Journal: Science (New York, N.Y.)

Article Title: JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications

doi: 10.1126/science.abc3393

Figure Lengend Snippet: (A) Schematic representation of the proteins in the PRC2-AEBP2-JARID2 complex. Newly modeled regions in EZH2, JARID2, and AEBP2 that contribute to the interaction with the nucleosome are marked by dashed boxes. (B) Coomassie-stained gel showing all of the PRC2-core subunits, cofactors JARID2 and AEBP2, and histone proteins in the sample used for structural studies. (C) (Top) Cryo-EM density map for PRC2-AJ1–450 bound to an H2AK119ub1-containing nucleosome. (Bottom) Atomic model of PRC2-AJ1–450 bound to an H2AK119ub1-containing nucleosome, with EZH2 (SANT1) highlighted in gold, EZH2 (SANT2) in hot pink, EZH2 (SET) in blue, EED in light blue, RBAP48 in light purple, SUZ12 in green, JARID2 in magenta, AEBP2 in red, ubiquitin in orange, histone H3 in light pink, H2A in salmon, H4 and H2B in khaki, and nucleosome DNA in cyan. The image shown is a composite map where the PRC2 and nucleosome core correspond to the local resolution-filtered map from multibody refinement, whereas the ubiquitin, JARID2 UIM, and AEBP2 zinc finger densities correspond to the local resolution-filtered map from the consensus refinement. This color coding also applies to movie S1.

Article Snippet: All human nucleosomes were purchased from Epicypher with the 5′ biotinylated 187-bp DNA sequence containing the 601-nucleosome positioning sequence: 5′(BioTEG)GGACCCTATACGCGGCCGCCCtggagaatcccggtctgcaggccgctcaattggtcgtagacagctctacgtggcgaatttgcgtgcatgcgcctgtcccccgcgttttaaccgccaaggggattactccctagtctccaggcacgtgtcagatatatacatcctgtGCCGGTCGCGAACAGCGACC 3′.

Techniques: Staining, Cryo-EM Sample Prep

Journal: Science (New York, N.Y.)

Article Title: JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications

doi: 10.1126/science.abc3393

Figure Lengend Snippet: (A) (Top) Cartoon representation of the newly defined EZH2 bridge helix (amino acids 497 to 513) that interacts with nucleosomal DNA and the H3 tail. (Left) Cryo-EM structure of the PRC2-AEBP2-JARID2 complex bound to an H2AK119ub1-containing nucleosome. The zoom out shows the bridge helix, with residues interacting with the EZH2 (SET) domain, nucleosomal DNA, and the histone tail depicted in stick representation. (Right) Helix wheel diagram for the bridge helix that shows the distribution of positively charged residues on the nucleosomal DNA interacting face (cyan), positive and hydrophobic residues on the EZH2 (SET) interacting face (blue), and the residues interacting with the backbone of the H3 tail (pink). aa, amino acids. (B) (Left) Density map of the histone H3 tail (amino acids 21 to 40) (contour level: 0.024) with the corresponding atomic model. Clear density for residues R26 and K27, which have been previously observed, as well as density for K23, K36, K37, and V35 allow us to define the full extent of interaction between EZH2 (SET) and the histone H3 tail. (Right) Extensive electrostatic and van der Waals interactions between the residues in EZH2 (SET) (blue; shown in green stick representation) and the histone H3 tail (black; shown in pink stick representation) guide the H3 tail into the catalytic site. (C) Close-up view of the interaction of EZH2 (SET and CXC) with the histone H3 tail (stick representation with corresponding cryo-EM density in transparency), which shows residues involved in either direct or indirect interaction with the histone H3 tail and nucleosome DNA that are also frequently found mutated in cancers as orange spheres [COSMIC database (57)]. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Article Snippet: All human nucleosomes were purchased from Epicypher with the 5′ biotinylated 187-bp DNA sequence containing the 601-nucleosome positioning sequence: 5′(BioTEG)GGACCCTATACGCGGCCGCCCtggagaatcccggtctgcaggccgctcaattggtcgtagacagctctacgtggcgaatttgcgtgcatgcgcctgtcccccgcgttttaaccgccaaggggattactccctagtctccaggcacgtgtcagatatatacatcctgtGCCGGTCGCGAACAGCGACC 3′.

Techniques: Cryo-EM Sample Prep

Journal: Science (New York, N.Y.)

