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

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

Research output: Contribution to journalArticlepeer-review

52 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)566-571
Number of pages6
JournalNature Methods
Volume11
Issue number5
DOIs
StatePublished - Jan 1 2014
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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