Promoter-proximal pausing by RNA Pol II is a rate-determining step in gene transcription that is hypothesized to be a prominent point at which regulatory factors act. The pausing factor NELF is known to induce and stabilize pausing, but not all kinds of pausing are NELF-mediated. Here, we find that NELF-depleted Drosophila melanogaster cells functionally recapitulate the NELF-independent pausing we previously observed in fission yeast (which lack NELF). Critically, only NELF-mediated pausing establishes a strict requirement for Cdk9 kinase activity for the release of paused Pol II into productive elongation. Upon inhibition of Cdk9, cells with NELF efficiently shutdown gene transcription, while in NELF-depleted cells, defective, non-productive transcription continues unabated. By introducing a strict checkpoint for Cdk9, the evolution of NELF was likely critical to enable increased regulation of Cdk9 in higher eukaryotes, as Cdk9 availability can be restricted to limit gene transcription without inducing wasteful, non-productive transcription.

Summary I present here the R/Bioconductor package BRGenomics, which provides fast and flexible methods for post-alignment processing and analysis of high resolution genomics data within an interactive R environment. Utilizing GenomicRanges and other core Bioconductor packages, BRGenomics provides various methods for data importation and processing, read counting and aggregation, spike-in and batch normalization, re-sampling methods for robust “metagene” analyses, and various other functions for cleaning and modifying sequencing and annotation data. Simple yet flexible, the included methods are optimized for handling multiple datasets simultaneously, make extensive use of parallel processing, and support multiple strategies for efficiently storing and quantifying different kinds of data, including whole reads, quantitative single-base data, and run-length encoded coverage information. BRGenomics has been used to analyze ATAC-seq, ChIP-seq/ChIP-exo, PRO-seq/PRO-cap, and RNA-seq data; is built to be unobtrusive and maximally compatible with the Bioconductor ecosystem; is extensively tested; and includes complete documentation, examples, and tutorials. Availability and Implementation BRGenomics is an R package distributed through Bioconductor (https://bioconductor.org/packages/BRGenomics). Full documentation with examples and tutorials are available online (https://mdeber.github.io).

Transcription is a fundamental process for all known organisms. Transcription by RNA Polymerase II (Pol II), the enzyme responsible for mRNA synthesis, is highly regulated by protein factors at every step. In multicellular eukaryotes, Pol II pauses 20‐60 bases downstream of the transcription start site in nearly all genes. This promoter‐proximal pausing is established as an important step in transcription regulation and is dependent on several pausing and elongation factors. The protein complexes DSIF (DRB Sensitivity Inducing Factor) and NELF (Negative Elongation Factor) bind to Pol II early in transcription to induce pausing. Pause release occurs upon phosphorylation of Pol II, DSIF and NELF by the Cdk9, Cyclin T1 heterodimer P‐TEFb (Positive Transcription Elongation Factor b). Although Pol II pausing is a key step in transcription regulation and has been extensively studied, the molecular mechanisms of pause and pause release are incompletely understood. Furthermore, recent studies have implicated controlled termination early in the gene as a mechanism to control transcription. A challenge to studying these key regulatory steps is that they occur in a narrow window: from initiation to Pol II pausing and release occurs in 20‐60 base range. Current approaches to study transcription factor interactions are unable to provide high enough resolution or sensitivity to address precisely where key transitions in transcription occur. To address this problem, I am developing an assay to measure the exact position of Pol II, when it is bound by a factor of interest. The technique couples our lab’s Precision Run‐on sequencing technique (PRO‐seq), which measures the position of active Pol II with single base‐pair resolution, and an immunoprecipitation enrichment step. Initial PRO‐IP‐seq (Precision Run‐on Immunoprecipitation sequencing) data shows that we can enrich for populations of Pol II bound by specific transcription factors. Utilizing this tool, we will be able to measure genomically where both stable and more transient transcription factors interact with Pol II at near single‐base resolution.