Journal: Cell & Bioscience

Article Title: RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells

doi: 10.1186/s13578-023-01018-2

Figure Lengend Snippet: H2BK120ub is deficient in the Sertoli cells in the Amh-Rnf20 −/− mice. a Mass spectrometry detection of the ubiquitination of the H2B at K120 with the peptide HAVSEGTK(120)AVTK in the Rnf20 Flox/Flox . The MQ software was used to analyze the data from mass spectrometry. X axis, m/z; Y axis, the intensity of ions; y, the C-terminal fragment ion (Y series). b Ubiquitinated peptide information at the position of the K120 in the Rnf20 Flox/Flox . The ubiquitination modification site was not detected in the Amh-Rnf20 −/− . c Immunofluorescent analysis of SOX9, H2BK120ub, and DMRT1 on serial paraffin-sections in the Rnf20 Flox/Flox and the Amh-Rnf20 −/− testes at 7 days after birth and adult mice. The nuclei were stained with DAPI. TRITC signals represent the localization of H2BK120ub, while FITC signals showed the localization of SOX9 or DMRT1. The white squares in the panels correspond to the enlarged panels. Sn, Sertoli cells; Sg, spermatogonia. Scale bar, 10 μm

Article Snippet: The following primary antibodies were used: Anti-RNF20 (21625-1-AP, Proteintech Group, Rosemont, IL, USA), Anti-H2BK120ub (Cat# 5546s, Cell Signaling Technology, Danvers, MA, USA), Anti-β-ACTIN (Cat# 66009-1-Ig, Proteintech Group, Rosemont, IL, USA), Anti-SOX9 (Cat# 82,630 S, Cell Signaling Technology, Danvers, MA, USA), Anti-Caspase3 (Cat# 19677-1-AP, Proteintech Group, Rosemont, IL, USA), Anti-Claudin 11 (Cat# 36-4500, Thermo Fisher, Waltham, MA, USA), Anti-N-Cadherin (Cat# WL01047, Wanleibio, Shenyang, China), Anti-β-Catenin (Cat# 51067-2-AP, Proteintech Group, Rosemont, IL, USA), and Anti-α-Catenin (Cat# GTX111168, GeneTex, Texas, USA).

Techniques: Mass Spectrometry, Software, Modification, Staining

Journal: Cell & Bioscience

Article Title: RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells

doi: 10.1186/s13578-023-01018-2

Figure Lengend Snippet: RNF20 deficiency in Sertoli cells impairs the Cldn11 transcription. a Scatterplots of differentially expressed genes. Red scatter, genes with significant up-regulated; blue scatter, genes with significant down-regulated; gray scatter, genes with no significant difference. X axis, Lg (WT FPKM) in the Rnf20 Flox/Flox ; Y axis, Lg (KO FPKM) in the Amh-Rnf20 −/− . b Gene ontology (GO) terms analysis of down-regulated genes in the Sertoli cells of the Amh-Rnf20 −/− testes. c, d Heatmaps showing the expression levels of down-regulated genes in the terms spermatogenesis ( c ) and cell adhesion ( d ) in the Sertoli cells of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . Color bar, Log 2 (FPKM). e Quantitative real-time PCR analysis of the genes Rnf20 and Cldn11 . The expression levels of the genes were related to Hprt expression. Relative levels, 2 −ΔCt ; T-tests were performed. *, p < 0.05, **, p < 0.01. f Western blot analysis of the expression levels of RNF20, CLDN11, and H2BK120ub proteins in adult mice. β-ACTIN was used as an internal control. g ChIP-PCR assays. The antibody specific for H2BK120ub was used in the ChIP analysis and primers were designed in the regions of promoter and exons of Cldn11 in the testes of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . The black graphs indicated the enriched levels in the Rnf20 Flox/Flox mice, while the white graphs indicated the levels in the Amh-Rnf20 −/− mice

