Study / S3W9BZQF82014-03-23
FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells
Matthew Murtha, Zeynep Tokcaer-Keskin, Zuojian Tang, Francesco Strino, Xi Chen et al.
About this study
Promoters and enhancers establish precise gene transcription patterns. The development of functional approaches for their identification in mammalian cells has been complicated by the size of these genomes. Here we report a high-throughput functional assay for directly identifying active promoter and enhancer elements called FIREWACh (Functional Identification of Regulatory Elements Within Accessible Chromatin), which we used to simultaneously assess over 80,000 DNA fragments derived from nucleosome-free regions within the chromatin of embryonic stem cells (ESCs) and identify 6,364 active regulatory elements. Many of these represent newly discovered ESC-specific enhancers, showing enriched binding-site motifs for ESC-specific transcription factors including SOX2, POU5F1 (OCT4) and KLF4. The application of FIREWACh to additional cultured cell types will facilitate functional annotation of the genome and expand our view of transcriptional network dynamics.
Full author list & citation
Matthew Murtha, Zeynep Tokcaer-Keskin, Zuojian Tang, Francesco Strino, Xi Chen, Yatong Wang, Xiangmei Xi, Claudio Basilico, Stuart Brown, Richard Bonneau, Yuval Kluger, Lisa Dailey. FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells. 2014-03-23. https://doi.org/10.1038/nmeth.2885
Experiments 1
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Accessible-chromatin (nucleosome-free region) fragments released from E14 mouse ESC nuclei with HaeIII or RsaI were cloned upstream of a minimal Fgf4 promoter-GFP lentiviral reporter, transduced at low copy, and selected by GFP FACS. The processed table contains the merged final FIREWACh interval set together with matched promoter-proximal/distal gene context and auxiliary luciferase-validation measurements where the source coordinates overlap the final set.