Article Title: JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications

doi: 10.1126/science.abc3393

Figure Lengend Snippet: (A) Bar graph showing the comparison of end-point, cumulative HMTase activity (H3K27me1/me2/me3) of PRC2-core, PRC2-AEBP2, PRC2-JARID2 (amino acids 106 to 450), PRC2-AEBP2ΔZn-JARID2 (amino acids 1 to 450), and PRC2-AEBP2-JARID2 (amino acids 1 to 450) on unmodified nucleosomes (gray), H3K4me3-containing (orange), H3K36me3-containing (green), and H2AK119ub1-containing nucleosomes (magenta). The differences in end-point measurements that are statistically significant are indicated by the P values. (B) Western blot analysis comparing the H3K27me3 production for unmodified nucleosomes versus the H3K4me3-containing, H3K36me3-containing, and H2AK119ub1-containing nucleosomes.

Article Snippet: All human nucleosomes were purchased from Epicypher with the 5′ biotinylated 187-bp DNA sequence containing the 601-nucleosome positioning sequence: 5′(BioTEG)GGACCCTATACGCGGCCGCCCtggagaatcccggtctgcaggccgctcaattggtcgtagacagctctacgtggcgaatttgcgtgcatgcgcctgtcccccgcgttttaaccgccaaggggattactccctagtctccaggcacgtgtcagatatatacatcctgtGCCGGTCGCGAACAGCGACC 3′.

Techniques: Activity Assay, Western Blot

Journal: Molecular cell

Article Title: Crystal structure of the LSD1/CoREST histone demethylase bound to its nucleosome substrate

doi: 10.1016/j.molcel.2020.04.019

Figure Lengend Snippet: Western blotting data for absence of H3K9me2 and H4K20me2 histone demethylation by LSD1 and LSD1+8a. (a) LSD1Δ1, LSD1 and LSD1+8a do not demethylate H3Kc9me2 nucleosomes, (b) ERRα does not enable LSD1Δ1 to demethylate H3Kc9me2 nucleosomes. 200 nM H3Kc9me2 197 bp nucleosomes were incubated with 67 nM LSD1Δ1 and 0, 67, 133 or 200 nM recombinant ERRα. (c) LSD1+8a does not demethylate H3K9me2 (Epicypher) or H3Kc9me2 nucleosomes with or without SVIL nuclear extracts. (d) LSD1/CoREST, LSD1+8a + CoREST and LSD1+8a/CoREST do not demethylate H4Kc20me2 147 bp or 197 bp nucleosomes. (e) LSD1+8a and LSD1+8a/CoREST complex do not demethylate H4Kc20me2 197 bp or H4K20me2 147 bp (Epicypher) nucleosomes. The reduction in H4Kc20me2 signal in lane 2 is not reproducible and also occurs when the non-catalytic CoREST subunit is added (lane 3).

Article Snippet: True H3K9me2 (#16–0324) and H4K20me2 (#16–0332) nucleosomes were purchased from Epicypher.

Techniques: Western Blot, Incubation, Recombinant

Journal: Molecular cell

Article Title: Crystal structure of the LSD1/CoREST histone demethylase bound to its nucleosome substrate

doi: 10.1016/j.molcel.2020.04.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: True H3K9me2 (#16–0324) and H4K20me2 (#16–0332) nucleosomes were purchased from Epicypher.

Techniques: Recombinant, Sequencing, Software

Journal: Communications biology

Article Title: A type II protein arginine methyltransferase regulates merozoite invasion in Plasmodium falciparum.

