H2A.Z deposition by SWR1C involves multiple ATP-dependent steps

Fan, Jiayi; Moreno, Andrew; Baier, Alexander; Loparo, Joseph J.; Peterson, Craig L.

doi:10.1038/s41467-022-34861-x

Cited by 18 publications

(18 citation statements)

References 52 publications

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“…To further investigate the role of the nucleosomal acidic patch for SWR1C-mediated dimer exchange, we explored how disruption of each acidic patch impacted the nucleosome binding affinity of SWR1C for nucleosomes, leveraging a fluorescence polarization assay. Using a Cy3labeled 77N0 nucleosome, we determined a Kd of 13.6 nM for an AB/AB nucleosome (Figure 2A), similar to prior studies 61 . However, disruption of even a single nucleosomal acidic patch severely weakened SWR1C binding, such that a Kd was unable to be determined (Figure 2B-D).…”

Section: The Nucleosomal Acidic Patch Is a Key Driver Of Swr1c Bindin...supporting

confidence: 75%

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

Peterson

Baier

Gioacchini

et al. 2023

Preprint

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The SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Numerous studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a “pincer-like” conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitinylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

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Section: The Nucleosomal Acidic Patch Is a Key Driver Of Swr1c Bindin...supporting

confidence: 75%

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

Peterson

Baier

Gioacchini

et al. 2023

Preprint

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“…Reactions to measure dimer exchange by SWR1C were performed under single turnover conditions (excess enzyme to nucleosomal substrate) and contained free H2A.Z/H2B (ZB) dimers which act as an essential co-substrate. Addition of SWR1C to a wildtype nucleosome led to a rapid drop in FRET, showing the biphasic kinetics consistent with the sequential exchange of the two nucleosomal H2A/H2B (AB) dimers 44,45 . Incubation of SWR1C with a nucleosome that lacks an intact acidic patch (AB-apm/AB-apm nucleosome) showed little decrease in the FRET signal, indicating that an intact nucleosomal acidic patch is essential for SWR1C to catalyze H2A/H2B eviction (Figure 1B).…”

Section: Dimer Exchange By Swr1c Requires the Nucleosomal Acidic Patchmentioning

confidence: 64%

“…Previously, we and others reported that SWR1C preferentially exchanges the linker distal H2A/H2B dimer, as monitored by either ensemble or single molecule FRET analyses 44,47,48 . The asymmetric nucleosome substrates, harboring only a single, labeled H2A/H2B dimer were leveraged to further investigate this preferential exchange.…”

Section: Dimer Exchange By Swr1c Requires the Nucleosomal Acidic Patchmentioning

confidence: 94%

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

Gioacchini

Peterson

2021

Preprint

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The SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Numerous studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated an essential role for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the nucleosomal acidic patch in the H2A.Z exchange reaction. Nucleosomes lacking acidic patch residues retain the ability to stimulate the ATPase activity of SWR1C, implicating a role in coupling the energy of ATP hydrolysis to H2A/H2B dimer eviction. A conserved arginine-rich region within the Swc5 subunit is identified that interacts with the acidic patch and is found to be essential for dimer exchange activity. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

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“…Single-molecule Stoichiometry Image Analysis. Channel alignment of the 532 nm and 641 nm channels was done as previously described (Fan et al, 2022). Briefly, a custom MATLAB script was used to identify fluorescent molecules in the 532 and 641 emission channels based on local maximum and particles were fit using a 2D Gaussian.…”

Section: Dna Pulldown Assaymentioning

confidence: 99%

Identification of the main barriers to Ku accumulation in chromatin

Bossaert,

Moreno,

Peixoto

et al. 2024

Preprint

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Repair of DNA double strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Given its high affinity for ends, multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ~1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unknown. Here we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process which actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism which operates in S-phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together our data shed light on the multiple layers of coordinated mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.

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H2A.Z deposition by SWR1C involves multiple ATP-dependent steps

Cited by 18 publications

References 52 publications

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

Identification of the main barriers to Ku accumulation in chromatin

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