Journal: PLoS ONE

Article Title: Nucleosome assembly and disassembly pathways in vitro

doi: 10.1371/journal.pone.0267382

Figure Lengend Snippet: Changes in the probability of YpY dimer formation are presented in terms of absolute values of log2 of the intensity ratios (|log2(IR)|) along the 601 sequence expressed in SHLs; they are given for decreasing (top panel) or increasing (bottom panel) ionic strengths, as indicated by the green and black arrow respectively. The IR quantities are the ratios calculated between the normalised peak heights associated with each YpY step in the histone plus DNA mixtures and those of DNA alone. Red and blue bars correspond to DNA residues involved in the interface with H3/H4 and H2A/H2B dimers, respectively; black bars correspond to dinucleotides contacted by the two dimers. Minor-groove inward facing regions observed in the nucleosome structures are represented by grey boxes. Error bars are first-order estimations of standard errors calculated on at least 3 independent experiments (see Material and Methods for details).

Article Snippet: Human recombinant histone H2A/H2B dimer (5.5 μg, 203 pmol) and histone (H3/H4) 2 tetramer (5.5 μg, 102 pmol) (New England BioLabs) were mixed with linearised 601 DNA fragments (10 μg, 23 pmol) in 30 μl of 2M salt buffer (18 mM Tris-HCl, 2 M NaCl, 0.9 mM DTT, 0.9 mM EDTA).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Nucleosome assembly and disassembly pathways in vitro

doi: 10.1371/journal.pone.0267382

Figure Lengend Snippet: This schematic representation shows those DNA regions that interact with the different histone dimers in the fully formed NCP. To distinguish the two copies of the H2A/H2B dimer, they are labelled 5’ (light blue, chains G, H) and 3’ (dark blue, chains C, D). Similarly, the two pairs of H3/H4 histones are labelled 5’ (pink, chains E, F) and 3’ (red, chains A, B). The DNA sequence is labelled as SHL (Super Helical Location, defined in Material and Methods). The denominations A, B, … of the histone chains are those commonly used in X-ray structures.

Article Snippet: Human recombinant histone H2A/H2B dimer (5.5 μg, 203 pmol) and histone (H3/H4) 2 tetramer (5.5 μg, 102 pmol) (New England BioLabs) were mixed with linearised 601 DNA fragments (10 μg, 23 pmol) in 30 μl of 2M salt buffer (18 mM Tris-HCl, 2 M NaCl, 0.9 mM DTT, 0.9 mM EDTA).

Techniques: Sequencing

Journal: bioRxiv

Article Title: GCNA is a histone binding protein required for spermatogonial stem cell maintenance

doi: 10.1101/2022.02.21.481287

Figure Lengend Snippet: I. Western blot of cell fractionations performed on BAC9 mESC. Chromatin fraction was washed with increasing concentrations of NaCl to release electrostatic interactions with the chromatin. The blot was probed with anti-GCNA (GCNA-1), anti-H3 and anti-tubulin antibodies. Data is representative from three independent experiments. B. Schematic representation of constructs used in (C), (D), (E) and (F). C. Western blot of the FLAG immunoprecipitation. 3T3 cells were transiently transfected with FLAG-mGCNA and a FLAG immunoprecipitation was performed on the soluble fraction. The blot was probed with anti-FLAG, anti-H3 and anti-beta-actin antibodies. Data is representative from three independent experiments. D. Western blot of the FLAG immunoprecipitation. 3T3 cells were transiently transfected with FLAG-mGCNA and a FLAG immunoprecipitation was performed on the soluble fraction. The blot was probed with anti-FLAG, anti-H2B and anti-beta-actin antibodies. Data is representative from three independent experiments. E. Western blot of the GFP immunoprecipitation. 293-T cells were transiently transfected with GFP-fused proteins and a GFP immunoprecipitation was performed on the soluble fraction. The blot was probed with anti-GFP, anti-H3 and anti-beta-actin antibodies. Data is representative from three independent experiments. F. Western blot of the GFP immunoprecipitation. 293-T cells were transiently transfected with GFP-fused proteins and a GFP immunoprecipitation was performed on the soluble fraction. The blot was probed with anti-GFP, anti-H2B and anti-beta-actin antibodies. Data is representative from three independent experiments. G. Proximity ligation assay performed with anti-H3 and anti-GCNA (Tra98) antibodies on wild-type and Gcna − /Y adult testis sections. Data is representative from two independent experiments.

Article Snippet: In a 100 µl reaction, 1.4 µg of H3-H4 (catalog no. M2509S, New England Biolabs) or H2A-H2B (catalog no. M2508S, New England Biolabs) were incubated with 2.5 µg of MBP or MBP-mGCNA in Binding buffer (25 mM Tris pH 7.5, 150 mM NaCl, 5 % glycerol).

Techniques: Western Blot, Construct, Immunoprecipitation, Transfection, Proximity Ligation Assay

Journal: bioRxiv

Article Title: GCNA is a histone binding protein required for spermatogonial stem cell maintenance

doi: 10.1101/2022.02.21.481287

Figure Lengend Snippet: A. Representative limited proteolysis assay performed with H3-H4, MBP and MBP-mGCNA. Proteins were incubated with trypsin for 0, 15, 45 and 90 min then analysed by SDS-PAGE followed by silver staining. Data is representative from three independent experiments. B. Representative limited proteolysis assay performed with H2A-H2B, MBP and MBP-mGCNA. Proteins were incubated with trypsin for 0, 2, 4 and 6 hours then analysed by SDS-PAGE followed by silver staining. Data is representative from two independent experiments. C. Schematic representation of the in vitro nucleosome assembly activity assay. A supercoiled plasmid is relaxed by TopoI then DNA was incubated with core histones and the histone chaperone. While nucleosomes are formed, supecoils are also formed into the plasmid and supercoiling is analysed by gel electrophoresis after deproteinisation of the plasmid. D. Representative in vitro nucleosome assembly activity assay with MBP and MBP-mGCNA. In this assay, MBP was used at a concentration of 0.66µM and MBP-mGCNA was used at 0.165, 0.33 or 0.66µM. Data is representative from three independent experiments. E. Quantification of the signal of the supercoiled plasmid in presence of histones and increasing concentrations of MBP-mGCNA (lanes 5 to 7 in (D)), relative to the signal with histones and MBP (lane 4 in (D)). Data represent the mean and S.D.. Data represent three independent experiments. P values were calculated by using an unpaired t-test.

Article Snippet: In a 100 µl reaction, 1.4 µg of H3-H4 (catalog no. M2509S, New England Biolabs) or H2A-H2B (catalog no. M2508S, New England Biolabs) were incubated with 2.5 µg of MBP or MBP-mGCNA in Binding buffer (25 mM Tris pH 7.5, 150 mM NaCl, 5 % glycerol).

Techniques: Proteolysis Assay, Incubation, SDS Page, Silver Staining, In Vitro, Activity Assay, Plasmid Preparation, Nucleic Acid Electrophoresis, Concentration Assay