Journal: Molecular Biology of the Cell

Article Title: Tip60 acetylation of histone H3K4 temporally controls chromosome passenger complex localization

doi: 10.1091/mbc.e21-06-0283

Figure Lengend Snippet: FIGURE 3: KAT5/Tip60 (Tip60) is the major histone acetyltransferase (HAT) for H3K4 in U2OS cells. (A) Histone H3 K4 acetylation detected by anti-H3K4ac immunoblot analysis from asynchronous and mitotic U2OS cells treated with Nu9056 inhibitor of Tip60’s acetyltransferase activity. Bottom cells arrested in nocodazole were treated with Nu9056 for 1 h. (B) Tip60 shRNA depleted cells show reduced level of histone H3K4ac. Cells were engineered to express an shRNA against Tip60 after addition of doxycyclin (Dox). (C) In vitro Tip60 HAT activity on histone H3 peptides was detected using histone H3K4ac immunoblot; 1 µg of each peptide was incubated with the specified reagents and then transferred to a membrane for immunodetection. (D) Targeting of Tip60 to centromeres increases histone H3K4ac to that region of mitotic chromosomes. Cells were engineered to express either CENP-B:GFP (CB-GFP) or CENP-B:GFP:Tip60 (CB-Tip60) after the addition of doxycyclin. Cells in doxycycline were stained for the indicated antibodies and mitotic cells were imaged by immunofluorescence. Dark and light blue dots in the plot represent independent experimental replicates. CB-GFP n = (11, 23); CB-Tip60 n = (16, 14) p value = (0.004; 0.005). For statistical analysis Welch’s t test was applied; **P < 0.01.

Article Snippet: Reaction was done in a buffer composed of 50 mM Tris, pH 8.0, 0.1 mM EDTA, and 5% glycerol and contained 0.01 μg/μl Tip60 (SignalChem, K314380G-05), 1 mM AcCoA (Sigma), 1 mM dithiothreitol, and 10 ηg/μl of BSA.

Techniques: Western Blot, Activity Assay, shRNA, In Vitro, Incubation, Membrane, Immunodetection, Staining, Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: Tip60 acetylation of histone H3K4 temporally controls chromosome passenger complex localization

doi: 10.1091/mbc.e21-06-0283

Figure Lengend Snippet: FIGURE 4: Targeting of Tip60 to centromeres dysregulates the localization of the Aurora B and other proteins of the Centromere Signaling Network (CSN). (A) Aurora B at centromeres is reduced when Tip60 is targeted to centromeres. Cells were arrested in mitosis with nocodazole and stained for the indicated antibody. CB-GFP n = (44, 16); CB-Tip60 n = (43, 13); p value = (0.0015; 0.033); scale bar, 5 µm. (B) Tip60 targeting affects many kinetochore/inner centromere events in an acetyltransferase dependent manner. Cells expressing CB-Tip60 were treated with either DMSO or the Tip60 acetyltransferase inhibitor Nu9056, and the localization of the indicated protein or PTM were imaged by immunofluorescence. Light and dark blue dots indicate two independent experimental replicates. Quantification of fluorescence signal of Aurora B nDMSO = (10, 9), nNU9056 = (11, 10), p value = (0.01, 0.0034); Sgo1 nDMSO = (10, 10), nNU9056 = (9, 11), p value = (0.049, 0.042); CENP-T nDMSO = (6, 11), nNU9056 = (10, 11), p value = (0.486, 0.051); Bub1 nDMSO = (11, 10), nNU9056 = (12, 11); p value = (0.4184, 0.6519); histone H3T3ph nDMSO = (8, 9), nNU9056 = (7, 9); p value (0.0018, 0.0025) and histone H2AT120ph nDMSO = (5, 9), nNU9056 = (6, 11) p value = (0.029, 0.0017) at centromeres in cells with Tip60 targeted to centromeres by CENP-B fusion and treated with Nu9016. For statistical analysis Welch’s t test was applied. ***P < 0.001, **P < 0.01, *P < 0.05, and ns P > 0.05.

