Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: Braf V600E signaling induces epigenetic silencing by increasing H3K27me3 abundance in NIH3T3 cells . ( A ) Western blot analysis of B-raf, phosphorylated (p-) and total forms of Erk1/2 and H3K27me3. GAPDH and histone H3 were used as loading controls. ( B ) Flow chart shows the experimental design. ( C ) Pie chart shows distribution of H3K27me3 enrichment across whole genome in Braf V600E cells relative to Braf WT cells. ( D ) Venn diagram illustrates that 150 loci were marked by H3K27me3 and downregulated by Braf V600E signaling.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the PierceTM Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Western Blot

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: Validation of mRNA expression and H3K27me3 of Braf V600E -repressed genes in vitro and in vivo . ( A ) qRT-PCR analysis of Abi3bp , Gimap6 , Hopx , Pdzd2 , Ptprd , Rspo2 , Mtss1 and Nrxn3 expression in Braf V600E cells relative to that in Braf WT cells. 18S rRNA was used as an endogenous control. Data were presented as mean ± SD from three independent experiments. ( B ) ChIP-qPCR analysis of promoter regions of the indicated genes in Braf V600E cells relative to that in Braf WT cells using anti-H3K27me3 antibody. Data were presented as mean ± SD from three independent assays. ( C ) Comparison of the levels of p-Erk1/2 and H3K27me3 between TPO-Cre/LSL-Braf V600E (Braf V600E ) and Braf wild-type mice by IHC analysis. The inserts show the magnified image of the area indicated by the red square. Scale bars, 200 μm. ( D ) qRT-PCR analysis of Abi3bp , Gimap6 , Hopx , Pdzd2 , Ptprd , Rspo2 , Mtss1 and Nrxn3 expression in Braf V600E mice relative to that in Braf wild-type mice. 18S rRNA was used as an endogenous control. Data were presented as mean ± SD. Statistically significant differences were indicated: *, P <0.05; **, P <0.01.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the PierceTM Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Biomarker Discovery, Expressing, In Vitro, In Vivo, Quantitative RT-PCR, Control, ChIP-qPCR, Comparison

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: The effect of inhibition of Braf V600E signaling on H3K27me3 levels and the expression of Braf V600E -repressed genes in Braf V600E mice. Comparison of tumor volume (A), weight (B), and the levels of p-Erk1/2 and H3K27me3 (C) between Braf V600E mice treated with a combination of PLX4720 and GSK1120212 (n=3) and vehicle (n=5), respectively. Scale bars, 200 μm. (D) qRT-PCR analysis of Abi3bp , Gimap6 , Hopx , Pdzd2 , Ptprd , Rspo2 , Mtss1 and Nrxn3 expression in combination-treated mice relative to that in vehicle-treated mice. DMSO was used as the vehicle control. 18S rRNA was used as an endogenous control. Data were presented as mean ± SD. Statistically significant differences were indicated: *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the PierceTM Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Inhibition, Expressing, Comparison, Quantitative RT-PCR, Control

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: Braf V600E increases H3K27me3 levels through regulating the PRC2 components via c-Myc . ( A ) Western blot analysis of B-raf, p-Erk1/2, total Erk1/2, c-Myc, Ezh2, Suz12, Jarid2 and H3K27me3 in VEC, Braf WT and Braf V600E cells, respectively. β-Actin and histone H3 were used as loading controls. ( B ) Western blot analysis of Ezh2, Suz12, Jarid2 and H3K27me3 upon c-Myc depletion in NIH3T3 cells. β-Actin and histone H3 were used as loading controls. si-NC, si-Myc1 and 2 represent control and specific c-Myc siRNAs, respectively. ( C ) Western blot analysis of Ezh2, Suz12, Jarid2 and H3K27me3 in Braf V600E cells relative to that in Braf WT cells. β-Actin and histone H3 were used as loading controls. ( D ) A comparison of the levels of c-Myc, Ezh2, Suz12, Jarid2 and H3K27me3 in the indicated mice by IHC staining. DMSO was used as the vehicle control. The insert shows the magnified image of the area indicated by the red square. Scale bars, 200 μm.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the PierceTM Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Western Blot, Control, Comparison, Immunohistochemistry

