Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites

Abstract

Chromatin remodeling complexes can translocate nucleosomes along the DNA in an ATP-dependent manner. Here, we studied autofluorescent protein constructs of the human ISWI family members Snf2H, Snf2L, the catalytically inactive Snf2L+13 splice variant, and the accessory Acf1 subunit in living human and mouse cells by fluorescence microscopy/spectroscopy. Except for Snf2L, which was not detected in the U2OS cell line, the endogenous ISWI proteins were abundant at nuclear concentrations between 0.14 and 0.83 μM. A protein interaction analysis showed the association of multimeric Snf2H and Acf1 into a heterotetramer or higher-order ACF complex. During the G1/2 cell cycle phase, Snf2H and Snf2L displayed average residence times <150 ms in the chromatin-bound state. The comparison of active and inactive Snf2H/Snf2L indicated that an immobilized fraction potentially involved in active chromatin remodeling comprised only 1-3%. This fraction was largely increased at replication foci in S phase or at DNA repair sites. To rationalize these findings we propose that ISWI remodelers operate via a "continuous sampling" mechanism: The propensity of nucleosomes to be translocated is continuously tested in transient binding reactions. Most of these encounters are unproductive and efficient remodeling requires an increased binding affinity to chromatin. Due to the relatively high intranuclear remodeler concentrations cellular response times for repositioning a given nucleosome were calculated to be in the range of tens of seconds to minutes.nucleosome translocation | fluorescence recovery after photobleaching | fluorescence correlation spectroscopy

Binding kinetics of human ISWI chromatin-remodelers to DNA repair sites elucidate their target location mechanism

“…Accordingly, the average residence time of ISWI remodelers increased to seconds and minutes at these sites. 1 This change in binding activity is in line with a 'release mechanism' proposed previously based on in vitro experiments: 2 Nucleosomes that are to be translocated by a given chromatin remodeling complex display a higher binding affinity than those at positions reached at the end points of the remodeling reaction.…”

“…The slow PCNA component is unlikely to represent chromatin dynamics since the diffusion coefficient was too large and the anomaly parameter was α ≈ 0.7 (for confined diffusion of chromatin fibers, an anomaly parameter larger than 1 is expected in the FCS fit of the data as observed for Snf2H with α = 2.1 ± 0.2, 1,21 ). We conclude that the equilibrium density of bound PCNA molecules along the chromatin fiber is insufficient to be detected in the correlation function.…”

“…Here, we used DNA damage sites induced via UV laser microirradiation as a prototypic system: By illuminating a small spot in the cell nucleus with high laser power at 405 nm for a few seconds, a large number of DNA lesions are produced, to which repair factors including chromatin remodelers are subsequently recruited. 1,8,17 Corresponding time series for GFP-tagged Snf2H, Snf2L, the inactive splice variant Snf2L+13 and Acf1 are shown in Figure 2A. As a marker for the DNA damage site PCNA-RFP was used.…”

“…In our recent work we found that the Snf2H-GFP construct behaved like endogenous Snf2H regarding intracellular localization, interaction with its Acf1 subunit and its in vitro nucleosome translocation activity. 1 To further test if the tagged protein is fully functional in living cells, a complementation approach was used here to rescue the S phase arrest reported previously for knockdown of endogenous Snf2H 16 ( Fig. 1).…”

“…By using a combination of advanced fluorescence microscopy and spectroscopy techniques we recently investigated the mobility and chromatin interactions of ISWI-type chromatin remodelers in living cells. 1 The study yielded short interaction times of Snf2H and Snf2L with their chromatin substrate with average residence times in the range of 10-150 ms. The concentrations of the endogenous proteins amounted to roughly 1 μM.…”