Article Snippet: Reaction was done in a buffer composed of 50 mM Tris, pH 8.0, 0.1 mM EDTA, and 5% glycerol and contained 0.01 μg/μl Tip60 (SignalChem, K314380G-05), 1 mM AcCoA (Sigma), 1 mM dithiothreitol, and 10 ηg/μl of BSA.

Techniques: Staining, Expressing, Immunofluorescence, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Tip60 acetylation of histone H3K4 temporally controls chromosome passenger complex localization

doi: 10.1091/mbc.e21-06-0283

Figure Lengend Snippet: FIGURE 5: The histone deacetylase HDAC3 activity opposes Tip60 activity to relocalize Aurora B from chromatin to inner centromeres. Cells treated to reduce HDAC3 by shRNA had reduced amounts of Aurora B but this was rescued by concurrent inhibition of Tip60 acetyltransferase activity. (A) Immunofluorescence images showing Aurora B inner centromeric localization in prometaphase cells treated with shHDAC3 knockdown with and without Tip60 inhibition. Note that most of the Aurora B remains on noninner centromeric (chromosome arm) regions after HDAC3 depletion. However, if Tip60 was inhibited in the previous S/G2 phase then HDAC3 is not required to localize Aurora B to inner centromeres; scale bar, 5 µm. (B) Quantification of Aurora B signal at centromeres after the indicated treatment: shCtrl n = (11, 13); shHDAC3 n = (13, 10); shHDAC3+Nu9056 n = (11, 5); p valueshCtrl-shHDAC3 = (0.0211, 0.0046); p valueshCtrl-shHDAC3+Nu9056 (0.49, 0.63). (C) Quantification of chromatin bound Aurora B signal after the centromeric signals have been subtracted to measure the amount of Aurora B on chromosome arms after the indicated treatments. shCtrl n = (11, 13); shHDAC3 n = (13, 10); shHDAC3+Nu9056 n = (11, 5); p valueshCtrl-shHDAC3 = (0.02678, 0.00065); p valueshCtrl-shHDAC3+Nu9056 (0.25, 0.41). For statistical analysis Welch’s t test was applied; ns > 0.05, *P < 0.05. Light and dark blue dots indicate two independent experimental replicates.

Article Snippet: Reaction was done in a buffer composed of 50 mM Tris, pH 8.0, 0.1 mM EDTA, and 5% glycerol and contained 0.01 μg/μl Tip60 (SignalChem, K314380G-05), 1 mM AcCoA (Sigma), 1 mM dithiothreitol, and 10 ηg/μl of BSA.

Techniques: Histone Deacetylase Assay, Activity Assay, shRNA, Inhibition, Immunofluorescence, Knockdown

Journal: Molecular Biology of the Cell

Article Title: Tip60 acetylation of histone H3K4 temporally controls chromosome passenger complex localization

doi: 10.1091/mbc.e21-06-0283

Figure Lengend Snippet: FIGURE 6: Inhibition of Tip60 promotes premature centromeric targeting of Aurora B in interphase cells. (A) Inhibition of Tip60 by Nu9056 leads to Aurora B localization at centromeres in interphase (S/G2) cells as shown by immunofluorescence; scale bar, 20 µm. (B) Quantification of S/G2 centromeric fluorescence signals for Aurora B in control cells (n = 9, 26) and in Tip60- inhibited cells (n = 26, 23) p value (2.833⋅10–7, 8.152⋅10–5). Aurora B signal that overlaps with an ACA mask was quantified in S/G2 cells as defined by Aurora B signal. G1 cells have low Aurora B signal since Aurora B is an APC substrate. For statistical analysis Welch’s t test was applied. ***P < 0.001. (C) A proposed model for Aurora B localization regulated by acetylation of H3K4 of H3 by Tip60. Our data suggest that histone H3K4ac by Tip60 during S/G2 prevents Haspin phosphorylation of H3T3 (arrow a) and Survivin binding to chromatin to inhibit CPC localization to centromeres (arrow c). During the middle of prophase HDAC3 removes H3K4ac mark to enable CPC accumulation at inner centromeres. Previous studies have shown that Tip60 inhibits Bub1 binding to H2AT120 (arrow b) (Lee et al., 2018) and together with our work it suggests that Tip60 is a master regulator of CPC localization. (D) A model to control the timing of CPC localization to inner centromeres through changes in chromatin PTMs. Tip60 acetylates histone H3 on K4 and histone H2A on K118 in S/G2 to prevent CPC localization. In midprophase HDAC1 and HDAC3 deacetylate these histone marks to enable Bub1 phosphorylation and subsequent Sgo1 recruitment, Haspin phosphorylation of histone H3T3 and subsequent Survivin binding of H3T3ph. Together these events drive CPC recruitment to inner centromeres in midprophase.

