TF-seq – Simultaneous Pathway Activity Inference and Gene Expression Analysis Using RNA Sequencing

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. Researchers from the Broad Institute have devloped 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, the researchers captured dynamic pathway activity changes underpinning the global transcriptional changes of the innate immune response. They 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.

Overview of Transcription Factor Activity Sequencing

(A) Transcription factor activity sequencing (TF-seq)’s 58 lentiviral reporter vectors are distinguished by the DNA response element (RE) driving Luc2P transcription, the RE sequence tags (RE tag), and their associated unique molecular identifiers (UMIs). Emerald-GFP (EmGFP) is downstream of the reporter gene with an invariant SV40 promoter and used to monitor the rate of lentiviral transduction.

(B) The 58 unique REs were created to represent more than 40 of the most widely studied signaling pathways, with a total degenerate vector complexity of 191,724 (n = 3 index-tagged PCR amplicons of the pooled TF-seq vector library; data points represent mean ± SD).

(C) To perform TF-seq, an equimolar ratio of all 58 TF-seq vectors is first transfected into HEK293T cells. After 48 hr, the conditioned media containing a pool of lentiviral particles representing the TF-seq reporter library are collected and transduced into a cell type of interest. After 72 hr, the heterogeneous population of cells transduced with TF-seq is collected and replated in 96-well microtiter plates for stimulation and assay collection.

(D) TF-seq libraries are prepared with gene-specific reverse transcriptase priming of the Luc2P reporter gene. Additional 5′ sequence is included in the TF-seq reverse transcriptase primers to add well-tags and a second UMI to approximate single-molecule mRNA counting and pool samples from a 96-well microtiter plate for Illumina adaptor PCR and multiplexed sequencing.

(E) Multiplexing pathway activity inferences using TF-seq correlate with luciferase pathway activity inferences (r = 0.68; p < 4.2 × 10⁻⁷ based on Spearman correlation test; n = 4 wells for luciferase measurements and n = 4 for the TF-seq measurements).

• Researchers have developed TF-seq to multiplex pathway-specific reporter gene assays using RNA-seq
• TF-seq pathway activity inferences cannot be detected with gene expression data alone
• TF-seq and global mRNA expression profiling can be performed on the same cells
• Integrative analysis associates transcription factors with direct target genes

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.