title: Function-based identification of mammalian enhancers using site-specific integration.
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.
authors.affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Diane E
authors.initials: DE
authors.lastname: Dickel
authors.affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Yiwen
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Alex S
authors.initials: AS
authors.lastname: Nord
authors.affiliation: 1] Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA. [2] Roddenberry Center for Stem Cell Biology and Medicine at Gladstone Institutes, San Francisco, California, USA.
authors.forename: John N
authors.initials: JN
authors.lastname: Wylie
authors.affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Akiyama
authors.affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Veena
authors.initials: V
authors.lastname: Afzal
authors.affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Plajzer-Frick
authors.affiliation: 1] Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. [2] Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
authors.forename: Aileen
authors.initials: A
authors.lastname: Kirkpatrick
authors.affiliation: 1] Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. [2] Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
authors.forename: Berthold
authors.identifier: 0000000163025705
authors.initials: B
authors.lastname: Göttgens
authors.affiliation: 1] Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA. [2] Roddenberry Center for Stem Cell Biology and Medicine at Gladstone Institutes, San Francisco, California, USA. [3] Department of Pediatrics, University of California, San Francisco, San Francisco, California, USA. [4] Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Benoit G
authors.initials: BG
authors.lastname: Bruneau
authors.affiliation: 1] Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. [2] US Department of Energy Joint Genome Institute, Walnut Creek, California, USA. [3] School of Natural Sciences, University of California, Merced, Merced, California, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Visel
authors.affiliation: 1] Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. [2] US Department of Energy Joint Genome Institute, Walnut Creek, California, USA.
authors.forename: Len A
authors.initials: LA
authors.lastname: Pennacchio
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002469:Cell Separation; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology*; D004742:Enhancer Elements, Genetic; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000166:cytology*; D058953:Neural Stem Cells / Q000166:cytology*; D010957:Plasmids / Q000378:metabolism; D017422:Sequence Analysis, DNA