Article Snippet: Reaction was done in a buffer composed of 50 mM Tris, pH 8.0, 0.1 mM EDTA, and 5% glycerol and contained 0.01 μg/μl Tip60 (SignalChem, K314380G-05), 1 mM AcCoA (Sigma), 1 mM dithiothreitol, and 10 ηg/μl of BSA.

Techniques: Inhibition, Immunofluorescence, Fluorescence, Control, Phospho-proteomics, Binding Assay

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative images and quantification of H3K9me3 by immunofluorescence staining in WT, LIPT1 −/− H460, and LIPT1 −/− H460 cells stably expressing Myc-LIPT1 cells. ( B ) Representative images and quantification of H3K9me3 by immunofluorescence staining in WT and LIPT1 −/− H157 cells. ( C and D ) Representative images and quantification of in situ proximity ligation assay (PLA, green dots) of interactions between histone H3 and TIP60 interaction (C) and between γH2AX and TIP60 (D) in WT and LIPT1 −/− H460 cells, with or without 10-Gy IR. ( E and F ) Representative images and quantification of in situ PLA (green dots) of interactions between histone H3 and TIP60 interaction (E) and between H2AX and TIP60 (F) in WT and LIPT1 −/− H157 cells with or without 10-Gy IR. For (A) to (F), nuclei were stained with Hoechst 33342. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. One-way ANOVA was used for the statistical analyses for (A), unpaired t test was used for (B), and two-way ANOVA was used for (C) to (F). **** P < 0.0001.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: Immunofluorescence, Staining, Stable Transfection, Expressing, In Situ, Proximity Ligation Assay, Imaging

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative images and quantification of in situ PLA (green dots) of ATM and TIP60 interaction in nonirradiated control and 1 hour after 4 Gy in WT, LIPT1 −/− H460, and LIPT1 −/− H460 reconstituted Myc-LIPT1 cells. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. ( B ) Quantification of in situ PLA (green dots) of ATM and TIP60 interaction in nonirradiated control and 1 hour after 4 Gy in WT and LIPT1 −/− H157 cells. ( C and D ) Immunoblotting analysis of ATM-pS1981, Ku70, γH2AX, and histone 3 in the soluble nuclear and chromatin fractions of WT and LIPT1 −/− in H460 (C) and in H157 (D) cells, with or without IR (0.5 hours post 10 Gy). Histone H3 and γH2AX served as chromatin markers. LE, long exposure; SE, short exposure. ( E and F ) Immunofluorescence images and quantification of colocalized ATM-pS1981 (red) and γH2AX (green) foci at 0.5 hours post-4 Gy in WT, LIPT1 −/− H460, and Myc-LIPT1–reconstituted H460 cells (E), as well as WT and LIPT1 −/− H157 cells (F). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. ( G and H ) Immunoblot analysis of ATM-pS1981, total ATM, Chk2-pT68, total Chk2, lipoyl-DLAT/DLST, and γH2AX in H460 (G) and H157 (H) cells at 0.5 hour post-10Gy. (A), (B), (E), and (F), quantification was performed on >100 cells per treatment. Two-way ANOVA was used for (A) and (B), one-way ANOVA was used for (E), and unpaired t test was used for (F). **** P < 0.0001.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: In Situ, Control, Staining, Western Blot, Immunofluorescence

