Journal: Nucleic Acids Research

Article Title: ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

doi: 10.1093/nar/gkaa815

Figure Lengend Snippet: ZFAT recruits BRD4 to centromeres. ( A ) Immunofluorescence images of ZFAT (HA) and BRD4 in HEK293 cells transiently expressing hZFAT-HA. The arrowhead indicates a transfected cell, as determined by the anti-HA antibody signal. ( B ) Immunofluorescence images of ZFAT, BRD4, CENP-A and DNA (DAPI) in HEK293 cells transiently expressing HA-hZFAT-EGFP. Enlarged images of the region enclosed by a white dotted line are shown. ( C ) Percentage of cells containing centromeric BRD4 foci in cells expressing EGFP or HA-hZFAT-EGFP, determined in (B). ( D ) ChIP-qPCR analysis of BRD4 at human repetitive DNA sequences using an anti-BRD4 antibody or control IgG in HEK293 cells expressing HA-hZFAT-EGFP or EGFP. The BRD4 protein levels are shown as the relative values to control IgG. (A, B) Scale bar, 5 μm. Data are representative of three independent experiments. (C, D) Data represent the mean ± SD of three independent experiments. * P < 0.05.

Article Snippet: The cDNA for human BRD4 (FHC11882) was purchased from Promega and cloned into an EGFP-C3 vector.

Techniques: Immunofluorescence, Expressing, Transfection

Journal: Nucleic Acids Research

Article Title: ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

doi: 10.1093/nar/gkaa815

Figure Lengend Snippet: ZFAT recruits BRD4 to centromeres through histone acetylation. ( A ) Schematic diagram of the BRD4 constructs used in (B). ( B ) Immunofluorescence images in HEK293 cells transiently expressing mCherry-hZFAT, and the full-length or deletion mutants of EGFP-BRD4. Enlarged images of the region enclosed by a white dotted line are shown. nls; nuclear localization signal. ( C ) Percentage of cells containing centromeric BRD4 foci in cells transfected with indicated expression vectors, determined in (B). ( D ) Immunofluorescence images of ZFAT (EGFP) and BRD4 in HEK293 cells expressing EGFP-hZFAT, treated with JQ1 or vehicle (Control). ( E ) Percentage of cells containing centromeric BRD4 foci in cells expressing EGFP-hZFAT, treated with JQ1 or vehicle, determined in (D). (B, D) Data are representative of three independent experiments. Scale bar, 5 μm. (C, E) Data represent the mean ± SD of three independent experiments.

Article Snippet: The cDNA for human BRD4 (FHC11882) was purchased from Promega and cloned into an EGFP-C3 vector.

Techniques: Construct, Immunofluorescence, Expressing, Transfection

Journal: Nucleic Acids Research

Article Title: ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

doi: 10.1093/nar/gkaa815

Figure Lengend Snippet: ZFAT recruits BRD4 to centromeres through KAT2B-mediated H4K8ac to promote the transcription of ncRNA. ( A ) ChIP-qPCR analysis of BRD4 at human repetitive DNA sequences using an anti-BRD4 antibody or control IgG in HEK293 cells transfected with indicated expression vectors and siRNAs. The BRD4 protein levels are shown as the relative values to control IgG. ( B ) Immunofluorescence images of ZFAT (HA) and BRD4 in HEK293 cells expressing the full-length or deletion mutants of hZFAT-HA. Enlarged images of the region enclosed by a white dotted line are shown. Scale bar, 5 μm. ( C ) Percentage of cells containing centromeric BRD4 foci in cells expressing the full length or deletion mutants of hZFAT-HA, determined in (B). ( D ) Immunoblotting analysis of ZFAT, KAT2B and BRD4 in HEK293 cells transfected with siRNAs for control or ZFAT (#1 and #5). ( E ) qRT-PCR analysis of centromeric ncRNA in HEK293 cells expressing EGFP or HA-hZFAT-EGFP (ZFAT), treated with JQ1 or vehicle. The RNA expression levels are shown as the relative values against those in vehicle-treated cells expressing HA-hZFAT-EGFP. ( F ) Immunoblotting analysis of ZFAT (HA) and BRD4 in HEK293 cells transfected with indicated expression vectors and siRNAs. N.S.; non-specific band. ( G ) qRT-PCR analysis of centromeric ncRNA in HEK293 cells transfected with indicated expression vectors and siRNAs. The RNA expression levels are shown as the relative values against those in cells transfected with control siRNA and HA-hZFAT-EGFP expression vector. ( H ) qRT-PCR analysis of centromeric ncRNA in HEK293 cells expressing EGFP or HA-hZFAT-EGFP (ZFAT), treated with α-amanitin or vehicle for 3 h. The RNA expression levels are shown as the relative values against those in vehicle-treated cells expressing HA-hZFAT-EGFP. (B, D, F) Data are representative of three independent experiments. (A, C, E, G, H) Data represent the mean ± SD of three independent experiments. * P < 0.05. (E, G, H) The RNA expression levels are normalized to those of β-actin.

Article Snippet: The cDNA for human BRD4 (FHC11882) was purchased from Promega and cloned into an EGFP-C3 vector.

Techniques: Transfection, Expressing, Immunofluorescence, Western Blot, Quantitative RT-PCR, RNA Expression, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

doi: 10.1093/nar/gkaa815

Figure Lengend Snippet: ZFAT is involved in chromosome segregation. ( A ) Immunofluorescence images of α-tubulin and DAPI in HT1080 cells transfected with indicated siRNAs. Data are representative of three independent experiments. Scale bar, 5 μm. ( B ) Percentage of cells with abnormal spindle morphology in mitotic cells transfected with indicated siRNAs, determined in (A). Data represent the mean ± SD of three independent experiments. ( C ) Model of ZFAT-regulated centromeric ncRNA transcription through the KAT2B–H4K8ac–BRD4 axis.

Article Snippet: The cDNA for human BRD4 (FHC11882) was purchased from Promega and cloned into an EGFP-C3 vector.

Techniques: Immunofluorescence, Transfection