Journal: Communications Biology

Article Title: Arabidopsis LSH10 transcription factor and OTLD1 histone deubiquitinase interact and transcriptionally regulate the same target genes

doi: 10.1038/s42003-023-04424-x

Figure Lengend Snippet: a Association of LSH10-His6 with the chromatin of the indicated target genes in the lsh10-1/LSH10-His6 plants. lsh10-1/LSH10-His6 , white bars; non-specific background signal obtained in the wild-type plants that do not express the His6 epitope, gray bars. The outside probe is the qChIP probe, depicted in Fig. , and located outside the EMSA probe locations. b Increase in H2B monoubiquitylation of the OSR2, WUS, ABI5 , and ARL promoter chromatin in the lsh10-1 mutant plants. Wild-type plants, dark gray bars; lsh10-1 , light gray bars; lsh10-2 , white bars. The chromatin association of LSH10-His6 and the degree of H2B monoubiquitylation were analyzed by qChIP with probes described in Fig. and listed in Supplementary Data . Error bars represent the SEM of n = 5 or n = 7 biological replicates with 3 technical repeats for each. The individual data points are indicated, and their numerical values are listed in Supplementary Data and Supplementary Data . Differences between mean values assessed by the two-tailed t-test are statistically significant for the p -values * p < 0.05 and ** p < 0.01; P ≥ 0.05 are not statistically significant (ns).

Article Snippet: 5 µl of the crude chromatin extracts were saved for use as an input control, and 100 µl were added into the microwell immobilized with the antibodies (non-specific rabbit IgG Isotype Control (Invitrogen, WB317638, 1 µg) as the negative control, specific rabbit anti-His-tag antibody (GenScript, A00174-40, 2.5 µg) or anti-monoubiquityl-histone H2B (Lys-120) (5546S, Cell Signaling Technology, Inc., 2.5 µg).

Techniques: Mutagenesis, Two Tailed Test

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: 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: iScience

Article Title: Histone malonylation is regulated by SIRT5 and KAT2A

doi: 10.1016/j.isci.2023.106193

Figure Lengend Snippet: Histone H2B is malonylated at K5 in SIRT5 KO mouse brain and liver (A) In vitro lysine malonyltransferase (KMaT) activity assay of the catalytic domains of KAT2A using synthesized histone peptides (20 amino acids at N terminus) as substrates at given concentrations in the presence of 50 μM malonyl-CoA. The reactions were stopped after incubation at 37°C for 20 min. KMaT activity was evaluated by measuring CoA-SH production using CPM at Ex = 390 nm/Em = 460 nm. (B and C) Non-enzymatic malonylation was examined by incubating synthesized histone peptides with 50 μM malonyl-CoA, and measuring the production of CoA-SH using CPM over time at Ex = 390 nm/Em = 460 nm (B). The slope was calculated to reflect the velocity of non-enzymatic of malonylation (C). (D) Extracted ion chromatograms of PDPA 6 KmalSAPAPK ( m / z 589.81, z = 2+) from mouse histone H2B type 2-B (Q64525) obtained by data-independent acquisition, that indicates that malonylation of K5 is increased in S5KO mouse brain tissues compared to wildtype (WT). (E) Quantification using the peak areas of four transitions of PDPA 6 KmalSAPAPK ( m / z 589.81, z = 2+) in four biological replicates of S5KO and WT mouse brain tissues, respectively. (F) Malonylated lysine sites on histones were previously identified in WT and S5KO mouse livers (Nishida, et al., Mol Cell. 2015). n = 5. Error bar: mean ± SD. ∗∗∗∗p < 10 −4 using Sidak multiple comparisons test following two-way ANOVA.

Article Snippet: Before SDS-PAGE, protein samples were heated at 70°C for 5 min. Antibodies used are listed as follows: anti-β-tubulin (Cell Signaling Technology, Cat. #2128), anti-SIRT5 (Cell Signaling Technology, Cat. #8782), anti-acetyl-lysine (Cell Signaling Technology, Cat. #9814), anti-malonyl-lysine (PTM Biolabs, Cat. #PTM-901), anti-succinyl-lysine (PTM Biolabs, Cat. # PTM-401), anti-glutaryl-lysine (PTM Biolabs, Cat. #PTM-1151), anti-ACC (Cell Signaling Technology, Cat. #3676), anti-phospho-ACC (Ser79) (Cell Signaling Technology, Cat. #3661), anti-H2B (Cell Signaling Technology, Cat. #12364), anti-KAT2A (Cell Signaling Technology, Cat. #3305), anti-HSP60 (Abcam, Cat. #ab59457), anti-Lamin A/C (Cell Signaling Technology, Cat. #2032), anti-COX IV (Abcam, Cat. #ab16056), and anti-GAPDH (Cell Signaling Technology, Cat. #5174).