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: The effect of inhibition of c-Myc by 10058-F4 on the levels of the PRC2 components and H3K27me3 in Braf V600E mice . Comparison of tumor volume ( A ), weight ( B ), and the levels of Ncl, Ezh2, Suz12, Jarid2 and H3K27me3 (C) between Braf V600E mice treated with 10058-F4 (n=3) and vehicle (n=3), respectively. DMSO was used as the vehicle control. The insert shows the magnified image of the area indicated by the red square. Scale bars, 200 μm. ( D ) qRT-PCR analysis of Abi3bp , Gimap6 , Hopx , Pdzd2 , Ptprd , Rspo2 , Mtss1 and Nrxn3 expression in 10058-F4-treated mice relative to that in vehicle-treated mice. DMSO was used as the vehicle control. 18S rRNA was used as an endogenous control. Data were presented as mean ± SD. Statistically significant differences were indicated: *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the PierceTM Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Inhibition, Comparison, Control, Quantitative RT-PCR, Expressing

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: The effect of inhibition of major signaling pathways on the levels of the PRC2 components and H3K27me3 in thyroid cancer and melanoma cells. Western blot was performed to evaluate the effect of the indicated inhibitors individually or in combinations on the levels of the PRC2 components and H3K27me3 in thyroid cancer cell lines BCPAP and K1 (A) and melanoma cell lines M14 and A375 (B). GAPDH and histone H3 were used as loading controls. DMSO was used as the vehicle control.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the PierceTM Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Inhibition, Protein-Protein interactions, Western Blot, Control

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: Schematic model of BRAF V600E signaling-induced epigenetic silencing by H3K27me3 in tumorigenesis . BRAF V600E mutation constitutively activates the MAPK/Erk signaling, resulting in upregulation of its downstream effector c-Myc in many types of cancer particularly thyroid cancer and melanoma. In general, c-Myc is degraded via the ubiquitin-proteasome system. However, this process can be inhibited by activated PI3K/Akt pathway in cancer cells. When c-Myc translocates into the nucleus, it regulates the expression of its target genes through binding to their regulatory elements via interaction with its obligate partner MAX or other unknown mechanisms. In the present study, c-Myc regulates the expression of the PRC2 components, contributing to epigenetic silencing through two distinct mechanisms. First, c-Myc transcriptionally regulates the expression of the PRC2 components through binding to the regulatory elements within their promoters. Second, c-Myc transcriptionally represses the expression of a panel of miRNAs, whereas some of them target and regulate the expression of the PRC2 components at post-transcriptional levels. In addition, BRAF V600E signaling also induces epigenetic silencing through Erk1/2-induced RNAPII poising and chromatin architecture.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the PierceTM Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Mutagenesis, Ubiquitin Proteomics, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

doi: 10.1101/2024.03.18.585588

Figure Lengend Snippet: A) A close-up view of the interactions between the N-terminal region of DNMT3A1 and acidic patch of the nucleosome. B) Top: amino acid sequence of the UDR peptide for WT, single acidic patch mutant (R181E), triple acidic patch mutant (R167E, R171E, R181E) and Ub mutant (W207A, L208A and W210A). dCypher-Luminex assay to measure interaction between 6xHis-tagged UDR peptides (WT or mutant as noted: the Queries) and a multiplexed panel of fully defined nucleosomes (the Targets; unmodified, with KUb (H2AK15Ub1, H2AK119Ub1, H2AK129Ub1, H2BK120Ub1, H3K14Ub1, or H2BK18Ub1). C) Left: Representative IF of FLAG-DNMT3A1 in 10T cells expressing DNMT3A1 WT , DNMT3A1 W330R (PWWP mutant),DNMT3A1 W330R+R181E or DNMT3A1 W330R+Ubmut . Right: Quantification of FLAG-DNMT3A1 IF in 10T cells expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E or DNMT3A1 W330R+Ub mut , shown as ratio of 10T cells displaying DNMT3A1 Xi accumulation compared to all 10T cells. Error bars represent standard deviation of five replicates. Student t test, ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: In brief, for each CUT&RUN reaction 500K cells [from 5 million cells/mL prepared in FBS with 10% DMSO] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con-A; EpiCypher #21-1401), and incubated overnight (4°C) with 0.5 µg of antibody (IgG, H3K4me3, H3K27me3, H3K36me2, H2AK119Ub1) (all antibodies validated to histone post-translation modification (PTM)-defined SNAP-ChIP nucleosome standards as previously ( )). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio.

