The NELF pausing checkpoint mediates the functional divergence of Cdk9.

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.

A functional proteomics platform to reveal the sequence determinants of lysine methyltransferase substrate selectivity.

Lysine methylation is a key regulator of histone protein function. Beyond histones, few connections have been made to the enzymes responsible for the deposition of these posttranslational modifications. Here, we debut a high-throughput functional proteomics platform that maps the sequence determinants of lysine methyltransferase (KMT) substrate selectivity without a priori knowledge of a substrate or target proteome. We demonstrate the predictive power of this approach for identifying KMT substrates, generating scaffolds for inhibitor design, and predicting the impact of missense mutations on lysine methylation signaling. By comparing KMT selectivity profiles to available lysine methylome datasets, we reveal a disconnect between preferred KMT substrates and the ability to detect these motifs using standard mass spectrometry pipelines. Collectively, our studies validate the use of this platform for guiding the study of lysine methylation signaling and suggest that substantial gaps exist in proteome-wide curation of lysine methylomes.