Journal: Nucleic acids research

Article Title: Chromatin accessibility and pioneer factor FOXA1 restrict glucocorticoid receptor action in prostate cancer.

doi: 10.1093/nar/gkad1126

Figure Lengend Snippet: Figure 3. Inhibition of p300’s enzymatic activity hinders cell proliferation and GR transcriptional regulation. ( A ) Depiction of chemical str uct ures of coregulator inhibitors. A-485, p300 / CBP histone acetyl transferase (HAT) domain inhibitor; CCS1477, p300 / CBP bromodomain inhibitor; I-BET762, bromodomain and extra terminal domain (BET) bromodomain inhibitor; BRM014, BRM / BRG1 ATP domain inhibitor. (B–E) Bar graphs depict KLK3 gene e xpression analy sis in V CaP ENZ 0 ( B ), V CaP ENZ 3w ( C ), 22Rv1 ENZ 0 ( D ), and 22Rv1 ENZ 3w ( E ) cells treated with or without Dex in the presence or absence of 0.1 or 1 μM of indicated inhibitor. Bars represent mean ± SD, n = 3. Statistical significance calculated using one-way ANO V A with Bonferroni post hoc test. (F, G) Line graphs depict change in cell proliferation as function of time in VCaP ( F ) and 22Rv1 ( G ) ENZ 0 and ENZ 3w cells with or without indicated chemical inhibitor treatment. Each data point represents mean ± SD, n = 3. Statistical significance calculated using Tw o-w a y ANO V A with Bonferroni post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For experiments with the coregulator inhibitors, the cells were treated with 0.1 μM or 1 μM A-485 p300 / CBP HAT ( Tocris, #6387 ) , CCS1477 p300 / CBP bromodomain ( MedChemExpress, #HY-111784 ) , I-BET762 BET bromodomain ( Sigma, #SML1272 ) , or BRM014 BRM / BRG1 ATP ( MedChemExpress, #HY-119374 ) inhibitors.

Techniques: Inhibition, Activity Assay

Journal: Nature Genetics

Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications

doi: 10.1038/s41588-024-01706-w

Figure Lengend Snippet: a , Schematic of the modular epigenetic editing platform. Upon DOX induction, dCas9 GCN4 recruits five copies of the CD of chromatin-modifying effector(s) or control GFP scFV to target loci via a specific gRNA. DNAme, DNA methylation. b , Relative abundance of the indicated histone modification at Hbb-y assayed by either CUT&RUN–qPCR or by chromatin immunoprecipitation followed by qPCR (ChIP–qPCR) (H3K36me3, H3K79me2), following epigenetic editing or control GFP scFV recruitment in ESCs for 7 d. Shown is the mean of three biologically independent experiments; error bars indicate s.d. Norm., normalized. c , Histogram showing mean DNA methylation installed at the unmethylated Col16a1 promoter, determined by bisulfite pyrosequencing in three biologically independent experiments; error bars indicate s.d. d – i , Relative abundance of the indicated histone modification (H3K4me3 ( d ), H3K27me3 ( e ), H2AK119ub ( f ), H3K27ac ( g ), H3K9me3 ( h ), H3K36me3 ( i )) across the Hbb-y locus after epigenetic programming with a specific CD scFV (Prdm9 ( d ), Ezh2 ( e ), Ring1b ( f ), p300 ( g ), G9a ( h ), Setd2 ( i ); red line) or control GFP scFV (gray line), assayed by CUT&RUN–qPCR. Mean enrichment across a ~14-kb region centered on the gRNA-binding site is shown for editing in biological triplicates as well as for endogenous positive (Pos1 and Pos2) and negative (Neg1 and Neg2) loci for each mark. NS, not significant. ND, not determined. j , Percentage of DNA methylation at CpG dinucleotides across the Col16a1 and Hand1 promoters in triplicate experiments. k , Scatterplots showing limited OFF-target gene expression changes following induction of the indicated epigenetic mark at Hbb-y for 7 d, relative to that of control GFP scFV . Differentially expressed genes are indicated in green or orange. Gray dots indicate unaffected genes. p300 , ep300 ; G9a , Ehmt2 ; Ring1b , Rnf2 . P values in all panels were calculated by one-tailed unpaired t -test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For experiments employing the p300 inhibitor A485, cells were stimulated with 100 ng ml −1 DOX for 3 d and, in parallel, treated with 3 μM A485 (Cayman Chemical, 24119).

