Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Sequence alignment of the C-terminal regions, beginning with the most C-terminal arginine residue, of selected variants of human histone H2A (top) and H2B (bottom). Canonical ubiquitination sites H2AK119ub and H2BK120ub are indicated. B) Schematic of spike-in experiment. Synthetic, isotope-labeled peptides were spiked into histone extracts before or after trypsin digestion and then analyzed by LC-MS/MS. *, heavy amino acid; [gg], diglycine remnant from ubiquitin; /, observed tryptic cleavage site. C) Extracted ion chromatograms of labeled synthetic peptides (blue lines) bearing H2AK118ub (bottom) or H2AK119ub (top) and their unlabeled endogenous forms (red lines) with spike-in occurring before or after digestion. The synthetic H2AK118ub and H2AK119ub peptides are distinguished by the presence of one (*) or two (**) isotopically labeled amino acids while the endogenous light forms of these sequences are isobaric (#). D) Extracted ion chromatograms of two synthetic peptides (blue lines) bearing H2BK120ub and their endogenous forms (red lines) with spike-in occurring before or after digestion.

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Sequencing, Residue, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A, B) Mixtures containing the H2AK119ub (A) and H2BK120ub (B) synthetic peptides were subjected to trypsin digestion with or without subsequent propionylation and then analyzed by LC-MS/MS. Extracted ion chromatograms are presented below each expected product sequence for the digestion with or without propionylation. *, heavy amino acid; [gg], diglycine remnant from ubiquitin; pr, propionyl group.

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Histone extracts were digested with or without subsequent propionylation. Extracted ion chromatograms are shown for the derivatized H2AK119ub peptide from the derivatized and underivatized conditions. The HCD IT MS/MS fragmentation spectrum is shown at right using the IPSA tool . [gg], diglycine remnant from ubiquitin; pr, propionyl group. B) As in A except for the derivatized and underivatized H2BK120ub peptide.

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Tandem Mass Spectroscopy

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Schematic of workflow for relative quantification of H2AK119ub and H2BK120ub by encoding samples and reference pools with either heavy (Pr-D5) or light (Pr-D0) propionic anhydride. Hypothetical mass spectra representing the precursor ion pairs of the H2AK119ub and H2BK120ub peptides (lower left) and the unmodified peptides from H2A and H2B (lower right) used for normalization are also shown. In this example, the sample appears in the light channel while the reference pool appears in the heavy channel. Based on the sample/pool ratio of the unmodified peptide, the normalized abundance of the ub-modified peptide is 0.5. B, C) Histone digests derivatized with either heavy or light propionic anhydride were combined in a 1:1 mixture and analyzed by PRM-based LC-MS/MS. Extracted ion chromatograms (left) and intact mass spectra (center) for the precursor ion pairs (light in red, heavy in blue) representing H2AK119ub (B) and H2BK120ub (C) are shown. Extracted ion chromatograms for selected fragments from the heavy and light precursors are also presented (right).

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Schematic of experiment in which the H2BK120ub synthetic peptide was mixed with the total H2B synthetic peptide at two-fold serially increasing molar ratios from 0.01 to 0.32 as indicated. Oppositely labeled samples and reference pools were combined for each of the two reciprocal labeling schemes and analyzed by PRM-based LC-MS/MS. B) The theoretical log2 change in the relative level of H2BK120ub, normalized to the 0.01 molar ratio, is plotted against the log2 of the observed sample/reference pool ratio for both the H2BK120ub peptide and the total H2B peptide. The color indicates whether the sample was labeled with heavy (H, red) or light (L, blue) propionic anhydride with the reference pool receiving the opposite isotopic label. C) The theoretical log2 change in the relative level of H2BK120ub is plotted against the observed log2 of the relative change after normalization to the total H2B peptide. As in B, the color indicates the isotope used for sample labeling. The line of best fit (solid, y = 1.134x + 0.089, R 2 = 0.994) after linear regression analysis is drawn against the expected trend (dashed, y = x).

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Histone extracts from parental (two left panels) and sgRING1A/B 10T1/2 cells (two right panels) were digested and derivatized for relative quantification of H2AK119ub and H2BK120ub. Extracted ion chromatograms, in which the light channel (L, red) encodes the reference pool and the heavy channel (H, blue) encodes individual samples, are shown for the paired precursor ions for the total H2A peptide and the H2AK119ub peptide. B) Relative quantification of H2AK119ub and H2BK120ub in RING1A/B-deficient cells and in response to inhibitors, including actinomycin D, panobinostat, Ei1, mitomycin C, and etoposide. 293T cells were treated with the indicated doses of inhibitors for 24 hrs.

