EnChAMP-seq genomic localization and footprinting of RNA Pol II and NELF-A

RNA Polymerase II (Pol II) transcription is highly regulated at two early steps in the transcription cycle: 1) Pre-Initiation Complex (PIC) assembly with its coupled initiation, and 2) promoter-proximal pausing with its controlled release. Here, we developed an optimized biochemical purification method that captures endogenously tagged chromatin-bound Pol II complexes under native conditions at these rate-limiting steps. We then identified a large set of Pol II interactors by mass spectrometry and determined the footprints of these assemblies on promoters with high resolution. Many well-known and new or understudied factors were identified as associated with the PIC and promoter proximal paused complexes, indicating that despite decades of efforts, these rate-limiting steps of the transcription cycle are far from being completely understood. The new and understudied factors implicate novel mechanisms of regulation that will need to be characterized to fully understand Pol II regulation. Overall design: EnChAMP-seq analysis of endogenously tagged GFP (control), GFP-RPB1 and NELFA-GFP HCT116 derived cell lines with replicates under no drug, +500nM NVP-2 for 1h, or +1uM Triptolide for 20min conditions.