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative immunoblots validating siRNA suppression of KDM4B in WT and LIPT1 −/− H460 cells, with GAPDH used as a loading control. ( B ) Representative images and quantification of H3K9me3 by immunofluorescence staining in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. ( C and D ) Representative images and quantification of in situ PLA (green dots) of interactions between γH2AX and TIP60 interaction (C), and between ATM and TIP60 (D) in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells, with or without 10- and 4-Gy IR. ( E ) Representative images and quantification of γH2AX foci by immunofluorescence staining in nonirradiated cells and at 6 and 24 hours after 4-Gy IR in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. ( F ) Representative images and quantification of RAD51 foci by immunofluorescence staining in nonirradiated cells and 6 hours after 4-Gy IR in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. For (B) to (F), nuclei were stained with Hoechst 33342. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. Two-way ANOVA was used for the statistical analyses. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: Western Blot, Control, Immunofluorescence, Staining, In Situ, Imaging

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative images and quantification of in situ PLA (green dots) of interactions between γH2AX and TIP60 in WT and LIPT1 −/− H460 cells with or without 1 mM dimethyl–α-KG and 10-Gy IR. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. Two-way ANOVA was used for the statistical analyses. **** P < 0.0001. ( B ) Immunoblotting analysis of ATM-pS1981 and histone H3 in the chromatin fractions of WT and LIPT1 −/− in H460 cells with or without 1 mM dimethyl–α-KG, 1 mM α-KG, and 10-Gy IR. Histone H3 was used as marker and internal control for the chromatin fraction. Protein levels of ATM-pS1981 were normalized with internal control histone H3. ( C ) Clonogenic assay of WT and LIPT1 −/− H460 cells with or without 1 mM dimethyl–α-KG after 2, 4, and 6 Gy. The surviving fraction was normalized to the corresponding sham control and survival curves were fitted using the linear-quadratic model. ( D ) Schematic illustrating how LIPT1 deficiency impairs the IR-induced, TIP60-ATM–mediated HR damage repair pathway due to a deficiency in α-KG–dependent demethylation.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: In Situ, Staining, Imaging, Western Blot, Marker, Control, Clonogenic Assay

Journal: Cell Regeneration

Article Title: MRG15 decline in aged/injured MuSCs hinders regeneration via differentiation defects

doi: 10.1186/s13619-026-00279-9

Figure Lengend Snippet: MRG15 interacts with MyoD to regulate histone acetylation and transcription of myogenic genes. A Metascape databases were used to predict the transcription factors interacting with MRG15. B Venn diagrams showing the overlapping (216 genes) between the H4K16 acetylation (ChIP-Seq) target (9930) and the down-regulated genes (1460) among RNA-seq. C GO analysis of the above 216 genes revealed an extreme enrichment of skeletal muscle tissue development terms. D Enrichment of canonical MyoD motif in the above binding regions. E and F Co-IP assays showing the interactions between MRG15 and MyoD ( E ), or TIP60 ( F ) in C2C12. G ChIP-PCR analysis of H4K16ac occupancy at the promoter regions of myogenic genes in sg Mrg15 versus sgCtrl-C2C12 cells. Data are presented as percentage of input (% Input). H qPCR analysis of myogenic genes expression in sg Mrg15 or sgCtrl-C2C12 ( n = 3). Means ± SEM are shown. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The whole-cell lysates obtained by centrifugation (with equal concentration of protein in different samples) were incubated with 1 μg of MyoD1 (M3512, Dako) or MRG15 (55257–1-AP, Proteintech), or TIP60 (10827–1-AP, Proteintech), antibody for 1.5 h at 4 °C with rotation followed by binding to Protein A/G PLUS-Agarose (sc-2003, Santa Cruz) for overnight at 4 °C.

Techniques: ChIP-sequencing, RNA Sequencing, Binding Assay, Co-Immunoprecipitation Assay, Expressing