doi: 10.1038/s42003-023-05038-z

Figure Lengend Snippet: Fig. 1 PfPRMT5 expression, localization, and its histone modifications during IDC. a PfPRMT5 expression at different stages of the IDC was analyzed by Western blots with anti-Protein C antibodies (targeting PTP tag) to detect the full-length PfPRMT5-PTP (~ 106 kDa) in the PfPRMT5::PTP parasite line. PfHSP70 was used as a loading control. R: ring, ET: early trophozoite, LT: late trophozoite, and S: schizont. The relative expression level of PfPRMT5 was normalized with PfHSP70. b Identification of PfPRMT5 in the parasite cytoplasmic (Cyto) and nuclear (Nuc) fractions (upper panel). Anti-H3 antibodies (middle panel) and anti-PfHLP antiserum (lower panel) are used as nuclear and cytoplasmic markers, respectively. c IFA with anti-Protein C and FITC- conjugated anti-rabbit IgG as primary and secondary antibodies, respectively, to detect the localization of PfPRMT5 in the PfPRMT5::PTP parasite line. Nuclei were stained with Hoechst 33342. The size of the scale bar is 5 μm. d Endogenous PfPRMT5 was used in the methylation assay with human mononucleosomes, bovine, and P. falciparum core histones as the substrates, and the reactions were separated by SDS-PAGE (15% gel). Left panel: Coomassie blue-stained gel. Right panel: fluorograph. e Western blots with anti-H3R2me2s, H3R8me2s, and H4R4me2s antibodies were conducted to detect PfPRMT5-dependent methylation on H3 and H4 in human mononucleosomes. H3 and H4 were used as loading controls. f The levels of H3R2me2s, H3R2me2a, H3R8me2s, and H4R4me2s in the WT parasite during the IDC were analyzed by Western blots with respective antibodies. H3 was used as a loading control and human mononucleosome (Mono) was used as a negative control without any modifications in the histones. g IFA with anti-H3R2me2s and Alexa fluor 488-conjugated anti-rabbit IgG as primary and secondary antibodies, respectively, to detect the localization of H3R2me2s in the WT 3D7 parasite. Nuclei were stained with DAPI. The size of the scale bar is 5 μm.

Article Snippet: Briefly, 2 μg of bovine core histones (H2A, H2B, H3, and H4) (Sigma), or purified P. falciparum core histones (H2A, H2B, H3, and H4)87,93,94, or human mononucleosome assembled from recombinant human histones without any PTM (EpiCypher #SKU: 16-0009) were incubated at 30 °C for 2 h within 20 μl of methylation assay buffer containing 1 μCi of [3H] S-adenosylmethionine37,87.

Techniques: Expressing, Western Blot, Control, Staining, Methylation, SDS Page, Negative Control

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: Nature

Article Title: Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases

doi: 10.1038/s41586-020-03069-8

Figure Lengend Snippet: Biochemical analysis of NSD3 and the overall structure of the NSD3-E1181K/T1232A-NCP complex. a , Domain architecture of the NSD family proteins. b , Catalytic activities of NSD3 C on 147-bp and 187-bp NCPs measured through an in vitro methylation method. Signals for 147-bp NCP were set as 100%. c , MST-based binding curves of NSD3 C with 147-bp and 187-bp NCPs. d , Michaelis-Menten titrations of NSD3 with147-bp and 187-bp NCPs. e , Gel-filtration profiles of NCPs with or without bound NSD3 C . SDS-PAGE gel is shown as an insert. For gel source data, see . The experiment has been repeated at least three times with similar results. f , Catalytic activities of wild-type (WT) NSD3 C and its E1181K/T1232A mutant on 187-bp NCPs measured through an in vitro methylation method. g , Side (left) and top (right) views of the sharpened cryo-EM density map of the 1:1 NSD3-NCP complex (contour at 3σ level). H3, H4, H2A, H2B, DNA and NSD3 are colored in blue, lime green, yellow, red, orange and grass green, respectively. h , Domain architecture of the AWS, SET and post-SET domains of NSD3, which are colored in magenta, green and cyan, respectively. Data in panels b, c, d and f are represented as means ± s.d. from n = 3 independent samples.

Article Snippet: Recombinant 147-bp human nucleosomes without modification (catalog: 16–0006) or with tri-methylation at Lys4 of H3 (H3K4me3, catalog: 16–0316), trimethylation at Lys27 of H3 (H3K27me3, catalog: 16–0317), mono-ubiquitination at Lys119 of H2A (H2AK119ub, catalog: 16–0363) or mono-ubiquitination at Lys120 of H2B (H2BK120ub, catalog: 16–0370) were purchased from EpiCypher company.

