Journal: Cell reports

Article Title: Coordinated neuron-specific splicing events restrict nucleosome engagement of the LSD1 histone demethylase complex

doi: 10.1016/j.celrep.2024.115213

Figure Lengend Snippet: (A) EMSA with Widom 601 DNA and PHF21A fragments. (B) EMSA with recombinant nucleosome core particles (NCPs). (C) EMSA assay using nucleosomal DNA extracted from 293T-derived mononucleosomes. (D) EMSA assay using 293T-derived mononucleosomes. (E) The amino acid sequence of PHF21A isoforms and the AT-hook-mutant. The core AT-hook motif amino acids GRP were replaced with ANE, the corresponding PHF21A-n sequence. Basic (+) and polar (*) amino acids that potentially contribute to DNA binding are denoted. (F) NCP-EMSA with PHD only, AT-hook only, AT-hook-PHD, and the AT-hook-mutant.

Article Snippet: Dimethylated nucleosomes (H3K4me2, H3K9me2, H3K27me2, H3K36me2, H4K20me2-containing nucleosomes) , Epicypher , 16–0334, 16–0324, 16–0339, 16–0319, 16–0332.

Techniques: Recombinant, Derivative Assay, Sequencing, Mutagenesis, Binding Assay

Journal: Cell reports

Article Title: Coordinated neuron-specific splicing events restrict nucleosome engagement of the LSD1 histone demethylase complex

doi: 10.1016/j.celrep.2024.115213

Figure Lengend Snippet: (A) Schematics of reconstituted complexes using protein prepared with Sf21 cells. (B) Input (lanes 1–5), LSD1, and CoREST were co-expressed in Sf21 cells where the LSD1 N terminus was fused to the MBP tag, pulled down by amylose resins, and further purified by a size-exclusion column. The bipartite LSD1-CoREST complex was then incubated with unmodified recombinant mononucleosomes (NCPs) with H3K4M substitution with or without recombinant PHF21A, pulled down with amylose resins, separated by SDS-PAGE, and visualized with Coomassie staining. (C) Quantification of pulled-down NCP. Pulled-down histones’ band intensity was normalized with the input histone signals ( n = 3, mean ± SEM). p values were calculated using one-way ANOVA followed by the Tukey test with multiple comparison corrections. * p < 0.002 and ** p < 0.0001. (D) EMSA with titration of LC-c and LC-n using H3K4M-Widom 601 mononucleosomes. (E) Quantification of the EMSA results in (D), n = 3 independent reactions, mean ± SEM. (F) EMSA with titration of PLC-c and PLC-n with a constant concentration of LC-c, LC-n, and the nucleosomes. (G) The crystal structure of the canonical LSD1 (cLSD1)-CoREST in the complex with the 191-bp nucleosome (PDB: 6VYP) visualized by PyMOL. Red: the position of the additional four residues (DTVK) of the LSD1-n, in which threonine is known to be phosphorylated (Ph).

Article Snippet: Dimethylated nucleosomes (H3K4me2, H3K9me2, H3K27me2, H3K36me2, H4K20me2-containing nucleosomes) , Epicypher , 16–0334, 16–0324, 16–0339, 16–0319, 16–0332.

Techniques: Purification, Incubation, Recombinant, SDS Page, Staining, Comparison, Titration, Concentration Assay

Journal: Cell reports

Article Title: Coordinated neuron-specific splicing events restrict nucleosome engagement of the LSD1 histone demethylase complex

doi: 10.1016/j.celrep.2024.115213

Figure Lengend Snippet: (A) Demethylation assays. The reconstituted complexes were incubated with the designer nucleosomes carrying the denoted histone di-methylation and analyzed by quantitative western blot using antibodies against corresponding methylations. (B) Quantification of the western blots. Signals were normalized to H3 levels measured by anti-pan H3 or H4 antibodies ( n = 3 independent reactions, mean ± SEM).

Article Snippet: Dimethylated nucleosomes (H3K4me2, H3K9me2, H3K27me2, H3K36me2, H4K20me2-containing nucleosomes) , Epicypher , 16–0334, 16–0324, 16–0339, 16–0319, 16–0332.

Techniques: Incubation, Methylation, Western Blot

Journal: Cell reports

Article Title: Coordinated neuron-specific splicing events restrict nucleosome engagement of the LSD1 histone demethylase complex

doi: 10.1016/j.celrep.2024.115213

Figure Lengend Snippet:

Article Snippet: Dimethylated nucleosomes (H3K4me2, H3K9me2, H3K27me2, H3K36me2, H4K20me2-containing nucleosomes) , Epicypher , 16–0334, 16–0324, 16–0339, 16–0319, 16–0332.

