Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: STAT3 localizes to promoters and regulates expression of KRAB-ZFP genes. (A and B) Levels of SZF1 (A) and ZNF557 (B) mRNAs in healthy-donor-derived LCLs and AD-HIES patient-derived LCLs were determined by qRT-PCR. (C to E) Levels of SZF1, ZNF557, and STAT3 mRNAs in EBV+ latent BL cells (C), healthy-donor-derived LCLs (D), and AD-HIES patient-derived LCLs (E) transfected with scrambled siRNA (open bars) or siSTAT3 (black bars) were quantitated by qRT-PCR. Data represent means of three independent experiments (C) or three separate LCLs (A, B, D, and E). (F and G) Relative amounts of SZF1 (F) and ZNF557 (G) promoter DNAs precipitated by anti-STAT3 antibody were determined by qPCR with normalization to input DNA. BL cells were left untreated or treated with NaB for 24 h and examined by qPCR using primers spanning bioinformatically predicted STAT3-binding sites in the SZF1 promoter (site 1, position −1463; site 2, position −1226; site 3, position −74 [relative to transcription start site]) or a non-STAT3-binding site on the SZF1 promoter (position −500 relative to the transcription start site) or the ZNF557 promoter (−255 relative to transcription start site). Data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05; NS, not significant; p, promoter).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Transfection, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: STAT3 functions via KRAB-ZFPs to regulate repression of lytic genes on the EBV episome. (A and B) EBV+ latent BL cells transfected with scrambled siRNA (open bars), siSZF1 (black bars), or siZNF557 (gray bars) were treated with NaB, and indicated EBV lytic transcripts were quantitated by qRT-PCR and immunoblot analysis with antibodies against β-actin and EBV lytic protein ZEBRA (A). BL cells transfected with indicated siRNAs and FITC-scrambled (Sc) siRNA (to mark transfected cells) were treated with NaB and percentages of lytic cells in FITC+ (i.e., transfected) cells (open bars) and FITC− (i.e., nontransfected) cells (black bars) were determined by flow cytometry (B). Flow cytometry was performed by using human sera to demarcate lytically active cells, as described previously (11), as well as antibodies against SZF1 or ZNF557. (C) BL cells transfected with empty vector (open bars), HA-SZF1 (black bars), or HA-ZNF557 (gray bars) were treated with NaB, and lytic transcripts were measured by qRT-PCR. (D) BL cells were transfected with indicated plasmids and FITC-scrambled siRNA, as described above for panel B, and treated with NaB, and percentages of lytic cells in FITC+ (i.e., transfected) cells (open bars) and FITC− (i.e., nontransfected) cells (black bars) were determined by flow cytometry, as described above for panel B. EV, empty vector. (E) Relative amounts of DNA precipitated by anti-SZF1 antibody in latent BL cells (open bars) versus NaB-treated BL cells (black bars) were determined by qPCR with normalization to input DNA. PCR primers flanked predicted binding sites for SZF1 on indicated EBV genes. p, promoter. (F) BL cells were transfected with control or STAT3 plasmid and indicated siRNAs, exposed to NaB, and examined by qRT-PCR for relative amounts of EBV lytic transcripts: BZLF1 (open bars), BRLF1 (black bars), and BMRF1 (gray bars). (G) BL cells were transfected with control or STAT3 plasmid and indicated siRNAs and examined by qRT-PCR for relative amounts of STAT3 (open bars), SZF1 (black bars), and ZNF557 (gray bars). Data in panels A, C, E, F, and G represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05). Data in panels B and D are representative of results from 2 independent experiments.

