Journal: Science advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1.

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: Fig. 2. SirT7 N189-dependent auto–ADP-ribosylation regulates SirT7 distribution and chromatin-binding dynamics. (A) In vitro auto-mADPRT assay as in Fig. 1 with bacterially expressed rSirT6 WT or N135Q. (B) Deacetylation reaction with the recombinant bacterial SirT6 WT, N135Q, and H133Y incubated with recombinant mononucleosomes acetylated in H3K18ac (*P < 0.05). Quantification of three experiments similar to the one shown in fig. S2A. The results are represented relative to WT SirT7 activity (100%). (C) ADP-ribosylation levels of SirT7 WT or N189A expressed in HEK293F cells monitored by far Western blot with anti–pan–ADP-ribose binding re- agent. HDAC, histone deacetylase. (D) Analysis of SirT7 auto-mADPRTion identified by MS. ADP-ribosylated peptides identified in SIRT7 WT or N189A after incubation with NAD+ were analyzed using high-energy collisional dissociation (HCD) and electron-transfer/higher-energy collision dissociation fragmentation methods. The most probable ADP-ribosylated sites are highlighted in red, as the modification cannot be localized with 100% confidence. Further information is included in table S1 and in Materials and Methods. (E) Structural model of the SirT7 catalytic domain indicating localization of the ADP-ribosylated peptides identified in (C). The ADP-ribosylation N189- dependent (magenta) and N189-independent (orange) residues, the N189/E185 cavity (blue), and the primary catalytic site bound to acetylated peptide (red) are shown. (F) IF assay of the indicated green fluorescent protein (GFP)–tagged SIRT7 proteins expressed in SirT7−/− MEFs. Nucleophosmin (B23) was included as a nucleolar marker. DAPI, 4′,6-diamidino-2-phenylindole. A representative image of the experiment is shown. Scale bar, 5 m. (G) Quantification of IF experiment in (F), indicating the percentage of GFP-positive cells with a regular nucleolar distribution (*P < 0.05). (H) Cellular distribution of SirT7 WT, H187Y (HY), N189A (NA), and N189Q (NQ) in whole-cell extract (WCE), cytoplasm, nucleoplasm, and chromatin in NIH3T3 cells. Controls for the nuclear fraction (fibrillarin), cytoplasm (tubulin), and chromatin (histone H3) are also shown.

Article Snippet: Deacetylation assays for SirT6 were performed in 50 mM trisHCl (pH 8.0), 150 mM NaCl, and 1 mM MgCl2 with 2 mM NAD+ (Sigma-Aldrich) in the presence of 3 g of rSirT6 and 1 g of semisynthetic recombinant mononucleosomes containing acetyl-lysine at position 18 of histone H3 (H3K18ac dNuc, EpiCypher) for 5 hours at 30°C.

Techniques: Binding Assay, In Vitro, Recombinant, Incubation, Activity Assay, Far Western Blot, Histone Deacetylase Assay, Modification, Marker

Journal: Science Advances

Article Title: The regulatory enzymes and protein substrates for the lysine β-hydroxybutyrylation pathway

doi: 10.1126/sciadv.abe2771

Figure Lengend Snippet: ( A ) Class I/II HDAC inhibitors (NaBu, TSA, and FK228), but not a class III HDAC inhibitor (NAM), elevate H3K9bhb and H3K9ac levels in HEK293 cells. DMSO, dimethyl sulfoxide. ( B ) TSA treatment increases histone Kbhb levels in HEK293 cells. Cells were treated with TSA at the indicated concentrations for 18 hours, and Kbhb and Kac levels were analyzed by immunoblotting with indicated antibodies. ( C ) Joint knockdown of HDAC1 and HDAC2 increases histone Kbhb levels in HEK293 and HeLa cells. Kbhb and Kac levels were detected by immunoblotting using indicated antibodies. Immunoblot of histone H3 was used as loading control. ( D ) An HDAC1/2/3 selective inhibitor, MS275, dose-dependently increases Kbhb and Kac levels in 293T cells. Cells were treated with MS275 for 24 hours, and the Kbhb and Kac levels were analyzed by immunoblotting with the indicated antibodies.

Article Snippet: The sequence-specific anti-H3K4bhb (PTM-1258), anti-H3K9bhb (PTM-1250), anti-H3K18bhb (PTM-1252), anti-H3K27bhb (PTM-1293), anti-H4K8bhb (PTM-1253), anti-H3K9ac (PTM-156), anti-H3K18ac (PTM-158), anti-H3K27ac (PTM-160), anti-H4K8ac (PTM-164), and pan anti-Kbhb (PTM-1201) antibodies were purchased from PTM Biolabs Inc. (Chicago, IL).

