Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Top 1000 genes ranked by enrichment or depletion. (b-c) Categorization of top 200 genes enriched (b) or depleted (c) in cardiac myocytes compared to undifferentiated hiPSCs by biological groups. (d-e) Gene ontology (GO) analysis of top enriched (d) or depleted (e) hits (see Methods for details). (f-g) Venn diagrams demonstrating the number of CHD (f) or Non-CHD (g) probands with predicted damaging DNVs in hits identified in our screen as enriching hiPSC:CM differentiation or depleting hiPSC:CM differentiation. Arrows depict Venn diagrams representing the number of probands from each cohort with predicted damaging DNVs identified in our screen that have mutations in known dominant CHD genes or where these candidate CHD genes may potentially be causative. (h) Venn diagram demonstrating the number of CHD probands with (purple) or without (brown) damaging DNVs in hits identified in our screen highlighting no enrichment for extracardiac anomalies (p=0.43) or neurodevelopmental delay (NDD; p=0.23) and a slight enrichment for conotruncal CHD (p=0.04). (i) TNNT2+ cells quantified by flow cytometry at day 10 of hiPSC to CM differentiation in WTC11 cells treated with 100nM JQ1 starting at day 6 (n=3 biologically independent samples). (j) MZ3 treatment (500 nM for 0, 3.5, 7, and 16 hours) effectively degrades BRD4 in SV20 hiPSCs as assessed by immunoblot analysis for BRD4 with β-actin expression as a loading control. In all graphs, error bars represent ±1 SEM. * represents p=0.0271 (two-tailed unpaired t test).

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Flow Cytometry, Western Blot, Expressing, Control, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: a-c, UMAP plots of single-cell gene expression of Brd4flox/flox (n = 2) and Mef2c-AHF-Cre; Brd4flox/flox (n = 2) embryos visualized by genotype (a), sample identity (b), or cluster identity (c). d, Gene expression analysis of single-cell data. Heat map shows genes most enriched in each cluster and their inferred identity. e, Violin plots showing gene expression of critical regulators in select clusters. f, Percentage of cells in each cluster in each genotype highlighting the expansion of cluster 0 (MSX1/2+ progenitors) upon BRD4 deletion. g,h, Multiple lineage and pseudotime trajectory inference of cells from control (Brd4flox/flox) (g) and mutant (Mef2c-AHF-Cre; Brd4flox/flox) (h) embryos. i, Proposed model for BRD4 action in SHF CPCs: BRD4 is critical for CM differentiation in a subset of SHF CPCs, with its loss leading to persistence of Wnt-activated, ISL1+, and MSX1/2+ CPCs. A, atrium; pSHF, posterior second heart field.

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Expressing, Control, Mutagenesis

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Image of Isl1Cre/+; Brd4flox/+ embryo appearing in Figure 2i with region microdissected for bulk RNA-seq highlighted in red. (b) Principal component analysis of RNA-seq from Isl1Cre/+; Brd4flox/+ (blue) and Isl1Cre/+; Brd4flox/flox (red) E9.5 embryos. (c) Volcano plots of E9.5 Isl1Cre/+; Brd4flox/+ vs. Isl1Cre/+; Brd4flox/flox embryonic hearts (same data appearing in Figure 4) with a subset of cardiac and Wnt-related genes annotated (see Methods for details). Isl1Cre/+; Brd4flox/+ (d,f) and Isl1Cre/+; Brd4flox/flox (e,g) E9.5 embryos at level of right ventricle stained with ISL1 (red, d-e), BRD4 (green, d-e), or AXIN2 (green, f-g). Note the expansion of ISL1- (arrow heads) and AXIN2- (dotted line) expressing cells into the RV from distal outflow tract in mutant embryos. (h-m) ISL1 and AXIN2 Immunohistochemistry of E10.5 Mef2c-AHF-Cre; Brd4flox/+ (h,k) and Mef2c-AHF-Cre; Brd4flox/flox (i,j,l,m) embryos at the level of outflow tract. (h-j, ISL1; k-m, AXIN2). Note the expansion of ISL1- (arrow heads) and AXIN2- (dotted line) expressing cells into the right ventricle from distal outflow tract in mutant embryos. (n-o) AXIN2 RNAscope of Brd4flox/flox (n,n′) and Isl1Cre/+; Brd4flox/flox (o,o′) E9.5 embryos at the level of the right ventricle (n′ and o′ are magnified images of n and o, respectively). (p-q) ISL1 representative immunofluorescence at day 8 of mESC-derived cardiac cultures treated with vehicle (DMSO; VEH) (p) or JQ1 (500 nM) (q) starting at day 5. (r) Isl1 expression in day 8–9 mESC-derived cardiac cultures treated with increasing doses of JQ1 (0–500 nM; JQ1 added at day 5; n=3 n=3 biologically independent samples per dose). (s-t) AXIN2 RNAscope of Mef2c-AHF-Cre; Brd4flox/+ (s,s′) and Mef2c-AHF-Cre; Brd4flox/flox (t,t′) E10.5 embryos at level of right ventricle (s′ and t′ are magnified images of s and t, respectively). For r, all comparisons are made relative to 0 nM compound. *** represents p=0.0007 (two-tailed unpaired t test). RV, right ventricle; OT, outflow tract. Scale Bar = 250 μm (a), 50 μm (d-m, n, o, s, t), 100 μm (p, q, n′, o′, s′, t′)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: RNA Sequencing Assay, Staining, Expressing, Mutagenesis, Immunohistochemistry, RNAscope, Immunofluorescence, Derivative Assay, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: Inhibi (a) Expression of Myh6, Nkx2–5, and Tnnt2 at day 7 of mESCs cardiac differentiation treated with JQ1 (100 nM) starting day 5 of differentiation (n=3 biologically independent samples). (b) TNNT2+ cells quantified by flow cytometry at d9 of mESC differentiation in CMV-CreERT2; Brd4flox/flox cells treated with 4-hydroxytamoxifen (TAM), JQ1 (250 nM) or MZ3 (500 nM) starting at day 5 (n=3 biologically independent samples). (c-d) Immunofluorescence of TNNT2 at day 9 of mESC to CM differentiation in wild type cells treated with JQ1 (100 nM) or vehicle starting at day 5. (e-f) Immunofluorescence of BRD4 in vehicle (ethanol, e-e′′) and TAM (f-f′′) treated undifferentiated mESCs. (g) Volcano plot showing RNA-seq from CMV-CreERT2; Brd4flox/flox (TAM vs. vehicle [VEH]) mESCs and gene ontology analysis of downregulated and upregulated genes (see Methods for details). (h) Heatmap showing expression of select transcription factor and muscle structural protein genes from RNA-seq in CMV-CreERT2; Brd4flox/flox (TAM vs. VEH) mESC-derived cardiac tissues (day 10; TAM or VEH added at day 5). In all graphs, error bars represent ±1 SEM. For a, all comparisons are made relative to 0 nM compound for each gene; * represents p<0.0493, ** represents p<0.0085 (two-tailed unpaired t test). For b, all comparisons are made relative to VEH for each condition; * represents p<0.0188 (two-tailed unpaired t test). Scale Bars = 100 μm (c, d, e, e′, e′′, f, f′, f′′)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, RNA Sequencing Assay, Derivative Assay, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: a-h, H&E staining (a,e) and immunohistochemistry (b-d, f-h) of Isl1Cre/+; Brd4flox/+ (b-d) and Isl1Cre/+; Brd4flox/flox (f-h) E14.5 hearts. Immunohistochemistry of Tnnt2 (red) and BRD4 (green). i,j, Isl1Cre/+; Brd4flox/+ (i,i′) and Isl1Cre/+; Brd4flox/flox (j,j′) E9.5 embryos. Regions in yellow boxes in i and j are shown in higher magnification in i′ and j′. k,l, H&E staining of a section through OFT and RV of Isl1Cre/+; Brd4flox/+ (k,k′) and Isl1Cre/+; Brd4flox/flox (l,l′) E9.5 embryos. Regions in yellow boxes in k and l are shown in higher magnification in k′ and l′. Scale bars, 200 μm (a,b,e,f), 100 μm (c,d,g,h,k,l), 250 μm (i,i′,j,j′) and 25 μm (k′,l′),

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Staining, Immunohistochemistry

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Lineage tracing of Isl1Cre/+; Brd4flox/+ and Isl1Cre/+; Brd4flox/flox with R26mTmG/+ allele. Immunohistochemistry of ISL1-derived cells (GFP) and TNNT2 or BRD4 (red) in heart and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes at E14 (5 sections from n=2 control embryos and 11 sections from n=4 mutant embryos). (b) Hematoxylin and eosin staining of a section through outflow tract and RV of Isl1Cre/+; Brd4flox/+ and Isl1Cre/+; Brd4flox/flox embryos at E12.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (10 sections from n=2 control embryos and 11 sections from n=2 mutant embryos). (c) Quantification of percentage phospho-histone H3-, cleaved caspase 3-, and TUNEL-positive cells in the RV of E12.5 and E14.5 embryos of indicated genotypes (n=2 biologically independent samples per genotype at E12.5; n=3–4 biologically independent samples per genotype at E14.5). (d) Hematoxylin and eosin staining of a section through outflow tract and RV of Isl1Cre/+; Brd4flox/+ and Isl1Cre/+; Brd4flox/flox embryos at E10 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (9 sections from n=3 control embryos and 8 sections from n=3 mutant embryos). (e) Quantification of percentage phospho-histone H3- and TUNEL-positive cells in the RV of E10 control (Isl1Cre/+; Brd4flox/+ or Brd4flox/flox) and Isl1Cre/+; Brd4flox/flox embryos (n=4 biologically independent samples per condition). (f) BRD4 and TNNT2 immunohistochemistry along with lineage tracing with R26mTmG/+ allele in Isl1Cre/+; Brd4flox/+ and Isl1Cre/+; Brd4flox/flox E9.5 embryos at level of right ventricle. Error bars represent ±1 SEM. All comparisons are made as indicated; * represents p=0.0091, ** represents p=0.0021, **** represents p<0.0001 (two-tailed unpaired t test). RV, right ventricle; LV, left ventricle; OT, outflow tract. Scale Bars = 100 μm (a, b, d, f)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: In Vivo, Immunohistochemistry, Derivative Assay, Control, Mutagenesis, Staining, TUNEL Assay, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: a,b, H&E staining of Mef2c-AHF-Cre; Brd4flox/+ (a) and Mef2c-AHF-Cre; Brd4flox/flox (b) E12.5 hearts. Note the RV hypoplasia in the mutant compared to control. c,d, Immunohistochemistry of BRD4 in Mef2c-AHF-Cre; Brd4flox/+ (c) and Mef2c-AHF-Cre; Brd4flox/flox (d) E10.5 OFTs. e,f, Mef2c-AHF-Cre; Brd4flox/+ (e) and Mef2c-AHF-Cre; Brd4flox/flox (f) E10.5 embryos. Scale bars, 100 μm (a-d) and 250 μm (e,f)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Staining, Mutagenesis, Control, Immunohistochemistry

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Hematoxylin and eosin staining of a section through outflow tract and RV of Mef2c-AHF-Cre; Brd4flox/+ and Mef2c-AHF-Cre; Brd4flox/flox embryos at E13.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (19 sections from n=3 control embryos and 18 sections from n=3 mutant embryos). (b) Quantification of percentage phospho-histone H3- and cleaved caspase 3-positive cells in the RV of E13.5 Mef2c-AHF-Cre; Brd4flox/+ and Mef2c-AHF-Cre; Brd4flox/flox embryos (n=3–4 biologically independent samples per genotype). (c) Hematoxylin and eosin staining of a section through outflow tract and RV of Mef2c-AHF-Cre; Brd4flox/+ and Mef2c-AHF-Cre; Brd4flox/flox embryos at E10.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (6 sections from n=3 control embryos and 6 sections from n=3 mutant embryos). (d) Quantification of percentage phospho-histone H3- and cleaved caspase 3-positive cells in the RV of E10.5 Mef2c-AHF-Cre; Brd4flox/+ and Mef2c-AHF-Cre; Brd4flox/flox embryos (n=3 biologically independent samples per genotype). Error bars represent ±1 SEM. All comparisons are made as indicated; * represents p=0.0489, ** represents p=0.0146, **** represents p<0.0001 (two-tailed unpaired t test). RV, right ventricle; LV, left ventricle. Scale Bars = 100 μm (a, c).

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: In Vivo, Staining, Control, Mutagenesis, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Attenuating Wnt signaling at the CPC stage (day 5) in mESC to CM differentiation by doubling the normal concentration of the small molecule Wnt inhibitor XAV939 concomitant with Brd4 genetic deletion by 4-hydroxytamoxifen treatment (TAM) in CMV-CreERT2; Brd4flox/flox mESCs partially normalizes expression of CPC markers and Msx1/2 by qRT-PCR (n=3–4 biologically independent samples per genotype). (b-e) Attenuating Wnt signaling at the CPC stage (day 5) in mESC to CM differentiation by doubling the normal concentration of the small molecule Wnt inhibitor XAV939 concomitant with Brd4 genetic deletion by 4-hydroxytamoxifen treatment (TAM) in CMV-CreERT2; Brd4flox/flox mESCs partially normalizes TNNT2 staining by immunofluorescence at day 9 of CM differentiation (for e, n=3 biologically independent samples per condition). (f,g) Attenuation of Wnt signaling at the CPC stage (day 6) in hiPSC to CM differentiation by addition of the small molecule Wnt inhibitor IWP4 (5 μM for low dose and 10 μM high dose) concomitant with BET inhibition using JQ1 (25 nM for low dose and 50 nM for high dose) increases the number of TNNT2+ cells as assessed by flow cytometry (n=3 biologically independent samples per condition). Error bars represent ±1 SEM. For a,e,f,g all comparisons are made with p values as indicated (two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Inhibition, Flow Cytometry

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: a, Heat maps showing enrichment of FLAG, BRD4 and H3K4Me3 CUT&RUN signals and H3K27Ac ChIP-seq34 enrichment from hiPSC-derived CPCs at day 6 ordered by FLAG intensity. b, Venn diagrams and table demonstrating co-occupancy of BRD4/FLAG and indicated histone modification (percentage of BRD4 peaks overlapping with indicated histone modification). c,d, Track view of AXIN2 (c) and CCND1 (d) loci showing indicated CUT&RUN factor occupancy or H3K27Ac ChIP-seq enrichment in hiPSC-derived CPCs. e, Metaplot demonstrating FLAG occupancy at genes associated with GO terms for Wnt signaling or contractility. ChIP-seq, chromatin immunoprecipitation followed by sequencing.

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Derivative Assay, Modification, ChIP-sequencing, Chromatin Immunoprecipitation, Sequencing

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Targeting strategy to introduce 3XFLAG epitope tag into the N-terminus of the endogenous BRD4 locus. (b) Karyotyping results of BRD4FLAG/FLAG hiPSC line. (c) Western blot analysis of protein lysates collected from BRD4FLAG/FLAG hiPSCs using FLAG antibody demonstrates expression of 3XFLAG-tagged BRD4 isoforms that are degraded upon addition of the PROTAC BET degrader dBET651. (d) Pearson correlation matrices demonstrating high reproducibility between replicate CUT&RUN datasets. (e-g) Track view of indicated loci showing CUT&RUN factor occupancy (FLAG, BRD4, H3K4Me3) or H3K27Ac ChIP-seq enrichment in hiPSC-derived CPCs.

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Introduce, Western Blot, Expressing, ChIP-sequencing, Derivative Assay

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: Mef2c-AHF-Cre; Brd4flox/+ (a) and Mef2c-AHF-Cre; Brd4flox/flox (b,c) E10.5 embryos at level of right ventricle stained with MSX1/2 (yellow); inset shows area indicated by arrowheads. RNAscope in Brd4flox/flox (d,e), Isl1Cre/+; Brd4flox/flox (f,g), Mef2c-AHF-Cre; Brd4flox/+ (h,i), and Mef2c-AHF-Cre; Brd4flox/flox (j,k) E9.5–10.5 embryos at level of right ventricle for MSX1 (d,f,h,j) or MSX2 (e,g,i,k). Regions in yellow boxes in d-k are shown in higher magnification in d′-k′. RV, right ventricle; OT, outflow tract. Scale Bars = 50 μm (a-c, d-k), 165 μm (d′, e′, f′, g′), 125 μm (h′, j′), 200 μm (i′, k′)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: In Vivo, Staining, RNAscope

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) Gene set enrichment plot showing that genes associated with high H3K9ac and H3K27ac are enriched for two independently defined pluripotency gene sets: Muller Plurinet (genes involved in the protein-protein network shared by diverse pluripotent cell types ) and Wong ESC Core (genes coordinately upregulated in mouse and human ESCs ). Data are derived from a single ChIP-Seq experiment . P values are calculated based on 1000 permutations by the GSEA algorithm and was not adjusted for multiple comparisons. ( b ) 2i increases acetylation at key pluripotency genes. H3K27ac (left) and H3K9ac (right) at enhancer (enh) or promoters of indicated genes as assessed by ChIP-qPCR. ( c ) ChIP-seq meta profile for Brd4 binding in ESCs cultured in S/L or S/L+2i. The metaprofile is centered on the midpoint of all Brd4 ChIP-seq peaks. ( d ) Brd4 ChIP-qPCR illustrating Brd4 binding in ESCs cultured in S/L (left) or S/L+2i (right) treated with DMSO (vehicle) or 500 nM JQ1 for 24 h. ( b,d ) Bars represent mean of n=3 technical replicates from one IP.

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Derivative Assay, ChIP-sequencing, ChIP-qPCR, Binding Assay, Cell Culture

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) Quantification of colony formation assay of cells cultured in S/L (left) or S/L+2i (right) transfected with Cas9 and the indicated sgRNA against a nongenic region of chromosome 8 (ch8, control) or exon 3 (ex3) or exon 4 (ex4) of Brd4 . Dotted line represents average of ch8 controls. Each bar represents quantification of a single well of a six-well plate; transfected samples were seeded in duplicate. ( b ) Alkaline phosphatase staining of colonies formed from single cells of clonal ESC lines edited with sgRNA against chromosome 8 (ch8) or Brd4 exon 3 and cultured in S/L or S/L+2i. One representative well of a six-well plate is shown. ( c ) Quantification of colony formation assay shown in ( b ). ( d ) Western blot depicting Brd4 levels in ESCs expressing doxycycline (dox)-inducible hairpins against Renilla (shRen) or Brd4 (shBrd4). Cells were cultured with or without dox for 48 h prior to harvest. Actin is used as a loading control. Western blot was performed two independent times. ( e ) Brightfield images of ESCs expressing shBrd4-2 cultured for 48 h with or without doxycycline (dox). ( f ) Population doublings of cells cultured for 72 h in doxycycline relative to controls grown without dox. ( g ) Alkaline phosphatase staining of colonies formed from single cells expressing the indicated hairpins grown in the presence or absence of doxycycline (dox) and cultured in S/L or S/L+2i. One representative well of a six-well plate is shown. All bars represent mean ±SEM ( c ) or ±SD ( f ) of n=3 independent samples. ****, P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparisons post test (shBrd4-1, P = 7.2e-10; shBrd4-2, P = 4.6e-8). Scale bar, 100 μm.

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Colony Assay, Cell Culture, Transfection, Control, Staining, Western Blot, Expressing

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) qRT-PCR on key pluripotency genes in ESCs cultured in S/L or S/L+2i treated with DMSO (veh) or 500 nM JQ1 for 24h. ( b ) qRT-PCR on key pluripotency genes in S/L or S/L+2i-cultured ESCs expressing dox-inducible hairpins against Renilla (shRen) or Brd4 (shBrd4) and treated with dox for 48 h. Shown are levels of Nanog and Esrrb in cells with dox relative to control S/L+shRen cells without dox. ( c ) Heat map shows expression of pluripotency associated genes measured by RNA-Seq in ESCs cultured in S/L or S/L+2i with vehicle (veh, DMSO) or 500 nM JQ1 for 72 h. Color scale represents Log 2 relative to mean expression level. ( d ) Nanog binding to key pluripotency loci in ESCs cultured in S/L or S/L+2i treated with DMSO or 500 nM JQ1 for 24h, assessed by ChIP-qPCR. Bar represents mean of n=3 technical replicates from one IP. ( e ) ATAC-Seq meta profile of chromatin accessibility in ESCs cultured in S/L (left) or S/L+2i (right) with DMSO or 500 nM JQ1. Data from 2 independent replicates are shown. ( f ) At baseline, 82% of Oct4, Sox2, Nanog (OSN) binding sites have an ATAC-Seq peak above background. OSN sites make up 60% (4,250/7,084) of the peaks that maintain accessibility despite JQ1 treatment in S/L+2i cultured ESCs but only 17% (93/545) of peaks lost in both conditions. ( g ) ChIP-seq meta profile for Med1 binding in ESCs cultured in S/L or S/L+2i treated 500 nM JQ1. ( h ) Box plot shows relative Med1 binding at n=635 genes that are downregulated > 2 fold, FDR < 5% by JQ1 in ESCs cultured in S/L but not in ESCs cultured in S/L+2i. Values from ChIP-Seq experiment shown in (g) with 1 ChIP per condition. Box, 25-75 th percentile; bar, median; whiskers, 5-95 th percentile. ****, P < 1e-15; ns, P = 0.27 by 2-way ANOVA with Tukey’s multiple comparisons post test. See . ( i ) Sites that gain Med1 binding in S/L+2i+JQ1 relative to S/L+JQ1 are mostly OSN binding sites. Data presented as mean ± SD of n=3 independent samples ( a , b ).

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Quantitative RT-PCR, Cell Culture, Expressing, Control, RNA Sequencing, Binding Assay, ChIP-qPCR, ChIP-sequencing

Journal: Pharmacological research

Article Title: BET degrader exhibits lower antiproliferative activity than its inhibitor via EGR1 recruiting septins to promote E2F1-3 transcription in triple-negative breast cancer.

doi: 10.1016/j.phrs.2024.107377

Figure Lengend Snippet: Fig. 3. The dissociation of BETs from E2F1–3 promoters upon MS645 and ARV771 treatments. a. Heat map of the normalized density of BRD4 in HCC1806 cells treated with MS645 or ARV771, centered at the TSS in a ±3 kb window. b. Genome browser views of the BRD4 peaks at E2F1–3 gene loci in HCC1806 cells treated with MS645 or ARV771. c. ChIP-qPCR analysis of BRD2, BRD3 and BRD4 occupancy at E2F1–3 gene loci in HCC1806 cells. d. ChIP-qPCR analysis of BRD2 and BRD4 occupancy at E2F1–3 gene loci in HCC1806 cells treated with MS645 or ARV771. e. Genome browser views of the H3K27ac peaks at E2F1–3 gene loci in HCC1806 cells treated with MS645 or ARV771. f and g. ChIP-qPCR analysis of H3K27ac (f), RNA polymerase II and MED1 (g) occupancy at E2F1–3 gene loci in HCC1806 cells treated with MS645 or ARV771. Statistical analysis was calculated by comparing DMSO to MS645 and ARV771 respectively. The data are shown as mean ± SEM from three biological replicates. Statistical analysis was performed using a two-tailed paired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.

Article Snippet: Antibodies against MED1 (A300–793A), BRD2 (A302–583A), BRD3 (A302–368A) and BRD4 (A301–985A100) were purchased from Bethyl Laboratories.

Techniques: ChIP-qPCR, Two Tailed Test

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a Scheme of screening using HiBiT-BRD4–expressing cells in ( d , e ). b , c MZ1-dependent decrease of HiBiT luminescence in a HCT116 cell line expressing HiBiT-BRD4. HiBiT-BRD4 cells were treated with the indicated concentration (nM) of MZ1 for 2 h ( b ) or for the indicated number of hours ( c ) before HiBiT luminescence analysis ( n = 5 ( b ) or 4 ( c ), biological replicates). d Heatmap presentation of chemicals that enhance or repress BRD4 degradation ( n = 3, biological replicates). HiBiT-BRD4 cells were treated with candidate chemicals for 6 h and with 30 nM MZ1 for 2 h. e Chemicals that enhance HiBiT-BRD4 degradation in the presence of MZ1. The data in ( d ) are presented to analyze chemicals that enhance/repress the BRD4 degradation in the presence ( y -axis) or absence ( x -axis) of MZ1 ( n = 3, biological replicates). f , g HiBiT-BRD4 cells were treated with PDD, GSK, or luminespib (1, 3, or 10 μM) for 4 h and with 30 nM MZ1 ( f ) or 50 nM dBET6 ( g ) for 2 h ( n = 4 ( f ) or 3 ( g ), biological replicates).

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Expressing, Concentration Assay

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a PDD promotes MZ1-induced degradation of BRD4 and BRD2. HeLa cells were treated with 3 μM PDD and/or 100 nM MZ1 for the indicated number of hours. The lower panel shows the band intensities of the blots from three biological replicates. P values in ANOVA are shown. b PDD promotes BRD4 degradation at a lower concentration than the concentration at which it exhibits cytotoxicity. (i) HiBiT-BRD4 cells were treated with the indicated concentration of PDD (6 h) together with MZ1 (2 h), and BRD4 degradation was quantified ( n = 6, biological replicates). Data were normalized to the MZ1 treatment alone. Asterisk: * P = 0.0003 or ** P < 0.0001 in ANOVA. (ii) The parental HCT116 cells were treated with the indicated concentration of PDD for 3 days, and cell viability was quantified ( n = 5, biological replicates). Asterisk: * P = 0.0003 or ** P < 0.0001 in ANOVA. c HCT116 cells were treated with 3 μM PDD for 6 h, and total RNA was isolated and subjected to RNA-sequencing analysis ( n = 3, biological replicates). d Knockdown of PARG promotes BRD4 degradation. HT1080 cells were transfected with the indicated siRNAs for 3 days, then treated with MZ1 as indicated. e , f PDD promotes BRD4 degradation induced by different PROTACs. HeLa ( e ) or HCT116 ( f ) cells were treated with PDD and/or ARV771 ( e ) or with the CRL4 CRBN -based dBET6 ( f ), as indicated. g , h Targeted degradation of ERα or MEK1 is not affected by PDD. MCF7 ( g ) or HCT116 ( h ) cells were treated with the indicated chemicals. i HiBiT-BRD4 cells were pre-treated with either PDD (4 h) and/or the indicated inhibitors (0.5 h) and then with 30 nM MZ1 for an additional 2 h ( n = 4, biological replicates).

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Concentration Assay, Isolation, RNA Sequencing, Knockdown, Transfection

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a PDD promotes ubiquitylation of BRD4 and BRD2. HeLa cells were treated with the indicated concentration of MZ1 for 1 h. MG132 was added 5 min prior to the treatment with MZ1. The ubiquitylated proteins were purified from the cell lysates using TUBE-conjugated agarose. Input or TUBE-pulldown samples were subjected to western blotting, as indicated. b TUBE pulldown using either K29-, K48-, or K63-specific TUBEs. c BRD4 was modified with K48- and K29-linked ubiquitin chains. Endogenous BRD4 was immunopurified from HCT116 cells treated with PDD (3 μM, 6 h) and MZ1 (100 nM, 1 h) together with MG132 (20 μM, 1 h) and subsequently subjected to PRM-based ubiquitin linkage quantification ( n = 2, biological replicates). d HCT116 cells were treated with PDD (3 μM, 6 h) and/or MZ1 (100 nM, 1 h), and BRD4-modified ubiquitin chains were analyzed using PRM. The data show abundance (normalized to the vehicle) of signature peptides for K29 ubiquitin linkages and total ubiquitin ( n = 2, biological replicates). e HT1080 cells transfected with the indicated siRNAs were treated as in ( a , b ) to pulldown K29-linked ubiquitin chains. f Knockdown of TRIP12 partially canceled PDD-dependent promotion of BRD4 degradation. HeLa cells were transfected with the indicated siRNAs and treated with PDD (3 μM, 6 h) and/or MZ1 (100 nM, 1 or 2 h). g BRD4–MZ1–CRL2 VHL ternary complex assembly. 293T cells were transfected with FLAG-BRD4 (lanes 2–5) and/or HA-VHL (lanes 1–5), and cell lysates were subjected to immunoprecipitation using anti-FLAG antibody. h The samples in ( g ) were subjected to LC-MS and label-free quantification ( n = 3, biological replicates, ANOVA). i Heatmaps of ChIP-seq signals of BRD4 in the cells treated either with control, PDD17273, or MZ1. BRD4-binding regions in control cells ( n = 3562) were subtracted for plotting, and peaks were divided into three clusters. j Percentage of BRD4-binding regions consisting of the three clusters. k Metaplots of ChIP-seq signals of BRD4 over the center of peaks. l Metaplots depicting H3K27ac ChIP-seq signals in HCT116 cells over BRD4-binding regions. m HCT116 cells were treated with PDD (5 μM, 4 h), and cell fractionation was performed. PARylation of chromatin fractions was analyzed.

