October 23, 2019  |  

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

Authors: Dickel, Diane E and Zhu, Yiwen and Nord, Alex S and Wylie, John N and Akiyama, Jennifer A and Afzal, Veena and Plajzer-Frick, Ingrid and Kirkpatrick, Aileen and Göttgens, Berthold and Bruneau, Benoit G and Visel, Axel and Pennacchio, Len A

The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro-differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.

Journal: Nature methods
DOI: 10.1038/nmeth.2886
Year: 2014

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