Singh RK et al. (2019), Transient Kinetic Analysis of SWR1C-Catalyzed H...

XB-ART-57114

Cell Rep 2019 Apr 09;272:374-386.e4. doi: 10.1016/j.celrep.2019.03.035.

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Transient Kinetic Analysis of SWR1C-Catalyzed H2A.Z Deposition Unravels the Impact of Nucleosome Dynamics and the Asymmetry of Histone Exchange.

Singh RK , Fan J , Gioacchini N , Watanabe S , Bilsel O , Peterson CL .

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The SWR1C chromatin remodeling enzyme catalyzes ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes such as transcription and DNA repair. Here, we investigate the transient kinetic mechanism of the histone exchange reaction, employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady-state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATP-dependent replacement of nucleosomal dimers, promoted by localized DNA unwrapping. Taken together, our transient kinetic studies identify intermediates and provide crucial insights into the SWR1C-catalyzed dimer exchange reaction and shed light on how the mechanics of H2A.Z deposition might contribute to transcriptional regulation in vivo.

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Species referenced: Xenopus laevis
GO keywords: nucleosome [+]

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	Figure 2. SWR1C Modulates the Conformational Fluctuations of the Nucleosome(A–F) The ratios of donor-acceptor cross-correlation to acceptor auto-correlation are plotted as a function of time under various experimental conditions. The experimental data were analyzed using either a single- or double-exponential rate equation, yielding the values of the kobs (t1/2 = 0.693/kobs) for the conformational fluctuation of the nucleosome.(A) Dynamics of an H2A-nucleosome.(B) The dynamics of the SWR1C-H2A nucleosome complex are 2 orders of magnitude faster than the free nucleosome.(C) Addition of AMP-PNP (a non-hydrolyzable analog of ATP) to the SWR1C-nucleosome complex induces additional nucleosome dynamics on the microsecond timescale.(D) Dynamics of the H2A.Z nucleosome.(E) The dynamics of the SWR1C-H2A.Z nucleosome complex are 2 orders of magnitude faster than the free nucleosome.(F) Addition of AMP-PNP to the SWR1C-H2A.Z nucleosome does not alter nucleosome dynamics.FCS curves were obtained after averaging at least 20–25 autocorrelation/cross-correlation curves.
	Figure 3. Transient Kinetics of ATP-Dependent Eviction of Two H2A-H2B Dimers from an H2A Nucleosome Are Asymmetric(A) Experimental strategy for monitoring the rate of eviction of nucleosomal H2A-H2B. The nucleosomal substrate contains a Cy3-labeled DNA end, and Cy5 is located on the H2A C terminus. Cy5 FRET signals were monitored over time in reactions that contained free H2A.Z-H2B dimers.(B) Representative kinetic trace for SWR1C-catalyzed eviction of H2A-H2B dimers from an H2A nucleosome. The experimental data were analyzed using adouble-exponential rate equation, yielding the kobs for the fast and slow phases as 0.33 ± 0.02 min−1 (half-life = 2.1 min) and 0.06 ± 0.01 min−1(half-life = 12.3 min), respectively.(C) The kinetic trace for SWR1C-catalyzed eviction of H2A-H2B dimers from an H2A nucleosome containing a 2-nt gap at both SHL+2.0 and SHL−2.0.(D) The kinetic trace for SWR1C-catalyzed eviction of the H2A-H2B dimer from an H2A nucleosome harboring a 2-nt gap at the linker-distal SHL+2.0. The monophasic trace was analyzed using a single-exponential rate equation, yielding the kobs as 0.06 ± 0.01 min−1(half-life = 12 min).(E) The kinetic trace for SWR1C-catalyzed eviction of the H2A-H2B dimer from an H2A nucleosome harboring a 2-nt gap at the linker-proximal SHL−2.0. The kinetic trace is monophasic; hence, it was analyzed using a single-exponential rate equation, yielding the observed rate as 0.12 ± 0.03min−1 (half-life = 6 min). At least 3–4 kinetic traces were collected for each experimental condition, and they were averaged. The resultant kinetic traces were analyzed using an exponential rate equation, and the error in the measurement represents the standard error of the parameter derived from non-linear regression analysis using the Origin software package (OriginLab).