Techniques: Sequencing, Mutagenesis, Luminex, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

doi: 10.1101/2024.03.18.585588

Figure Lengend Snippet: Related to . A) Left: Dnmt3l locus showing the location of sgRNA. Middle: Western blot of DNMT3L in QKO and TKO mESCs with total H3 as loading control. Right: Validation of the Dnmt3l KO clone (QKO mESC) by TIDE (Tracking of Indels by Decomposition) analysis to quantify the frequency of small insertions or deletions. B) Western blot of DNMT3A in QKO mESCs and QKO mESCs expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E or DNMT3A1 W330R+Ub mut . C) Correlation matrix of DNMT3A1 ChIP in QKO mESC expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E or DNMT3A1 W330R+Ub mut , as well as H3K27me3, H2AK119Ub and H3K36me2 CUT&RUN in DNMT3A1 WT QKO mESC over 10kb bins. D) Percent DNA methylation measured by RRBS in DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E or DNMT3A1 W330R+Ub mut TKO mESC per 10k bins grouped and ranked by H3K36me2 CUT&RUN enrichment (Q1 lowest to Q4 highest). E) Metagene plot showing enrichment of DNMT3A1 ChIP-seq reads in DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E or DNMT3A1 W330R+Ub mut QKO mESC, centered at CGIs ± 5 kb and grouped by either H3K27me3 (top 20% vs. bottom 80%) or H3K4me3 (four quadrants). F) Left: Boxplot showing percent of promoter methylation in QKO mESC and QKO mESC expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E or DNMT3A1 W330R+Ub mut for both H3K27me3/H2AK119Ub-positive and -negative promoters. Right: Boxplot showing percent of promoter methylation in TKO mESC expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E or DNMT3A1 W330R+Ub mut for both H3K27me3/H2AK119Ub-positive and -negative promoters.

Article Snippet: In brief, for each CUT&RUN reaction 500K cells [from 5 million cells/mL prepared in FBS with 10% DMSO] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con-A; EpiCypher #21-1401), and incubated overnight (4°C) with 0.5 µg of antibody (IgG, H3K4me3, H3K27me3, H3K36me2, H2AK119Ub1) (all antibodies validated to histone post-translation modification (PTM)-defined SNAP-ChIP nucleosome standards as previously ( )). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio.

Techniques: Western Blot, Control, Expressing, DNA Methylation Assay, ChIP-sequencing, Methylation

Journal: bioRxiv

Article Title: Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

doi: 10.1101/2024.03.18.585588

Figure Lengend Snippet: A) Genome browser view of DNMT3A1 ChIP–seq reads for DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 R181E and DNMT3A1 Ub mut QKO mESC at chromosome 1: 137.7–139.8 Mb, GRCm38. Additionally, the Cut and Run reads for the histone modifications H3K4me3, H3K27me3, H2AK119Ub and H3K36me2 in DNMT3A1 WT QKO mESC are shown. Genes from the RefSeq database are annotated at the bottom. B) Cut and run reads per 1000 averaged 1kb bin for H3K27me3 and ChIP-seq reads per 1000 averaged 1kb bin for DNMT3A1 in DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 R181E and DNMT3A1 Ub mut QKO mESC. To generate bins, 1kb genomic tiles were ranked by H3K36me2 enrichment in DNMT3A1 WT QKO mESC and grouped into 1000 rank-ordered bins. C) Percent of DNA methylation (EMseq) in DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 R181E and DNMT3A1 Ub mut QKO mESC per 10k bins grouped by H3K36me2 CUT&RUN enrichment (Q1 low to Q4 highest). D) Enrichment heat map of H3K27me3 and H3K36me2 CUT&RUN reads in DNMT3A1 WT QKO mESC and DNMT3A1 ChIP-seq reads in DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 R181E and DNMT3A1 Ub mut QKO mESC sorted by H3K27me3 centered at CGIs ± 5 kb. E) Enrichment plot of ChIP-seq reads of DNMT3A1 in DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 R181E and DNMT3A1 Ub mut QKO mESC centered at CGIs ± 5 kb and grouped highest 20% H2AK119Ub enriched CGIs (H2AK119Ub+) and lowest 80% H2AK119Ub enriched CGIs (H2AK119Ub-).

