Diane E. Dickel, Yiwen Zhu, Alex S. Nord, John N. Wylie, Jennifer A. Akiyama et al.
The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe Site-specific Integration FACS-sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Pluripotent cell reporter assays, targeted single-copy genomic integration, and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. We demonstrate the utility of this method by functionally interrogating >500 kb of mouse and human sequence for enhancer activity and identifying embryonic stem (ES) cell enhancers at pluripotency loci including NANOG. We also demonstrate the effectiveness of the approach in differentiated cell populations through the identification of cardiac enhancers from cardiomyocytes and neuronal enhancers from neural progenitors. SIF-seq is a powerful and flexible method for the de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.
Diane E. Dickel, Yiwen Zhu, Alex S. Nord, John N. Wylie, Jennifer A. Akiyama, Veena Afzal, Ingrid Plajzer-Frick, Aileen Kirkpatrick, Berthold Göttgens, Benoit G. Bruneau, Axel Visel, Len A. Pennacchio. Function-based Identification of Mammalian Enhancers Using Site-Specific Integration. 2014-03-23. https://doi.org/10.1038/nmeth.2886
A human BAC spanning NANOG was tested in mouse ES cells with the H19-insulated Venus targeting vector. Single-copy integrated constructs were compared between fluorescent sorted and unsorted input populations by amplicon sequencing.
A human BAC spanning MYH6 and MYH7 was randomly sheared and integrated as single-copy Venus reporter constructs in mouse ES cells. Fluorescent sorted and unsorted input populations were sequenced to identify ES-cell-active regions.
The same human NANOG BAC region was tested in mouse ES cells with the comparison Venus targeting vector lacking the H19 insulator. Single-copy integrated constructs were compared between fluorescent sorted and unsorted input populations by amplicon sequencing.
The human MYH6/MYH7 BAC library was differentiated from mouse ES cells into cardiomyocytes before the fluorescent sort. Sequencing of fluorescent and unsorted input populations identified cardiomyocyte-enriched promoters and candidate heart enhancers.
A pooled library of previously characterized human ultraconserved noncoding elements was integrated as single-copy Venus reporter constructs in mouse ES cells and then differentiated to Nestin-positive neural progenitors. Fluorescent sorted and unsorted input libraries were sequenced to identify elements enriched for neural-progenitor enhancer activity.
A randomly sheared mouse BAC spanning Nanog and neighboring genes was tested in mouse ES cells using single-copy Hprt-targeted Venus reporter constructs. Fluorescent sorted and unsorted input populations were sequenced to identify enriched enhancer regions.
A randomly sheared mouse BAC spanning the Sall1 locus was cloned beside a minimal hsp68-Venus reporter and integrated as single-copy Hprt-targeted constructs in mouse ES cells. Fluorescent sorted and unsorted input populations were sequenced to identify enriched enhancer regions.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 13 files (ZIP)ena_read_run.tsvREADME.txtreference_sequences/hg19_chr12_7832184_7996058.fareference_sequences/hg19_chr14_23767304_23990800.fareference_sequences/mm9_chr6_122573551_122806765.fareference_sequences/mm9_chr8_91399959_91630935.fareference_sequences/ultraconserved_vista_annotations.tsvreference_sequences/ultraconserved_vista_sequences_hg38.fasra_runinfo.csvsupplementary_sources/Dickel2014_supplementary_information.pdfsupplementary_sources/Dickel2014_supplementary_software.zipsupplementary_sources/Pennacchio2006_MOESM1_Supplementary_Table_1.xlssupplementary_sources/Visel2008_MOESM4_Supplementary_Table_4.xls