	Figure 4. SWR1C Catalyzes ATP-Dependent Unwrapping of Nucleosomal DNA during Dimer Exchange(A) The emission spectra under Cy3 excitation at 530 nm of 77N0-Cy3 H2A-Cy5 nucleosomes incubated with ATP (black), SWR1C (red), SWR1C and ATP (green), or SWR1C and AMP-PNP (blue).(B) Normalized Cy5 FRET trace of 77N3-Cy3 H3-Cy5 nucleosomes incubated under saturating nucleotide concentrations with ATP (black); SWR1C and ATP (red); SWR1C and AMP-PNP (blue); or SWR1C, H2A.Z-H2B dimers, and ATP (green).(C) Normalized Cy5 FRET trace of 77N3-Cy3 H3-Cy5 nucleosomes bound to SWR1C under low nucleotide concentrations with H2A.Z-H2B dimers and ATP (black), dimers and AMP-PNP (red), or no dimers and ATP (green).(D) Normalized Cy5 signal under direct excitation at 650 nm showing no ATP-dependent change in the Cy5 environment for 77N3-Cy3 H3-Cy5 nucleosomes during SWR1C dimer exchange with low ATP concentration (black) compared with AMP-PNP (red).The emission spectra in (A) were taken after 35 min of incubation, except for the reaction with the nucleosome and SWR1C, which was adjusted for photobleaching using the spectra from the nucleosome and ATP reaction pre- and post-incubation. Spectra were collected in triplicates. FRET reaction time course traces were collected in at least duplicates, averaged, and fit to a linear regression or single-exponential decay model. The y-intercept of each fit was normalized to 1.
	Figure 5. Transient Kinetics of ATP-Dependent Deposition of Two H2A.Z-H2B Dimers Is Asymmetric(A) Experimental strategy for monitoring the rate of deposition of H2A.Z-H2B. The nucleosomal substrate contains only the Cy3-labeled DNA end, and Cy5 islocated on the free H2A.Z-H2B dimer (H2A.Z-C125-Cy5).(B) Kinetic trace for the SWR1C-catalyzed deposition of the H2A.Z-H2B dimer to the intact H2A nucleosome. The biphasic trace was analyzed using a double-exponential rate equation, yielding the kobs for the fast and slow phases as 0.31 ± 0.01 min−1 (half-life = 2.2 min) and 0.04 ± 0.01 min−1(half-life = 16.6 min), respectively.(C) Reactions as in (B), but the nucleosome contained 2-nt gaps at both SHL+2.0 and SHL−2.0.(D) Same as in (B), but the reactions contained a nucleosome with a 2-nt gap at the linker-distal SHL+2.0. The monophasic trace was analyzed using a single-exponential rate equation, yielding the kobs as 0.04 ± 0.01min−1(half-life = 16 min).(E) Reactions as in (B), but the nucleosome harbors a 2-nt gap at the linker-proximal SHL−2.0. The monophasic trace was analyzed using a single-exponential rate equation, yielding the observed rate as 0.14 ± 0.02 min−1(half-life = 5 min).At least 3–4 kinetic traces were collected for each experimental condition, and they were averaged. The resultant kinetic traces were analyzed using an exponential rate equation, and the error in the measurement represents the standard error of the parameter derived from non-linear regression analysis using the Origin software package (OriginLab).
	Figure 6. Nucleosome Determinants of Asymmetric Dimer Exchange(A) Normalized Cy5 FRET signal comparing theSWR1C-dependent kinetics of H2A-Cy5 eviction from nucleosomes with the linker on the TA-poor (black) or TA-rich side (red) of the 601 nucleosome position sequence.(B) Normalized Cy5 FRET signal showing biphasic kinetics of H2A-H2B dimer eviction from center-positioned 55N78 H3-Cy3 H2A-Cy5 nucleosomes by SWR1C and the H2A.Z-H2B dimer upon addition of ATP (black) compared with the negative controls of AMP-PNP (red) or nucleosome alone plus ATP (green). The half-lives of the fast and slow phase are 0.6 min and 9.4 min, respectively, slightly faster than the rates of dimer eviction on the asymmetric 55N0 nucleosome.Traces were collected in triplicates, averaged, and fit to a double-exponential decay model. The y-intercept of each fit was normalized to 1.
	Figure 7. Kinetic Model of the SWR1C-Catalyzed Histone Dimer Exchange Reaction(1) The engagement of SWR1C to the H2A-nucleosome enhances the unwrapping and/or rewrapping kinetics of the nucleosomal DNA on the millisecond timescale. (2) Binding of ATP to the SWR1C-engaged nucleosome further affects its dynamics on the microsecond timescale. (3) SWR1C and free H2A.Z-H2B dimers catalyze translocation of nucleosomal DNA, leading to unwrapping of DNA from the linker-distal nucleosome edge. We propose that this is the power stroke of the reaction. (4) Unwrapping of nucleosomal DNA leads to eviction and replacement of the distal H2A-H2B dimer. (5) SWR1C remains engaged with the H2A-H2A.Z heterotypic nucleosome and catalyzes the slower replacement of the linker-proximal H2A-H2B dimer, utilizing the H2A.Z-H2B-mediated second round of the power stroke.

References [+] :

Adkins, Nucleosome dynamics regulates DNA processing. 2013, Pubmed