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 1 PHF8 and TIP60 colocalize and recruit each other in neuronal interchromatin space. A, Endogenous PHF8 immunostained with anti-PHF8 antibody (ab36068; Abcam) forms hundreds of discrete puncta that specifically localize to the interchromatin space in hippocampal neurons (representative z-slice of a hippocampal neuronal nucleus; left, widefield; right, SIM). Scale bar, 1 m. B, Endogenous TIP60 forms puncta of roughly the same caliber as those of PHF8 above (left, hippocampal nucleus in widefield; right, SIM), which also localize to the interchromatin space. Scale bar, 1 m. C, A representative field of hippocampal neurons stained with an antibody against the phosphorylated CTD of RNA polymerase II (YSPTSPS phospho S5, abbreviated to S5P), showing that S5P, a marker of the transcription initiation complex, localizes to the same nuclear compartment as PHF8 in the nucleus. Scale bar, 1 m. D, A hippocampal neuronal nucleus outlined in blue, showing the localization of spectrally distinct PHF8tdTomato and TIP60CFP pseudo-colored in green and red, respectively, which overlapped completely in the nuclear interchromatin space (merge channel, yellow pixels indicate colocalization). DAPI was used to stain the DNA (blue). Scale bar, 0.2 m. E, When TIP60 is overexpressed by itself in hippocampal neurons (middle, red), endogenous PHF8 (left, green) is seen to be recruited to the TIP60 puncta in hippocampal neurons (right, merge). DAPI was used to stain the DNA (blue). Scale bar, 0.2 m. Asterisk () indicates endogenous protein staining. F, When PHF8 is overexpressed by itself (left, green) in hippocampal neurons, endogenous TIP60 (middle) is seen to be recruited to the PHF8 puncta (right, merge). DAPI was used to stain the DNA (blue). Scale bar, 0.2 m. Asterisk () indicates endogenous protein staining. G. Endogenous TIP60 is located within 30 nm of PHF8 as shown by P-LISA, showing distinct areas where PHF8 interacts with endogenous TIP60 (red spots) on the border with DAPI-dense regions (blue). Scale bar, 0.5 m. H, Two Hek293 nuclei are shown, one is positive for PHF8 shRNA (outlined in red) while the other is not (outlined in green). Positive PHF8TIP60 interaction hotspots were stained as red punctae. Scale bar, 0.5 m. I, Quantification of the number of hotspots found in cells transfected with PHF8, PHF8 shRNA, the mutant PHF8 F279S, or, in F279S-transfected cells expressing PHF8, shRNA were quantified using Blobfinder, and the means and standard errors are displayed in a bar grapht (triple asterisks indicating statistical significance using the unpaired t test; p 0.0001). J, Double immunofluorescence confirming the existence of PHF8 in the identified PLA hotspots where PHF8 and TIP60 interact. Scale bar, 0.5 m.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Staining, Marker, shRNA, Transfection, Mutagenesis, Expressing, Immunofluorescence

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 2 PHF8 and TIP60 physically associate to form a dual function chromatin-modifying complex. A, Coimmunoprecipitation of PHF8 and TIP60 in HEK293T nuclear extracts, where TIP60YFP was pulled down with anti-GFP antibody and PHF8FLAG was detected with anti-FLAG by Western blotting. B, Pulldown of PHF8YFP showed that TIP60FLAG was detected in the IP fraction but not in the YFP-only control lane. C, Endogenous coimmunoprecipitation of PHF8 and TIP60 in DIV12 cortical neuronal nuclear extracts, showing that PHF8 is able to be pulled down by both the anti-PHF8 antibody and anti-TIP60 antibody, but not the anti-GFP antibody. D, E, Truncated constructs of TIP60 protein (A to F) containing the indicated TIP60 domain (E) were fused to YFP and then cotransfected with full-length PHF8FLAG and immunoprecipitated with an anti-GFP antibody. Western analysis was performed to detect PHF8FLAG in the immunoprecipitates using the anti-FLAG antibody. A negative control of YFP only is denoted by (-), whereas full-length TIP60 served as a positive control (). F, G, Total histones from HEK293 cells overexpressing PHF8, TIP60, or both were separated on TAU gels (F) or conventional SDS-PAGE (G). Overexpression of TIP60 alone increases H3K9 acetylation in HEK293 cells for both the H3.1 and H3.3 isoforms, whereas acetylation of the non-TIP60 substrate H2BK5 was not affected. Coexpression of PHF8 and TIP60 increases H3.3K9 acetylation to even higher levels. H, Chromatin immunoprecipitation using an antibody specific to H3K9me2, showing that overexpression of wild-type PHF8 but not the clinical mutant F279S (U, unbound or input levels of H3K9me2; B, bound or immunoprecipitated H3K9me2). I, J, ChIP assays of HEK293T cells transfected with PHF8, TIP60, or both analyzing histone tails positive for H3K4me3, the transcriptionally-activating histone mark that is known to be bound by PHF8, show that the increase in H3K9ac (I) and H3K14ac (J) is specific to histones carrying H3K4me3, and that this histone population was enriched in H3.3 (as shown by the more intense staining of this isoform on the TAU gel; asterisk). Western blot of the same lysates using an H3.3 antibody serves as loading control. The right panel shows bar graphs quantifying the increase in H3.3K9 and H3.3K14 acetylation, relative to the untransfected control (n 3; p 0.19 for PHF8 only, 0.04 for TIP60 only, 0.02 for PHF8TIP60; asterisks indicates statistical significance: p 0.05).

