A dual role for H2A.Z.1 in modulating the dynamics of RNA Polymerase II initiation and elongation

RNAPII pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step at most metazoan genes that allows fine-tuning of gene expression in response to diverse signals 1–5 . During pause-release, RNA Polymerase II (RNAPII) encounters an H2A.Z.1 nucleosome 6–8 , yet how this variant contributes to transcription is poorly understood. Here, we use high resolution genomic approaches 2,9 (NET-seq and ChIP-nexus) along with live cell super-resolution microscopy (tcPALM) 10 to investigate the role of H2A.Z.1 on RNAPII dynamics in embryonic stem cells (ESCs). Using a rapid, inducible protein degron system 11 combined with transcriptional initiation and elongation inhibitors, our quantitative analysis shows that H2A.Z.1 slows the release of RNAPII, impacting both RNAPII and NELF dynamics at a single molecule level. We also find that H2A.Z.1 loss has a dramatic impact on nascent transcription at stably paused, signal-dependent genes. Furthermore, we demonstrate that H2A.Z.1 inhibits re-assembly and re-initiation of the PIC to reinforce the paused state and acts as a strong additional pause signal at stably paused genes. Together, our study suggests that H2A.Z.1 fine-tunes gene expression by regulating RNAPII kinetics in mammalian cells.