Techniques: In Vitro, Methylation, Binding Assay, Filtration, SDS Page, Mutagenesis, Cryo-EM Sample Prep

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) MA plots of DESeq2 results from E16.5 mouse brains comparing WT and Phf21a -KO cortices. Differentially expressed genes (DEGs) are defined as a q <0.05. Upregulated DEGs are shown in orange (Up), downregulated DEGs in blue (Down), and non-significant genes in gray (NS). The top 8 DEGs based on log2 fold change (log2FC) are annotated. (B) Top 5 Gene Ontology (GO) terms associated with DEGs. (C and D) UCSC genome browser view of two genes, Tead3 (upregulated in Phf21a -KO vs. WT) and Cdk12 (constant gene). (E) Metagenomic plot of histone methylation distribution: H3K4me2 around transcription start sites (TSSs) ± 30 kb, H3K9me2 around transcription end sites (TESs) ± 30 kb, and H4K20me2 around TES ± 30 kb. Signals are normalized to control immunoglobulin (Ig)G. (F) Venn diagram comparing E18.5 Myt1l −/− DEGs (blue) and E16.5 Phf21a −/− DEGs (green). (G) Scatterplot comparing the log2FC of all DEGs found in either E18.5 Myt1l −/− or E16.5 Phf21a −/− cortices. Blue dots: DEGs only found in Myt1l −/− . Green dots: DEGs only found in E16.5 Phf21a −/− . Orange: DEGs common in the two mutants.

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

Techniques: Methylation, Control

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: bioRxiv

Article Title: Decoding the Protein Composition of Whole Nucleosomes with Nuc-MS

doi: 10.1101/2020.09.08.287656

Figure Lengend Snippet: (a) MS 1 : full charge state distribution of a synthetic mono-ubiquitinated nucleosome (O, observed average mass and precision at 1σ; T, theoretical mass for a homotypic nucleosome carrying two mono-ubiquitins on H2AK119). (b) MS 2 : spectral region reporting monoisotopic neutral masses for the ubiquitinated histone H2A and ejected proteoforms of the other core histones. (c) MS 3 : graphical fragment map of ubiquitinated H2A, consistent with mono-ubiquitination at lysine 119. Fragments were manually validated using TDValidator. P-scores were calculated using ProSight Lite.

Article Snippet: The nucleosome ubiquitinated at H2AK119 ( EpiCypher #16-0363) and mono- and di-methylated at K36 ( EpiCypher #16-0322 and #16-0319) were desalted 10 times into 150 mM ammonium acetate using 30 kDa MWCO spin filters prior to MS analysis.

Techniques:

Journal: Molecular Metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: FASN is required for H3K9 butyrylation in the absence of fatty acids. A–B. Hap1 and Hap1ΔFASN cells were cultured to near confluency before the fetal bovine serum was removed and the cells were maintained in A., C., and E. DMEM with 17.5 mM of glucose or B., D., and F. DMEM (no glucose) with 100 μM of palmitate-BSA. A–B. After ∼20 h, the protein was extracted, fractionated, and analyzed by Western blotting as described in A. C–F. Cells maintained in C. and E. glucose only or D. and F. palmitate only were subjected to a Mito Stress Test using an extracellular flux analyzer (XF e 96). Oxygen consumption rates (OCR) were measured in live cells before and after sequentially treating the cells with oligomycin (oligo), FCCP, and anti-mycin A and rotenone (AA/Rtn) as indicated by the arrows shown on the curve in E . C–D. The curves represent the real-time measurements of the OCR ( p mole/min, Y axis) vs time (min, X axis). E–F. The bar graphs represent the mitochondrial basal OCR (last rate measured before oligo injection minus non-mitochondrial respiration rate), SRC (the difference maximum [which was the maximum measurement after FCCP injection minus the non-mitochondrial respiration rate] and basal respiration), proton leak (minimum rate measured after oligo injection minus non-mitochondrial respiration), and ATP production levels (last rate measured before oligo injection minus the minimum rate after oligo injection). G. Western blotting signals for H3K9Bu in A. and B. were quantitated, normalized to H3K9Ac, averaged, and graphed. Error bars represent S.E.M. and p ≤ 0.05 vs Hap1.

Article Snippet: Recombinant nucleosomes with modified histones H3K9Ac (cat# 16-0314), H3K9Bu (cat# 16-0371), and H3K18Bu (cat# 16-0373) were purchased from EpiCypher, Inc., and analyzed by Western blotting with the corresponding antibodies.

