Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Singh RK

PROVIDER: S-EPMC6545893 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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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 Raushan K RK Fan Jiayl J Gioacchini Nathan N Watanabe Shinya S Bilsel Osman O Peterson Craig L CL

Cell reports 20190401 2

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 ...[more]

PMID: 30970243

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Project description:The incorporation of histone variant H2A.Z into nucleosomes creates specialized chromatin domains that regulate DNA-templated processes, such as gene transcription. In Saccharomyces cerevisiae, the diverging H2A.Z C terminus is thought to provide the H2A.Z exclusive functions. To elucidate the roles of this H2A.Z C terminus genome-wide, we used derivatives in which the C terminus was replaced with the corresponding region of H2A (ZA protein), or the H2A region plus a transcriptional activating peptide (ZA-rII'), with the intent of regenerating the H2A.Z-dependent regulation globally. The distribution of these H2A.Z derivatives indicates that the H2A.Z C-terminal region is crucial for both maintaining the occupation level of H2A.Z and the proper positioning of targeted nucleosomes. Interestingly, the specific contribution on incorporation efficiency versus nucleosome positioning varies enormously depending on the locus analyzed. Specifically, the role of H2A.Z in global transcription regulation relies on its C-terminal region. Remarkably, however, this mostly involves genes without a H2A.Z nucleosome in the promoter. Lastly, we demonstrate that the main chaperone complex which deposits H2A.Z to gene regulatory region (SWR1-C) is necessary to localize all H2A.Z derivatives at their specific loci, indicating that the differential association of these derivatives is not due to impaired interaction with SWR1-C. IMPORTANCE We provide evidence that the Saccharomyces cerevisiae C-terminal region of histone variant H2A.Z can mediate its special function in performing gene regulation by interacting with effector proteins and chaperones. These functional interactions allow H2A.Z not only to incorporate to very specific gene regulatory regions, but also to facilitate the gene expression process. To achieve this, we used a chimeric protein which lacks the native H2A.Z C-terminal region but contains an acidic activating region, a module that is known to interact with components of chromatin-remodeling entities and/or transcription modulators. We reasoned that because this activating region can fulfill the role of the H2A.Z C-terminal region, at least in part, the role of the latter would be to interact with these activating region targets.

Dataset Information

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

Publications

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

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