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Concentration Assay, Purification, Western Blot, Modification, Ubiquitin Proteomics, Transfection, Knockdown, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, ChIP-sequencing, Control, Binding Assay, Cell Fractionation

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a PERK inhibitors promote BRD4 degradation. HCT116 cells were treated with either GSK, GSK2656157, or AMG-PERK (10 μM, 6 h) and/or MZ1 (100 nM, 1 or 2 h). b HSP90 inhibitors promote BRD4 degradation. HCT116 cells were treated with either GSK, luminespib, or 17-AAG (10 μM, 6 h) and/or MZ1 (100 nM, 1 or 2 h). c BRD4 degradation in the presence of GSK is proteasome-dependent. HCT116 cells were treated with either GSK (10 μM, 6 h), MG132 (20 μM, 2 h), or MZ1 (100 nM, 1 or 2 h). d HCT116 cells were treated with either PDD, GSK, or luminespib (10 μM, 14 h) and/or 50 ng/mL CHX for the indicated number of hours. Total cell lysates were subjected to Western blotting. e , f HCT116 cells were transfected with the indicated siRNAs and treated with MZ1 (100 nM, 1 or 2 h). (Right) BRD4 band intensities were quantified ( n = 3 ( d ) or 2 ( e ), biological replicates). Asterisk: * P = 0.0014 or ** P = 0.0002 in ANOVA. g , h HCT116 cells were treated with either GSK, luminespib, or 17-AAG (10 μM, 6 h) and/or MZ1 (100 nM, 1 h). Cell lysates were subjected to pulldown using the indicated TUBEs. i 293T cells were transfected with FLAG-BRD4 (lanes 2–6) and/or HA-VHL (lanes 1–6) and then treated with the indicated chemicals; co-immunoprecipitation was subsequently performed.

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Western Blot, Transfection, Immunoprecipitation

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a , b PDD or GSK promotes BRD4 degradation induced by SIM1. HeLa ( a ) or HCT116 ( b ) cells were treated with PDD, GSK, and/or MZ1 as indicated, and cell lysates were subjected to western blotting. c HiBiT-BRD4 cells were treated as indicated, and luminescence was measured ( n = 3, biological replicates). d , e PDD or GSK promotes cell death induced by SIM1. HeLa cells were treated with PDD, GSK, and/or MZ1 for 3 days, as indicated. Asterisk: ( d ) * P = 0.0006 or ** P < 0.0001 in ANOVA ( n = 5, biological replicates). e ** P < 0.0001 in ANOVA ( n = 5, biological replicates). f , h – j HeLa ( f ), HCT116 ( h ), MCF7 ( i ), or MDA-MB231 ( j ) cells were treated with the indicated chemicals for 6 h or for the indicated number of hours (100 nM ThalSNS, 30 nM ARV471, 0.5 μM SJF8240). Total cell lysates were subjected to Western blotting. The lower panel shows the band intensities of the blots from two biological replicates. g HeLa cells were treated with the indicated chemicals for 3 days, and cell viability was quantified. Asterisk: * P = 0.026 or ** P < 0.0001 in ANOVA ( n = 5, biological replicates). k Schematic model. Various cell-intrinsic pathways spontaneously counteract target degradation at multiple steps. Inhibitors to PARG, PERK, or HSP90 robustly enhance the targeted degradation of BRD4 as well as BRD2/3 and sensitize cells to PROTAC-induced apoptosis. PARG inhibition promotes TRIP12-mediated K29/K48-branched ubiquitylation of BRD4 by facilitating the BRD4-PROTAC-CRL2 VHL ternary complex, while HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step.

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Western Blot, Inhibition

Journal: Nucleic Acids Research

Article Title: A histidine cluster determines YY1-compartmentalized coactivators and chromatin elements in phase-separated enhancer clusters

doi: 10.1093/nar/gkac233

Figure Lengend Snippet: YY1 compartmentalizes additional coactivators to nuclear puncta. ( A and B ) Colocalization of BRD4, MED1 and CDK9 with endogenous YY1 (A) or with Flag-YY1 (B) in nuclear puncta in MDA-MB-231 cells. In (B), endogenous YY1 was knocked down by sh-YY1-3′-UTR. Endogenous proteins were detected by their corresponding antibodies. Flag-YY1 was detected by a Flag epitope antibody. Nuclei were visualized by DAPI staining. Line scans of the colocalization images are depicted by white arrows with quantification shown at right. ( C ) Quantified average sizes of merged puncta in MDA-MB-231 cells with endogenous YY1 (A) and Flag-YY1 (B). Data are mean ± s.e.m. of puncta in 6 fields in each group. ( D and E ) Colocalization of active RNA Pol II with YY1 (D) or Flag-YY1 (E) in nuclear puncta of MDA-MB-231 cells. In (E), endogenous YY1 was knocked down by sh-YY1-3′-UTR. Active RNA Pol II was detected by antibodies for phosphorylation of Ser 5 (S5P) or Ser 2 (S2P), with nuclei detected by DAPI. Line scans of colocalization images are depicted by white arrows with quantification shown at right. ( F ) Quantification of average sizes of merged puncta in MDA-MB-231 cells with endogenous YY1 (D) and Flag-YY1 (E). Data are presented as mean ± s.e.m. from puncta of six fields in each group. ( G ) Representative images of droplet formation of mCherry-YY1 with EGFP-MED1-IDR, EGFP-BRD4-IDR, or EGFP-Pol II-IDR. ( H and I ) Localization of active (H3K27ac, H3K4me1 and H3K4me3) and repressive (H3K9me3) histone markers with endogenous YY1 (H) or Flag-YY1 (I) in MDA-MB-231 cells. In (I), endogenous YY1 was knocked down by sh-YY1-3′-UTR. Histone markers were determined using corresponding antibodies. Nuclei were detected by DAPI. Line scans of colocalization images are depicted by white arrows with quantification shown at right. ( J ) Quantified average sizes of merged puncta in MDA-MB-231 cells with endogenous YY1 (H) and Flag-YY1 (I). Data are presented as mean ± s.e.m. of puncta in six fields in each group. All experiments in this figure were independently repeated at least 6 times with similar results. In (C), (F) and (J), P values are indicated on top of the quantification analyses. n.s.: not significant.

Article Snippet: Reagents and antibodies used in this study include: YY1 (H-10) (Santa Cruz, cat# sc-7341, 1:1000 for western blot (WB), 1:300 for immunofluorescent (IF) staining); YY1 (H414) (Santa Cruz, cat# sc-1703, 1:50 for chromatin immunoprecipitation (ChIP)); FOXM1 (Thermo Fisher Scientific, cat# 702664, 1:5000 for WB); Flag (Sigma, cat# F1804, 1:300 for IF); EP300 (Cell Signaling Technology, cat# 86377, 1:500 for IF, 1:50 for ChIP); BRD4 (Santa Cruz, cat# sc-518021, 1:200 for IF, 1:50 for ChIP); MED1 (Santa Cruz, cat# sc-74475, 1:200 for IF, 1:50 for ChIP); CDK9 (Cell Signaling Technology, cat# 2316, 1:100 for IF); RNA Pol II-S2P (Millipore, cat# 04-1571, 1:200 for IF); RNA Pol II-S5P (Millipore, cat# 04-1572, 1:200 for IF); H3K4me1 (Cell Signaling Technology, cat# 5326, 1:500 for IF); H3K4me3 (Cell Signaling Technology, cat# 9751, 1:200 for IF); H3K27ac (Abcam, cat# ab4729, 1:500 for IF); H3K9me3 (Cell Signaling Technology, cat# 13969, 1:500 for IF); pAKT-Thr308 (Cell Signaling Technology, cat# 13038S, 1:1000 for WB); pAKT-Ser473 (Cell Signaling Technology, cat# 4060S, 1:1000 for WB); AKT (Cell Signaling Technology, cat# 4685S, 1:1000 for WB); Ki-67 (Thermo Fisher Scientific, cat# 710229, 1:100 for IF); GAPDH (Acton, cat# 10R-G109A, 1:1000 for WB); Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, cat# A32731, 1:500 for IF); Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific, cat# A32742, 1:500 for IF); Goat anti-mouse IgG-HRP (Santa Cruz, cat# sc-2005,1:5000 for WB); Goat anti-rabbit IgG-HRP (Santa Cruz, cat# sc-2004, 1:5000 for WB); JQ-1 carboxylic acid (MedChemExpress, cat# 202592-23-2); 1,6-hexanediol (Aladdin, cat# H103708).

Techniques: FLAG-tag, Staining, Phospho-proteomics

Journal: Nucleic Acids Research

Article Title: A histidine cluster determines YY1-compartmentalized coactivators and chromatin elements in phase-separated enhancer clusters

doi: 10.1093/nar/gkac233

Figure Lengend Snippet: YY1 binds to the FOXM1 promoter and activates its expression. ( A ) YY1 and FOXM1 expression in mammary cell lines. Nontumorigenic MCF-10A cells, and three indicated breast cancer cell lines were analyzed by Western blot using indicated antibodies (top row). YY1 and FOXM1 mRNA levels were quantified by RT-qPCR (bottom row). ( B ) Effects of YY1 on FOXM1 in mammary cells. MCF-10A cells were infected by lentivirus expressing YY1, while MDA-MB-231 and MCF-7 cells were infected by lentivirus carrying sh-YY1. YY1 and FOXM1 protein and mRNA levels were analyzed by Western blot (top row) and RT-qPCR (bottom row). ( C ) Mapping the YY1-regulated region in the FOXM1 promoter. Conserved YY1 binding elements with a core sequence of ATGG (Jaspar Matrix ID: MA0095.2, top row). Reporters with different FOXM1 promoter lengths driving Gluc were generated. Data of reporter assays in response to ectopic YY1 in HeLa cells were examined (bottom row). Data represent mean ± s.d. ( n = 3). ( D ) Evaluation of putative YY1 binding sites in the FOXM1 promoter. Putative YY1 binding sites S1–S5 were identified in the 1141-bps region upstream of the transcription start site (TSS) of the FOXM1 gene, with WT and mutants presented (top panel). Data of reporter assays in response to YY1 in HeLa cells are presented in the bottom row. ( E ) ChIP-qPCR assays to examine YY1 binding to the FOXM1 promoter. The FOXM1 promoter is presented in the top row with five qPCR primer pairs. YY1 antibody and a control IgG were used in ChIP assays. Data quantification and agarose gels of ChIP-qPCR in MDA-MB-231 and MCF-7 cells were presented in the middle and bottom rows, respectively. ( F ) EMSA to test YY1 binding to the FOXM1 promoter. His × 6-YY1 was incubated with Cy5-labeled S2, S4, S5 probes, and mutants (S2M, S4M and S5M) with a CDC6 promoter probe and its mutant as positive and negative controls, respectively. YY1-probe complex and free probe positions are labeled on the left. ( G ) Left: representative images of droplet formation by EGFP-YY1 with Cy5-labeled probes in Figure and . Right: schematic model of droplet formation promoted by DNA probes. ( H ) Image of live MDA-MB-231 and MCF-7 cells transfected with EGFP-YY1(ΔZF) plasmid. ( I ) ChIP-qPCR assays to test the effects of YY1(H-A) mutant expression on coactivators’ binding to the FOXM1 promoter. MDA-MB-231 cells with endogenous YY1 knocked down by sh-YY1-3′-UTR and infected by lentivirus carrying an empty vector, or expressing Flag-YY1 WT or its (H-A) mutant were collected. EP300, BRD4 and MED1 antibodies, and a control IgG were used in ChIP assays, and qPCR primers are shown in . Both quantification and agarose gels of ChIP-qPCR were presented. ( J and K ) Evaluation of YY1 mutations’ effects on the FOXM1 promoter. In (J), FOXM1 promoter reporter (pFOXM1-prmt-Gluc) and pCMV-SEAP vector were cotransfected with YY1 WT and mutant vectors into HeLa (left) and HEK-293T (right) cells in 24-well plates. Gluc activity in each well was measured and normalized against its SEAP activity. In (K), YY1 vectors were transfected into MDA-MB-231 (left) and MCF-7 (right) cells with endogenous YY1 knockdown, followed by RT-qPCR to quantify FOXM1 mRNA levels. Western blot analyses showing shRNA-mediated endogenous YY1 knockdown are presented at bottom panels.

Article Snippet: Reagents and antibodies used in this study include: YY1 (H-10) (Santa Cruz, cat# sc-7341, 1:1000 for western blot (WB), 1:300 for immunofluorescent (IF) staining); YY1 (H414) (Santa Cruz, cat# sc-1703, 1:50 for chromatin immunoprecipitation (ChIP)); FOXM1 (Thermo Fisher Scientific, cat# 702664, 1:5000 for WB); Flag (Sigma, cat# F1804, 1:300 for IF); EP300 (Cell Signaling Technology, cat# 86377, 1:500 for IF, 1:50 for ChIP); BRD4 (Santa Cruz, cat# sc-518021, 1:200 for IF, 1:50 for ChIP); MED1 (Santa Cruz, cat# sc-74475, 1:200 for IF, 1:50 for ChIP); CDK9 (Cell Signaling Technology, cat# 2316, 1:100 for IF); RNA Pol II-S2P (Millipore, cat# 04-1571, 1:200 for IF); RNA Pol II-S5P (Millipore, cat# 04-1572, 1:200 for IF); H3K4me1 (Cell Signaling Technology, cat# 5326, 1:500 for IF); H3K4me3 (Cell Signaling Technology, cat# 9751, 1:200 for IF); H3K27ac (Abcam, cat# ab4729, 1:500 for IF); H3K9me3 (Cell Signaling Technology, cat# 13969, 1:500 for IF); pAKT-Thr308 (Cell Signaling Technology, cat# 13038S, 1:1000 for WB); pAKT-Ser473 (Cell Signaling Technology, cat# 4060S, 1:1000 for WB); AKT (Cell Signaling Technology, cat# 4685S, 1:1000 for WB); Ki-67 (Thermo Fisher Scientific, cat# 710229, 1:100 for IF); GAPDH (Acton, cat# 10R-G109A, 1:1000 for WB); Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, cat# A32731, 1:500 for IF); Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific, cat# A32742, 1:500 for IF); Goat anti-mouse IgG-HRP (Santa Cruz, cat# sc-2005,1:5000 for WB); Goat anti-rabbit IgG-HRP (Santa Cruz, cat# sc-2004, 1:5000 for WB); JQ-1 carboxylic acid (MedChemExpress, cat# 202592-23-2); 1,6-hexanediol (Aladdin, cat# H103708).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Infection, Binding Assay, Sequencing, Generated, ChIP-qPCR, Control, Incubation, Labeling, Mutagenesis, Transfection, Plasmid Preparation, Activity Assay, Knockdown, shRNA

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone demethylase KDM9 activates the CCND1/AKT pathway to promote the malignant progression of gliomas through interaction with BRD2

doi: 10.1007/s00018-025-05900-9

Figure Lengend Snippet: KDM9 regulates CCND1 transcription through interaction with BRD2. ( A ) Protein silver staining of IP via KDM9 antibody enrichment in LN229 cells. ( B ) Transcription factors in the protein mass spectrometry data are shown. Exogenous and endogenous IP experiments were performed to analyze the interaction between KDM9 and BRD4 ( C ) or BRD2 ( D ). ( E ) The IF assay was used to detect the expression and co-localization of KDM9 and BRD2 in LN229 cells. ( F ) After the transfection of pcDNA3.1-BRD2-His into LN229-shKDM9 cells for 48 h, ChIP-qPCR was performed to detect the enrichment of H4K20me2 at the CCND1 P4 promoter region (with IgG as negative control). ( G ) After the transfection of wild-type or mutant KDM9 plasmids into U251 cell lines for 48 h, luciferase reporter assays were conducted to detect CCND1 transcription activity (Left). After the transfection of pcDNA3.1-BRD2-His into LN229-shKDM9 cells for 48 h, luciferase reporter assays were performed to detect CCND1 transcription activity (Right). ( H ) After the transfection of shBRD2 plasmids into U251-KDM9-WT cell lines for 48 h, luciferase reporter assays were conducted to detect CCND1 transcription activity. ( I ) After the transfection of pcDNA3.1-BRD2-His into shKDM9 glioma cells for 48 h, a Western blot experiment was conducted to detect the protein expression of KDM9, BRD2, CCND1, AKT/P-AKT, mTOR/P-mTOR, and CD133. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: CD133 (18470–1-AP, 1:1000), Histone H4 (16047–1-AP, 1:500), CCND1 (60186–1-Ig, 1:2000), SMAD2 (12570–1-AP, 1:2000), P-AKT (66444–1-IG, 1:1000), AKT (60230–2-IG, 1:1000), Flag-Tag (66008–4-Ig, 1:5000), BRD2 (22236–1-AP, 1:1000), and BRD4 (28486–1-AP, 1:1000) were purchased from Proteintech (Chicago, IL).

Techniques: Silver Staining, Mass Spectrometry, Expressing, Transfection, ChIP-qPCR, Negative Control, Mutagenesis, Luciferase, Activity Assay, Western Blot

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113 physically interacts with ZRF1 and BRD4 in NB cells. a Volcano plots showing differentially expressed genes (fold change> 1.5, P < 0.05) in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1 . b Coomassie blue staining (left panel) and Venn diagram (right panel) revealing identification of p113-interacting proteins pulled down by p113 or Flag-tag antibody in SH-SY5Y cells stably transfected with 3Flag-tagged p113 , and those overlapped with transcription factors (TF) or epigenetic factors derived from ChIP-X and EpiFactors databases. c Co-IP and western blot assays indicating the interaction among p113, ZRF1, and BRD4 in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , scramble shRNA (sh-Scb), or sh-ecircCUX1. d Secondary co-IP assays showing protein interaction among p113, ZRF1, and BRD4 in SH-SY5Y cells stably transfected with HA-tagged p113 , Flag-tagged ZRF1 , and His-tagged BRD4 . e BiFC assay revealing the interaction of p113 with ZRF1 or BRD4 (arrowheads) in SH-SY5Y cells stably transfected with indicated constructs, with nuclei stained by DAPI. Scale bars: 10 μm. f and g Western blot assay (g) validating the knockdown of ZRF1 or BRD4 in SH-SY5Y cells stably transfected with scramble (Scb) or specific sgRNA for CRISPR interference (CRISPRi, f). Wild type (WT) cells were taken as negative controls. h Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with CRISPRi sgRNA specific against ZRF1 or BRD4 . i Schematic illustration of protein interaction among p113, ZRF1, and BRD4. j Dual-luciferase assay showing the activity of ZRF1 in NB cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1 ( n = 5). Fisher’s exact test for overlapping analysis in b . ANOVA compared the difference in j . * P < 0.05 vs. mock or sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c - e , g , h and j

Article Snippet: Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [ , , ].

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Staining, FLAG-tag, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, shRNA, Bimolecular Fluorescence Complementation Assay, Construct, CRISPR, Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113/ZRF1/BRD4 complex promotes lipid metabolic reprogramming and mitochondrial complex I activity in NB cells. a Heatmap, distribution, and binding motif of ChIP-Seq (left panel) assay revealing genomic enrichment of ZRF1 in SH-SY5Y cells, while Venn diagram, heatmap, and GO pathway (right panel) showing identification of p113/ZRF1/BRD4 target genes by overlapping analysis of RNA-seq results upon p113 over-expression and ChIP-seq peaks of ZRF1 or BRD4. b ChIP-seq assay showing the binding peak of BRD4 or ZRF1 on promoter regions of ALDH3A1 , NDUFA1 , or NDUFAF5 in SH-SY5Y cells. c Western blot assay indicating the expression of ALDH3A1, NDUFA1, or NDUFAF5 in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi. d Schematic illustration showing the involvement of ALDH3A1, NDUFA1, or NDUFAF5 in lipid metabolic reprogramming and mitochondrial respiratory activity. e Relative OCR levels in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi ( n = 5). f Relative fatty acid levels, complex I activity, NAD + /NADH ratio, and ATP levels in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi ( n = 5). ANOVA compared the difference in e and f . * P < 0.05 vs. mock+CRISPRi-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c , e and f

Article Snippet: Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [ , , ].

Techniques: Activity Assay, Binding Assay, ChIP-sequencing, RNA Sequencing Assay, Over Expression, Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: Therapeutic blocking p113-ZRF1 interaction inhibits NB progression. a 3D structure and sequences of inhibitory peptides (ZIP-12) for blocking interaction between p113 and ZRF1, and those of mutant control (Ctrl) peptides. b Confocal images showing the distribution of synthesized FITC-labeled Ctrl or ZIP-12 peptides (20 μmol·L − 1 , arrowheads) within cultured BE(2)-C cells, with nuclei and cellular membranes staining with DAPI or Dil. Scale bars: 10 μm. c Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L − 1 ) for 24 h. d Relative fatty acid levels, complex I activity, NAD + /NADH ratio, and ATP levels in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L − 1 ) for 24 h. e In vivo images (left upper panel), growth curve (right panel), and weight at the end points (right panel) of xenografts formed by subcutaneous injection of BE(2)-C cells in nude mice ( n = 5 per group) that were treated with intravenous injection of Ctrl or ZIP-12 peptides (5 mg·kg − 1 ) as indicated (left lower panel). f In vivo imaging (left panel), lung metastatic colonization (right lower panel), and Kaplan–Meier curves (right lower panel) of nude mice ( n = 5 for each group) treated with tail vein injection of BE(2)-C cells, Ctrl or ZIP-12 peptides (5 mg·kg − 1 ) as indicated (right upper panel). Student’s t test or ANOVA compared the difference in d - f . Log-rank test for survival comparison in f . * P < 0.05, ** P < 0.01 vs. Ctrl. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in b - d

Article Snippet: Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [ , , ].

Techniques: Blocking Assay, Mutagenesis, Synthesized, Labeling, Cell Culture, Staining, Co-Immunoprecipitation Assay, Western Blot, Activity Assay, In Vivo, Injection, In Vivo Imaging

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: CUX1 , ZRF1 , BRD4 and target genes are associated with poor outcome of NB patients. a Kaplan–Meier curves indicating overall survival of 498 well-defined NB cases (GSE62564) with high or low expression of CUX1 (cutoff value = 32.233), ZRF1 (cutoff value = 21.749), BRD4 (cutoff value = 652.58), ALDH3A1 (cutoff value = 1.181), NDUFA1 (cutoff value = 22.511), or NDUFAF5 (cutoff value = 7.964). b The positive expression correlation between ZRF1 and ALDH3A1 , NDUFA1 , or NDUFAF5 in 498 well-defined NB cases (GSE62564). c The mechanisms underlying p113-faciliated NB progression: as a novel protein encoded by ecircCUX1 , p113 cooperates with ZRF1 and BRD4 to activate the transcription of ALDH3A1 , NDUFA1 , or NDUFAF5 , resulting in promoted conversion of fatty aldehydes into fatty acids, fatty acid β-oxidation, mitochondrial complex I activity, growth, and aggressiveness of NB cells. Meanwhile, inhibitory peptides (ZIP-12) blocking p113-ZRF1 interaction suppresses tumor progression. Log-rank test for survival comparison in a . Pearson’s correlation coefficient for b

Article Snippet: Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [ , , ].

Techniques: Expressing, Activity Assay, Blocking Assay

Journal: European journal of medicinal chemistry

Article Title: DW71177: A novel [1,2,4]triazolo[4,3-a]quinoxaline-based potent and BD1-Selective BET inhibitor for the treatment of acute myeloid leukemia.

doi: 10.1016/j.ejmech.2023.116052

Figure Lengend Snippet: Fig. 3. Structural basis of the BD1 selectivity of compound 3. (A and B) Surface representation of the BRD4-BD1 with bound compound 3 (green sticks). (A) The protein surface is colored by conservation scores using the ConSurf server. (B) The protein surface is colored according to the YRB scheme, which highlights both hydrophobicity (yellow) and charge (red or blue) in protein structures. The residues constituting the rim of the binding pocket are labeled. (C) A closer view of the BRD4-BD1 inhibitor binding site. Compound 3 is presented in green sticks and BD1 in gray ribbons. (D and E) Superposition of the BD1-compound 3 structure onto human apo-BRD4-BD1 (cyan, PDB ID 2OSS) (D) and apo-BRD4-BD2 (pink, PDB ID 2OUO) (E). Water molecules and hydrogen bonds are shown as red spheres and orange dotted lines, respectively. (F) Sequence alignment of human bromodomain and extraterminal domain (BET) family proteins. Fully and partially conserved residues are shaded in blue and gray, respectively. Residues interacting with compound 3 are indicated by filled circles on the top. Sequence positions corresponding to Asp144 and Ile146 of BRD4 are highlighted in red letters and circles, respectively.

Article Snippet: The genes encoding the bromodomains of human BRD2, BRD3, BRD4 and BRDT were PCR-amplified from the plasmids obtained from Addgene (plasmid no. 65376, 65377, 65378 and 65381, respectively).

Techniques: Binding Assay, Labeling, Sequencing

Journal: European journal of medicinal chemistry

Article Title: DW71177: A novel [1,2,4]triazolo[4,3-a]quinoxaline-based potent and BD1-Selective BET inhibitor for the treatment of acute myeloid leukemia.

doi: 10.1016/j.ejmech.2023.116052

Figure Lengend Snippet: Fig. 5. Transcriptome analyses of DW-71177-driven BD1-selective inhibition. (A) Venn diagrams showing the number of genes that were more than 1.5-fold downregulated (left) or upregulated (right) when MV4-11 cells were treated with OTX-015 (300 nM), DW-71177 (300 nM), DW-71374 (300 nM), or RVX-208 (5 μM) for 24 h. (B) Clustering analysis of compound-induced transcriptional alterations. (C) Fold change (FC) in gene expression upon treatment of BET in hibitors (BETis) was examined using the gene sets selected with BRD4 ChIP-seq data analysis. (D) Fold change in the mRNA expression of acute myeloid leukemia (AML) oncogenes and tumor suppressor genes (TSGs) upon treatment with BETis. (E) Fold change in the mRNA expression of housekeeping genes upon treatment with BETis.

Article Snippet: The genes encoding the bromodomains of human BRD2, BRD3, BRD4 and BRDT were PCR-amplified from the plasmids obtained from Addgene (plasmid no. 65376, 65377, 65378 and 65381, respectively).

Techniques: Inhibition, Gene Expression, ChIP-sequencing, Expressing

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 1. JNK directly interacts with and phosphorylates BRD4 (A) BRD4 co-localizes with kinase active JNK. Proximity ligation assays (PLAs) with anti-BRD4 and anti-pJNK on fixed HCT116 cells. Negative control; anti- nucleolin, and anti-BRD4 (scale bars, 20 mM). (B) JNK co-immunoprecipitates with BRD4. BRD4 was immunoprecipitated from HeLa nuclear extract using anti-BRD4 and immunoblotted with anti-JNK. (C) BRD4 binds JNK directly. Recombinant JNK1 (0.1 and 0.2 mg) was pulled down with 0.5 mg rBRD4 immobilized on FLAG beads. (D) JNK phosphorylates BRD4. Upper: map of BRD4 and deletion mutants. Lower: autoradiograph of kinase assays with GST-JNK1 and WT-BRD4 or deletion mutants. (E) JNK phosphorylation sites on BRD4. Upper: JNK consensus phosphorylation sites located on human/mouse BRD4. Lower: autoradiograph of kinase assays with His-JNK1 and BRD4 WT or the point mutants. (F) BRD4 is phosphorylated at Thr1186 and Thr1212 JNK activation. HCT116 cells were treated with anisomycin, heat shock, LPS treatment, or UV stress. BRD4 phosphorylation was assessed by immunoblotting (upper) and densitometric quantification (lower). (G) BRD4’s interaction with JNK is abrogated by phosphorylation. CoIP of JNK with BRD4 following anisomycin treatment of WT- and 3A-BRD4-expressing HCT116 cells. See also Figures S1 and S2.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Ligation, Negative Control, Immunoprecipitation, Recombinant, Autoradiography, Phospho-proteomics, Activation Assay, Western Blot, Expressing

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 2. JNK phosphorylation of BRD4 releases it from chromatin (A) BRD4 is released from the chromatin upon JNK activation by anisomycin. Immunoblots of chromatin-free (CF) and chromatin-bound (CB) BRD4 in HCT116 cells following treatment with anisomycin. (B) Inhibition of JNK kinase blocks BRD4’s release from chromatin. Immunoblots of CF and CB BRD4 in HCT116 cells transfected with WT JNK1 or JNK1 and dominant-negative kinase mutants JNK1/JNK2 (APF) individually or in combination, followed by anisomycin treatment. (C) BRD4 is released from the chromatin upon JNK activation by heat shock. Left: immunoblots of CF and CB BRD4 in HCT116 cells grown at 37C or heat shocked at 42C for 15 min in the presence or absence of JNK inhibitor SP600125. Right: immunoblots showing pJNK levels under the above conditions. (D) JNK preferentially phosphorylates BRD4 bound to mononucleosomes. Anti-BRD4 pT1212 and anti-BRD4 immunoblots of kinase assays with recombinant JNK1 and BRD4 after pre-incubating BRD4 with or without assembled mononucleosomes (MN) for 10 or 20 min. (E) Mutation of BRD4 phosphorylation sites prevents BRD4 release from chromatin. Left: immunoblots of CF and CB BRD4 in HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock treatment. Right: immunoblots showing pJNK levels following heat shock in WT- and 3A-BRD4-expressing cells. (F) JNK activation results in global loss of CB BRD4. Total BRD4 peaks detected in BRD4 ChIP-seq of control-untreated and anisomycin-treated DLD1 BRD4- IAA7 cells expressing endogenous BRD4 or exogenous WT or 3A-BRD4 following auxin treatment. (G) Loss of JNK-phosphorylated BRD4 from chromatin is widespread. Distribution of BRD4 ChIP-seq peaks across the genomes of cells described in (F).

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Inhibition, Transfection, Dominant Negative Mutation, Recombinant, Mutagenesis, Expressing, ChIP-sequencing, Control

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 3. JNK-mediated BRD4 release from chromatin disrupts its nucleosome clearance function (A) BRD4 binding to mononucleosomes is abrogated upon phosphorylation by JNK. Anti-histone H3 immunoblot of assembled mononucleosomes pulled down by recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK and immobilized on FLAG beads. (B) JNK phosphorylation of BRD4 inhibits H3K122 acetylation. Anti-histone H3K122ac immunoblot of assembled mononucleosomes subjected to an in vitro HAT assay with recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK. (C) H3K122 acetylation is reduced in vivo upon JNK activation. Immunoblots of whole-cell extracts (WCEs) of HCT116 cells that were untreated (control) or treated with either DMSO (mock) or anisomycin. (D) In vivo H3K122 acetylation by BRD4 is regulated by JNK. Immunoblots of WCEs of HCT116 cells that were transfected with WT or 3A-BRD4 and treated with or without anisomycin. (E) In vivo H3K122 acetylation is regulated by JNK kinase activity. Immunoblots of WCEs of HCT116 cells that were transfected with WT JNK1, JNK2, or their respective dominant-negative mutants (JNK APF), either individually or together, and treated with or without anisomycin. Densitometric quantification of H3K122ac levels is shown below. (F) Nucleosome clearance activity by BRD4 is controlled by JNK phosphorylation. Autoradiograph of an in vitro nucleosome clearance assay showing disso- ciation of assembled mononucleosomes by unphosphorylated or JNK pre-phosphorylated (*) WT or 3A-BRD4 upon being subjected to a HAT assay in the presence or absence of AcCoA. See also Figure S4.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Binding Assay, Phospho-proteomics, Western Blot, Recombinant, In Vitro, HAT Assay, In Vivo, Activation Assay, Control, Transfection, Activity Assay, Dominant Negative Mutation, Autoradiography

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 4. JNK-mediated BRD4 release from chromatin activates BRD4 kinase (A) JNK activation induces phosphorylation of BRD4 kinase substrates. Immunoblots of WCEs of HCT116 cells that were treated with DMSO (mock), anisomycin, or anisomycin with JNK peptide inhibitor D-JNK1. (B) Blocking JNK activity inhibits induction of BRD4 kinase. Immunoblots of WCEs of HCT116 cells that were transfected with FLAG-tagged WT JNK1, JNK2, or respective dominant-negative mutants (JNK APF), either individually or together, and treated with or without anisomycin. (C) JNK phosphorylation of BRD4 is necessary for induction of BRD4 kinase. Immunoblots of WCEs of HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock. (D) BRD4 phosphorylation regulates Myc stability. Immunofluorescence images showing Myc levels in HCT116 cells transfected with either WT or 3A-BRD4 or empty vector (control) and subjected to heat shock (scale bars, 25 mM). See also Figure S5.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Blocking Assay, Activity Assay, Transfection, Dominant Negative Mutation, Plasmid Preparation, Control

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 5. JNK phosphorylation toggles BRD4 enzymatic activities and is reversed by PP4 phosphatase (A) BRD4 kinase and HAT activities are cross-regulated by its substrates. Top: autoradiograph of an in vitro kinase assay with BRD4 and RNA Pol II CTD in the presence or absence of assembled mononucleosomes. Bottom: immunoblots of an in vitro HAT assay with BRD4 and histone H3 in the presence or absence of RNA Pol II CTD. (B) JNK activation enhances the interaction between BRD4 and its kinase substrates. Immunoblots showing co-immunoprecipitated total and T1212-phos- phorylated BRD4 from HCT116 cells treated with or without anisomycin. Top: BRD4 co-immunoprecipitated with RNA Pol II CTD. Bottom: BRD4 co-immu- noprecipitated with CDK9. (C) JNK-mediated phosphorylation of BRD4 is transient. Immunoblots of WCEs of HCT116 cells grown under normal conditions, subjected to heat shock or heat shocked and then rescued for 20 min. (D) Inhibition of phosphatases enhances phosphorylated BRD4 levels. Immunoblots of WCEs of HCT116 cells that were treated with or without anisomycin alone or anisomycin with phosphatase inhibitor, nodularin. (E) Phosphatase PP4 dephosphorylates JNK-phosphorylated BRD4. Immunoblots of WCEs of HCT116 cells that were transfected with either control, PP2Ac, or PP4c siRNA and treated with or without anisomycin. (F) BRD4’s interaction with RNA Pol II CTD is modulated by PP4. Immunoblots showing total and pT1212 BRD4 co-immunoprecipitated with RNA Pol II CTD from HCT116 cells transfected with either control or PP4c siRNA and treated with anisomycin.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Phospho-proteomics, Autoradiography, In Vitro, Kinase Assay, Western Blot, HAT Assay, Activation Assay, Immunoprecipitation, Inhibition, Transfection, Control

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 6. JNK-mediated BRD4 release from chromatin activates transcription (A) JNK activation enhances expression of BRD4-regulated genes. Volcano plots showing differential gene expression observed in RNA-seq analysis of WT- and 3A-BRD4-expressing HCT116 cells after anisomycin treatment. (B) Inflammatory and immune response pathways are enriched among the BRD4-regulated genes induced by JNK activation. GO analysis of genes induced in anisomycin-treated WT-BRD4-expressing cells relative to control HCT116 cells. (C) Induction of key inflammatory and immune response genes depends on JNK phosphorylation of BRD4. RT-qPCR of cDNA from anisomycin-treated WT- and 3A-BRD4-expressing cells relative to control cells. Error bars, SEM (n = 3 independent experiments; *p < 0.001 by two-tailed Student’s t tests). (D) JNK activation leads to increased BRD4-RNA Pol II interaction at BRD4-regulated inflammatory and immune response genes. Sequential-ChIP assays showing RNA Pol II and RNA Pol II-bound BRD4 at the promoter and gene body regions of CCL20, CXCL1, BIRC3, and control Myc genes. Error bars, SEM (n = 4 technical replicates from 2 independent experiments; *p < 0.05 by two-tailed Student’s t tests). See also Figure S6.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Activation Assay, Expressing, Gene Expression, RNA Sequencing, Control, Phospho-proteomics, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 7. BRD4 phosphorylation and chromatin release are correlated with thymocyte activation and EMT (A) Thymocyte activation correlates with JNK phosphorylation of BRD4. Left: flow cytometry profiles of thymocytes activated by 0.3 ng PMA/0.3 mg ionomycin or by 10 ng PMA/3.75 mg ionomycin. FACS analysis of CD4/CD8 (upper) and CD69 expression (lower). Right: immunoblots of WCEs from unstimulated and stimulated thymocytes. Densitometric quantification of relative BRD4 phosphorylation levels is shown below. (B) Thymocyte activation is correlated with JNK-mediated release of BRD4 from chromatin. Immunoblot of chromatin-free (CF) and chromatin-bound (CB) BRD4 in thymocytes unstimulated or stimulated as described above. Densitometric quantification of CF:CB BRD4 ratio is shown below. Anti-histone H3 immunoblot monitors purity of separation. (C) Immunoblots of WCEs from PC3 cells at day 0 and day 5 of treatment with or without EMT-inducing media supplement. (D) EMT induction correlates with JNK-mediated release of BRD4 from chromatin. Immunoblot of CF and CB BRD4 in PC3 cells after 5 days of treatment with or without (control) EMT-inducing media. (E) EMT induction and BRD4 phosphorylation are both dependent on JNK activity. Immunoblots of WCEs from PC3 cells that were treated, or not, with EMT- inducing media alone or in combination with JNK peptide inhibitor D-JNK-1. (F) EMT induction and expression of EMT regulators are dependent on BRD4 phosphorylation. Immunoblots of WCEs from PC3 cells that were treated, or not, with EMT-inducing media and transfected with WT-BRD4, 3A-BRD4, or empty vector control on day 3 of treatment.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Phospho-proteomics, Activation Assay, Cytometry, Expressing, Western Blot, Control, Activity Assay, Transfection, Plasmid Preparation

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 8. Model of BRD4-JNK interaction and the switching of BRD4 functions BRD4 primarily functions as a chromatin regulator by acetylating H3K122 and dissociating nucleosomes. Upon activation, JNK phosphorylates BRD4, releasing it from chromatin and activating its kinase. Chromatin-free BRD4 is then dephosphorylated by PP4, enhancing its interaction with and phosphorylation of RNA Pol II CTD, PTEFb, and Myc, thereby activating transcription at specific genes. A portion of dephosphorylated BRD4 returns to chromatin to renew its chromatin regulatory function.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Activation Assay, Phospho-proteomics

Journal: Nature methods

Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

doi: 10.1038/nmeth.3022

Figure Lengend Snippet: Preparation of DNL and its use in in vitro ChIP-Seq experiments. ( a ) Modified histone variants prepared by protein semi-synthesis are assembled with the respective barcoded DNA into a barcoded nucleosome (‘NUC’) library (‘DNL’). After biochemical assays with a writer, reader or nuclear extract, the binders and reaction products are isolated by affinity- or immunoprecipitation, followed by DNA experiment multiplexing. NUC identity and abundance is analyzed by next generation sequencing (NGS). ( b ) Combinations of histone modifications (‘mod’) selected for the first version of the library (‘DNL-1’). Unmodified (‘–mod’) or modified H3 proteins (vertical axis) were combined with otherwise unmodified histones (‘–mod’), H2AK119ub, H2BK120ub, or mono-/hyperacetylated H4 (horizontal axis). Additionally, a NUC bearing H2BK120ub and H4Kac 5 was prepared. Asterisk: this variant was employed in the Brd4 experiment . ( c ) Analysis of the combined DNL-1 by native gel electrophoresis and ethidium bromide (EtBr) DNA staining. The bands from NUCs containing combinations of unmodified, Kac or Kme3 histones overlap, whereas the shifted fainter upper band represents NUCs containing ubiquitylated H2A or H2B. For details on modified histones, DNA preparation, NUC assembly and NGS, see .

Article Snippet: pFlag-CMV2-Brd4 (1–1362) encoding the cDNA of full-length human Brd4 protein was obtained from Addgene (plasmid #22304).

Techniques: In Vitro, ChIP-sequencing, Modification, Isolation, Immunoprecipitation, Multiplexing, Next-Generation Sequencing, Variant Assay, Nucleic Acid Electrophoresis, Staining

Journal: Nature methods

Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

doi: 10.1038/nmeth.3022

Figure Lengend Snippet: Profiling the substrate specificity of histone mark readers. ( a ) Left: Model showing bivalent nucleosomal recognition behavior of BPTF. Right: Immobilized GST-tagged BPTF-PHD (gray) and BPTF-PHD-BD (blue) were incubated with DNL-1, followed by DNA isolation and experiment multiplexing. The resulting DNA was analyzed by next generation sequencing, and the raw sequencing reads were normalized against input and the H3K4me3-containing variant, set as 1 (red asterisks). Values (mean ± SD, n=3) for the respective NUC variants are plotted as a 3D bar graph using the same grid as shown in . ( b ) Left: Model depicting potential binding modes of p300. Right: Library incubation of resin-bound GST-tagged p300-BD (gray) and p300-BD-PHD (blue). All subsequent steps were performed as in ( a ). Internal normalization: H4Kac 5 variant, set as 1 (red asterisks). Values are shown as (mean ± SD, n=3). ( c ) Left: Model depicting potential interaction between Brd4 and acetylated NUC. Right: Library incubation of resin-bound Flag-Brd4-BD1-BD2. All subsequent steps were performed as in ( a ) except for pulldown with an anti-Flag antibody. Internal normalization: H4Kac 5 variant, set as 1 (red asterisk). Experiment was performed in duplicate (replicate shown in ). See Source Data Table 1 for input-normalized sequencing reads.

Article Snippet: pFlag-CMV2-Brd4 (1–1362) encoding the cDNA of full-length human Brd4 protein was obtained from Addgene (plasmid #22304).

Techniques: Incubation, DNA Extraction, Multiplexing, Next-Generation Sequencing, Sequencing, Variant Assay, Binding Assay

Journal: bioRxiv

Article Title: Loss of NAT10 disrupts enhancer organization via p300 mislocalization and suppresses transcription of genes necessary for metastasis progression

doi: 10.1101/2024.01.24.577116

Figure Lengend Snippet: (a) Morphology of Nat10 shRNA knockdown (top) and Nat10 KO 4T1 cells grown on different plates (bottom). Scale bar = 10 μm. (b) Hypothetical model of the interaction between NAT10 and the NUP210-bound mechanosensitive protein complex at the nuclear pore. (c) Co-IP showing the interaction of NAT10 with Myc-tagged NUP210 and SIPA1 in 4T1 cells. (d) Co-IP showing the interaction of Flag-tagged NAT10 and BRD4 isoforms in 4T1 cells. (e) and (f) Reciprocal Co-IP showing the interaction of Myc-tagged NAT10 and Flag-tagged BRD4 isoforms in human 293FT cells. (g) Proximity ligation assay showing the interactions (red dots) of NUP210 with NAT10 and SIPA1. Scale bar = 5 μm. (h) Western blot showing the level of NUP210 and ITGB1 protein in Nat10 KO 4T1 cells. (i) qRT-PCR showing the level of NUP210-dependent mechanosensitive, inflammatory response genes in Nat10 KO 4T1 cells, multiple t-test, mean ± s.e.m. (j) Immunofluorescence showing the distribution of histone H3.1/3.2 and H3K9me3 heterochromatin markers in Nat10 KO 4T1 cells. Scale bar = 10 μm. (k) Western blot showing the levels of NAT10 and associated mechanosensitive proteins in 4T1 cells grown on plates with soft (0.2kPa) and stiff (plastic dish, stiffness > GPa) matrices coated with either fibronectin or type I collagen.

Article Snippet: Following antibodies and dilutions were used: rabbit NAT10 (1:5000, Abcam), mouse Nat10 (1:5000, Proteintech), mouse β-actin (1:10,000; Abcam), rabbit NUP210 (1:1000, Fortis Life Sciences), rabbit SIPA1 (1:1000, Abcam), rabbit pan-BRD4 (1:5000, Cell Signaling Technology), mouse Flag-tag (1:5000, Millipore-Sigma), rabbit Flag-tag (1:1000, Cell Signaling Technology), Mouse Myc-tag (1:5000, Cell Signaling Technology), rabbit ITGB1 (1:1000, Cell Signaling Technology), mouse H3.1/3.2 (1:5000, Active Motif), rabbit RRP1B (1:5000, Millipore-Sigma), mouse NPM1 (1:1000, Abcam), rabbit α/β-tubulin (1:5000, Cell Signaling Technology), rabbit H3.3 (1:5000, Abcam), mouse pan-H4 (1:1000, Cell Signaling Technology), rabbit H4K12ac (1:5000, Cell Signaling Technology), rabbit H4K20ac (1:5000, Millipore-Sigma), rabbit H4K5ac (1:1000, Active Motif), rabbit H4K8ac (1:1000, Abcam), rabbit H3K27ac (1:1000, Cell Signaling Technology), mouse pan-H3 (1:10000, Cell Signaling Technology), rabbit H4K16ac (1:1000, Cell Signaling Technology), rabbit pan-H4ac (1:1000, Active Motif), rabbit pan-ac-K (1:1000, Cell Signaling Technology), Rabbit p300 (1:5000, Cell Signaling Technology), rabbit ac-p300 (K1499)/ac-CBP (K1535) (1:5000, Cell Signaling Technology), rabbit phospho-p300 (S89) (1:300, Aviva), rabbit c-MYC (1:5000, Cell Signaling Technology), rabbit EIF2α (1:1000, Cell Signaling Technology), rabbit phospho-EIF2α (1:1000, Cell Signaling Technology), rabbit p70 S6 Kinase (1:1000, Cell Signaling Technology), rabbit phospho-p70 S6 Kinase (T421/S424) (1:1000, Cell Signaling Technology).

Techniques: shRNA, Knockdown, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of NAT10 disrupts enhancer organization via p300 mislocalization and suppresses transcription of genes necessary for metastasis progression

doi: 10.1101/2024.01.24.577116

Figure Lengend Snippet: (a) Co-IP showing the interaction of Flag-tagged and endogenous NAT10 with p300 in 4T1 cells. (b) Immunofluorescence showing the colocalization of NAT10 with p300 and enhancer mark H3K27ac, Scalebar = 5 μm. NAT10-Ms, mouse NAT10 antibody; NAT10-Rb, rabbit NAT10 antibody. (c) Proximity ligation assay showing the interaction of NAT10 with H3K27ac and H4K20ac marks in 4T1 cells, Scalebar = 10 μm. (d) 3D reconstruction showing NAT10 interactions with H3K27ac and H4K20ac within the nucleus of 4T1 cells, Scalebar = 2 μm. (e) Normalized ChIP-seq profile of NAT10 enrichment with active enhancer marks (H3K27ac and H4K20ac) in 4T1 cells. (f) IGV plot showing the ChIP enrichment of NAT10 with members of the nuclear pore-associated mechanosensitive protein complex at the Myc super-enhancer region. BRD4_short_V5 = V5-tagged short isoform of BRD4, RRP1B_HA = HA-tagged RRP1B, RRP1B_endo = endogenous RRP1B.

Article Snippet: Following antibodies and dilutions were used: rabbit NAT10 (1:5000, Abcam), mouse Nat10 (1:5000, Proteintech), mouse β-actin (1:10,000; Abcam), rabbit NUP210 (1:1000, Fortis Life Sciences), rabbit SIPA1 (1:1000, Abcam), rabbit pan-BRD4 (1:5000, Cell Signaling Technology), mouse Flag-tag (1:5000, Millipore-Sigma), rabbit Flag-tag (1:1000, Cell Signaling Technology), Mouse Myc-tag (1:5000, Cell Signaling Technology), rabbit ITGB1 (1:1000, Cell Signaling Technology), mouse H3.1/3.2 (1:5000, Active Motif), rabbit RRP1B (1:5000, Millipore-Sigma), mouse NPM1 (1:1000, Abcam), rabbit α/β-tubulin (1:5000, Cell Signaling Technology), rabbit H3.3 (1:5000, Abcam), mouse pan-H4 (1:1000, Cell Signaling Technology), rabbit H4K12ac (1:5000, Cell Signaling Technology), rabbit H4K20ac (1:5000, Millipore-Sigma), rabbit H4K5ac (1:1000, Active Motif), rabbit H4K8ac (1:1000, Abcam), rabbit H3K27ac (1:1000, Cell Signaling Technology), mouse pan-H3 (1:10000, Cell Signaling Technology), rabbit H4K16ac (1:1000, Cell Signaling Technology), rabbit pan-H4ac (1:1000, Active Motif), rabbit pan-ac-K (1:1000, Cell Signaling Technology), Rabbit p300 (1:5000, Cell Signaling Technology), rabbit ac-p300 (K1499)/ac-CBP (K1535) (1:5000, Cell Signaling Technology), rabbit phospho-p300 (S89) (1:300, Aviva), rabbit c-MYC (1:5000, Cell Signaling Technology), rabbit EIF2α (1:1000, Cell Signaling Technology), rabbit phospho-EIF2α (1:1000, Cell Signaling Technology), rabbit p70 S6 Kinase (1:1000, Cell Signaling Technology), rabbit phospho-p70 S6 Kinase (T421/S424) (1:1000, Cell Signaling Technology).

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Proximity Ligation Assay, ChIP-sequencing