Techniques: Cell Culture, Western Blot, Injection

Journal: Molecular Metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: ACAA2 is associated with promoters and required for H3K9 butyrylation. Twelve-week-old C57BL/6J mice were maintained on a low-fat (10 Kcal %) or high-fat (60 Kcal %) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diets for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled (n = 3 each). Chromatin was extracted from the heart tissue and subjected to anti-ACAA2 ChIP–Seq. A–B. The average signal (Avg Signal) of the chromatin-bound ACAA2 sequence reads (Y axis) assembled at gene promotors (−2 Kb to +2 Kb from the transcription start sites [TSS], X axis) were graphed for each condition (see color keycode at top) or C–D. all reads were represented by heatmaps aligned across the same region. Each input was generated from a pool of Sham and TAC heart chromatin. E. ACAA2-bound sequence fragments (Y axis) aligned to chromosome coordinates (X axis) are shown for Clasp1 , Arpc4 , and Usp1 genes. Each ChIP–Seq reaction was conducted with a pool of 3 hearts (LV). The anti-ACAA2 ChIP–Seq was 2x for the low-fat diet with similar results (1 set, sham and TAC, in this figure and 1 set previously published, GEO accession # GSE119391 ) and 1x for the high-fat diet. F. A diagram of the β-oxidation pathway and the enzymes that catalyze it depicted in red. Deletion of ACADS (ΔACADS), HADHA (ΔHADHA), or ACAA2 (ΔACAA2) and its consequences on the intermediate metabolites are shown, where the upward arrow signifies an increase and the downward arrow signifies a decrease in the indicated intermediates. Accumulation of acetoacetyl-CoA upon deletion of ACAA2 inhibited the trifunctional enzyme activity. Short-chain acyl-CoA was salvaged by ketogenesis as depicted in light blue. G. Recombinant H3K9Ac, H3K9Bu, and H3K18Bu nucleosomes (25, 50, and 75 ng of protein) were analyzed by Western blotting using the corresponding antibodies. H. Protein from human Hap1, Hap1ΔACAA2, J. Hap1ΔHADHA, or L. ΔHap1ΔACADS cells was extracted, fractionated into cytosol (Cyto), membrane/mitochondria (Mem/mito), nucleoplasm (Nuc), and analyzed by Western blotting for the molecules listed on the right of each panel (n = 3–6 each). I., K., and M. H3K9Bu or H3K9Cr signals were normalized to H3 or POL II, averaged, and the relative values calculated and graphed after adjusting one of the Hap1 sample levels to 1. Error bars represent S.E.M. The brackets encompass the values that were statistically compared for significance, with the p values listed above each.

Article Snippet: Recombinant nucleosomes with modified histones H3K9Ac (cat# 16-0314), H3K9Bu (cat# 16-0371), and H3K18Bu (cat# 16-0373) were purchased from EpiCypher, Inc., and analyzed by Western blotting with the corresponding antibodies.

Techniques: Isolation, ChIP-sequencing, Sequencing, Generated, Activity Assay, Recombinant, Western Blot

Journal: Molecular Metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: Higher levels of H3K9Bu correlate with blunted stress- and diet-induced changes in gene expression. Twelve-week-old C57BL/6J mice were fed a fat-free (0 Kcal % fat) or high-fat (60 kcal % fat) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diets for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled for ChIP–Seq assay with anti-H3K9Bu or anti-H3K9Ac (n = 1, pool of 3 hearts each) or the RNA was extracted for RNA-Seq (n = 3 independent hearts each). A–F. The results were sorted according to mRNA of genes that were significantly (p ≤ 0.05) A–C. upregulated (2,221 genes) or D–F. downregulated (2,242 genes) during TAC and a 60 kcal % fat diet (Fat 60 diet). a. and d. Violin plots showing the median and quartiles of H3K9Bu and B. and E. H3K9Ac average values of reads at the promoter regions (−1 kb to +1 kb) after subtracting the input for each. c. and F. Violin plots of the median and quartiles of the shrunken log 2 fold change (LFC) of the RNA-Seq for fat-free (Fat 0) TAC/Sham, Fat 60 TAC/Sham, Fat 60 TAC/Fat 0 TAC, and Fat 60 Sham/Fat 0 Sham. G. and H. MA plots of the DeSeq2 shrunken LFC (Y axis) vs mean of normalized counts (X axis) of the TAC/Sham RNA-Seq data with the different diets. I. A gene enrichment plot of GO term fatty acid β-oxidation generated from the heart tissue RNA-Seq data (n = 3) from the mice maintained on the low-fat (10% fat) vs high-fat (60% fat) diet for 11 days. Heatmap of the differentially expressed genes is shown on the right. The red asterisks indicate p < 0.05. J. RNA-Seq reads for ACADS mRNA from the heart tissue of the mice on the indicated diets were averaged and plotted. Error bars represent S.E.M. The brackets encompass the values that were statistically compared for significance, with the p values listed above each. K–L. RNA-Seq results for K. Nppb, Ankrd1, and Acta1, or L. Tbx20, Rasl11b, and Grk5 were averaged (n = 3) and plotted as bar graphs (see color keycode at top). ∗ indicates p ≤ 0.05 vs Sham and # indicates p ≤ 0.05 vs TAC. M–N. H3K9Bu- and H3K9Ac-bound sequence fragments (Y axis) aligned to the chromosome coordinates (X axis) are shown for the genes in ( K–L. ). O. Gene enrichment plots of GO term respirasome generated from the Sham vs TAC heart tissue RNA-Seq data (n = 3, each) from the mice maintained on a 0, 10, or 60 kcal % fat diets, with a heatmap of the differentially expressed genes shown on the right of each.