Article Snippet: In brief, for each CUT&RUN reaction 500K cells [from 5 million cells/mL prepared in FBS with 10% DMSO] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con-A; EpiCypher #21-1401), and incubated overnight (4°C) with 0.5 µg of antibody (IgG, H3K4me3, H3K27me3, H3K36me2, H2AK119Ub1) (all antibodies validated to histone post-translation modification (PTM)-defined SNAP-ChIP nucleosome standards as previously ( )). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio.

Techniques: ChIP-sequencing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

doi: 10.1101/2024.03.18.585588

Figure Lengend Snippet: A) Scatter plots showing % DNAme (measured by RRBS) at all promoters (TSS ± 500bp) in DNMT3A1 W330R , DNMT3A W330R+181E and DNMT3A1 W330R+Ub mut versus DNMT3A1 WT -expressing 10T cells. Each dot represents a single promoter. Significantly hyper- (>10% and q<0.01) and hypo-methylated (>-10% and q<0.01) promoters are colored in red and blue, respectively. B) Metagene plot showing % DNAme over CGIs ± 5 kb (left), promoters (TSS± 5 kb) (middle) and Polycomb+ promoters (H3K27me3 + and H2AK119Ub + ) (right) in DNMT3A1 W330R , DNMT3A W330R+R181E and DNMT3A1 W330R+Ub mut compared to DNMT3A1 WT 10T cells. C) Barplot showing the representations among all promoters, or 10T DNMT3A W330R hypermethylated promoters, for each group of promoters defined by different histone modificaiton status in control cells as summarized in . D) Metagene plot showing enrichment of H2AK119Ub, H3K27me3 and H3K4me3 CUT&RUN reads from DNMT3A1 WT , DNMT3A1 W330R , DNMT3A W330R+R181E and DNMT3A1 W330R+Ub mut 10T cells, centered at TSS ± 5 kb and grouped by all, hypermethylated and hypomethylated promoters. E) Genome browser view at Chromosome 2, 74.5–75.0Mb, showing differences in % DNA methylation (measured by RRBS) between various DNMT3A1 mutant versus DNMT3A1 WT 10T cells. Bottom shows H2AK119Ub, H3K27me3 and H3K4me3 CUT&RUN reads from DNMT3A1 WT and DNMT3A1 W330R 10T cells. Genes from RefSeq database are annotated at bottom.

Article Snippet: In brief, for each CUT&RUN reaction 500K cells [from 5 million cells/mL prepared in FBS with 10% DMSO] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con-A; EpiCypher #21-1401), and incubated overnight (4°C) with 0.5 µg of antibody (IgG, H3K4me3, H3K27me3, H3K36me2, H2AK119Ub1) (all antibodies validated to histone post-translation modification (PTM)-defined SNAP-ChIP nucleosome standards as previously ( )). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio.