Techniques: Western Blot, Knockdown, Control

Journal: bioRxiv

Article Title: High-Throughput Epigenetic Profiling Immunoassays for Accelerated Disease Research and Clinical Development

doi: 10.1101/2024.12.05.626944

Figure Lengend Snippet: A. Quantification of Histone H3 Acetylation (H3K27Ac, H3K9Ac, H3K18Ac) and Methylation Levels (H3K9Me3, H3K27Me3) using Nu.Q ® Immunoassays (ratio (%) of assay-specific PTMs to the Nu.Q ® H3.1 level) in HeLa cells treated with NaB (Red) vs untreated cells (Gray). Each bar represents the average PTM levels across the three replicates, with error bars indicating standard deviation (SD)(n= 3, Multiple unpaired t-test; ** p < 0.01; *** p < 0.001; ns: not significant). B. Western Blot analysis of Histone H3 Acetyl and Methyl PTMs with a representative result for Histone H3 (H3). The C-terminal end of histone H3 (H3) were included in all WB used as loading control. Each line represents a cropped gel/blot picture (upper/lower marker: 20kDa-15kDa) ofWB . Each column represents a different HeLa cell chromatin extract preparation from cells untreated (first #1-#3) or treated with NaB (last #1-#3lanes, +NaB). The quantitative analysis of the Western blot results is shown alongside in Supplementary Figure 2.

Article Snippet: The PVDF membranes was then incubated 2 hours with antibodies against H3K27Me3 (Cell Signaling Technology; #9733BF – lot: 25), H3K36Me3 (Active Motif; #80218 – lot: 2280106), H3K9Me3 (Abcam; #232324 – lot: GR3452452-1), H3K4Me2 (Volition; #3349 lot: rr260604a-6240), H3R8Cit (Abcam; #232939 – lot: GR3402039-3), H3K27Ac (Volition; #3118– lot: rr200819b-6298), H3K18Ac (Synabs; #FYN213R CL3 lot: 10487), H3K9Ac (Active Motif; #91103 – lot: 22420006), H3K9Me1 (Cell Signaling Technology; #14186BF – lot: 5), H3K4Me1 (Cell Signaling Technology; #5326BF – lot: 4), diluted on Tris Buffer Saline, 1% of Casein (Biorad; #1706435) + 0.1% Tween ® 20 (Merck Life Sciences BV; #T2700) at 2µg/ml and incubated for 2H at RT or overnight at 4°C.

Techniques: Methylation, Standard Deviation, Western Blot, Control, Marker

Journal: Molecular cell

Article Title: Myc regulates chromatin decompaction and nuclear architecture during B cell activation

doi: 10.1016/j.molcel.2017.07.013

Figure Lengend Snippet: Key Resources Table

Article Snippet: h3k18ac , Diagenode , C15410139.

Techniques: Recombinant, Cell Isolation, Knock-In, Plasmid Preparation

Journal: bioRxiv

Article Title: Matrix metalloproteinase-2 mediates ribosomal RNA transcription by cleaving nucleolar histones

doi: 10.1101/2020.02.21.958280

Figure Lengend Snippet: A. Western blots using antibodies against N-terminal (left) and C-terminal (right) specific epitopes in recombinant human histone H3 isoforms after incubation (1 h, 37°C), with increasing molar ratios of recombinant MMP-2 to histone (1:1000 to 1:50). In the right lane of each panel, using the highest molar ratio, 10 µM ARP-100 (MMP-2 preferring inhibitor) was added to confirm MMP-2-dependent histone clipping. Arrowheads show the uncleaved histone band. Molecular weight markers are shown at left, n = 3 independent experiments. B. Left: In vitro degradation of recombinant human histone H3.3 (rH3.3) by MMP-2 at 1:150 (MMP-2:histone) molar ratio for 1 h at 37°C. Arrow shows the abundant ≈17 kDa cleavage product of rH3.3 by MMP-2. Right: Edman degradation sequencing of this band shows an MMP-2 cleavage site in rH3.3 between K18 and Q19 residues, n = 3 independent experiments. C. Histones purified from isolated U2-OS nucleoli were incubated with MMP-2 (1 h, 37°C) with or without 10 µM ARP-100. Immunoblot using anti-H3 K18ac does not reveal the 17 kDa cleavage product, verifying that MMP-2 cleavage of histone H3.3 occurs at its N-terminus, n = 3 independent experiments.

Article Snippet: The following reagents and antibodies were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), Biowhittaker; fetal bovine serum (FBS), Harlan-Seralab; anti-FUS/TLS (ab23439), anti-α-tubulin (ab4074), anti-fibrillarin (ab5821), anti-MMP-2 (ab92536 for WB, IP), anti-H3 C-terminal (ab12079) and anti-H4 C-terminal (ab31827), Abcam; anti-H3 N-terminal (05-499), Upstate Biotechnology; anti-H4 N-terminal (AHP413), Biolegend; anti-H3 K18ac (AR-0103) and anti-H3 K23ac (AR-0104), Upstate Biotechnology; recombinant human histones, New England Biolabs; 72 kDa MMP-2 and anti-MMP-2 (AB-19015, IF), EMD Millipore; anti-CTCF (#2899), Cell Signaling; anti-UBF (sc-13125x), Santa Cruz.

Techniques: Western Blot, Recombinant, Incubation, Molecular Weight, In Vitro, Sequencing, Purification, Isolation