Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: MV histone doublets are re-localized to viral factories (VFs) (A) Histone dimer pairs (H2B-H2A and H4-H3) within Eukarya were aligned against the doublet Marseilleviridae histones using HHpred’s multiple sequence alignment tool (ClustalΩ). Known α helices from the histone fold (HF) domain in Eukarya are dark-colored tubes (H2B, red; H2A, yellow; H4, green; H3, blue; and additional helices, gray). Predicted α helices in MV histones were generated using HHrped’s Quick 2D prediction web server (shown in lighter coloration) within the Marseilleviridae histone doublets. Known R-T pairs and DNA binding residues are shown in Eukarya histones along with their conservation within Marseilleviridae histones; additional predicted DNA-binding residues are shown (positions demonstrated by lollipops). (B) Light microscopy fluorescence images (scale bar, 10 μm) of A. castellanii cells transfected with GFP- A. castellanii -H2A, MV-H2B-H2A-GFP, and MV-H3-H4-GFP, non-infected and infected with MV at 4 h PI. While GFP- A. castellanii -H2A concentrates only in the nucleus (N) of the non-infected cells, MV-H2B-H2A-GFP and MV-H3-H4-GFP are scattered in the entire cell (including the nucleus). Upon virus infection, GFP- A. castellanii -H2A remains in the nucleus (yellow arrows), while MV-H2B-H2A-GFP and MV-H3-H4-GFP re-localize to the VF (cyan arrows). DAPI staining remains in the nucleus all along the infection, but the intense fluorescence in the late VF hides the staining of the nucleus at 4 h PI. See also Figure S1 .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Sequencing, Generated, Binding Assay, Light Microscopy, Fluorescence, Transfection, Infection, Virus, Staining

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae , the host Acanthamoeba castellanii , and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP- A. castellanii -H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to and .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Labeling, Virus, Infection, Sequencing, Generated, Residue, Binding Assay, Light Microscopy, Fluorescence, Transfection, Staining

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: Mass spectrometry proteomics of the purified MV virions

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Mass Spectrometry, Purification, Membrane

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: MV-histones form nucleosome-like particles (A) SDS-PAGE of purified Melbournevirus (MV) histone doublets. (B) Sucrose gradient sedimentation of MV NLPs with 147 or 207 bp DNA. The compositions of MV NLPs were analyzed by native- and SDS-PAGE. (C) MV-NLP and eNuc were reconstituted on Widom ‘601’ DNA and Melbournevirus native DNA, respectively. The MV-NLP and eNuc were heat treated at 37 and 55°C. (D) Native PAGE of reconstituted MV mini-NLP (mini H2B-H2A instead of H2B-H2A) with 147 bp DNA. (E) GraFix of MV-NLPs with 207 bp DNA (top two panels) and 147 bp DNA (bottom two panels. Native- and SDS- PAGE of the crosslinked MV-NLP fractions representing successful crosslinking, compared to the native MV-NLP input. (F) Representative AFM images: Samples were diluted in TCS buffer and applied to APTES coated mica, rinsed with water and imaged on a NanoWizard Sa with a TAP300-GD cantilever. Samples include 147 bp DNA only, eNuc 147 , MV-H4-H3 with 147 bp DNA, MV-NLP 147 , MV-NLP 207 and MV-NLP 207 GraFix. Related to Figure 3 .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: SDS Page, Purification, Sedimentation, Clear Native PAGE

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: S values (S (20,W) ), frictional ratios (f/f0), and calculated molecular weights (including confidence intervals) of histone-DNA complexes derived from SV-AUC

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Derivative Assay, Molecular Weight

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: Cryo-EM reveals that MV-histone doublets form nucleosome-like structures with asymmetrically extending DNA (A) Overview of MV-NLP 147 and electron density. The equivalent regions of MV H3, H4, H2A, and H2B are shown in blue, green, yellow, and red, respectively. (B) Overlay of MV-NLP 147 (blue) with eNuc (gray). Only 80 bp of DNA with associated histones are shown for clarity. Superhelix locations (SHLs) are numbered from 0 to 6 starting from the nucleosome dyad (φ). (C) Comparison of the DNA path of eNuc 147 (gray ribbon diagram), MV-NLP 147 (blue electron density), and MV-NLP 207 (green electron density). (D) Charged surface representation of the histones for MV-NLP 147 and eNuc 147 . Coordinates for eNuc 147 were taken from 3LZ0. See also A and S3B and .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: Comparison of MV-NLP and eNuc histone structures (A) Superposition of two MV-H4-H3 doublets (green and blue) with the eukaryotic (H3-H4) 2 tetramer in gray (left), and a close-up of MV-H4-H3 doublet connector (right). Interactions of the MV-H4 N-terminal tail with the nucleosome are also shown. (B) A comparison of the four-helix bundle structure formed by H4 and H2B. (C) Superposition of two MV-H2B-H2A doublets (in red and yellow) with eukaryotic H2A-H2B dimers in gray (left), and a close-up of the MV-H2B-H2A doublet connector and docking domain. See for detailed information. Additional possible configurations for both connectors are shown in Figure S4 .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Comparison

