SLAM-seq Identifies Direct Transcriptional Targets of BRD4 and MYC

BET bromodomain inhibitors (BETi) have demonstrated antitumor activity in preclinical models of leukemia and other tumor types. The therapeutic effects of BETi are thought to derive from inhibition of BRD4, displacing it from acetylated histones to repress its target genes, including MYC. BRD4 and MYC are central transcriptional regulators in cancer, but defining their direct functions in gene regulation has proven challenging. To investigate direct transcriptional targets of BRD4 and MYC and the effects of BETi, Muhar and colleagues used thiol(SH)-linked alkylation of the metabolic sequencing of RNA (SLAM-seq). This technique allows for quantification of newly synthesized mRNAs by labeling them with 4-thiouridine (4sU); subsequent alkylation of 4sU in a simple chemical reaction results in misincorporation of a G during reverse transcription and a T to C conversion in 3′-end mRNA sequencing. SLAM-seq was combined with the auxin-inducible degron system which allowed for rapid indole-3-acetic acid–induced degradation of endogenous BRD4 and MYC within 30 minutes. This approach revealed that BRD4 is a general coactivator of RNA polymerase II (Pol2)–dependent transcription, acting globally to enhance gene transcription. Whereas high-dose BETi treatment recapitulated the effects of BRD4 degradation, lower doses, which are therapeutically active in leukemia, triggered repression of a select set of BETi hypersensitive target genes including MYC. In contrast to the broad transcriptional activation induced by BRD4, MYC acted primarily as a transcriptional activator of a specific set of target genes, in contrast to the broad transcriptional activation induced by BRD4. MYC degradation predominantly downregulated genes involved in protein and nucleotide biosynthesis and rapidly suppressed these processes, providing an explanation for previously reported effects of MYC on global cellular RNA and protein levels. In addition to demonstrating that SLAM-seq can be utilized to identify direct transcriptional targets, these findings provide insight into the molecular mechanisms by which the BRD4–MYC axis contributes to tumorigenesis.

Muhar M, Ebert A, Neumann T, Umkehrer C, Jude J, Wieshofer C, et al. SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis. Science 2018 Apr 5 [Epub ahead of print].

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