Article Snippet: Recombinant nucleosomes with modified histones H3K9Ac (cat# 16-0314), H3K9Bu (cat# 16-0371), and H3K18Bu (cat# 16-0373) were purchased from EpiCypher, Inc., and analyzed by Western blotting with the corresponding antibodies.

Techniques: Expressing, Isolation, ChIP-sequencing, RNA Sequencing Assay, Generated, Sequencing

Journal: Molecular Metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: H3K9Bu encompasses TSSs and is modulated by dietary fat and stress. Twelve-week-old C57BL/6J mice were maintained on A., D., H., and K. a fat-free (0 Kcal %), B., E., I., and L. low-fat (10 Kcal %), or C., F., J., and M. high-fat (60 Kcal %) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diets for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled (n = 3 each). Chromatin was extracted from the heart tissue and subjected to A–G. anti-H3K9Bu or H–M. anti-H3K9Ac ChIP–Seq (n = 1, pool of 3 hearts each). A–C and H–J. Curves representing the average signal (Avg Signal) of the chromatin-bound sequence reads assembled at all the gene promoters (−2 kb to +2 kb from the TSS, see color keycode at top). Each input was generated from a pool of Sham and TAC heart chromatin. D–F. and K–M. Heatmaps representing the sequence reads aligned across the same region. g. H3K9Bu-bound sequence fragments aligned (Y axis) to chromosome coordinates (X axis) are shown for Clasp1 , Arpc4 , and Usp1 genes.

Article Snippet: Recombinant nucleosomes with modified histones H3K9Ac (cat# 16-0314), H3K9Bu (cat# 16-0371), and H3K18Bu (cat# 16-0373) were purchased from EpiCypher, Inc., and analyzed by Western blotting with the corresponding antibodies.

Techniques: Isolation, ChIP-sequencing, Sequencing, Generated

Journal: Molecular Metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: ACADS is a mitochondrial and nuclear protein and its deficiency is associated with a specific increase in H3K9Bu. A–B. Cardiac myocytes were isolated and cultured for 24 h before they were supplemented with Ad-tGFP, Ad-ACADS-tGFP, or Ad-NLS-mutant ACADS (Ad-mt-ACADS) for an additional 24 h before the cells were harvested. C–D. Twelve-week-old Balc/cJ and Balb/cByJ (ACADS-deficient) mice were maintained on a high-fat (60 Kcal %) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diet for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated. A. and C. Protein from isolated cardiac myocytes or heart tissue was fractionated into cytosol (Cyto) membranes, including mitochondria (Mem/Mito), nucleoplasm (Nuc), and chromatin (Chrom). The protein was analyzed by Western blotting with antibodies for the molecules listed on the right of the panels or A. with anti-tGFP (top panel) or anti-ACADS (second from the top panel). The first and last lanes show protein standards, and their sizes are listed on the left of each panel. B. and D . Western blotting signals were quantitated, and cytosolic proteins were normalized to AKT1, membrane/mitochondrial protein to VDAC1, nucleoplasm proteins to Pol II, and H3K9Bu to H3 or Pol II (n = 4–6 each). D . The results were plotted relative to a cJ-Sham signal adjusted to 1. B. Signals for tGFP, ACADS-tGFP, and mt-ACADS-tGFP in the Cyto and Nuc fractions were plotted each relative to its corresponding Mem/Mito signal adjusted to 1. Error bars represent S.E.M. The brackets encompass the values that were statistically compared for significance, with the p values listed above each.

