Cell-type-specific RNA polymerase II activity maps in intact tissues provide a gateway to mammalian gene regulatory mechanisms in vivo.

Accessing ongoing RNA polymerase II (RNA Pol II) activity in specific cell types within intact tissue is critical to reveal regulatory mechanisms of development. We developed precision run-on in cell-type-specific in vivo system followed by sequencing (PReCIS-seq), a method combining Cre-inducible GFP tagging of endogenous RNA Pol II with transcriptional run-on and GFP immunoprecipitation, to map transcriptionally engaged RNA Pol II genome-wide in targeted cell types of mouse tissues. Applied to keratinocytes within intact skin, PReCIS-seq demonstrates that transcriptionally activated functions of biological transitions generally employ both RNA Pol II promoter-recruitment and promoter-proximal pause-release mechanisms. A global RNA Pol II regulatory polarization features extreme pausing levels at cellular safeguarding vs. lineage identity genes across development and homeostasis. This polarization is associated with distinct proximal-promoter structures, distinguishing high-paused genes with restricted RNA Pol II pause-release from low-paused genes undergoing rapid RNA Pol II firing into productive elongation. PReCIS-seq also identifies active enhancers based on divergent transcription. This approach enables high-resolution, cell-type-specific analysis of RNA Pol II dynamics in intact tissues across mammalian development, homeostasis, and disease.