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Western Blot, Control, Construct, Immunoprecipitation, Negative Control, Positive Control, SDS Page, Over Expression, Chromatin Immunoprecipitation, Mutagenesis, Transfection, Staining

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 4 Neuronal activity reorganizes PHF8 and TIP60 in the nucleus and effectuate histone methylation and acetylation changes. A, A representative image of a pair of hippocampal neuronal nuclei during the first 5 min of 4APBicFors treatment and then at 45 min, showing the activity-dependent increase of PHF8 and TIP60 protein in the nucleus. B, Neural network activity visualized by Ca2

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Activity Assay, Methylation

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 5 PHF8 and TIP60 modulate neuronal activity-induced histone acetylation at H3K9acS10P and activation of the Arc gene. A, Representative microscopic field of hippocampal neurons after 1 h of network activation by chemLTP, showing a positive correlation between the expression of Arc (red) and Tip60 (blue) with the phosphoacetylation mark H3K9acS10P (green). The bottom panels show three different neurons that induced varying amounts of ARC protein. The neuron expressing the highest amount of ARC (3) also has high amounts of H3K9acS10P. B, Quantification of 20 immunofluorescence-analyzed fields exemplified in A, showing a statistically significant increase in H3K9acS10P as well as endogenous TIP60 in ARC-expressing neurons (n 347 neurons; p 0.00001). C, Fusion constructs of PHF8 and its mutant F279S were individually expressed in hippocampal neurons and the next day the neuronal network was activated using ChemLTP (4APBicFors). After 1 h of upregulated synaptic activity, the expression of PHF8, but not its mutant F279S, significantly increases histone acetylation at H3K9acS10P (n 397 neurons; p 0.00001). D, A representative microscopic field of neuronal nuclei after 1 h of ChemLTP, with neuronal nuclei stained by DAPI outlined in magenta,

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Activity Assay, Activation Assay, Expressing, Immunofluorescence, Construct, Mutagenesis, Staining

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 7 PHF8 and TIP60 are actively recruited to specific neuronal gene promoters. A, B, Within minutes of synaptic activation (x-axis: time of increased network activity, in minutes), time-course ChIP shows an early detectable decrease in the chromatin mark H3K9me2 at the Arc TSS (A), which is mirrored by a concomitant, highly transient increase in the levels of H3K9acS10P at the Arc TSS (B). This increase in H3K9acS10P was specific to the Arc promoter as analyses of Rpl19, JunB, Synaptophysin, Homer1A, BDNF promoter 1, Txnip, Gapdh, and Fos intergenic region (Negative Ctrl) did not show an activity-dependent increase. C, D, Time-course ChIP followed by qRT-PCR using primers against the transcriptional start site regions of the Arc gene, arc synaptic response element, ribosomal protein L19 (Rpl19), neuronal PAS domain protein 4 (Npas4), and synaptophysin. Both TIP60 (C) and PHF8 (D) are recruited to the Arc TSS within minutes of activation of the neuronal network, but not to the Rpl19, Npas4, or Synaptophysin transcriptional start sites.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Activation Assay, Activity Assay, Quantitative RT-PCR

