Journal: Clinical Cancer Research

Article Title: Zinc Finger MYND-Type Containing 8 (ZMYND8) Is Epigenetically Regulated in Mutant Isocitrate Dehydrogenase 1 (IDH1) Glioma to Promote Radioresistance

doi: 10.1158/1078-0432.ccr-22-1896

Figure Lengend Snippet: Figure 4. ZMYND8 knockout GCCs display reduced viability to irradiation, which is further enhanced when combined with BRD4 and HDAC inhibition. A, Experimental model in which ZMYND8 lentiviral particles were generated to knockout (KO) ZMYND8 expression in human and mouse GCCs mediated by CRISPR-Cas9-sgRNAs. ZMYND8 KO GCCs were selected based on resistance to 10 mg/mL puromycin for 1 week in mouse mIDH1 GCCs and 2 weeks for human mIDH1 GCCs. Representative Western blot quantification of ZMYND8 KO for (B) SF10602, (C) MGG119, and (D) NPAI mouse NS. Cellular viability of ZMYND8 WT, shown by a black line vs. ZMYND8 KO, represented by the red line, was assessed 72 hours after irradiation (IR) exposure using CellTiter-Glo assay in the (E) SF10602, (F) MGG119, and (G) NPAI. Results are expressed in relative luminescence units (RLU) to control nonirradiated (0 Gy) cells. H, Working model of ZMYND8-interacting partner bromodomain-containing protein (BRD4), where ZMYND8 is recruited to enhancer regions marked by H3K4me1 and contributes to the regulation of cancer cell survival and proliferation-associated genes. BRD4 inhibitors, JQ1 and I-BET-762, can disrupt this interaction. I, Working model of ZMYND8-interacting partner HDAC1/2 (histone deacetylase 1/2); a component of the NuRD complex, where ZMYND8 binds H3K14ac residues present at damaged chromatin regions and recruits HDAC along with other NuRD subunits: MDB2, CHD4, and GATAD2A. Panobinostat inhibits HDAC1/2 and prevents histone deacetylation mediated by HDAC1/2, which is required for transcriptional repression at regions of DNA damage. J, Representative bar graph of cellular viability measured in RLU, which shows the effect of BRD4 (JQ1, I-BET-762) or HDAC (panobinostat) inhibition alone (IR) or in combination with irradiation (þIR) in mouse NPAI ZMYND8 WT in blue vs. NPAI ZMYND8 KO in red. Errors bars represent SEM from independent biological replicates (n ¼ 3). ns, not significant; , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001; unpaired t test.

Article Snippet: After blocking, membranes were incubated with primary anti-phospho gH2AX (1:1,000; Cell Signaling Technologies, cat. #9718S), anti-gH2AX (1:1,000; Cell Signaling Technologies, cat. #2595S), primary antibody ZMYND8 (1:2,000; Bethyl Laboratories, cat. #A302-089A), primary antibody TIMELESS (1:1,000; Bethyl Laboratories, cat. #A300-960A), primary antibody proliferating cell nuclear antigen (PCNA) (1:1,000; Cell Signaling Technologies, cat. #2586T), primary antibody TREX1 (1:1,000; Abcam, cat. #ab185228), primary antibody BRD4 (1:1,000; Cell Signaling Technology, cat. #83375B), primary antibody HDAC1 (1:1,000; Cell Signaling Technology, cat. #2062S), primary antibody HDAC2 (1:1,000; Cell Signaling Technology, cat. # 57156S), Vinculin (1:3,000; Thermo Fisher Scientific, cat. #7,000,062), or b-tubulin antibodies (1:4,000; Sigma-Aldrich, cat. #A1978) overnight at 4 C. The next day, blots were washed with TBS-0.1% tween-20 and incubated with secondary (1:4,000) antibodies [Dako, Agilent Technologies, goat anti-rabbit 1:4,000 (cat. # P0448), rabbit anti-mouse 1:4,000 (cat. # P0260)] for 1 hour at RT.

