Fig. 4. Single-particle tracking of H2B inside of H3K27ac-enriched chromatin regions.

(A) Top row, left: Sample single frame of H3K27ac Fab in living RPE1 cells. Middle: Sample single frame of single-molecule Halo-H2B in the same cell. Right: Colored composite of H3K27ac and H2B channels in green and orange, respectively. Bottom row, left: Outlines of H3K27ac local adaptive binarization mask overlaid upon the 100-frame average image from which the mask was extracted. Middle: Single local adaptive binarization mask of H3K27ac-enriched regions. Right: H3K27ac mask with overlaid Halo-H2B tracks in orange. (B) Selected zoom from (A) showing sample full traces of Halo-H2B tracks occurring both inside and outside RNAP2-Ser5ph–enriched regions. (C) Sample single-cell fit for the ensemble MSD of all H2B tracks lasting 10 frames or longer, localized in a randomized mask control (gray, n = 1892 total tracks across 10 randomizations) or in H3K27ac-enriched chromatin (green, n = 445 tracks). Determined diffusion coefficient (K_α) and alpha coefficient (α) for this single-cell fit are labeled, which contribute a single point to the violin plot in (D). Error bars are SEM. (D) Difference in K (ΔK) and α (Δα) for RNAP2-Ser5ph–enriched regions from a randomized mask control. Each data point represents the difference in fit coefficients for a single-cell fit (n = 30 cells), relative to the mean, with each cell comprising thousands of H2B tracks. Significance determined via Student’s t test.