PRO-IP-seq Tracks Molecular Modifications of Engaged Pol II Complexes at Nucleotide Resolution

Abstract

RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we developed Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq) and tracked phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncovered precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide, through early and late promoter-proximal pause, and into productive elongation. Pol II CTD was predominantly unphosphorylated in the early pause-region, whereas serine-2- and serine-5-phosphorylations occurred preferentially in the later pause-region. Serine-7-phosphorylation dominated after the pause-release in a region where Pol II accelerates to its full elongational speed. Interestingly, tracking transcription upon heat-induced reprogramming demonstrated that Pol II with phosphorylated CTD remains paused on heat-repressed genes.

Characterization of gene regulatory elements and dynamic antimicrobial immune responses in mosquito cells using PRO-seq

“…When assessing RNA polymerase positioning across individual genes, we found a predominant accumulation at promotor-proximal regions from where polymerase is primarily released into the gene body (30). The two main peaks around +30 and +50 nt suggest an early and late pausing site, as also reported for Drosophila melanogaster and Homo sapiens (31, 45, 48). Noteworthy, only little transcription is seen at divergent regions, indicating that most Ae.…”

“…In contrast, the PRO-seq reads located at the start of a gene often converge in a single dominant peak allowing a highly accurate estimate of the exact TSN location. An overall deviation between annotated TSSs and the exact TSN, with a downstream shift of the TSN, has also been reported for Drosophila melanogaster and Homo sapiens (31,32,45).…”

“…2B, C). More specifically, two main peaks around +30 nt and +50 nt are seen, which likely reflect the early and late pause site (31). Interestingly, however, only little divergent transcription upstream of the TSS is seen (Fig.…”