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Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells.
Shiqi Xie1, Jialei Duan1, Boxun Li1
1Laboratory of Regulatory Genomics, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Molecular Cell
|April 19, 2017
Summary
Mosaic-seq enables precise measurement of enhancer activity at single-cell resolution. This new method reveals that only a few key enhancer elements significantly impact gene expression, with combinatorial effects being substantial.
Area of Science:
- Molecular Biology
- Genomics
- Epigenetics
Background:
- Quantifying endogenous enhancer activity is crucial but challenging due to limited methodologies.
- Understanding enhancer function is key to deciphering gene regulation.
Purpose of the Study:
- To develop a novel method, Mosaic-seq, for systematically perturbing and measuring enhancer activity at single-cell resolution.
- To identify key constituent enhancers within super-enhancers and characterize their regulatory roles.
Main Methods:
- Mosaic-seq utilizes a CRISPR barcoding system to simultaneously measure transcriptomes and sgRNA modulators in single cells.
- CRISPR interference (CRISPRi) with dCas9-KRAB was employed to repress enhancer activity.
- Analysis was performed on 51,448 sgRNA-induced transcriptomes from 71 enhancers across 15 super-enhancers.
Main Results:
- Only a subset of constituent enhancers significantly affects target gene expression.
- Binding of p300 and RNA Polymerase II (RNAPII) are characteristic features of major effector enhancers.
- Key parameters of enhancer activity, including population penetrance and individual cell contribution, were determined.
- Simultaneous repression of multiple weak enhancers can have a synergistic effect on super-enhancer activity.
Conclusions:
- Mosaic-seq provides a powerful tool for dissecting enhancer function at an unprecedented resolution.
- Super-enhancers are composed of a few critical elements and many minor contributors.
- Combinatorial regulation by weak enhancers plays a significant role in modulating gene expression.