PRO-IP-seq Tracks Molecular Modifications of Engaged Pol II Complexesat 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/527107v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1cd08c7org.highwire.dtl.DTLVardef@1fb7507org.highwire.dtl.DTLVardef@1c05c1borg.highwire.dtl.DTLVardef@1deffdf_HPS_FORMAT_FIGEXP M_FIG C_FIG