Article Snippet: The following primary antibodies were used: Anti-RNF20 (21625-1-AP, Proteintech Group, Rosemont, IL, USA), Anti-H2BK120ub (Cat# 5546s, Cell Signaling Technology, Danvers, MA, USA), Anti-β-ACTIN (Cat# 66009-1-Ig, Proteintech Group, Rosemont, IL, USA), Anti-SOX9 (Cat# 82,630 S, Cell Signaling Technology, Danvers, MA, USA), Anti-Caspase3 (Cat# 19677-1-AP, Proteintech Group, Rosemont, IL, USA), Anti-Claudin 11 (Cat# 36-4500, Thermo Fisher, Waltham, MA, USA), Anti-N-Cadherin (Cat# WL01047, Wanleibio, Shenyang, China), Anti-β-Catenin (Cat# 51067-2-AP, Proteintech Group, Rosemont, IL, USA), and Anti-α-Catenin (Cat# GTX111168, GeneTex, Texas, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: bioRxiv

Article Title: A system for in vivo evaluation of protein ubiquitination dynamics using deubiquitinase-deficient strains

doi: 10.1101/2023.06.18.545485

Figure Lengend Snippet: (A-B) Immunoblots for histone H2BK123ub1 and/or histone H3K4 methylation (mono, me1; di, me2; tri, me3) in extracts prepared from A) strains lacking Rad6, Bre1 or Lge1 and B) strains lacking Rad6, Bre1 or Lge1 in the background of ubp8Δubp10Δ double deletion mutant. Ponceau S staining and histone H3 levels served as loading controls. Histone H2BK123ub1 was detected using anti-H2B or anti-H2BK120 ubiquityl antibody. Molecular weights of the protein standards used as size markers (kDa) are indicated.

Article Snippet: The following antibodies were used in immunoblotting: anti-Flag M2 (F3165; Sigma), anti-V5 (46-0708; Invitrogen); anti-HA (39628; Active Motif); anti-Pgk1 (459250; Invitrogen), anti-H2B (39237; Active Motif), anti-H3 (ab1791; Abcam), anti-H3K4me1 (39297; Active Motif), anti-H3K4me2 (399141; Active Motif), anti-H3K4me3 (39159; Active Motif), anti-ubiquityl-Histone H2B (Lys120) (D11) XP® (5546; Cell Signaling); anti-mono- and polyubiquitinylated conjugates monoclonal antibody (clone FK2) (HRP conjugate) (BML-PW0150; Enzo Life Sciences), anti-PCNA/Pol30 (ab221196; Abcam).

Techniques: Western Blot, Methylation, Mutagenesis, Staining

Journal: bioRxiv

Article Title: A system for in vivo evaluation of protein ubiquitination dynamics using deubiquitinase-deficient strains

doi: 10.1101/2023.06.18.545485

Figure Lengend Snippet: (A) Sequences of the distal end of WT H2B C-terminal helix and of mutants. Lysine 123, the site of monoubiquitination is indicated. (B) Growth assay conducted by spotting 10-fold serial dilutions of indicated strains on synthetic medium lacking histidine (-HIS) or lacking histidine and containing 5-fluoroorotic acid (-HIS+FOA). (C-D) Left: Immunoblots for H2BK123ub1 in extracts prepared from C) UBP8UBP10 or D) ubp8Δubp10Δ strains expressing Flag epitope-tagged WT or mutant histone H2B. Ponceau S staining served as loading control. Right: Fold-change in H2BK123ub1 levels in the indicated mutants relative to WT H2B (set as 1). For densitometry quantitation, the signals for H2BK123ub1 in WT or mutant H2B were initially normalized to the signals for Ponceau S-stained proteins. Plotted are means ± SEM from three independent experiments. ns, not significant; *, p -value <0.05 (Student’s t-test).