Article Snippet: Recombinant nucleosomes with modified histones H3K9Ac (cat# 16-0314), H3K9Bu (cat# 16-0371), and H3K18Bu (cat# 16-0373) were purchased from EpiCypher, Inc., and analyzed by Western blotting with the corresponding antibodies.

Techniques: Isolation, Cell Culture, Mutagenesis, Western Blot

Journal: Molecular Metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: Deletion of ACADS reverses stress-induced downregulation of H3K9Bu in the heart. Twelve-week-old Balc/cJ and Balb/cByJ (ACADS-deficient) mice were maintained on a low-fat (10% of calories) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diet for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled (n = 3, each). Chromatin was extracted from the heart tissue and subjected to anti-H3K9Bu ChIP–Seq (n = 1, a pool of 3 hearts each). A–B. The average signal (Avg Signal) of the chromatin-bound H3K9Bu reads assembled at all of the gene promotors (−2 Kb to +2 Kb from the TSS) were plotted for each condition (see color keycode at top) C–D. or all sequence reads were represented by a heatmap across the same region. E–F. H3K9Bu-bound sequence fragments (Y axis) aligned to chromosome coordinates (X axis) are shown for Clasp1 , Arpc4 , Usp1 , and Myl2 genes. g. Violin plots showing the median and quartiles of H3K9Bu average values of sequence reads within gene bodies in 3,242 genes (+1 Kb from TSS to gene end). H–I. The average signal (Avg Signal) of the chromatin-bound H3K9Ac reads assembled at all of the gene promotors (−2 Kb to +2 Kb from the TSS) graphed for each condition (see color keycode at top) or J–I. all reads were represented by heatmaps across the same region. The input was generated from a pool of Balb/cJ and Balb/cByJ, Sham, and TAC heart chromatin.

Article Snippet: Recombinant nucleosomes with modified histones H3K9Ac (cat# 16-0314), H3K9Bu (cat# 16-0371), and H3K18Bu (cat# 16-0373) were purchased from EpiCypher, Inc., and analyzed by Western blotting with the corresponding antibodies.

Techniques: Isolation, ChIP-sequencing, Sequencing, Generated

Journal: Molecular Metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: Deletion of ACADS blunts stress-induced changes in gene expression. Twelve-week-old Balc/cJ and Balb/cByJ (ACADS-deficient) mice were maintained on a low-fat (10% of calories) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diet for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled (n = 3 each) for ChIP–Seq with anti-H3K9Bu or anti-H3K9Ac (n = 1, a pool of 3 hearts each) or the RNA was extracted for RNA-Seq (n = 3 independent hearts each). A–F. The results were sorted according to mRNA of genes that were significantly (p ≤ 0.05) A–C. upregulated (1,272 genes) during TAC in the Balb/cJ mice (cJ) or D–F. downregulated (1,016) during TAC in the cJ mice. A. and D. Violin plots showing the median and quartiles of H3K9Bu and B. and E. H3K9Ac average values of sequence reads at the promoter regions (−1,000 to +1,000) after subtracting the input for each. C. and F. Violin plots of the median and quartiles of the shrunken log 2 fold change (LFC) of the RNA-Seq for cJ-TAC/Sham, cByJ-TAC/Sham, cJ-TAC/cByJ-TAC, and cJ-Sham/cByJ-Sham. G. and H. MA plots of the DeSeq2 shrunken LFC (Y axis) vs mean of normalized counts (X axis) of the TAC/Sham RNA-Seq data for Balb/cJ and Balb/cByJ. I–J. Enrichment plots for GO terms generated from the Sham and TAC and heart RNA-Seq results of the Balb/cJ (cJ) and Balb/cByJ (cByJ) mice.

Article Snippet: Recombinant nucleosomes with modified histones H3K9Ac (cat# 16-0314), H3K9Bu (cat# 16-0371), and H3K18Bu (cat# 16-0373) were purchased from EpiCypher, Inc., and analyzed by Western blotting with the corresponding antibodies.

Techniques: Expressing, Isolation, ChIP-sequencing, RNA Sequencing Assay, Sequencing, Generated