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

Nat Methods. 2014 May;11(5):566-71. doi: 10.1038/nmeth.2886. Epub 2014 Mar 23.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Differentiation
  • Cell Separation
  • Chromosomes, Artificial, Bacterial / genetics
  • Embryonic Stem Cells / cytology*
  • Enhancer Elements, Genetic*
  • Flow Cytometry
  • Gene Expression Regulation
  • Gene Library
  • Genes, Reporter
  • Genetic Vectors
  • Genomics
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Mice
  • Mice, Transgenic
  • Myocytes, Cardiac / cytology*
  • Neural Stem Cells / cytology*
  • Plasmids / metabolism
  • Sequence Analysis, DNA

Associated data

  • SRA/SRP034877