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: MV-nucleosome histone dimer arrangement and histone connectors (A) Description of ϕ, the pseudo-dihedral angle measuring the relative orientations of the H2B-H2A subunits, between MV-NLP and eNuc. Larger magnitude values correspond to a dimer arrangement that is angled further away from the dyad axis. Fictitious particle locations are shown as black spheres, and histones (H3 in blue, H4 in green, H2A in yellow, and H2B in red) and DNA (silver and gold ribbons) are semi-transparent to allow for better visibility of each particle’s location. (B) Overlay of de novo loop models with feasible conformations connecting H2B to H2A and (C) H4 to H3 in the MV-histone doublets. Loop configurations were generated using Modeler (v9.20) and then placed in the nucleosome context. After manually removing physically non-relevant (knots) conformations and clashes identified with a cutoff distance of 0.8 Å using CPPTRAJ of the Amber MD package (v18), the three best loops are shown in different colors (cross correlation range against the simulated map is 0.614-0.623). Related to Figure 5 .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Generated

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet:

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Virus, Recombinant, Transfection, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Regulation of ATAD2B bromodomain binding activity by the histone code

doi: 10.1101/2022.11.14.516501

Figure Lengend Snippet: (A) A surface representation of the ATAD2B BRD in complex with the H4K12ac ligand (residues 4-17) (PDB ID: 8EOQ). The H4K12ac ligand residues are shown as orange sticks, while the cartoon representation of BRD (cyan) highlights the ZA and BC loops in green and pink, respectively. (B) An isolated image of the simulated annealing composite omit map (gray) around the histone ligand H4K12ac (orange) contoured at 1σ. The composite omit map was calculated before building the histone ligand into the structure. All figures were made using the PyMOL Molecular Graphics system using version 2.3, Schrödinger, LLC. (C) Coordination of the H4K12ac ligand by the ATAD2B BRD. Residues lining the BRD pocket that are involved in ligand coordination are displayed in cyan, while the H4K12ac ligand residues are in orange. Hydrogen bond contacts are represented by a solid black line, and hydrophobic interactions are shown by dashed black lines. All contacts were determined using the PLIP program .

Article Snippet: CUT&RUN experiments were performed in the MCF7 cell line, using specific antibodies for the following proteins: ATAD2B (Invitrogen, Catalog # PA5-83233, Lot:), histone H4K5ac (Invitrogen, Ref # MA-32009; Lot:), histone H4K8ac (Epicypher Ab -Ref. 13-0036; Lot:), and histone H4K12ac (EpiCypher, SKU:13-0037, Lot:).

Techniques: Isolation

Journal: bioRxiv

Article Title: Regulation of ATAD2B bromodomain binding activity by the histone code

doi: 10.1101/2022.11.14.516501

Figure Lengend Snippet: (A) Immunoblot showing the interaction of GST-tagged ATAD2B BRD (41 kDa) with endogenous histone H4 (15 kDa) from MCF7 cells. Recombinant GST alone was used as a control. Specific antibodies were used for the GST tag, and the H4K5ac and H4K12ac modifications. (B) Euler plot showing strong replicate concordance for ATAD2B CUT&RUN samples. A total of 30,636 high-confidence peaks were identified across two biological replicates for ATAD2B. (C) Correlation heatmap based on comparing ATAD2B and our H4Kac PTM CUT&RUN data against all public ChIP-seq data for histone modifications in the breast cancer cell line MCF7. Clustering is derived from the Euclidian distance of signalValue for each factor and shows a strong correlation between ATAD2B and the H4Kac PTMs. (D) Signal heat map of CUT&RUN signals for all factors at the ATAD2B high-confidence peaks, showing strong signal correlation for histone H4Kac PTMs with the signal at ATAD2B peaks. (E) UCSC genome browser snapshots of CUT&RUN signal (reads per kilo-million) on multiple breast cancer-related genes (MYC, E2F1, BRCA1).

Article Snippet: CUT&RUN experiments were performed in the MCF7 cell line, using specific antibodies for the following proteins: ATAD2B (Invitrogen, Catalog # PA5-83233, Lot:), histone H4K5ac (Invitrogen, Ref # MA-32009; Lot:), histone H4K8ac (Epicypher Ab -Ref. 13-0036; Lot:), and histone H4K12ac (EpiCypher, SKU:13-0037, Lot:).

Techniques: Western Blot, Recombinant, Control, ChIP-sequencing, Derivative Assay