Techniques: Expressing, Methylation, Control, DNA Methylation Assay, Mutagenesis

Journal: bioRxiv

Article Title: Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

doi: 10.1101/2024.03.18.585588

Figure Lengend Snippet: Related to and . A) Boxplot showing percent of global DNAme (500kb bins, measured by RRBS) in 10T cells expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E and DNMT3A1 W330R+Ub mut . B) Heat map showing enrichment of H3K27me3, H2AK119Ub and H3K4me3 CUT&RUN reads at all promoters (TSS± 5kb) in 10T control cells. Promoters are classified into seven groups: H3K4me3 + ; H3K27me3 + ; H2AK119Ub + ; H3K4me3 + and H2AK119Ub + ; H3K27me3 + and H2AK119Ub + ; H3K27me3 + and H3K4me3 + ; H3K4me3 + and H3K27me3 + and H2AK119Ub + used in analysis for . C) Scatterplot comparing the changes in % DNAme (y-axis) and H3K27me3 (CUT&RUN, x-axis) between 10T cells expressing DNMT3A1 W330R and DNMT3A1 WT for all gene promoters positive for H3K27me3 in at least one condition. D) Boxplot comparing the differences in % promoter DNAme for both upregulated and downregulated genes between 10T cells expressing DNMT3A1 W330R and DNMT3A1 WT . E) Barplot showing selected GO terms, ranked by -log(p-value) for, top: Upregulated genes in day 6 DNMT3A1 WT adipocytes compared to undifferentiated DNMT3A1 WT 10T cells (DEseq2, q>0.01 and LFC>2). Bottom: Downregulated genes in day 6 DNMT3A1 W330R vs. day 6 DNMT3A1 WT adipocytes (DEseq2, q>0.01 and LFC< -2). F) MA-plot showing log10 of average normalized RNA-seq reads for each gene (x-axis) and log2 fold change (LFC) of gene expression (y-axis) between (left) DNMT3A1 W330R and DNMT3A1 WT 10T cells on day 6 of adipocyte differentiation; (right) DNMT3A1 W330R+R181E and DNMT3A1 WT 10T cells on day 6 of adipocyte differentiation. Significantly differentially expressed genes (by DEseq2) are highlighted in green (LFC>2 and q<0.01) or red (LFC<-2 and q<0.01). G) Violin plots showing the distribution of % promoter DNAme (measured by RRBS) in 10T cells expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E for all promoters, or promoters of genes upregulated during adipocyte differentiation. H) Heatmap showing % promoter methylation (measured by RRBS) in 10T cells expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E for promoters of genes upregulated during adipocyte differentiation.

Article Snippet: In brief, for each CUT&RUN reaction 500K cells [from 5 million cells/mL prepared in FBS with 10% DMSO] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con-A; EpiCypher #21-1401), and incubated overnight (4°C) with 0.5 µg of antibody (IgG, H3K4me3, H3K27me3, H3K36me2, H2AK119Ub1) (all antibodies validated to histone post-translation modification (PTM)-defined SNAP-ChIP nucleosome standards as previously ( )). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio.

Techniques: Expressing, Control, RNA Sequencing Assay, Methylation

Journal: bioRxiv

Article Title: Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

doi: 10.1101/2024.03.18.585588

Figure Lengend Snippet: A) Schematic of normal esophagus (EN) and 4NQO-induced esophageal squamous cell carcinoma (ESCC) organoid collection. Right: Representative images of EN and ESCC organoid histology. B) Scatter plots showing % DNAme (measured by RRBS) at all promoters (TSS ± 500bp) in ESCC versus EN organoids. Each dot represents a single promoter. Significantly hyper- (>10% and q<0.01) and hypo-methylated (>-10% and q<0.01) promoters are colored in red and blue, respectively. C) Heat map showing enrichment of H3K27me3, H2AK119Ub and H3K4me3 CUT&TAG reads at all promoters (TSS± 5kb) in EN organoids. Promoters are classified into six groups: H3K4me3 + ; H3K27me3 + ; H2AK119Ub + ; H3K4me3 + and H2AK119Ub + ; H3K27me3 + and H2AK119Ub + ; H3K4me3 + and H3K27me3 + and H2AK119Ub + . D) Barplots showing the representations among all promoters, or ESCC hypermethylated promoters, for each of the six groups of promoters as defined in . E) Heatmap showing % promoter methylation (measured by RRBS) in EN organoids, ESCC organoids, and EN organoids expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E for all ESCC hypermethylated promoters. F) Genome browser view of Cdkn2a gene (chromosome 4: 89.187 Mb -89.216 Mb, GRCm39), showing %DNAme (measured by RRBS) in EN organoids, ESCC organoids, and EN organoids expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E . Bottom shows H3K27me3 and H2AK119Ub CUT&TAG reads in EN and ESCC organoids. Right panel: Average DNA methylation shown at the Cdkn2a promoter (chromosome 4: 89.200.200 bp -89.200.300 bp, GRCm39). Genes from RefSeq database are annotated at the bottom.

Article Snippet: In brief, for each CUT&RUN reaction 500K cells [from 5 million cells/mL prepared in FBS with 10% DMSO] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con-A; EpiCypher #21-1401), and incubated overnight (4°C) with 0.5 µg of antibody (IgG, H3K4me3, H3K27me3, H3K36me2, H2AK119Ub1) (all antibodies validated to histone post-translation modification (PTM)-defined SNAP-ChIP nucleosome standards as previously ( )). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio.

