Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Histone H3 , Cell Signaling , Cat #9715; RRID: AB_331563.

Techniques: Plasmid Preparation, Derivative Assay, Recombinant, In Situ, Staining, RNA Sequencing Assay, ChIP-sequencing, shRNA, Software

Journal: mBio

Article Title: HDA-2-Containing Complex Is Required for Activation of Catalase-3 Expression in Neurospora crassa

doi: 10.1128/mbio.01351-22

Figure Lengend Snippet: HDA-2-containing complex can bind to the cat-3 locus and regulate H4 acetylation. (A) Schematic depiction of cat-3 (NCU00355) on the Neurospora genome. (primer pairs 5 to 8) under the schematic indicate the regions tested by ChIP-qPCR. TSS, transcription start site; ORF, open reading frame. (B and C) ChIP assays showing the binding levels of HDA-2 (B) and SIF-2 (C) at the cat-3 locus in the WT, hda-2 KO (B), and sif-2 KO (C) strains. (D and E) ChIP assays showing the acetylation of H4 at the cat-3 locus in the WT, hda-2 KO (D), and sif-2 KO (E) strains. (F and G) ChIP assays showing the acetylation of H3 at the cat-3 locus in the WT, hda-2 KO (F), and sif-2 KO (G) strains. (H and I) ChIP assays showing the occupancy levels of H2B (H) and H3 (I) at the cat-3 locus in the WT, hda-2 KO , and sif-2 KO strains. Error bars show SD ( n = 3). Significance was assessed by using a two-tailed t test. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001.

Article Snippet: The ChIP was carried out with 3 μL of anti-H4ac antibody (06-866; Millipore), 3 μL of anti-H3ac antibody (06-599; Millipore), 3 μL of anti-H3 antibody (2650; CST), 2 μL of anti-H2B antibody (1790; abcam), 10 μL of anti-HDA-2 antibody, 10 μL of anti-SIF-2 antibody, 10 μL of anti-TFIIB antibody, 10 μL of anti-RPB-1 antibody, 10 μL of anti-H2A.Z antibody, 10 μL of anti-INO80 antibody, 10 μL of anti-ARP8 antibody.

Techniques: Binding Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Cannabinoid CB2 receptors are upregulated via bivalent histone modifications and control primary afferent input to the spinal cord in neuropathic pain

doi: 10.1016/j.jbc.2022.101999

Figure Lengend Snippet: Nerve injury alters activating and repressive histone modifications at the Cnr2 promoter in the DRG. A , top , schematic representation of the rat Cnr2 gene exon and intron composition of different transcript variants. The coding sequence (starting with ATG) of the same 360 amino acid (aa) long CB2 protein (rCB2A) is localized solely within the common Exon 2 of both transcript variant 1 and variant 2. Transcript variant 3 (Exon 1–3), although 100% identical to Exon 2, encodes a longer (410 aa) CB2 protein (rCB2B). Bottom , alignment of rat CB2 protein isoforms with corresponding human (hCB2) and mouse (mCB2) isoforms shows the presence of a distinct C-terminal sequence in the rCB2B isoform, whereas the rCB2A isoform shares a high degree of similarity with hCB2 and mCB2. B , real-time qPCR analysis of Cnr2 mRNA levels using specific primers for each indicated Exon in rat DRG tissues 21 days after sham or SNL surgery. Data are expressed as mean ± SEM (n = 6 independent experiments; p < 0.0001, F(5,30) = 53.36). ∗∗∗ p < 0.001 compared with the sham group of Exon 1 (one-way ANOVA followed by Dunnett’s post hoc test). C , ChIP-qPCR quantification shows the enrichment of histone marks at -463 bp to -373 bp, -115 bp to -15 bp, -53 bp to +28 bp, and +117 bp to +219 bp regions flanking the transcription start site of the rat Cnr2 gene on chromosome 5. DRG tissues were obtained from rats 21 days after sham or SNL surgery and subjected to ChIP-qPCR to quantify the immunoprecipitated DNA using the antibodies against H3K9ac, H3K4me3, H3K9me2, H3K27me3, and pan-histone H3. The percentage of their enrichment at each indicated region on the Cnr2 promoter was normalized to the input sample and corrected with that of pan-histone H3 values. Rabbit IgG was used as a negative control in immunoprecipitations. The rat negative control primers spanning a gene desert on rat chromosome 3 was used to confirm the specificity of ChIP-qPCR. Data are expressed as means ± SEM (n = 6 independent experiments per group; DRG tissues from three rats were pooled as one sample). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with the sham group (two-tailed Student’s t test). CB2, Type-2 cannabinoid receptors; ChIP-qPCR, chromatin immunoprecipitation–quantitative PCR; DRG, dorsal root ganglion; SNL, spinal nerve ligation.

Article Snippet: The chromatin was cleared from insoluble materials by centrifugation and further diluted prior to overnight immunoprecipitation using the protein G magnetic beads and 5 μg of the following antibodies: rabbit anti-histone H3 (#2650S, Cell Signaling Technology), rabbit anti-H3K4me3 (#9751S, Cell Signaling Technology), rabbit anti-H3K9ac (#9649S, Cell signaling Technology), rabbit anti-H3K9me2 (#4658S, Cell Signaling Technology), and rabbit anti-H3K27me3 (#9733S, Cell Signaling Technology).

Techniques: Sequencing, Variant Assay, Immunoprecipitation, Negative Control, Two Tailed Test, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Ligation