Article Snippet: The following antibodies were used in immunoblotting: anti-Flag M2 (F3165; Sigma), anti-V5 (46-0708; Invitrogen); anti-HA (39628; Active Motif); anti-Pgk1 (459250; Invitrogen), anti-H2B (39237; Active Motif), anti-H3 (ab1791; Abcam), anti-H3K4me1 (39297; Active Motif), anti-H3K4me2 (399141; Active Motif), anti-H3K4me3 (39159; Active Motif), anti-ubiquityl-Histone H2B (Lys120) (D11) XP® (5546; Cell Signaling); anti-mono- and polyubiquitinylated conjugates monoclonal antibody (clone FK2) (HRP conjugate) (BML-PW0150; Enzo Life Sciences), anti-PCNA/Pol30 (ab221196; Abcam).

Techniques: Growth Assay, Western Blot, Expressing, FLAG-tag, Mutagenesis, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: A system for in vivo evaluation of protein ubiquitination dynamics using deubiquitinase-deficient strains

doi: 10.1101/2023.06.18.545485

Figure Lengend Snippet: (A) The steady-state levels of ubiquitination of a protein in vivo are maintained by the actions of ‘writer’ E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases and ‘eraser’ DUBs. (B) In yeast S. cerevisiae , the in vivo steady-state levels of H2BK123ub1 in a nucleosome are maintained by the ‘writer’ complex comprised of Rad6 (E2), Bre1 (E3) and accessory/adapter protein Lge1, and two ‘eraser’ DUBs, Ubp10 and the SAGA complex-associated Ubp8. IDR, intrinsically disordered region; cc, coiled-coil domain. (C) In the ubp8Δubp10Δ double null mutant strain, H2BK123ub1 accumulates due to ubiquitin addition and absence of deubiquitination. (D) High levels of H2BK123ub1 are not observed in the ubp8Δubp10Δ mutant strain when either the IDR or coiled-coil domain of Lge1 or the C-terminal acidic tail of Rad6 are deleted. Thus, the absence of relevant DUBs revealed the roles for various regions or domains of proteins involved in the ubiquitin-conjugation step. (E) Residues of the H2B C-terminal helix (Cα) impact the activity of the E2-E3 complex and the DUBs by influencing their access to substrate K123 or its ubiquitin conjugated form, respectively. Aspartate substitution at residue 120 in H2B Cα inhibits the Rad6-Bre1-Lge1-mediated monoubiquitination of H2BK123 in both WT and ubp8Δubp10Δ strains. In contrast, arginine substitution at position 120 in H2B Cα promotes removal of the conjugated ubiquitin by Ubp8 and Ubp10, as evidenced by the reduced H2BK123ub1 in the H2BA120R mutation in a strain expressing these two DUBs and not in their absence. Thus, the use of the DUB deletion strain informed on the dynamics of deubiquitination in addition to the ubiquitin conjugation.

Article Snippet: The following antibodies were used in immunoblotting: anti-Flag M2 (F3165; Sigma), anti-V5 (46-0708; Invitrogen); anti-HA (39628; Active Motif); anti-Pgk1 (459250; Invitrogen), anti-H2B (39237; Active Motif), anti-H3 (ab1791; Abcam), anti-H3K4me1 (39297; Active Motif), anti-H3K4me2 (399141; Active Motif), anti-H3K4me3 (39159; Active Motif), anti-ubiquityl-Histone H2B (Lys120) (D11) XP® (5546; Cell Signaling); anti-mono- and polyubiquitinylated conjugates monoclonal antibody (clone FK2) (HRP conjugate) (BML-PW0150; Enzo Life Sciences), anti-PCNA/Pol30 (ab221196; Abcam).

Techniques: In Vivo, Mutagenesis, Conjugation Assay, Activity Assay, Expressing

Journal: iScience

Article Title: Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation

doi: 10.1016/j.isci.2023.106743

Figure Lengend Snippet:

Article Snippet: Mouse anti-Histone H2B , Cell Signaling Technology , Cat #2934 RRID: AB_2295301.

Techniques: Recombinant, Mutagenesis, Software, CRISPR

Journal: bioRxiv

Article Title: Structure of the human Bre1 complex bound to the nucleosome

doi: 10.1101/2023.03.31.535082

Figure Lengend Snippet: a, H3. b , H4. c , H2A. d , H2B. e , DNA. f , RING domain bound to the acidic patch (modeled here as RING A ). g , RING domain bound to the DNA phosphates (modeled here as RING B ). h , Close-up view near the arginine anchor. i , Close-up view of the RING B -DNA interface.

