Summary Various factors differentially recognize trimethylated histone H3 lysine 4 (H3K4me3) near promoters, H3K4me2 just downstream, and promoter-distal H3K4me1 to modulate gene expression. This methylation “gradient” is thought to result from preferential binding of the H3K4 methyltransferase Set1/COMPASS to promoter-proximal RNA polymerase II. However, other studies have suggested that location-specific cues allosterically activate Set1. ChIP-Seq experiments show that H3K4 methylation patterns on active genes are not universal or fixed, and change in response to both transcription elongation rate and frequency, as well as reduced COMPASS activity. Fusing Set1 to RNA polymerase II results in H3K4me2 throughout transcribed regions, and similarly extended H3K4me3 on highly transcribed genes. Tethered Set1 still requires histone H2B ubiquitylation for activity. These results show higher-level methylations reflect not only Set1/COMPASS recruitment, but also multiple rounds of transcription. This model provides a simple explanation for non-canonical methylation patterns at some loci or in certain COMPASS mutants.

Summary Dynamic interactions between RNA polymerase II and various mRNA processing and chromatin modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code".

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.

Peptidyl-prolyl cis / trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity, and cell permeability to give BJP-06–005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.

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