Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

Methods
Movies with two dimensional single molecule data were analyzed by DiaTrack Version 3.05 (Vallotton and Olivier, 2013), which determines the precise position of single molecules by Gaussian intensity fitting and assembles particle trajectories over multiple frames. In Diatrack remove blur was set to 0.1, remove dim set at 70 and max jump set at 6 pixels, where each pixel was 107 nm. Datasets contain session files from Diatrack with single molecule localization, intensity of single molecules, frame numbers and single molecule tracking information from recorded videos.