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Flow Cytometry, Plasmid Preparation, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs regulate persistence of KSHV, the other oncogenic human herpesvirus. (A) Levels of SZF1, ZNF557, and STAT3 mRNAs in KSHV+ primary effusion lymphoma (BCBL-1) cells transfected with scrambled siRNA (open bars) or siSTAT3 (black bars) were quantitated by qRT-PCR. BCBL-1 cells were transfected with scrambled siRNA or siSTAT3 and harvested for immunoblotting with indicated antibodies. (B and C) BCBL-1 cells were transfected with empty vector and scrambled siRNA, SZF1 (B) or ZNF557 (C) plasmid and scrambled siRNA, or SZF1 (B) or ZNF557 (C) plasmid and siTRIM28, exposed to VPA, and harvested for determination of the relative amounts of transcripts from the KSHV latency-to-lytic-cycle switch gene ORF50 and the early lytic gene ORF59 by qRT-PCR. All data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05). (D and E) BCBL-1 cells were transfected with combinations of empty vector, HA-tagged KRAB-ZFP plasmids, scrambled siRNA, and siTRIM28, treated with VPA, and harvested for immunoblotting with indicated antibodies. Data are representative of results from two independent experiments. (F) Model of oncogenic human herpesvirus persistence depicting that STAT3 transcriptionally activates the KRAB-ZFPs SZF1 and ZNF557, which localize to EBV and KSHV genomes. Bound SZF1 functions directly through TRIM28 to repress lytic genes despite the presence of lytic triggers, while bound ZNF557 functions in a TRIM28-independent manner (?), thereby maintaining latency and promoting virus persistence.

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: STAT3 localizes to promoters and regulates expression of KRAB-ZFP genes. (A and B) Levels of SZF1 (A) and ZNF557 (B) mRNAs in healthy-donor-derived LCLs and AD-HIES patient-derived LCLs were determined by qRT-PCR. (C to E) Levels of SZF1, ZNF557, and STAT3 mRNAs in EBV+ latent BL cells (C), healthy-donor-derived LCLs (D), and AD-HIES patient-derived LCLs (E) transfected with scrambled siRNA (open bars) or siSTAT3 (black bars) were quantitated by qRT-PCR. Data represent means of three independent experiments (C) or three separate LCLs (A, B, D, and E). (F and G) Relative amounts of SZF1 (F) and ZNF557 (G) promoter DNAs precipitated by anti-STAT3 antibody were determined by qPCR with normalization to input DNA. BL cells were left untreated or treated with NaB for 24 h and examined by qPCR using primers spanning bioinformatically predicted STAT3-binding sites in the SZF1 promoter (site 1, position −1463; site 2, position −1226; site 3, position −74 [relative to transcription start site]) or a non-STAT3-binding site on the SZF1 promoter (position −500 relative to the transcription start site) or the ZNF557 promoter (−255 relative to transcription start site). Data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05; NS, not significant; p, promoter).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Transfection, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs repress EBV lytic genes through the transcriptional corepressor TRIM28. (A and B) BL cells were transfected with empty vector and scrambled siRNA, SZF1 (A) or ZNF557 (B) plasmid and scrambled siRNA, or SZF1 (A) or ZNF557 (B) plasmid and siTRIM28. Cells were exposed to NaB and harvested for determination of relative amounts of transcripts from the EBV latency-to-lytic-cycle switch gene BZLF1 and the early lytic gene BMRF1 by qRT-PCR. Data represent results from two independent experiments with 3 technical replicates each; error bars indicate standard errors of the means (*, p ≤ 0.05). Samples were also harvested for immunoblotting and reacted with indicated antibodies. Representative data from two independent experiments are shown. (C and D) ChIP was performed on BL cells containing doxycycline (dox)-inducible shTRIM28 cassette. Cells were left untreated (open bars) or treated with doxycycline (black bars) for 12 h. DNA precipitated by using anti-H3K9Ac and anti-H3K9Me3 antibodies was analyzed via qPCR using primers spanning predicted SZF1-binding sites in the promoters of lytic genes BZLF1 (C) and BMRF1 (D) and normalized to input. Immunoblot analysis of TRIM28 after doxycycline induction of shTRIM28 was performed to assess knockdown efficiency. Data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Binding Assay, Knockdown

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: STAT3 functions via KRAB-ZFPs to regulate repression of lytic genes on the EBV episome. (A and B) EBV+ latent BL cells transfected with scrambled siRNA (open bars), siSZF1 (black bars), or siZNF557 (gray bars) were treated with NaB, and indicated EBV lytic transcripts were quantitated by qRT-PCR and immunoblot analysis with antibodies against β-actin and EBV lytic protein ZEBRA (A). BL cells transfected with indicated siRNAs and FITC-scrambled (Sc) siRNA (to mark transfected cells) were treated with NaB and percentages of lytic cells in FITC+ (i.e., transfected) cells (open bars) and FITC− (i.e., nontransfected) cells (black bars) were determined by flow cytometry (B). Flow cytometry was performed by using human sera to demarcate lytically active cells, as described previously (11), as well as antibodies against SZF1 or ZNF557. (C) BL cells transfected with empty vector (open bars), HA-SZF1 (black bars), or HA-ZNF557 (gray bars) were treated with NaB, and lytic transcripts were measured by qRT-PCR. (D) BL cells were transfected with indicated plasmids and FITC-scrambled siRNA, as described above for panel B, and treated with NaB, and percentages of lytic cells in FITC+ (i.e., transfected) cells (open bars) and FITC− (i.e., nontransfected) cells (black bars) were determined by flow cytometry, as described above for panel B. EV, empty vector. (E) Relative amounts of DNA precipitated by anti-SZF1 antibody in latent BL cells (open bars) versus NaB-treated BL cells (black bars) were determined by qPCR with normalization to input DNA. PCR primers flanked predicted binding sites for SZF1 on indicated EBV genes. p, promoter. (F) BL cells were transfected with control or STAT3 plasmid and indicated siRNAs, exposed to NaB, and examined by qRT-PCR for relative amounts of EBV lytic transcripts: BZLF1 (open bars), BRLF1 (black bars), and BMRF1 (gray bars). (G) BL cells were transfected with control or STAT3 plasmid and indicated siRNAs and examined by qRT-PCR for relative amounts of STAT3 (open bars), SZF1 (black bars), and ZNF557 (gray bars). Data in panels A, C, E, F, and G represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05). Data in panels B and D are representative of results from 2 independent experiments.

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Flow Cytometry, Plasmid Preparation, Binding Assay, Control

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: Cells with latent/episomal EBV express higher levels of KRAB-ZFPs than do lytic cells in culture and blood. (A to C) Flow cytometric analysis of latent BL cells that were left untreated (A and B, left) or treated with NaB for 24 h (A and B, right), followed by staining with human serum containing antibodies to EBV lytic antigens (x axis) and anti-SZF1 antibody (y axis) (A) or anti-ZNF557 antibody (y axis) (B), and cells that were stained with human serum lacking antibodies to EBV lytic antigens and isotype control antibody (C). Latent/refractory and lytic cells are indicated; data are representative of results from 3 independent experiments. (D to F) Peripheral blood B cells (D and E, left) and non-B cells (D and E, right) from an immunosuppressed peripheral blood stem cell transplant recipient with EBV viremia were stained with antibodies to EBV lytic antigen ZEBRA (x axis) and KRAB-ZFPs (y axis) (SZF1 [D] and ZNF557 [E]), and B cells were stained with control antibodies (F). Latent (and uninfected) cells as well as lytic cells are indicated.

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Staining, Control

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: SZF1 and TRIM28 interact directly and colocalize on EBV lytic promoters preferentially in quiescent/latent cells. (A) Coimmunoprecipitation assays using control rabbit IgG or anti-SZF1, anti-ZNF557, or anti-TRIM28 antibody were performed on BL cells that were cotransfected with HA-SZF1 and HA-ZNF557 plasmids. Precipitates were analyzed by probing with anti-TRIM28 or anti-HA antibody to detect pulled-down endogenous TRIM28 or overexpressed, HA-tagged SZF1 or ZNF557, respectively. IP, immunoprecipitation; IB, immunoblotting. (B) Proximity ligation assays using antibodies targeting SZF1 and TRIM28 (left) or ZNF557 and TRIM28 (right) were performed on NaB-treated BL cells. Lytic cells were identified by immunofluorescence staining with anti-ZEBRA antibody. Representative fields from 3 independent experiments are shown. (C) Thirty lytic (ZEBRA+) and 70 refractory (ZEBRA−) nuclei were examined, and the numbers of PLA foci per ZEBRA+ and ZEBRA− nucleus were determined. (D) PLAs using control goat IgG plus control rabbit IgG (−/−), control goat IgG plus rabbit anti-SZF1 antibody (−/SZF1), goat anti-TRIM28 antibody plus control rabbit IgG (TRIM28/−), or goat anti-TRIM28 plus rabbit anti-SZF1 antibodies (TRIM28/SZF1) were performed on BL cells. Fluorescent green foci indicate in situ interactions between TRIM28 and SZF1. Data are representative of results from 3 independent experiments. (E to G) Chromatin precipitated from BL cells using anti-SZF1, anti-TRIM28, or a control rabbit IgG antibody was subjected to qPCR analysis (S, SZF1; T, TRIM28) using primers spanning bioinformatically predicted SZF1-binding sites within BZLF1p (E), BRLF1 (F), and BMRF1p (G) and normalized to input or subjected to a second round of ChIP using anti-TRIM28 or anti-SZF1 antibody (S/T and T/S), respectively, before qPCR analysis. Negligible amounts of DNA were pulled down by using rabbit IgG control antibody, and values were subtracted from experimental results prior to normalization. (H to J) ChIP–re-ChIP was performed, as described above for panels E to G, on untreated and NaB-treated BL cells. Data represent the averages of results from three independent experiments; error bars indicate the standard errors of the means (*, p ≤ 0.05).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Control, Immunoprecipitation, Western Blot, Ligation, Immunofluorescence, Staining, In Situ, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs regulate persistence of KSHV, the other oncogenic human herpesvirus. (A) Levels of SZF1, ZNF557, and STAT3 mRNAs in KSHV+ primary effusion lymphoma (BCBL-1) cells transfected with scrambled siRNA (open bars) or siSTAT3 (black bars) were quantitated by qRT-PCR. BCBL-1 cells were transfected with scrambled siRNA or siSTAT3 and harvested for immunoblotting with indicated antibodies. (B and C) BCBL-1 cells were transfected with empty vector and scrambled siRNA, SZF1 (B) or ZNF557 (C) plasmid and scrambled siRNA, or SZF1 (B) or ZNF557 (C) plasmid and siTRIM28, exposed to VPA, and harvested for determination of the relative amounts of transcripts from the KSHV latency-to-lytic-cycle switch gene ORF50 and the early lytic gene ORF59 by qRT-PCR. All data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05). (D and E) BCBL-1 cells were transfected with combinations of empty vector, HA-tagged KRAB-ZFP plasmids, scrambled siRNA, and siTRIM28, treated with VPA, and harvested for immunoblotting with indicated antibodies. Data are representative of results from two independent experiments. (F) Model of oncogenic human herpesvirus persistence depicting that STAT3 transcriptionally activates the KRAB-ZFPs SZF1 and ZNF557, which localize to EBV and KSHV genomes. Bound SZF1 functions directly through TRIM28 to repress lytic genes despite the presence of lytic triggers, while bound ZNF557 functions in a TRIM28-independent manner (?), thereby maintaining latency and promoting virus persistence.

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Virus

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs repress EBV lytic genes through the transcriptional corepressor TRIM28. (A and B) BL cells were transfected with empty vector and scrambled siRNA, SZF1 (A) or ZNF557 (B) plasmid and scrambled siRNA, or SZF1 (A) or ZNF557 (B) plasmid and siTRIM28. Cells were exposed to NaB and harvested for determination of relative amounts of transcripts from the EBV latency-to-lytic-cycle switch gene BZLF1 and the early lytic gene BMRF1 by qRT-PCR. Data represent results from two independent experiments with 3 technical replicates each; error bars indicate standard errors of the means (*, p ≤ 0.05). Samples were also harvested for immunoblotting and reacted with indicated antibodies. Representative data from two independent experiments are shown. (C and D) ChIP was performed on BL cells containing doxycycline (dox)-inducible shTRIM28 cassette. Cells were left untreated (open bars) or treated with doxycycline (black bars) for 12 h. DNA precipitated by using anti-H3K9Ac and anti-H3K9Me3 antibodies was analyzed via qPCR using primers spanning predicted SZF1-binding sites in the promoters of lytic genes BZLF1 (C) and BMRF1 (D) and normalized to input. Immunoblot analysis of TRIM28 after doxycycline induction of shTRIM28 was performed to assess knockdown efficiency. Data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: SZF1 and TRIM28 interact directly and colocalize on EBV lytic promoters preferentially in quiescent/latent cells. (A) Coimmunoprecipitation assays using control rabbit IgG or anti-SZF1, anti-ZNF557, or anti-TRIM28 antibody were performed on BL cells that were cotransfected with HA-SZF1 and HA-ZNF557 plasmids. Precipitates were analyzed by probing with anti-TRIM28 or anti-HA antibody to detect pulled-down endogenous TRIM28 or overexpressed, HA-tagged SZF1 or ZNF557, respectively. IP, immunoprecipitation; IB, immunoblotting. (B) Proximity ligation assays using antibodies targeting SZF1 and TRIM28 (left) or ZNF557 and TRIM28 (right) were performed on NaB-treated BL cells. Lytic cells were identified by immunofluorescence staining with anti-ZEBRA antibody. Representative fields from 3 independent experiments are shown. (C) Thirty lytic (ZEBRA+) and 70 refractory (ZEBRA−) nuclei were examined, and the numbers of PLA foci per ZEBRA+ and ZEBRA− nucleus were determined. (D) PLAs using control goat IgG plus control rabbit IgG (−/−), control goat IgG plus rabbit anti-SZF1 antibody (−/SZF1), goat anti-TRIM28 antibody plus control rabbit IgG (TRIM28/−), or goat anti-TRIM28 plus rabbit anti-SZF1 antibodies (TRIM28/SZF1) were performed on BL cells. Fluorescent green foci indicate in situ interactions between TRIM28 and SZF1. Data are representative of results from 3 independent experiments. (E to G) Chromatin precipitated from BL cells using anti-SZF1, anti-TRIM28, or a control rabbit IgG antibody was subjected to qPCR analysis (S, SZF1; T, TRIM28) using primers spanning bioinformatically predicted SZF1-binding sites within BZLF1p (E), BRLF1 (F), and BMRF1p (G) and normalized to input or subjected to a second round of ChIP using anti-TRIM28 or anti-SZF1 antibody (S/T and T/S), respectively, before qPCR analysis. Negligible amounts of DNA were pulled down by using rabbit IgG control antibody, and values were subtracted from experimental results prior to normalization. (H to J) ChIP–re-ChIP was performed, as described above for panels E to G, on untreated and NaB-treated BL cells. Data represent the averages of results from three independent experiments; error bars indicate the standard errors of the means (*, p ≤ 0.05).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Immunoprecipitation, Western Blot, Ligation, Immunofluorescence, Staining, In Situ, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs regulate persistence of KSHV, the other oncogenic human herpesvirus. (A) Levels of SZF1, ZNF557, and STAT3 mRNAs in KSHV+ primary effusion lymphoma (BCBL-1) cells transfected with scrambled siRNA (open bars) or siSTAT3 (black bars) were quantitated by qRT-PCR. BCBL-1 cells were transfected with scrambled siRNA or siSTAT3 and harvested for immunoblotting with indicated antibodies. (B and C) BCBL-1 cells were transfected with empty vector and scrambled siRNA, SZF1 (B) or ZNF557 (C) plasmid and scrambled siRNA, or SZF1 (B) or ZNF557 (C) plasmid and siTRIM28, exposed to VPA, and harvested for determination of the relative amounts of transcripts from the KSHV latency-to-lytic-cycle switch gene ORF50 and the early lytic gene ORF59 by qRT-PCR. All data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05). (D and E) BCBL-1 cells were transfected with combinations of empty vector, HA-tagged KRAB-ZFP plasmids, scrambled siRNA, and siTRIM28, treated with VPA, and harvested for immunoblotting with indicated antibodies. Data are representative of results from two independent experiments. (F) Model of oncogenic human herpesvirus persistence depicting that STAT3 transcriptionally activates the KRAB-ZFPs SZF1 and ZNF557, which localize to EBV and KSHV genomes. Bound SZF1 functions directly through TRIM28 to repress lytic genes despite the presence of lytic triggers, while bound ZNF557 functions in a TRIM28-independent manner (?), thereby maintaining latency and promoting virus persistence.

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs repress EBV lytic genes through the transcriptional corepressor TRIM28. (A and B) BL cells were transfected with empty vector and scrambled siRNA, SZF1 (A) or ZNF557 (B) plasmid and scrambled siRNA, or SZF1 (A) or ZNF557 (B) plasmid and siTRIM28. Cells were exposed to NaB and harvested for determination of relative amounts of transcripts from the EBV latency-to-lytic-cycle switch gene BZLF1 and the early lytic gene BMRF1 by qRT-PCR. Data represent results from two independent experiments with 3 technical replicates each; error bars indicate standard errors of the means (*, p ≤ 0.05). Samples were also harvested for immunoblotting and reacted with indicated antibodies. Representative data from two independent experiments are shown. (C and D) ChIP was performed on BL cells containing doxycycline (dox)-inducible shTRIM28 cassette. Cells were left untreated (open bars) or treated with doxycycline (black bars) for 12 h. DNA precipitated by using anti-H3K9Ac and anti-H3K9Me3 antibodies was analyzed via qPCR using primers spanning predicted SZF1-binding sites in the promoters of lytic genes BZLF1 (C) and BMRF1 (D) and normalized to input. Immunoblot analysis of TRIM28 after doxycycline induction of shTRIM28 was performed to assess knockdown efficiency. Data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Binding Assay, Knockdown

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: SZF1 and TRIM28 interact directly and colocalize on EBV lytic promoters preferentially in quiescent/latent cells. (A) Coimmunoprecipitation assays using control rabbit IgG or anti-SZF1, anti-ZNF557, or anti-TRIM28 antibody were performed on BL cells that were cotransfected with HA-SZF1 and HA-ZNF557 plasmids. Precipitates were analyzed by probing with anti-TRIM28 or anti-HA antibody to detect pulled-down endogenous TRIM28 or overexpressed, HA-tagged SZF1 or ZNF557, respectively. IP, immunoprecipitation; IB, immunoblotting. (B) Proximity ligation assays using antibodies targeting SZF1 and TRIM28 (left) or ZNF557 and TRIM28 (right) were performed on NaB-treated BL cells. Lytic cells were identified by immunofluorescence staining with anti-ZEBRA antibody. Representative fields from 3 independent experiments are shown. (C) Thirty lytic (ZEBRA+) and 70 refractory (ZEBRA−) nuclei were examined, and the numbers of PLA foci per ZEBRA+ and ZEBRA− nucleus were determined. (D) PLAs using control goat IgG plus control rabbit IgG (−/−), control goat IgG plus rabbit anti-SZF1 antibody (−/SZF1), goat anti-TRIM28 antibody plus control rabbit IgG (TRIM28/−), or goat anti-TRIM28 plus rabbit anti-SZF1 antibodies (TRIM28/SZF1) were performed on BL cells. Fluorescent green foci indicate in situ interactions between TRIM28 and SZF1. Data are representative of results from 3 independent experiments. (E to G) Chromatin precipitated from BL cells using anti-SZF1, anti-TRIM28, or a control rabbit IgG antibody was subjected to qPCR analysis (S, SZF1; T, TRIM28) using primers spanning bioinformatically predicted SZF1-binding sites within BZLF1p (E), BRLF1 (F), and BMRF1p (G) and normalized to input or subjected to a second round of ChIP using anti-TRIM28 or anti-SZF1 antibody (S/T and T/S), respectively, before qPCR analysis. Negligible amounts of DNA were pulled down by using rabbit IgG control antibody, and values were subtracted from experimental results prior to normalization. (H to J) ChIP–re-ChIP was performed, as described above for panels E to G, on untreated and NaB-treated BL cells. Data represent the averages of results from three independent experiments; error bars indicate the standard errors of the means (*, p ≤ 0.05).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Control, Immunoprecipitation, Western Blot, Ligation, Immunofluorescence, Staining, In Situ, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs regulate persistence of KSHV, the other oncogenic human herpesvirus. (A) Levels of SZF1, ZNF557, and STAT3 mRNAs in KSHV+ primary effusion lymphoma (BCBL-1) cells transfected with scrambled siRNA (open bars) or siSTAT3 (black bars) were quantitated by qRT-PCR. BCBL-1 cells were transfected with scrambled siRNA or siSTAT3 and harvested for immunoblotting with indicated antibodies. (B and C) BCBL-1 cells were transfected with empty vector and scrambled siRNA, SZF1 (B) or ZNF557 (C) plasmid and scrambled siRNA, or SZF1 (B) or ZNF557 (C) plasmid and siTRIM28, exposed to VPA, and harvested for determination of the relative amounts of transcripts from the KSHV latency-to-lytic-cycle switch gene ORF50 and the early lytic gene ORF59 by qRT-PCR. All data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05). (D and E) BCBL-1 cells were transfected with combinations of empty vector, HA-tagged KRAB-ZFP plasmids, scrambled siRNA, and siTRIM28, treated with VPA, and harvested for immunoblotting with indicated antibodies. Data are representative of results from two independent experiments. (F) Model of oncogenic human herpesvirus persistence depicting that STAT3 transcriptionally activates the KRAB-ZFPs SZF1 and ZNF557, which localize to EBV and KSHV genomes. Bound SZF1 functions directly through TRIM28 to repress lytic genes despite the presence of lytic triggers, while bound ZNF557 functions in a TRIM28-independent manner (?), thereby maintaining latency and promoting virus persistence.

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Virus