Article Snippet: Ubiquitinated H2BK120 was detected using ubiquityl-histone H2B (Lys120) (D11), with XPR Rabbit mAb as the primary antibody (No. 5546; Cell Signaling), goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz Biotechnology) as the secondary antibody, and ECL Prime (Cytiva) as the chemiluminescent reagent.

Techniques:

Journal: bioRxiv

Article Title: Structure of the human Bre1 complex bound to the nucleosome

doi: 10.1101/2023.03.31.535082

Figure Lengend Snippet: a , Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b , Close-up view of ubiquitin and H2B. Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c , Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.

Article Snippet: Ubiquitinated H2BK120 was detected using ubiquityl-histone H2B (Lys120) (D11), with XPR Rabbit mAb as the primary antibody (No. 5546; Cell Signaling), goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz Biotechnology) as the secondary antibody, and ECL Prime (Cytiva) as the chemiluminescent reagent.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Epigenetic mechanisms to propagate histone acetylation by p300/CBP

doi: 10.1101/2023.03.31.535039

Figure Lengend Snippet: a Schematic representation of the domain architecture of human p300. NRID, nuclear receptor interaction domain; TAZ1, transcriptional adaptor zinc-finger domain 1; KIX, kinase-inducible domain of CREB-interacting domain; BD, bromodomain; RP, the RING and PHD zinc-fingers; HAT, histone acetyltransferase domain; ZZ, ZZ-type zinc-finger; TAZ2, transcriptional adaptor zinc-finger domain 2; and IBiD, IRF3-binding domain. The positions of the N- and C-termini and the start/end residues of the major domains are shown at the top. The positions of the start/end residues of the construct used in this study ( i.e ., p300 BRPHZT ) are shown at the bottom. b In vitro acetyltransferase activity of p300 BRPHZT toward an H4-di-acetylated nucleosome. The histone and residue for which acetylation was detected by immunoblotting are shown above each panel. Color code: H2A, yellow; H2B, red; H3, blue; H4, green. Nucleosome (Nuc): un, unmodified; ac (green), H4K12/K16-acetylated. p300 BRPHZT (p300): -, none; +, 1 μM. CBP30: -, none; +, 10 μM. The y-axis indicates the immunoblotting signal intensity at 1 min after the reaction. Means ± SD ( N = 3). Statistical significance was assessed by a two-sample one-sided Welch’s t -test (NS, P ≥ 0.05; * P < 0.05; ** P < 0.01). The alternative hypothesis is as follows: lane 4, increase vs. lane 3; lane 5, decrease vs. lane 3; lane 6, decrease vs. lane 4. c Structure of p300 BRPH bound to H2BNT and acetylated H4NT delineated by cryo-electron microscopy (cryo-EM). Left, top view; right, side view. p300 BRPH (#1 in Supplementary Fig. 8) binds to H4acNuc in a Slinky-like bent conformation via bromodomain and HAT. d Overall structure of p300 H2B (#1) with H4-di-acetylated nucleosome in cartoon presentation. Color code: orange, p300 BD; cyan, p300 RP; magenta, p300 HAT; green, K12/K16-acetylated H4; red, H2B. e Close-up view of the binding mode of p300 bromodomain (BD, #1) to the H4-di-acetylated nucleosome (H4K12acK16ac). The map corresponding to H4NT is colored light blue. f Superposition of the cryo-EM structure of p300 BRPH (#4) and the crystal structure of p300 BRPH lacking AIL (5LKU). The magenta region circled in black is the substrate-binding site of HAT. g Close-up view of the cryo-EM map (#4) and the structure of H2BNT. h Close-up view of H2BNT (#4) shown as a cartoon representation.