Techniques: Knock-Out, Irradiation, Inhibition, Generated, Expressing, CRISPR, Western Blot, Glo Assay, Control, Histone Deacetylase Assay

Journal: Nature

Article Title: Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.

doi: 10.1038/s41586-024-08533-3

Figure Lengend Snippet: Fig. 3 | Structural mechanisms and amino acid preferences of functional KBTBD4 mutations. a, Cryo-EM map of LHC-bound KBTBD4 mutants with the two KBTBD4 protomers (slate and green), HDAC1 (pink), CoREST (orange) and InsP6 (red). Left, KBTBD4-PR; right, KBTBD4-TTYML. b, Ribbon diagram of the KBTBD4-PR–HDAC1–CoREST–InsP6 complex. Subunits of the complex are coloured the same way as in a. The hotspot arginine residue is shown in space filling model mode. InsP6 is shown in cyan and red sticks. c, Close-up view of the 4b-4c loops of KBTBD4-PR-A (slate) and KBTBD4-PR-B (green) after the β-propeller domain of the latter is superimposed onto that of the former. Side chains of two phenylalanine residues in the 4b-4c loop of KBTBD4-PR-A are shown in sticks. d, Close-up view of the 2b-2c loops of KBTBD4-A (slate) and KBTBD4-B (green) after the β-propeller domain of the former is superimposed

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used LSD1 (Bethyl Laboratories, A300-215A, Lot no. 2) RCOR1 (Cell Signaling Technology, #14567, Lot no. 1) GAPDH (Santa Cruz Biotechnology, sc-477724, Lot no. G2920; RRID: AB_627678) HA (Cell Signaling Technology, #3724, Lot no. 10) FLAG (Sigma-Aldrich, F1804, Lot no. #SLCN3722) KBTBD4 (Novus Biologicals, NBP1-88587, Lot no. A116815 ) HDAC1 (Cell Signaling Technology, #34589, Lot no. 4) HDAC2 (Cell Signaling Technology, #57156, Lot no. 1) Anti-Rabbit IgG HRP conjugate (Promega, Cat#W4011, RRID:AB_430833) Anti-Mouse IgG HRP conjugate (Promega, Cat#W4021, RRID:AB_430834) PierceTM Anti-HA Magnetic Beads (Thermo Scientific, #88837) Validation All antibodies used were commercial and validated for the appropriate application.

Techniques: Functional Assay, Cryo-EM Sample Prep, Residue

Journal: Nature

Article Title: Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.

doi: 10.1038/s41586-024-08533-3

Figure Lengend Snippet: Fig. 4 | Converging mechanism between KBTBD4 cancer mutations and UM171. a, Simplified ligand plot of UM171–KBTBD4–HDAC1 interactions. HDAC1 and KBTBD4 residues are denoted by pink and green circles, respectively. b, Superposition analysis of the β-propellers in protomer-B of the KBTBD4-WT and KBTBD4-PR dimers. The structural differences at several top surface loops are indicated by arrows. The 2b-2c loop is labelled. c, A comparison of UM171 (yellow and blue sticks), the side chain of Tyr312 of KBTBD4-TTYML-B (cyan and red sticks) and the side chain of Arg312 of KBTBD4-PR-B (green and blue sticks) at the active site pocket of HDAC1 (pink). The three complex structures are superimposed through HDAC1. Two phenylalanine residues outlining the entrance of the HDAC1 active site tunnel are shown in sticks. d, A comparison between UM171 (yellow and blue sticks) and the 2b-2c loop of KBTBD4-PR-B with the KBTBD4–UM171–HDAC1 structure superimposed with the KBTBD4- PR–HDAC1 structure through HDAC1. The side chains of key residues at the interface are shown in sticks. e, A comparison between UM171 (yellow and blue sticks) and the 2b-2c loop of KBTBD4-TTYML-B with the KBTBD4–UM171– HDAC1 structure superimposed with the KBTBD4-TTYML–HDAC1 structure through HDAC1. The side chains of key residues at the interface are shown in sticks. f, A close-up view of the inter-molecular interfaces among KBTBD4- TTYML (green), HDAC1 (pink, surface representation), CoREST (orange, surface representation) and InsP6 (cyan, orange and red sticks). The side chains of key KBTBD4-TTYML residues involved in InsP6 interaction and at the nearby 2b-2c loop are shown in sticks. Zn, zinc.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used LSD1 (Bethyl Laboratories, A300-215A, Lot no. 2) RCOR1 (Cell Signaling Technology, #14567, Lot no. 1) GAPDH (Santa Cruz Biotechnology, sc-477724, Lot no. G2920; RRID: AB_627678) HA (Cell Signaling Technology, #3724, Lot no. 10) FLAG (Sigma-Aldrich, F1804, Lot no. #SLCN3722) KBTBD4 (Novus Biologicals, NBP1-88587, Lot no. A116815 ) HDAC1 (Cell Signaling Technology, #34589, Lot no. 4) HDAC2 (Cell Signaling Technology, #57156, Lot no. 1) Anti-Rabbit IgG HRP conjugate (Promega, Cat#W4011, RRID:AB_430833) Anti-Mouse IgG HRP conjugate (Promega, Cat#W4021, RRID:AB_430834) PierceTM Anti-HA Magnetic Beads (Thermo Scientific, #88837) Validation All antibodies used were commercial and validated for the appropriate application.