Techniques: In Vitro, Activity Assay, Synthesized, Incubation

Journal: iScience

Article Title: Histone malonylation is regulated by SIRT5 and KAT2A

doi: 10.1016/j.isci.2023.106193

Figure Lengend Snippet:

Article Snippet: Before SDS-PAGE, protein samples were heated at 70°C for 5 min. Antibodies used are listed as follows: anti-β-tubulin (Cell Signaling Technology, Cat. #2128), anti-SIRT5 (Cell Signaling Technology, Cat. #8782), anti-acetyl-lysine (Cell Signaling Technology, Cat. #9814), anti-malonyl-lysine (PTM Biolabs, Cat. #PTM-901), anti-succinyl-lysine (PTM Biolabs, Cat. # PTM-401), anti-glutaryl-lysine (PTM Biolabs, Cat. #PTM-1151), anti-ACC (Cell Signaling Technology, Cat. #3676), anti-phospho-ACC (Ser79) (Cell Signaling Technology, Cat. #3661), anti-H2B (Cell Signaling Technology, Cat. #12364), anti-KAT2A (Cell Signaling Technology, Cat. #3305), anti-HSP60 (Abcam, Cat. #ab59457), anti-Lamin A/C (Cell Signaling Technology, Cat. #2032), anti-COX IV (Abcam, Cat. #ab16056), and anti-GAPDH (Cell Signaling Technology, Cat. #5174).

Techniques: Recombinant, Plasmid Preparation, Software

Journal: iScience

Article Title: Histone malonylation is regulated by SIRT5 and KAT2A

doi: 10.1016/j.isci.2023.106193

Figure Lengend Snippet: Histone H2B is malonylated at K5 in SIRT5 KO mouse brain and liver (A) In vitro lysine malonyltransferase (KMaT) activity assay of the catalytic domains of KAT2A using synthesized histone peptides (20 amino acids at N terminus) as substrates at given concentrations in the presence of 50 μM malonyl-CoA. The reactions were stopped after incubation at 37°C for 20 min. KMaT activity was evaluated by measuring CoA-SH production using CPM at Ex = 390 nm/Em = 460 nm. (B and C) Non-enzymatic malonylation was examined by incubating synthesized histone peptides with 50 μM malonyl-CoA, and measuring the production of CoA-SH using CPM over time at Ex = 390 nm/Em = 460 nm (B). The slope was calculated to reflect the velocity of non-enzymatic of malonylation (C). (D) Extracted ion chromatograms of PDPA 6 KmalSAPAPK ( m / z 589.81, z = 2+) from mouse histone H2B type 2-B (Q64525) obtained by data-independent acquisition, that indicates that malonylation of K5 is increased in S5KO mouse brain tissues compared to wildtype (WT). (E) Quantification using the peak areas of four transitions of PDPA 6 KmalSAPAPK ( m / z 589.81, z = 2+) in four biological replicates of S5KO and WT mouse brain tissues, respectively. (F) Malonylated lysine sites on histones were previously identified in WT and S5KO mouse livers (Nishida, et al., Mol Cell. 2015). n = 5. Error bar: mean ± SD. ∗∗∗∗p < 10 −4 using Sidak multiple comparisons test following two-way ANOVA.

Article Snippet: Rabbit monoclonal anti-H2B , Cell Signaling Technology , Cat. #12364, RRID: AB_2714167.

Techniques: In Vitro, Activity Assay, Synthesized, Incubation

Journal: iScience

Article Title: Histone malonylation is regulated by SIRT5 and KAT2A

doi: 10.1016/j.isci.2023.106193

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-H2B , Cell Signaling Technology , Cat. #12364, RRID: AB_2714167.