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques:

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet:

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques:

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: The Journal of Cell Biology

Article Title: Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells

doi: 10.1083/jcb.201911130

Figure Lengend Snippet: In vitro characterization of sensors binding to Ub–nucleosomes. (A–H) Histone octamers containing nonhydrolyzable Ub-histone mimics were combined with 147-mer Widom 601 DNA to reconstitute mononucleosomes, whose quality were monitored by (A) native PAGE and (C) SDS-PAGE. Similarly, tri-nucleosome arrays containing H2AK15Ub, H2AK119Ub, and H2BK120Ub were reconstituted with non-linker-ended tri-nucleosomes (NLE-tri) DNA ( ; ), digested with EcoRI, and analyzed by (B) native PAGE and (D) SDS-PAGE. (E) Alexa Fluor 488–labeled Reader1.0 (molecular weight = 14.3 kD) and IE1-tSR (pan-Ub–nucleosome sensor; molecular weight = 18.1 kD) were analyzed by SDS-PAGE and detected by fluorescence or Coomassie staining as indicated. (F) IE1-tSR affinities for mono- or tri-nucleosomes containing H2AK119Ub and mononucleosomes containing H2AK129Ub were determined by measuring fluorescence changes in Alexa Fluor 488–labeled IE1-tSR upon titration with increasing concentrations of the indicated Ub–nucleosomes. Affinities of (G) Reader1.0 and (H) Reader2.1 for mono- and tri-nucleosome arrays containing H2AK15Ub, H2AK119Ub, and H2BK120Ub. Tables in G and H show the results from fitting the data with a single-site binding model. K i values were determined from half-maximal inhibitory concentrations (IC 50 ) using the equation.

Article Snippet: Next, cells were immunostained with a rabbit mAb against H2BK120Ub (CST; mAb; 5546; diluted 1:800 with 1% BSA and 0.1% Triton X-100 in PBS) for 2 h and with an Alexa Fluor 568–conjugated goat anti-rabbit IgG (Thermo Fisher Scientific; diluted 1:500 with 1% BSA and 0.1% Triton X-100 in PBS) for 1 h. U-2 OS cells stably expressing Reader1.0-eGFP were treated with 0 or 100 ng/ml Dox for 24 h before being exposed to IR (1.5 Gy) using a 137 Cs gamma-ray source.

Techniques: In Vitro, Binding Assay, Clear Native PAGE, SDS Page, Labeling, Molecular Weight, Fluorescence, Staining, Titration

Journal: The Journal of Cell Biology

Article Title: Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells

doi: 10.1083/jcb.201911130

Figure Lengend Snippet: MD simulations of Ub–nucleosomes guided design of sensors for H2BK120Ub. (A) Starting structures for all-atom MD simulations of H2AK15Ub– and H2BK120Ub–nucleosomes included one H2A/H2B dimer, the surrounding DNA double helix, and Ub linked through an isopeptide bond to either H2AK15 or H2BK120. DNA is shown as a gray molecular surface; the histone octamer is dark gray, except for the H2A/H2B dimer used for simulations (H2A, light orange; H2B, pale green). Ub is shown as an orange (H2AK15Ub) or green (H2BK120Ub) surface. (B) Percentage of MD conformations accessible with the indicated anchors and UBDs after alignment to a reference nucleosome, docking, and filtering to remove steric clashes. (C) Positions of Ub residue I44 in all allowable conformers of H2AK15Ub (orange) or H2BK120Ub (green) when both the IE1 anchor and UBQ1 UBA UBD are docked. (D) For the MD conformers in C, distributions of distances between the nucleosome acidic patch (H2AE61, δ carbon) and the Ub hydrophobic patch (I44, β carbon) revealed a cluster of conformers unique to H2BK120Ub. (E) Reader2.0/2.1 were generated by connecting the IE1 anchor to a UBD (UBQ1 UBA-WT or UBQ1 UBA-A556E ) without a linker. (F) Schematic of the competition assays used to measure affinities between Reader2.0/2.1 and Ub–nucleosomes in vitro. (G) Reader2.0/2.1 binding was measured with the indicated Ub–nucleosomes; the affinities are shown in H as K i (nM) values. conf., conformations; R2.0, Reader2.0; R2.1, Reader2.1.

Article Snippet: Next, cells were immunostained with a rabbit mAb against H2BK120Ub (CST; mAb; 5546; diluted 1:800 with 1% BSA and 0.1% Triton X-100 in PBS) for 2 h and with an Alexa Fluor 568–conjugated goat anti-rabbit IgG (Thermo Fisher Scientific; diluted 1:500 with 1% BSA and 0.1% Triton X-100 in PBS) for 1 h. U-2 OS cells stably expressing Reader1.0-eGFP were treated with 0 or 100 ng/ml Dox for 24 h before being exposed to IR (1.5 Gy) using a 137 Cs gamma-ray source.