Techniques: Comparison

Journal: Nature

Article Title: Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.

doi: 10.1038/s41586-024-08533-3

Figure Lengend Snippet: Fig. 5 | HDAC1/2 inhibitors block the neomorphic activity of KBTBD4 mutants. a, Steric clash between SAHA (yellow) and the central arginine residue at the 2b-2c loop of KBTBD4-PR-B. The KBTBD4-PR–HDAC1 complex structure is superimposed with the HDAC2–SAHA complex structure (PDB 4LXZ) through the HDAC subunits. b, Flow cytometry quantification of GFP+ cells for KBTBD4-null CoREST–GFP cells pre-treated with DMSO, CI-994 (10 µM), SAHA (10 µM) or RBC1HI (10 µM) for 1 h followed by dox-inducible overexpression of the indicated KBTBD4 variant. Data are mean ± s.d. of n = 3 biological replicates. c, Immunoblots of HA IP from 293T cells transfected with the indicated HA–KBTBD4 variant, pre-treated with MLN4924 (1 µM) for 3 h, and then treated with DMSO, UM171 (1 µM) or SAHA (10 µM) for 1 h. d, TR-FRET signal between fluorescein–LHC and anti-His CoraFluor-1-labelled antibody with indicated His–KBTBD4 mutant in the presence of DMSO, SAHA (10 µM), CI-994 (10 µM) or RBC1HI (10 µM) (n = 2 biological replicates). e, Ex vivo proliferation for ICB1572 (KBTBD4-PR), MED411FH (KBTBD4-WT) and RCMB28 (KBTBD4-WT) cells with RBC1HI treatment at indicated doses for 72 h. Data are mean ± s.d. across biological replicates from PDX cells derived from n = 5 (ICB1572), n = 3 (RCMB28) and n = 2 (MED411FH) implanted mice. f, Immunoblots showing LSD1, CoREST and GAPDH in ICB1572 after 24 h treatment with MLN4924 or RBC1HI at the indicated doses. Data in b and d and immunoblots in c and f are representative of two independent experiments. FACS-gating schemes and uncropped blots can be found in Supplementary Figs. 1a and 3, respectively.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used LSD1 (Bethyl Laboratories, A300-215A, Lot no. 2) RCOR1 (Cell Signaling Technology, #14567, Lot no. 1) GAPDH (Santa Cruz Biotechnology, sc-477724, Lot no. G2920; RRID: AB_627678) HA (Cell Signaling Technology, #3724, Lot no. 10) FLAG (Sigma-Aldrich, F1804, Lot no. #SLCN3722) KBTBD4 (Novus Biologicals, NBP1-88587, Lot no. A116815 ) HDAC1 (Cell Signaling Technology, #34589, Lot no. 4) HDAC2 (Cell Signaling Technology, #57156, Lot no. 1) Anti-Rabbit IgG HRP conjugate (Promega, Cat#W4011, RRID:AB_430833) Anti-Mouse IgG HRP conjugate (Promega, Cat#W4021, RRID:AB_430834) PierceTM Anti-HA Magnetic Beads (Thermo Scientific, #88837) Validation All antibodies used were commercial and validated for the appropriate application.

