Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.

Summary Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukemia (AML)1,2, BET inhibitors are being explored as promising therapeutic avenue in numerous cancers3–5. While clinical trials have reported single-agent activity in advanced hematologic malignancies6, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukemia, we performed a chromatin-focused RNAi screen in a sensitive MLL/AF9; NrasG12D -driven AML model, and investigated dynamic transcriptional profiles in sensitive and resistant murine and human leukemias. Our screen reveals that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodeling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukemias regardless of their sensitivity, resistant leukemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signaling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic ChIP- and STARR-seq enhancer profiles reveal that BET-resistant states are characterized by remodeled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signaling as a driver and candidate biomarker of primary and acquired BET resistance in leukemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.