Techniques: Methylation, Expressing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

doi: 10.1101/2024.03.18.585588

Figure Lengend Snippet: Related to . A) Left: Boxplot showing percent of global DNAme (500kb bins, measured by RRBS) in 3x EN and 3x ESCC organoids. Right: Boxplot showing percent of global DNAme (500kb bins, measured by RRBS) in EN organoids expressing DNMT3A1 WT , DNMT3A1 W330R , DNMT3A1 W330R+R181E . B) Scatterplot comparing the changes in % DNAme (y-axis) and H3K27me3 (CUT&RUN, x-axis) between EN and ESCC organoids for all gene promoters positive for H3K27me3 in at least one condition. C) Scatter plots showing % DNAme (measured by RRBS) at all promoters (TSS ± 500bp) in EN organoids expressing DNMT3A1 W330R or DNMT3A1 W330R+R181E versus EN organoids expressing DNMT3A1 WT . Each dot represents a single promoter. Significantly hyper- (>10% and q<0.01) and hypo-methylated (>-10% and q<0.01) promoters are colored in red and blue, respectively. D) Boxplot showing the differences in % DNAme between ESCC and EN organoids (purple), EN organoids expressing DNMT3A1 W330R and DNMT3A1 WT (red), EN organoids expressing DNMT3A1 W330R+R181E and DNMT3A1 WT (blue), for all ESCC hypermethylated promoters. Student t test, ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001. E) Venn diagrams showing the overlap between hypermethylated promoters in EN organoids expressing DNMT3A1 W330R and hypermethylated promoters in HNSC (top) or ESCA (bottom) patient samples.

Article Snippet: In brief, for each CUT&RUN reaction 500K cells [from 5 million cells/mL prepared in FBS with 10% DMSO] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con-A; EpiCypher #21-1401), and incubated overnight (4°C) with 0.5 µg of antibody (IgG, H3K4me3, H3K27me3, H3K36me2, H2AK119Ub1) (all antibodies validated to histone post-translation modification (PTM)-defined SNAP-ChIP nucleosome standards as previously ( )). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio.

Techniques: Expressing, Methylation

Journal: Addiction biology

Article Title: Dysregulation of the histone demethylase KDM6B in alcohol dependence is associated with epigenetic regulation of inflammatory signaling pathways.

doi: 10.1111/adb.12816

Figure Lengend Snippet: FIGURE 5 Chromatin immunoprecipitation sequencing (ChIP‐seq) analysis of H3K27me3 in control compared with postdependent (PD) nucleus accumbens. A, A ChIP quantitative real‐time polymerase chain reaction (qPCR) experiment demonstrated that the H3K27me3 ChIP antibody showed high specificity for the target PTM when tested against a panel of spike‐in recombinant nucleosomes bearing related methylation states that were distinguished by primers specific to unique DNA barcodes. B, Optimization of ChIP‐seq protocol in rodent brain shows enrichment of the antibody signals over IgG controls. H3K27me3, a repressive epigenetic modification, is highly associated with a gene that is suppressed in the brain (BRDT) and lowly associated with an actively transcribed gene (ACTB). In contrast, H3K4me3, a mark associated with transcriptional activation, is enriched for ACTB over BRDT. C, ChIP‐seq analysis shows that the majority of H3K27me3‐associated genomic regions (71%) are enriched in control rats as compared with PD rats (n = 4)

Article Snippet: Specificity of the H3K27me3 antibody was confirmed by ChIP quantitative real‐time PCR (qPCR), where the SNAP‐ChIP K‐MetStat Panel (EpiCypher #19‐1001) of DNA‐barcoded recombinant nucleosomes was spiked into a ChIP experiment prior to chromatin fragmentation.37 Recovery of on‐target nucleosomes (H3K27me3) compared with related lysine methylation states was deciphered by TaqMan qPCR using the SNAP‐ChIP Dual Labeled Hydrolysis Probe (EpiCypher #18‐6001) and the Full Panel Primer Set (EpiCypher #18‐ 6101).

Techniques: ChIP-sequencing, Control, Real-time Polymerase Chain Reaction, Recombinant, Methylation, Modification, Activation Assay