Techniques: Blocking Assay, Activity Assay, Residue, Flow Cytometry, Over Expression, Variant Assay, Western Blot, Transfection, Mutagenesis, Ex Vivo, Derivative Assay

Journal: Cell reports

Article Title: PRMT1 promotes epigenetic reprogramming associated with acquired chemoresistance in pancreatic cancer

doi: 10.1016/j.celrep.2024.114176

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-HDAC1 , Proteintech , Cat#10197–1-AP; RRID:AB_2920338.

Techniques: Recombinant, cDNA Synthesis, SYBR Green Assay, Bicinchoninic Acid Protein Assay, DNA Extraction, Sequencing, Gene Expression, Plasmid Preparation, Software

Journal: Experimental & Molecular Medicine

Article Title: Canonical Wnt signaling regulates Mbd3 protein stability during neurogenesis

doi: 10.1038/s12276-025-01510-4

Figure Lengend Snippet: a – c ChIP–qPCR analysis of Mbd3 ( a ), HDAC1 ( b ) and MTA1 ( c ) occupancy at Mbd3-binding loci in undifferentiated and differentiated cells in different stimuli for the Wnt signaling cascade ( N = 3). Data are presented as mean ± s.d.; one-way ANOVA was performed to calculate the significance (* P < 0.05, ** P < 0.01, *** P < 0.001). d A model of the regulatory role of canonical Wnt signaling on cell-fate determination during neurogenesis, in which Mbd3–NuRD functions downstream to suppress the transcription of neural differentiation genes, thereby maintaining the stem cell pool.

Article Snippet: To deplete specific target genes, GSK3β siRNA (sc-35525, Santa Cruz Biotechnology), Mbd3 siRNA (sc-35868, Santa Cruz Biotechnology) and HDAC1 siRNA (sc-29344, Santa Cruz Biotechnology) were used and tested against a control siRNA construct (sc-37007).

Techniques: ChIP-qPCR, Binding Assay

Journal: Experimental & Molecular Medicine

Article Title: Canonical Wnt signaling regulates Mbd3 protein stability during neurogenesis

doi: 10.1038/s12276-025-01510-4

Figure Lengend Snippet: a – c , ChIP–qPCR analysis of Mbd3 ( a ), HDAC1 ( b ) and MTA1 ( c ) occupancy at Mbd3-binding loci in undifferentiated and differentiated cells with or without CHIR treatment ( N = 3). Data are presented as mean ± s.d.; one-way ANOVA was performed to calculate the significance (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: To deplete specific target genes, GSK3β siRNA (sc-35525, Santa Cruz Biotechnology), Mbd3 siRNA (sc-35868, Santa Cruz Biotechnology) and HDAC1 siRNA (sc-29344, Santa Cruz Biotechnology) were used and tested against a control siRNA construct (sc-37007).

Techniques: ChIP-qPCR, Binding Assay

Journal: Experimental & Molecular Medicine

Article Title: Canonical Wnt signaling regulates Mbd3 protein stability during neurogenesis

doi: 10.1038/s12276-025-01510-4

Figure Lengend Snippet: a , b Reciprocal ChIP–qPCR targeting Mbd3 ( a ) and HDAC1 ( b ) in Mbd3-knockdown cells, with or without Wnt3a supplementation ( N = 3). c , d Reciprocal ChIP–qPCR targeting Mbd3 ( c ) and HDAC1 ( d ) in HDAC1-knockdown cells, with or without Wnt3a supplementation ( N = 3). Data are presented as mean ± s.d.; one-way ANOVA was performed to calculate the significance (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: To deplete specific target genes, GSK3β siRNA (sc-35525, Santa Cruz Biotechnology), Mbd3 siRNA (sc-35868, Santa Cruz Biotechnology) and HDAC1 siRNA (sc-29344, Santa Cruz Biotechnology) were used and tested against a control siRNA construct (sc-37007).

Techniques: ChIP-qPCR, Knockdown