Figure 2: HP1α dynamically interacts with chromatin fibres.
From: Multivalency governs HP1α association dynamics with the silent chromatin state
(a) HP1α fused to NpuN is expressed, bound to NpuC-beads, cleaved in ligation buffer and reacted with P1 in situ, yielding labelled HP1α (HP1α(A532)). (I=input; FT=flowthrough; W1-3=washes; E1-3=elutions). (b) Left: single chromatin arrays are detected by Atto647N emission. Right: HP1α interaction dynamics are monitored by Atto532 emission. Scale bar, 5 μm. (c) Time trace of fluorescence intensity (blue) from a single chromatin array showing transient HP1α binding events, fitted by a step function (red). Inset: determination of bound (on-) and unbound (off-) times by a thresholding algorithm. (d) Dissociation kinetics: cumulative histogram of binding intervals (tbright) for 100 chromatin arrays (105 frames each), fitted by a double-exponential function (fit: τoff,1=0.25±0.03 s, τoff,2=2.26±1.22 s). (e) Association kinetics: cumulative histogram of intervals between binding events (tdark) over 30 chromatin arrays, fitted by a single-exponential function (fit: λon=22.9±9.8 s). (f) The HP1α residence time (τoff,1) depends on H3K9me3 density. Numbers indicate % amplitude of the fast phase (errors: s.e.m.; n =2–16 replicates). (g) The HP1α residence time (τoff,1) decreases as a function of HP1α concentration due to local competition. The indicated concentration of unlabelled HP1α is added to 1 nM Atto532-labelled HP1α. Numbers indicate % amplitude of the fast phase (errors: s.e.m., n=3–16 replicates). (d–g) For all fit results, see Table 1.
