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

  • Author(s): Dickel, Diane E;
  • Zhu, Yiwen;
  • Nord, Alex S;
  • Wylie, John N;
  • Akiyama, Jennifer A;
  • Afzal, Veena;
  • Plajzer-Frick, Ingrid;
  • Kirkpatrick, Aileen;
  • Göttgens, Berthold;
  • Bruneau, Benoit G;
  • Visel, Axel;
  • Pennacchio, Len A
  • et al.
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.

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