Techniques: Recombinant, Plasmid Preparation, Software

Journal: Communications Biology

Article Title: A maladaptive feedback mechanism between the extracellular matrix and cytoskeleton contributes to hypertrophic cardiomyopathy pathophysiology

doi: 10.1038/s42003-022-04278-9

Figure Lengend Snippet: Immunoblot analysis of L-type calcium channel ( a , b ) and β 1 integrin ( c , d ) protein expression performed on total heart homogenate pooled from groups of 5 pre- (10–15-wk-old) or post-cardiomyopathic (30–50-week-old) cTnI-G203S mice and age-matched wt counterparts. Representative immunoblots probed with L-type calcium channel α 1C subunit (Ca V 1.2, a ) or β 1 integrin ( c ) antibody, then GAPDH monoclonal antibody. Densitometry analysis of Ca V 1.2 ( b ) or β 1 integrin ( d ) protein expression, normalized to associated GAPDH expression. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( b ) or Kruskal-Wallis test ( d ) determined statistical significance. Densitometry analysis of relative mTOR expression (calculated as Phospho-mTOR/Total mTOR) performed on cytoplasmic ( e ) and nuclear ( f ) fractions pooled from groups of five pre- or post-cardiomyopathic cTnI-G203S mice and age-matched wt counterparts. β-tubulin and histone H2B antibodies were used as loading controls for cytoplasmic and nuclear fractions respectively. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( e ) or Kruskal-Wallis test ( f ) determined statistical significance. g Schematic indicating structural-functional link between the L-type calcium channel, cytoskeletal network, mitochondria, integrin and the extracellular matrix in wt and cTnI-G203S cardiac myocytes. A disrupted cytoskeletal architecture in cTnI-G203S cardiac myocytes may trigger a maladaptive feedback mechanism between increased cytoskeletal and extracellular matrix stiffness, resulting in a hypermetabolic mitochondrial state.

Article Snippet: Histone H2B (D2H6, rabbit mAb, Cell Signaling Technology, 12364): Species: H, M, R, Mk; Applications: WB, IHC, ChiP; Validated by Cell Signaling Technology.

Techniques: Western Blot, Expressing, Functional Assay

Journal: Science Advances

Article Title: ZBTB18 restricts chromatin accessibility and prevents transcriptional adaptations that drive metastasis

doi: 10.1126/sciadv.abq3951

Figure Lengend Snippet: ( A ) PCA of RNA-seq data indicates that E0771GFP, M11GFP, and M12GFP have distinct gene expression patterns ( n = 3 biological replicates per cell line). ( B ) Differential gene expression for M11GFP versus M12GFP and M11GFP versus E0771GFP. Only genes with significant differential expression ( P < 0.05) for both comparisons are shown. ( C ) Numbers of transcription factors with increased target gene expression ( P < 0.05) when comparing RNA-seq data obtained from E0771GFP, M11GFP, and M12GFP cell lines, as determined using GSEA analysis with the MSigDB C3 TFT (transcription factor target) collection. Genes with an FPKM of >1 were included in this analysis. ( D ) GSEA enrichment plot for ZBTB18 target gene expression in M11GFP cells compared with E0771GFP cells (left) and M12GFP cells (right). NES, normalized enrichment score. ( E ) Relative Zbtb18 mRNA levels in E0771GFP versus M11GFP (left; n = 3 biological replicates) and 67NR versus 4T1 (right; n = 4 biological replicates) cells, as determined by RT-qPCR. ( F ) ZBTB18 protein levels, detected by immunoblotting. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. Two biological replicates are shown. Right: Relative ZBTB18 expression levels calculated after normalization with GAPDH ( n = 4 biological replicates). ( G and H ) Relative expression levels of selected ZBTB18 target genes in E0771GFP and M11GFP (G) ( n = 3 biological replicates) or 67NR and 4T1 (H) ( n = 4 biological replicates) cells, as determined by RT-qPCR. ( I ) ZBTB18 protein expression levels, in whole-cell lysates and nuclear fractions, detected by immunoblotting. Nuclear histone H2B (H2B) and cytoplasmic GAPDH were used as controls. Relative ZBTB18 expression levels calculated after normalization with H2B are shown. ( J ) Immunofluorescence staining for ZBTB18 in E0771GFP and M11GFP cells. Cell nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bar, 50 μm. (E to H) Means ± SEM, unpaired two-sided t test. ns P > 0.05, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The primary antibodies used were anti-ZBTB18 (ZNF238; Proteintech, #12714-1-AP; RRID:AB_2218388; Atlas Antibodies, HPA074019), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore, #MAB374; RRID:AB_2107445), anti–histone H2B (H2B; Cell Signaling Technology, #12364;; RRID:AB_2714167), anti–phospho-Smad2 (Cell Signaling Technology, #3108, RRID:AB_490941), anti-Smad2/3 (Cell Signaling Technology, #8685; RRID:AB_10889933), and anti-TGFBR2 (Cell Signaling Technology, #79424; RRID:AB_2799933).

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining