Classic functional genomics assays, whereby changes in gene expression are monitored after a factor of interest is disrupted, cannot distinguish between direct and indirect effects. Muhar et al. now present a strategy that combines rapid chemical-genetic perturbation with measurements of immediate gene expression changes to preclude indirect effects.

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a strategy that combines rapid chemical-genetic perturbation with measurements of immediate gene expression changes

The authors set out to identify targets of transcriptional regulators such as bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal motif (BET) protein family. BRD4 is reversibly bound by BET inhibitors (BETi), which prevents its interaction with acetylated histones and leads to repression of its target genes.

BRD4 expression was perturbed in K562 cancer cells using the auxin-inducible degron (AID) system, whereby the degradation of AID-tagged target proteins can be rapidly induced. The authors then applied an optimized version of thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), a previously established genome-wide method for measuring nascent mRNAs. They observed overall transcriptional downregulation and accumulation of RNA polymerase II (Pol II) at transcription start sites, confirming that BRD4 exerts transcriptional control by releasing paused Pol II.

The effects of high-dose BETi treatment mirrored those observed after BRD4 degradation, but applying lower therapeutically active BETi doses revealed a selective transcriptional response that affected only a small set of hypersensitive genes. Applying their strategy to the hypersensitive target MYC, the team introduced an AID-tag into the endogenous MYC locus of two independent cancer cell lines and used SLAM-seq to quantify changes in mRNA output following MYC degradation. In contrast to BRD4, the transcriptional responses observed after MYC degradation in both cell lines were highly specific rather than global, suggesting that MYC activates the transcription of specific target genes rather than functioning as a general transcriptional activator or repressor.

Taken together, mapping direct transcriptional responses to the degradation of AID-tagged proteins provides a scalable method to explore the gene regulatory landscape.