Techniques: Strep-tag, Virus, Expressing, Recombinant, Construct, Clone Assay, Software

Journal: The American journal of the medical sciences

Article Title: WITHDRAWN: Demethylation of Di-Methylation of Lysine 4 on Histone 3 Is Inhibited by General Control Nondepressible 5-Induced Acetylation of Lysine-Specific Demethylase 1.

doi: 10.1016/j.amjms.2020.01.002

Figure Lengend Snippet: FIGURE 3. Acetylation-induced impairment of affinity between lysine-specific demethylase 1 (LSD1) and nucleosomes contributed to a decrease in demethylation of di-methylation of lysine 4 on his- tone 3 (H3K4me2). A, LSD1 acetylation attenuated its affinity to nucleosomes in vitro. Nucleosomes were reconstituted with recom- binant core histones and biotin-labeled DNA (Bio-nucl). Bio-nucl was then incubated with HA-LSD1 (WT, K-Q or K-R) proteins and then isolated with streptavidin beads. LSD1 K-Q was used to mimic LSD1 acetylation. LSD1 K-R was resistant to acetylation. The asso- ciated proteins were examined by immunoblotting with antibodies against HA and LSD1. B, LSD1 acetylation did not affect its ability to de-methylate H3K4me2 in bulk histones in vitro. Bulk histones were incubated with HA-LSD1 (WT, K-R or K-Q), followed by immunoblot- ting with antibodies against H3K4me2 and HA. H3K4me3 was used as the loading control. C, LSD1 acetylation abolished its demethyla- tion activity in H3K4me2. Mononucleosomes from SW48 cells were incubated with HA-tagged LSD1 (WT, K-R and K-Q). Immunoblot assay was performed to detect H3K4me2, H3K4me3 and HA.

Article Snippet: Separation by SDS-PAGE and detection by immunoblotting were carried out to detect histone H3K4me2 and H3K4me3 with antibodies (orb229069; orb229070; Biorbyt).

Techniques: Methylation, In Vitro, Labeling, Incubation, Isolation, Western Blot, Control, Activity Assay

Journal: Molecular cell

Article Title: Examining the roles of H3K4 methylation states with systematically characterized antibodies

doi: 10.1016/j.molcel.2018.08.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-H3K4me2 antibody Epicypher 13-0013, Lot Number 14247001. , Epicypher , Cat#13-0013, RRID:AB_.

Techniques: Virus, Recombinant, Synthesized, Peptide Microarray, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Transcriptional effects of actin-binding compounds: the cytoplasm sets the tone

doi: 10.1007/s00018-018-2919-4

Figure Lengend Snippet: Primary antibodies

Article Snippet: Table 1 Antibody Supplier MRTF-A (G-8) mouse mAb IgG 2a , sc-390324 Santa Cruz Biotechnology, Dallas, TX, USA YAP (B-8) mouse mAb IgG 2a , sc-398182 Santa Cruz Biotechnology, Dallas, TX, USA YAP (D8H1X) XP ® rabbit mAb Cell Signaling Technology, Danvers, MA, USA Angiomotin (D2O4H) rabbit mAb Cell Signaling Technology, Danvers, MA, USA Monoclonal Anti-BrdU Clone BU-33 Sigma-Aldrich, St. Louis, MO, USA HA-Tag (6E2) mouse mAb Cell Signaling Technology, Danvers, MA, USA Histone H3 (acetyl K9), mouse mAb IgG 1 , ab12179 Abcam, Cambridge, UK Histone H3, ab1791 Abcam, Cambridge, UK Histone H3 (trimethyl K4), mouse mAb IgG 1 , ab12209 Abcam, Cambridge, UK Histone H3 (dimethyl K4), mouse mAb IgG 1 , 25254 BPS Bioscience, CA, USA POLR1A, rabbit mAb IgG 1 , HPA031513 Sigma-Aldrich, St. Louis, MO, USA Anti-ACTB, mouse mAb IgG 1 , AMAB91241 Sigma-Aldrich, St. Louis, MO, USA Open in a separate window Primary antibodies.

Techniques:

Journal: Cells

Article Title: Transcriptional Control of Subcutaneous Adipose Tissue by the Transcription Factor CTCF Modulates Heterogeneity in Fat Distribution in Women

doi: 10.3390/cells13010086

Figure Lengend Snippet: Apple and pear enriched CTCF binding sites target different genomic regions. ( A ) Proportional Venn diagrams showing the number of genes with pear enriched (yellow), apple enriched (blue), and common CTCF binding sites (intersection) in ABD ADSCs. ( B ) Bar graph representing the genomic distribution of the body shape-enriched CTCF binding sites in ABD ADSCs. ( C ) Heatmap of the emission parameters in which each column corresponds to a different state and each row corresponds to a different mark for four histone modifications (H3K4me2, H3K4me3, H3K27me3, and H3K27ac), ATAC-seq, RNAPII, and CTCF. ( D ) ChromHMM heat map representation showing signals around the apple (left side) and pear (right side) enriched CTCF binding sites in apple and pear samples ( n = 6). The colors referred to the different states in ( C ).

Article Snippet: The following ChIP grade antibodies were used: rabbit anti-H3K4me3 (Diagenode—C15410003), rabbit anti-H3K4me2 (DIagenode—pAb-035-050), rabbit anti-H3K27me3 (Diagenode—C15410069), rabbit anti-H3K27Ac (Abcam—ab4729), anti-CTCF antibody (Actif Motif—61311), and rabbit anti-RNAPII (Abcam—ab5095) to study RNA Polymerase II binding.

Techniques: Binding Assay