Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H acetylation requires TIP60 and is recognized by BRD4. ( A ) Lysates of HEK293T, Jurkat, HeLa and NK-92 cells were incubated with an acetyl-lysine antibody or normal rabbit IgG (rIgG). Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H or SSRP1 antibodies. ( B ) Lysates of HEK293T cells were incubated with a TIP60 antibody or normal mouse IgG (mIgG). Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H or TIP60 antibodies. ( C ) HEK293T cells stably expressing TIP60 or non-targeting (NT) shRNA were subjected to protein immunoblotting and RT-qPCR analyses of TIP60 expression. RT-qPCR results were calculated from three independent experiments (**** P < 0.0001, Student's t -test). ( D ) Lysates of HEK293T cells in ( C ) were incubated with an acetyl-lysine antibody. Immunoprecipitated protein samples were analyzed by protein immunoblotting using a SUPT16H antibody. ( E ) Lysates of HEK293T cells treated with a TIP60-specific inhibitor MG149 were incubated with an acetyl-lysine antibody. Immunoprecipitated protein samples were analyzed by protein immunoblotting using a SUPT16H antibody. ( F ) Lysates of HEK293T cells were incubated with a BRD4 antibody (C-terminus) or rIgG. Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H, SSRP1, or BRD4 (C-terminus) antibodies. ( G ) Lysates of HEK293T cells were incubated with a SUPT16H antibody or mIgG. Immunoprecipitated protein samples were analyzed by protein immunoblotting using BRD4 (C-terminus), BRD2, SSRP1 or SUPT16H antibodies. The higher band of BRD4 in ( F ) and ( G ) labeled with the asterisk was BRD4L, while the lower one was its degraded product. ( H ) Lysates of HEK293T cells treated with a BETi JQ1 (1 μM) were adjusted to assure the same input of SUPT16H, and incubated with a BRD4 antibody (C-terminus). Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H or BRD4 (C-terminus) antibodies. ( I ) Lysates of HEK293T cells in ( C ) were incubated with a BRD4 antibody (C-terminus). Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H or BRD4 (C-terminus) antibodies. (Int: intensity)

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Incubation, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Labeling

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H is acetylated at K674 of the middle domain (MD). ( A ) Schematic illustration of defined domains of SUPT16H protein. NTD: N-terminal domain; DD: dimerization domain; MD: middle domain; CTD: C-terminal domain. ( B ) Lysates of HEK293T cells transfected with the vector expressing indicated FLAG-tagged protein domains of SUPT16H were incubated with an acetyl-lysine or FLAG antibody, and immunoprecipitated protein samples were analyzed by protein immunoblotting using FLAG or acetyl-lysine antibodies, respectively. ( C ) HEK293T cells stably expressing TIP60 or NT shRNA were transfected with the vector expressing FLAG-tagged MD of SUPT16H. Cell lysates were incubated with acetyl-lysine or FLAG antibodies, and immunoprecipitated protein samples were analyzed by protein immunoblotting using FLAG or acetyl-lysine antibodies, respectively. ( D ) Lysates of HEK293T cells in ( C ) were incubated with a BRD4 antibody (C-terminus). Immunoprecipitated protein samples were analyzed by protein immunoblotting using FLAG or BRD4 (C-terminus) antibodies. ( E ) Prediction of acetylated lysine sites in the MD of SUPT16H by PhosphoSitePlus®. Lysine site K674 has the highest likelihood to be acetylated. ( F ) Lysates of HEK293T cells transfected with FLAG-tagged, wild-type (WT) or K674R MD of SUPT16H, were incubated with an acetyl-lysine antibody. Immunoprecipitated protein samples were analyzed by protein immunoblotting using a FLAG antibody. ( G ) Lysates of HEK293T cells in ( F ) were incubated with a BRD4 antibody (C-terminus). Immunoprecipitated protein samples were analyzed by protein immunoblotting using FLAG or BRD4 (C-terminus) antibodies.

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation, Western Blot, Stable Transfection, shRNA

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H-BRD4 interaction prevents SUPT16H from protein degradation. ( A ) HEK293T, Jurkat, HeLa and NK-92 cells treated with JQ1 were subjected to protein immunoblotting analysis using a SUPT16H antibody. ( B ) The similar analysis as in (A) was performed for HEK293T cells treated with UMB-136 for 48 h. ( C, D ) HEK293T cells stably expressing BRD4 or NT shRNA were subjected to mRNA RT-qPCR analysis of BRD4 or SUPT16 expression ( C ) or protein immunoblotting analysis using a SUPT16H antibody (D). ( E, F ) The similar analysis as in (C, D) was performed for HEK293T cells transfected with BRD4 or NT siRNAs. Results were calculated from three independent experiments (**** P < 0.0001, Student's t test for C, one-way ANOVA for E). ( G ) Lysates of HEK293T cells treated with JQ1 (1 μM) were incubated with a SUPT16H antibody. Immunoprecipitated protein samples were analyzed by protein immunoblotting using K48-ubquitin or SUPT16H antibodies.

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Transfection, Incubation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H-BRD4 associates with HDAC1 and EZH2 contributing to gene silencing. (A, B) Lysates of HEK293T cells transfected with the vector expressing V5-tagged HDAC1 ( A ) or EZH2 ( B ) were incubated with a V5 antibody or mIgG. Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H, BRD4 (C-terminus), or V5 antibodies. (C–E) Lysates of HEK293T cells were incubated with HDAC1 ( C ), EZH2 ( D ) or SUPT16H ( E ) antibodies. Immunoprecipitated protein samples were analyzed by protein immunoblotting using the indicated antibodies. (F–H) HEK293T cells treated with JQ1 ( F ), UMB-136 ( G ) or CBL0137 (500 nM) ( H ) were subjected to protein immunoblotting analysis of SUPT16H, HDAC1 and phospho-HDAC1 (ser421/423). (I–L) HEK293T cells stably expressing BRD4 or NT shRNA ( I ) were subjected to protein immunoblotting analysis of SUPT16H, HDAC1 and phospho-HDAC1 (ser421/423). The similar analysis as in ( I ) was performed for HEK293T cells transfected with BRD4 ( J ) or SUPT16H ( L ) siRNAs. Knockdown of SUPT16H by its siRNAs was confirmed by RT-qPCR to measure its mRNA level (K). ( M ) HEK293T cells treated with CBL0137 at the increasing doses (100, 200, 500 nM) were subjected to protein immunoblotting analysis using histone H3ac or total H3 antibodies. ( N, O ) Lysates of HEK293T cells transfected with SUPT16H or BRD4 siRNAs were crosslinked and incubated with HDAC1, phospho-HDAC1 (ser421/423), EZH2 antibodies, or control IgG. Immunoprecipitated DNA samples were analyzed by qPCR analysis using primers targeting the promoter region of IFI44L (N) or MX2 (O). Results were calculated from three independent experiments (**** P < 0.0001, one-way ANOVA for K, two-way ANOVA for N, O).

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation, Western Blot, Stable Transfection, shRNA, Knockdown, Quantitative RT-PCR, Control

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H-BRD4 suppresses gene expression of IFN signaling. (A, B) HEK293T cells transfected with BRD4 ( A ) or SUPT16H ( B ) siRNAs were subjected to mRNA RT-qPCR analysis of IFNβ and ISG15 expression. ( C ) HEK293T cells transfected with SUPT16H or NT siRNAs were subjected to mRNA RT-qPCR analysis of IL-4/6/8 expression. (D, E) NK-92 cells treated with CBL0137 (500 nM) were subjected to mRNA RT-qPCR ( D ) or protein immunofluorescence ( E ) analysis of IL-4/6/8 expression. (F, G) HEK293T cells transfected with SUPT16H or NT siRNAs were further transfected with an ISRE-driven ( F ) or GAS-driven ( G ) firefly luciferase reporter plus TK-driven Renilla luciferase control vectors, which were stimulated by Type I (IFNα) or II (IFNγ) IFNs, respectively. Relative luciferase unit, RLU (firefly/ Renilla luciferase activity) from above cells was calculated and normalized to that of siNT-transfected, IFN-treated cells. (H, I) Lysates of HEK293T ( H ) or NK-92 ( I ) cells treated with CBL0137 (500 nM) were crosslinked and incubated with H3ac, H3K9me3, H3K27me3 antibodies or normal IgGs. Immunoprecipitated DNA samples were analyzed by qPCR analysis using primers targeting the promoter region of ISGs (IFI44L, MX2). Results were calculated from three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-way ANOVA).

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Gene Expression, Transfection, Quantitative RT-PCR, Expressing, Immunofluorescence, Luciferase, Control, Activity Assay, Incubation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H–BRD4 function of gene suppression in antiviral IFN signaling. FACT subunit SUPT16H undergoes protein acetylation at K674 of MD domain, catalyzed by TIP60, which is recognized by BRD4. Such SUPT16H–BRD4 interaction prevents the protein degradation of SUPT16H. SUPT16H–BRD4 physically associates with epigenetic silencing enzymes (HDAC1, EZH2), and further affects their activation status and/or promoter association as well as modulates the histone marks (H3ac, H3K9me3, H3K27me3), which overall results in the functional overlaps of SUPT16H and BRD4 to suppress gene expression, including HERVs, non-coding repetitive elements, IFNs, and ISGs, in multiple types of cells, such as epithelial and NK cells. Furthermore, the SUPT16H inhibitor CBL0137 is potent to induce IFN signaling in both epithelial and NK cells, forming two host defense layers against viral infections.

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Activation Assay, Functional Assay, Gene Expression

Journal: STAR Protocols

Article Title: Cloning BRD4 long isoform into overexpression vectors for stable overexpression of BRD4-L in mammalian cells

doi: 10.1016/j.xpro.2022.101785

Figure Lengend Snippet:

Article Snippet: Download the appropriate sequences. a. Download the Reference Sequence for Homo sapiens bromodomain containing 4 (BRD4), transcript variant long, mRNA (GenBank: NM_058243.3) from the NCBI’s website, using the GenBank data base ( BRD4-L RefSeq ). b. Download the sequence for LentiV_Blast (Addgene, cat# 111887) from Addgene’s website ( LentiV_Blast ).

Techniques: Virus, Recombinant, Cloning, Gel Extraction, Plasmid Preparation, Sequencing, Over Expression, Expressing, Software, Imaging

Journal: eLife

Article Title: A twin UGUA motif directs the balance between gene isoforms through CFIm and the mTORC1 signaling pathway

doi: 10.7554/eLife.85036

Figure Lengend Snippet:

Article Snippet: The following antibodies are used for western blotting: mouse monoclonal anti-beta actin (ProteinTech, 66009-1-Ig), mouse monoclonal anti-alpha tubulin (Millipore, CP06), mouse monoclonal anti-HA (clone 16B12, BioLegend, 901501), rabbit polyclonal anti-TRIM9 (ProteinTech, 10786-1-AP), rabbit monoclonal anti-S6 ribosomal protein (Cell Signaling Technology, 2217), rabbit monoclonal anti-Phospho-S6 ribosomal protein (Ser240/244) (Cell Signaling Technology, 5364), rabbit polyclonal anti-CPSF6 (Bethyl Labs, A301-356A), rabbit polyclonal anti-BRD4 (Bethyl Labs, A301-985A), and rabbit monoclonal anti-BRD4 (Abcam, ab128874).

Techniques: cDNA Synthesis, Mutagenesis, PCR Cloning, SYBR Green Assay, Sequencing, Recombinant, Generated, Software

Journal: STAR Protocols

Article Title: Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation

doi: 10.1016/j.xpro.2022.102004

Figure Lengend Snippet: Analysis of BRD4 accumulation in SE element of miRNA encoding gene (A and B) The alteration of BRD4 accumulation according to the different antibody and antigen ratio and (B) different stimulation conditions- US-unstimulated, INF- infected, LPS- Lipopolysaccharide and JQ1 treated macrophages. (C) Semi-quantitative PCR of BRD4-pulled miR146a-5p SE DNA. Percent accumulation of BRD4 protein upon miR146a-5p SE elements on- US-unstimulated, INF- infected, LPS- Lipopolysaccharide and JQ1 treated macrophages. Each experiment has been performed in triplicates. Statistical significance (∗∗P<0.01, ∗∗∗P<0.001) was calculated using one-way ANOVA and data are represented as mean± SD (Graph pad prism 5.0).

Article Snippet: Brd4 , Cell Signaling Technology , BRD4 (E2A7X) Rabbit mAb.

Techniques: Infection, Real-time Polymerase Chain Reaction

Journal: STAR Protocols

Article Title: Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation

doi: 10.1016/j.xpro.2022.102004

Figure Lengend Snippet: Calculation of percent input (% BRD4 accumulation in SE of miR146a-5p) and possible outcomes

Article Snippet: Brd4 , Cell Signaling Technology , BRD4 (E2A7X) Rabbit mAb.

Techniques: Infection

Journal: STAR Protocols

Article Title: Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation

doi: 10.1016/j.xpro.2022.102004

Figure Lengend Snippet:

Article Snippet: Brd4 , Cell Signaling Technology , BRD4 (E2A7X) Rabbit mAb.

Techniques: Recombinant, Protease Inhibitor, SYBR Green Assay, Purification, Bradford Assay, Software