Techniques: Control, DNA Methylation Assay, Modification, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Targeted Gene Expression, One-tailed Test

Journal: Nature Genetics

Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications

doi: 10.1038/s41588-024-01706-w

Figure Lengend Snippet: ( a ) Dot plot of genome-wide H3K4me3 enrichment across sliding 3 kb tiles by calibrated Cut&Run. Shown is genome-wide H3K4me3 upon epigenetic editing at the Hbby locus with Prdm9-CD scFV relative to control ESC with GFP scFV , demonstrating the vast majority of the genome does not acquire H3K4me3 peaks (minimal OFF-targeting). (b) Genome tracks showing ON-target enrichment of programmed H3K4me3 across the Hbby locus by Prdm9-CD scFV mediated epigenetic editing. (c) Correlation matrix of replicate transcriptomes (RNA-seq) following induction of the indicated epigenetic editing system with DOX. We routinely observed high correlation (>0.98) between global gene expression, with few OFF-target genes mis-ex- pressed, indicative of preferential ON-target activity. The exception is p300 scFV , and we therefore reduced the DOX concentration to mitigate indirect effects. (d) ESC proliferation following a titration of p300-CD scFV induction levels with DOX. (e) Schematic and fluorescent images of ESC carrying the reference (REF) reporter knocked-in to distinct genomic locations; a permissive locus for transcription (left) and a non-permissive locus (right). Images were captured in two independent experiments with similar results. (f) Quantification of baseline H3K4me3 and H3K27me3 at identical reference reporters located within the each genomic context.

Article Snippet: For experiments employing the p300 inhibitor A485, cells were stimulated with 100 ng ml −1 DOX for 3 d and, in parallel, treated with 3 μM A485 (Cayman Chemical, 24119).

Techniques: Genome Wide, Control, RNA Sequencing, Gene Expression, Activity Assay, Concentration Assay, Titration

Journal: Nature Genetics

Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications

doi: 10.1038/s41588-024-01706-w

Figure Lengend Snippet: a , H3K4me3 enrichment over the transcriptional start site (TSS) ±5 kb in wild-type and Mll2 CM/CM ESCs, stratified according to H3K4me3 changes in Mll2 CM/CM ESCs. b , MA plot of expression change for each gene in Mll2 CM/CM ESCs, colored by whether the promoter loses H3K4me3 (green) or retains H3K4me3 (red). WT, wild type. c , Bar plots showing expression of the indicated genes in wild-type, Mll2 CM/CM and Mll2 CM/CM + Prdm9 scFV ESCs, in which H3K4me3 has been programmed back to a repressed promoter that previously lost H3K4me3. Shown is the mean of three biological replicates assayed by qPCR with reverse transcription (RT–qPCR). Error bars represent s.d., and significance of rescue was calculated by two-tailed unpaired t -test. d , Bar plots of endogenous gene expression in wild-type ESCs and upon programming H3K4me3 with Prdm9 scFV or control mut-Prdm9 scFV . Data are the mean of biological triplicates; error bars represent s.d. Significance was calculated by one-way ANOVA with Tukey’s correction. O ct6 ( Pou3f1 ). e , Dot plots showing single-cell expression of the OFF reporter after targeting with different H3K4me3 effectors: Prdm9 scFV (left) or Setd1a scFV (right). n = 500 individual cells; bars denote the geometric mean. Reading was performed for three independent experiments. f , Bar plots of mean gene expression in wild-type ESCs targeted with Setd1a scFV or untargeted (−DOX), assayed by RT–qPCR from biological triplicates. Error bars, s.d. with significance calculated by two-tailed unpaired t -test. g , Epigenetic landscape response at the OFF reporter before (−DOX) and after (+DOX) targeted H3K4me3 programming. Histone modification enrichment is indicated across ~2 kb. n = 3 independent experiments with significance calculated by two-tailed unpaired t -test. h , Left: bar plots showing that the mean percentage of mCherry-positive cells is restricted after (+DOX) H3K4me3 installation by Prdm9 scFV in the presence or absence of the p300 inhibitor (inh) A485. Con, control. Data are biological triplicates; error bars represent s.d. P values were calculated by two-way ANOVA with Tukey’s correction. Right: relative abundance of the indicated histone modifications after programming H3K4me3 (+DOX) in the presence of A485. n = 3 independent experiments, with significance calculated by two-tailed unpaired t -test. i , Schematic of the strategy and scatterplot showing genes that depend on MLL2-mediated promoter H3K4me3 for upregulation (up) during the ESC transition to EpiLCs. Significant genes are colored. j , Dot plots showing normalized log expression of each gene ( n = 498) that is normally activated in wild-type EpiLCs but fails to be upregulated in Mll2 CM/CM cells. Where indicated, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For experiments employing the p300 inhibitor A485, cells were stimulated with 100 ng ml −1 DOX for 3 d and, in parallel, treated with 3 μM A485 (Cayman Chemical, 24119).

