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: 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: Journal of Pediatric Hematology/Oncology

Article Title: Interaction of E2F3a and CASP8AP2 Regulates Histone Expression and Chemosensitivity of Leukemic Cells

doi: 10.1097/MPH.0000000000002558

Figure Lengend Snippet: E2F3a and CASP8AP2 collaboratively affected H2A and H2B expression. A, Overexpression of E2F3a increased the expression of H2A and H2B in HEK-293T cells. B, E2F3a knockdown decreased the expression of H2A and H2B in HEK-293T cells. C, Overexpression of CASP8AP2 enhanced the expression of H2A and H2B. D, Downregulation of CASP8AP2 reduced the expression of histones in HEK-293T cells. E, The reduction in histones induced by the E2F3a knockdown could be reversed by the overexpression of CASP8AP2.

Article Snippet: A Western blot was carried out with the following antibodies: histone H2A and H2B antibodies (CST, USA), GAPDH antibody (SAB, USA), anti-FLAG antibody (Sigma, USA), and anti-GST antibody (CST).

Techniques: Expressing, Over Expression

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

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet:

Article Snippet: H2BK120ub (rabbit) , Cell Signaling Technology , Cat no. 5546; RRID: AB_10693452.

Techniques: Recombinant, Purification, Western Blot, Software

Journal: Molecular Therapy. Nucleic Acids

Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

doi: 10.1016/j.omtn.2023.02.014

Figure Lengend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

Techniques: RNA Sequencing Assay, Infection, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

doi: 10.1016/j.omtn.2023.02.014

Figure Lengend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

Techniques: Expressing, Infection, Staining, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

doi: 10.1016/j.omtn.2023.02.014

Figure Lengend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

Techniques: Expressing, ChIP-sequencing, Infection, Binding Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

doi: 10.1016/j.omtn.2023.02.014

Figure Lengend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

doi: 10.1016/j.omtn.2023.02.014

Figure Lengend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

Techniques: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

Journal: Molecular Therapy. Nucleic Acids

Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

doi: 10.1016/j.omtn.2023.02.014

Figure Lengend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

Techniques: