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 journalArticle

48 Citations (Scopus)

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

Fingerprint

Sorting
Assays
Flow Cytometry
Throughput
High-Throughput Nucleotide Sequencing
Flow cytometry
DNA sequences
Stem cells
Cardiac Myocytes
Screening
Stem Cells
Genes
Fluorescence
Cells
Genome
DNA
In Vitro Techniques
Mouse Embryonic Stem Cells

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Dickel, D. E., Zhu, Y., Nord, A., Wylie, J. N., Akiyama, J. A., Afzal, V., ... Pennacchio, L. A. (2014). Function-based identification of mammalian enhancers using site-specific integration. Nature Methods, 11(5), 566-571. https://doi.org/10.1038/nmeth.2886

Function-based identification of mammalian enhancers using site-specific integration. / Dickel, Diane E.; Zhu, Yiwen; Nord, Alexander; Wylie, John N.; Akiyama, Jennifer A.; Afzal, Veena; Plajzer-Frick, Ingrid; Kirkpatrick, Aileen; Göttgens, Berthold; Bruneau, Benoit G.; Visel, Axel; Pennacchio, Len A.

In: Nature Methods, Vol. 11, No. 5, 01.01.2014, p. 566-571.

Research output: Contribution to journalArticle

Dickel, DE, Zhu, Y, Nord, A, Wylie, JN, Akiyama, JA, Afzal, V, Plajzer-Frick, I, Kirkpatrick, A, Göttgens, B, Bruneau, BG, Visel, A & Pennacchio, LA 2014, 'Function-based identification of mammalian enhancers using site-specific integration', Nature Methods, vol. 11, no. 5, pp. 566-571. https://doi.org/10.1038/nmeth.2886
Dickel, Diane E. ; Zhu, Yiwen ; Nord, Alexander ; Wylie, John N. ; Akiyama, Jennifer A. ; Afzal, Veena ; Plajzer-Frick, Ingrid ; Kirkpatrick, Aileen ; Göttgens, Berthold ; Bruneau, Benoit G. ; Visel, Axel ; Pennacchio, Len A. / Function-based identification of mammalian enhancers using site-specific integration. In: Nature Methods. 2014 ; Vol. 11, No. 5. pp. 566-571.
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