Abstract Post-translational protein modifications play an important role in the regulation of gene dynamics. Certain modifications, such as histone acetylation and RNA polymerase II phosphorylation, are associated with transcriptionally active chromatin. However, the spatial and temporal relationship between chromatin and post-translational protein modifications, and how these dynamics facilitate selective gene expression, remain poorly understood. In this study, we address this problem by developing a general methodology for quantifying in live cells the dynamics of chromatin across multiple time and length scales in the context of residue-specific protein modifications. By combining Fab-based labeling of endogenous protein modifications with single-molecule imaging, we track the dynamics of chromatin enriched with histone H3 Lysine-27 acetylation (H3K27ac) and RNA polymerase II Serine-5 phosphorylation (RNAP2-Ser5ph). Our analysis reveals chromatin enriched with H3K27ac is separated from chromatin enriched with RNAP2-Ser5ph. Furthermore, in these separated sites, we show the presence of the two modifications are inversely correlated with one another on the minutes timescale. We then track single nucleosomes in both types of sites on the sub-second timescale and again find evidence for distinct and opposing changes in their diffusive behavior. While nucleosomes diffuse ∼15% faster in chromatin enriched with H3K27ac, they diffuse ∼15% slower in chromatin enriched with RNAP2-Ser5ph. Taken together, these results argue that high levels of H3K27ac and RNAP2-Ser5ph are not often present together at the same place and time, but rather each modification marks distinct sites that are transcriptionally poised or active, respectively.

Live-cell imaging uncovers the relationship between histone acetylation, transcription initiation, and nucleosome mobility