Article Snippet: Membranes were incubated for 20–40 min at 25 °C with Bullet ImmunoReaction Buffer (Nacalai Tesque, 18439-85) containing the following antibodies at the indicated dilution rate: H2AK5ac (Abcam, ab45152, 1/3,000), H2B (Cell Signaling, #12364, 1/1,000), H2BK12ac (Abcam, ab40883, 1/500), H2BK15ac (Abcam, ab62335), H2BK16ac (Abcam, ab177427, 1/1,000), H2BK20ac (Abcam, ab177430, 1/500), H2BK23ac (Abcam, ab222770, 1/1,000), the C-terminus of H3 (Merck, 07-690, 1/3,000), H3K14ac (Merck, 07-353, 1/1,000), H3K18ac (Abcam, ab1191, 1/1,000), H3K23ac (Merck, 07-355, 1/1,000), H3K27ac (Merck, 07-360, 1/3,000), the C-terminus of H4 (Abcam, ab10158, 1/1,000), H4K5ac (MABI, 0405, 1/500), H4K8ac (MABI, 0408, 1/500), H4K12ac (MABI, 0412, 1/500), or H4K16ac (MABI, 0416, 1/500).

Techniques: Zinc-Fingers, Binding Assay, Construct, In Vitro, Activity Assay, Western Blot, Electron Microscopy, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Epigenetic mechanisms to propagate histone acetylation by p300/CBP

doi: 10.1101/2023.03.31.535039

Figure Lengend Snippet: a Various conformations of p300 BRPH with the H4-di-acetylated nucleosome (H4acNuc) shown by cryo-electron microscopy (cryo-EM) maps and structural modelling: left, P300 H3-I (#5 in Supplementary Fig. 8); center, P300 H3-II (#6); right, P300 H2A (#7). (See for color coding). b The positions of the superhelical location (SHL) at which p300 bromodomain (BD) interacts. Complex structures showing (top) superimposition of p300 H2B -H4acNuc (#4) and P300 H2A -H4acNuc (#7) and (bottom) superimposition of p300 H3-I -H4acNuc (#5) and p300 H3-II -H4acNuc (#6). The respective regions where p300 BD interacts with DNA are indicated by black squares and are shown on the right in close-up, displaying p300 (ribbon diagram) and nucleosome (surface diagram). c Basic patches interacting with DNA at p300 HAT. In the top left panel (#5), two basic patches are circled in blue. One basic patch (K1456, K1459, K1461, and R1462) is located around the β κ -α j loop (KJ basic patch) and the other (K1488, R1494, and K1592) is located in α K and α N (KN basic patch). The K/R residues involved in the interaction with DNA are shown in blue. The other three panels show the surface electrostatic potential of p300 BRPH for each complex structure, with surfaces charged positively in blue or negatively in red. Other panels (#5–#7): surface electrostatic potential of p300 BRPH for each complex structure. Positively charged surfaces are colored in blue and negatively charged surfaces in red. d Close-up views of the density and model structure of each NT in the H4acNuc complex. From left to right, the HAT catalytic center of p300 or CBP is shown in close proximity to H3NT (H3-I, #2), H3NT (H3-II, #10), H2ANT (#7), and H2BNT (#4) in H4acNuc. The rightmost panel showing H2BNT is another angle of . Color codes of NT: blue: H3NT, yellow: H2ANT, red: H2BNT; cyan, p300 RP; magenta, p300 HAT.

Article Snippet: Membranes were incubated for 20–40 min at 25 °C with Bullet ImmunoReaction Buffer (Nacalai Tesque, 18439-85) containing the following antibodies at the indicated dilution rate: H2AK5ac (Abcam, ab45152, 1/3,000), H2B (Cell Signaling, #12364, 1/1,000), H2BK12ac (Abcam, ab40883, 1/500), H2BK15ac (Abcam, ab62335), H2BK16ac (Abcam, ab177427, 1/1,000), H2BK20ac (Abcam, ab177430, 1/500), H2BK23ac (Abcam, ab222770, 1/1,000), the C-terminus of H3 (Merck, 07-690, 1/3,000), H3K14ac (Merck, 07-353, 1/1,000), H3K18ac (Abcam, ab1191, 1/1,000), H3K23ac (Merck, 07-355, 1/1,000), H3K27ac (Merck, 07-360, 1/3,000), the C-terminus of H4 (Abcam, ab10158, 1/1,000), H4K5ac (MABI, 0405, 1/500), H4K8ac (MABI, 0408, 1/500), H4K12ac (MABI, 0412, 1/500), or H4K16ac (MABI, 0416, 1/500).

