CRE-target gene interactions was established, revealing regulatory networks.
- The findings highlight CRE111 and Lrrc31 as critical regulators of the LIF-JAK1-STAT3 pathway, essential for stem cell maintenance.
