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Function-based identification of mammalian enhancers using site-specific integration.

Diane E Dickel¹, Yiwen Zhu¹, Alex S Nord¹

¹Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

|March 25, 2014

Summary

We developed site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq) to discover functional enhancers in mammalian genomes. This method efficiently identifies enhancers in various cell types, including stem cells and differentiated cells.

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Area of Science:

Genomics
Molecular Biology
Epigenetics

Background:

Identifying functional regulatory sequences in mammalian genomes is a significant challenge.
Enhancers are crucial DNA elements that regulate gene expression but are difficult to locate comprehensively.

Purpose of the Study:

To introduce and validate site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq) as a novel method for discovering distant-acting enhancers.
To demonstrate the utility of SIF-seq for functional interrogation of large genomic regions across different cell types.

Main Methods:

SIF-seq combines targeted single-copy genomic integration, reporter assays, and flow cytometry with high-throughput DNA sequencing.
This approach enables parallel screening of numerous DNA sequences for enhancer activity.

Main Results:

SIF-seq successfully identified enhancers in mouse and human sequences, including novel enhancers near pluripotency genes like NANOG in mouse embryonic stem cells.
The assay also detected cardiac enhancers in differentiated cardiomyocytes and neuronal enhancers in neural progenitor cells.

Conclusions:

SIF-seq is a powerful, flexible, and unbiased method for the de novo functional identification of mammalian enhancers.
This technique has broad applicability for enhancer discovery across a wide range of cell types.