Techniques: Generated, In Vitro, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells

doi: 10.1083/jcb.201911130

Figure Lengend Snippet: U-2 OS cells expressing Reader2.0 and Reader2.1 at high levels showed an increase in H2BK120Ub. (A) U-2 OS cells transfected with Reader1.0/2.0/2.1-eGFP were stained with an antibody to H2BK120Ub. Cell nuclei were counterstained with DAPI. Scale bar, 5 µm. (B) MFI of H2BK120Ub and Reader1.0/2.0/2.1-eGFP signals were measured using the ZEN 2.3 imaging software. Cells were clustered into three groups according to sensors’ expression levels: Reader-eGFP (-) or nontransfected controls (MFI < 1,000); Reader-eGFP low (MFI between 1,000 and 10,000); and Reader-eGFP high (MFI > 10,000). Between 30 and 63 cells were analyzed per condition. Bars show mean ± SD. Statistical analyses are as described in Materials and methods. (C–E) Correlation between H2BK120Ub levels and Reader expression levels in cells expressing the indicated sensor at high levels (MFI > 10,000). (F) MFI of Reader1.0/2.0/2.1-eGFP in cells analyzed by FRAP . (G) Nuclear mobilities of Reader1.0/2.0/2.1-eGFP and their mutant variants were assessed by FRAP (ROI-1, gray circle). Fluorescence recoveries were monitored at 0.5-s intervals, background-corrected (ROI-2, yellow square), and normalized to pre-bleach fluorescence intensities. ROI-3 (blue square) and ROI-4 (green rectangle) were used for monitoring unintentional bleaching and for image acquisition, respectively. Scale bar, 5 µm. R1.0, Reader1.0; R2.0, Reader2.0; R2.1, Reader2.1; t, time.

Article Snippet: Next, cells were immunostained with a rabbit mAb against H2BK120Ub (CST; mAb; 5546; diluted 1:800 with 1% BSA and 0.1% Triton X-100 in PBS) for 2 h and with an Alexa Fluor 568–conjugated goat anti-rabbit IgG (Thermo Fisher Scientific; diluted 1:500 with 1% BSA and 0.1% Triton X-100 in PBS) for 1 h. U-2 OS cells stably expressing Reader1.0-eGFP were treated with 0 or 100 ng/ml Dox for 24 h before being exposed to IR (1.5 Gy) using a 137 Cs gamma-ray source.

Techniques: Expressing, Transfection, Staining, Imaging, Software, Mutagenesis, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells

doi: 10.1083/jcb.201911130

Figure Lengend Snippet: Reader2.0 and Reader2.1 detect H2BK120Ub in the nucleus. (A) Reader1.0/2.0/2.1-eGFP and constructs mutated to eliminate interactions with Ub (Anchor), nucleosome acidic patch (UBD), or both (NB) are listed. Images show nuclear localization of the proteins expressed in U-2 OS cells. Scale bar, 5 µm. (B) FVP or C1 was used to deplete, respectively, H2BK120Ub or all histone Ub conjugation. U-2 OS cells treated with 5 µM FVP or 10 µM C1 for the indicated times were analyzed by Western blotting. (C and D) Live-cell FRAP measurements of Reader1.0/2.0/2.1-eGFP variants expressed in U-2 OS cells with or without 5 µM FVP or 10 µM C1 pretreatment for 1 h. FRAP kinetics were fit best by a single fast recovery rate for R1.0/2.1-NB and R1.0/2.1-UBD (C), or two exponential components for all other constructs (D). Note that Reader2.0/2.1 have the same anchor, which is referred to as R2.1-Anchor. (E) Fractions (%) of the fast and slow components of FRAP recoveries were determined from fits to the data in C and D; calculated recovery t 1/2 values are in . (F) Models depicting the origins of fast and slow components of the FRAP recoveries. R1.0, Reader1.0; R1.1, Reader1.1; R2.0, Reader2.0; R2.1, Reader2.1.

Article Snippet: Next, cells were immunostained with a rabbit mAb against H2BK120Ub (CST; mAb; 5546; diluted 1:800 with 1% BSA and 0.1% Triton X-100 in PBS) for 2 h and with an Alexa Fluor 568–conjugated goat anti-rabbit IgG (Thermo Fisher Scientific; diluted 1:500 with 1% BSA and 0.1% Triton X-100 in PBS) for 1 h. U-2 OS cells stably expressing Reader1.0-eGFP were treated with 0 or 100 ng/ml Dox for 24 h before being exposed to IR (1.5 Gy) using a 137 Cs gamma-ray source.

Techniques: Construct, Conjugation Assay, Western Blot