title: Simultaneous Pathway Activity Inference and Gene Expression Analysis Using RNA Sequencing.
abstract: Reporter gene assays are a venerable tool for studying signaling pathways, but they lack the throughput and complexity necessary to contribute to a systems-level understanding of endogenous signaling networks. We present a parallel reporter assay, transcription factor activity sequencing (TF-seq), built on synthetic DNA enhancer elements, which enables parallel measurements in primary cells of the transcriptome and transcription factor activity from more than 40 signaling pathways. Using TF-seq in Myd88(-/-) macrophages, we captured dynamic pathway activity changes underpinning the global transcriptional changes of the innate immune response. We also applied TF-seq to investigate small molecule mechanisms of action and find a role for NF-κB activation and coordination of the STAT1 response in the macrophage reaction to the anti-inflammatory natural product halofuginone. Simultaneous TF-seq and global gene expression profiling represent an integrative approach for gaining mechanistic insight into pathway activity and transcriptional changes that result from genetic and small molecule perturbations.
authors.affiliation: Program in Medical and Population Genetics, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: O'Connell
authors.affiliation: Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Raivo
authors.initials: R
authors.lastname: Kolde
authors.affiliation: Program in Medical and Population Genetics, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Sooknah
authors.affiliation: Program in Medical and Population Genetics, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Graham
authors.affiliation: Center for the Science of Therapeutics, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Sundberg
authors.affiliation: Program in Medical and Population Genetics, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Isabel
authors.initials: I
authors.lastname: Latorre
authors.affiliation: Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Tarjei S
authors.initials: TS
authors.lastname: Mikkelsen
authors.affiliation: Program in Medical and Population Genetics, Broad Institute, Cambridge, MA 02142, USA.|Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA.|Center for the Science of Therapeutics, Broad Institute, Cambridge, MA 02142, USA.|Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Ramnik J
authors.initials: RJ
authors.lastname: Xavier
mesh_terms: D001483:Base Sequence; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing; D016328:NF-kappa B; D012313:RNA; D017423:Sequence Analysis, RNA; D059467:Transcriptome
chemical_list: D016328:NF-kappa B; D012313:RNA