Techniques: Electron Microscopy, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Epigenetic mechanisms to propagate histone acetylation by p300/CBP

doi: 10.1101/2023.03.31.535039

Figure Lengend Snippet: a In vitro acetyltransferase activity of p300 BRPHZT toward the H4-di-acetylated nucleosome. The histone and its residue at which acetylation was detected by immunoblotting are shown above each panel. Nucleosome (Nuc): un, unmodified; ac (green), H4K12/K16-acetylated. p300 BRPHZT (p300): WT, wild-type; 4A, with mutations of R1133A, K1134A, R1137A, and K1140A; 4E, with mutations of R1133E, K1134E, R1137E, and K1140E. CBP30: -, none; +, 10 μM. The y-axis indicates the immunoblotting signal intensity at 1 min after the reaction. Means ± SD ( N = 3). Statistical significance was assessed by a two-sample one-sided Welch’s t -test (NS, P ≥ 0.05; * P < 0.05; ** P < 0.01). The alternative hypothesis is as follows: lane 4, increase vs. lane 3; lanes 5, 7, and 11, decrease vs. lane 3; lanes 6, 8, and 12, decrease vs. lane 4). b In vitro acetyltransferase activity of p300 BRPHZT toward the H2B-tetra-acetylated nucleosome. Columns marked ac (in red) indicate the H2BK12/K15/K20/K23-acetylated nucleosome. Other indications are the same as in a . c In vitro acetyltransferase activity of p300 BRPHZT toward the H3-di-acetylated nucleosome. Columns marked ac (blue) indicate the H3K14/K18-acetylated nucleosome. d Thermal stability assay of the H2B-acetylated nucleosome. Mean values of thermal denaturation curves from 60.0 °C to 90.0 °C for derivative fluorescence intensity are plotted for the unmodified nucleosome (black line) and the H2BK12/K15/K20/K23-acetylated nucleosome (red line). The temperature at which the H2A-H2B dimer or the H3-H4 tetramer dissociates from the nucleosome is shown at the bottom. Means ± SD ( N = 3). e ‘Epi-central’ model of histone acetylation signalling. Arrows indicate the flow of information, with acetylation information in red. f Hypothetical logic of context-dependent gene expression in metazoans. The symbol in the center indicates a triple-input AND logic gate.

Article Snippet: Membranes were incubated for 20–40 min at 25 °C with Bullet ImmunoReaction Buffer (Nacalai Tesque, 18439-85) containing the following antibodies at the indicated dilution rate: H2AK5ac (Abcam, ab45152, 1/3,000), H2B (Cell Signaling, #12364, 1/1,000), H2BK12ac (Abcam, ab40883, 1/500), H2BK15ac (Abcam, ab62335), H2BK16ac (Abcam, ab177427, 1/1,000), H2BK20ac (Abcam, ab177430, 1/500), H2BK23ac (Abcam, ab222770, 1/1,000), the C-terminus of H3 (Merck, 07-690, 1/3,000), H3K14ac (Merck, 07-353, 1/1,000), H3K18ac (Abcam, ab1191, 1/1,000), H3K23ac (Merck, 07-355, 1/1,000), H3K27ac (Merck, 07-360, 1/3,000), the C-terminus of H4 (Abcam, ab10158, 1/1,000), H4K5ac (MABI, 0405, 1/500), H4K8ac (MABI, 0408, 1/500), H4K12ac (MABI, 0412, 1/500), or H4K16ac (MABI, 0416, 1/500).

Techniques: In Vitro, Activity Assay, Western Blot, Stability Assay, Fluorescence, Expressing