Techniques: Expressing, Reverse Transcription, Quantitative RT-PCR, Two Tailed Test, Gene Expression, Control, Modification

Journal: Nature Genetics

Article Title: Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications

doi: 10.1038/s41588-024-01706-w

Figure Lengend Snippet: ( a ) Genome view of replicate assays showing genes that specifically lose promoter H3K4me3 in Mll2 CM/CM ESC (red), which is linked with strong expression downregulation (green). ( b ) qRT-PCR showing re-targeting H3K4me3 back to endogenous promoters that have lost H3K4me3 in Mll2 CM/CM ESC partially rescues their expression level (see also Fig. ). The control Pldn gene exhibits no initial loss of H3K4me3 (indirectly affected), and accordingly was not rescued by deposition of further H3K4me3. Bar plots show the mean of n = 3 biologically independent experiments. Error bars represent S.D. Significance of rescue is calculated by two-tailed unpaired t-test. ( c ) Silent endogenous genes targeted for H3K4me3 epigenetic editing that do not exhibit significant transcriptional responses. Bar plots show the mean of N = 3 biologically independent experiments. Error bars represent S.D. Significance by one-way ANOVA with Tukey’s multiple test correction. ( d ) Comparison of two independent H3K4me3 effectors for epigenetic editing (Prdm9-CD scFV and Setd1a-CD scFV ). Upper: CUT&RUN-qPCR showing the level of H3K4me3 deposited at the OFF reporter promoter by each effector and their respective catalytic-mutant controls. Note Prdm9-CD scFV deposits significantly higher levels of H3K4me3 than Setd1a-CD scFV . Lower: transcriptional impact of H3K4me3 programming in single cells to each effector reveals a dose-dependent response. Bar plots show the mean of N = 3 biologically independent experiments. Error bars represent S.D. P-values are calculated by one-way ANOVA with Tukey’s multiple test correction. ( e ) Flow cytometry plot at day 3 of Prdm9 scFV induction, showing ~half the population have initiated a transcriptional response (activation). Active (ON) and inactive (OFF) populations were purified and the level of deposited H3K4me3 assayed by CUT&RUN-qPCR. Whilst all cells are enriched with H3K4me3, those with the higher levels are active, indicating a threshold level of H3K4me3 is necessary to trigger transcriptional activation. Bar plots show the mean of N = 3 biologically independent experiments. Error bars represent S.D. ( f ) Representative flow cytometry histogram showing that programming H3K4me3 no longer activates expression in the presence of an acetylation inhibitor (A485) - compare with short-term induction plot above with no A485. Shown right is CUT&RUN-qPCR confirming H3K4me3 is programmed in the presence of A485 but cannot elicit downstream effects on transcription. Significance calculated by one-tailed unpaired t-test. *P < 0.05 **P < 0.01, ***P < 0.001 .

Article Snippet: For experiments employing the p300 inhibitor A485, cells were stimulated with 100 ng ml −1 DOX for 3 d and, in parallel, treated with 3 μM A485 (Cayman Chemical, 24119).

Techniques: Expressing, Quantitative RT-PCR, Control, Two Tailed Test, Comparison, Mutagenesis, Flow Cytometry, Activation Assay, Purification, One-tailed Test