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 8 Common interacting partners between PHF8 and TIP60 function primarily in transcription and mRNA processing. Top, A Venn diagram showing several interacting partners of PHF8 and TIP60 as identified by immunoprecipitation followed by mass spectrometry. The overlapped region in the middle represents common partners that interact with PHF8 and TIP60, which include the splicing factor SFPQ (PSF) and its partner NONO, as well as several ATP-dependent RNA helicases, and the histone chaperone nucleolin. Proteins that have known acetylation sites are marked by a triangle (Choudhary et al., 2009). Arrows indicate known functional interactions between identified proteins. Font size indicates the percentage of the total protein that the identified MS/MS peptides covered (large font: 25% coverage; medium font: 5 25% coverage; small font: 5% coverage). Histone proteins identified in the IPMS are in bold. Bottom, A listing of the top eight biological functions attributed to the proteins identified in the IPMS of both PHF8 and TIP60 in order of abundance, as computed by the software DAVID (http://david.abcc.ncifcrf.gov/home.jsp), with the associated p value and Benjamini factor, showing that interactors of PHF8 and TIP60 are enriched in the functions of RNA processing, RNA splicing, and mRNA processing.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Immunoprecipitation, Mass Spectrometry, Functional Assay, Tandem Mass Spectroscopy, Software

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 9 Endogenous TIP60 is located within 30 nm of PHF8 in the activated hippocampal neuronal nucleus. A, A maximum intensity projection of a dual color 3D STORM image of a hippocampal neuronal nucleus that has undergone 1 h of chemLTP. The neuron has been labeled for endogenous TIP60 (red) and endogenous PHF8 (green), showing that the two molecules closely interact in various localized puncta in the nucleus. Scale bar, 1 m. The insets on the right show six representative complexes at higher magnification (scale bar, 50 nm). B, A highly magnified view of two endogenous PHF8TIP60 complexes shown in the outlined area in A. The insets on the right show three projections of the single-molecule interaction between PHF8 and TIP60 viewing down the x-, y-, and z-axes, demonstrating that the complexes formed between these two chromatin-modifying enzymes have well-defined spatial relationship. Each dot corresponds to the localization of a single molecule. Scale bars, 50 nm.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Labeling

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 10 PHF8 and TIP60 form a tripartite complex with the splicing factor PSF and associates with newly transcribed nascent RNA. A, A maximum intensity projection of a 3D STORM image of an activated hippocampal neuronal nucleus. Single-molecule imaging of endogenous PHF8 (green), endogenous TIP60 (red), and PTB-associated splicing factor (blue), with the corresponding single- channel views. Each dot corresponds to the localization of a single molecule. Scale bar, 500 nm. B, C, Four representative higher magnification views of the neuronal nucleus depicted in A, showing that PSF (blue) forms a tailing structure within the interface between PHF8 (green) and TIP60 (red) viewed axially B or longitudinally C as a recognizable tripartite complex. Scale bar, 50 nm.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Imaging

Journal: Journal of Biological Chemistry

Article Title: Identification of New Subunits of the Multiprotein Mammalian TRRAP/TIP60-containing Histone Acetyltransferase Complex

doi: 10.1074/jbc.c300389200

Figure Lengend Snippet: FIG. 2. Analytical TSK SP-NPR HPLC of the TRRAP/TIP60 complex. A, cofractionation of DMAP1 with components of the TRRAP/ TIP60 complex. Anti-FLAG-agarose eluates prepared as described in the legend of Fig. 1A from HeLa cells stably expressing FLAG-MRGBP were applied to a TSK SP-NPR HPLC column (4.6 35 mm, Toso Haas) pre-equilibrated in buffer containing 20 mM Hepes-NaOH (pH 7.9), 0.5 mM EDTA, 1 mM DTT, 10% (v/v) glycerol, and 0.1 M KCl. The column was eluted at 0.2 ml/min with a 6-ml linear salt gradient from 0.1 to 0.6

Article Snippet: Anti-TIP60 antibodies were from Upstate-Cell Signaling Solutions.

Techniques: Stable Transfection, Expressing

Journal: Journal of Biological Chemistry

Article Title: Identification of New Subunits of the Multiprotein Mammalian TRRAP/TIP60-containing Histone Acetyltransferase Complex

doi: 10.1074/jbc.c300389200

Figure Lengend Snippet: FIG. 3. Analytical Superose 6 gel filtration of the TRRAP/TIP60 complex. MRGBP-associating proteins were prepared as described in the legend of Fig. 1B by chromatography on consecutive anti-FLAG- agarose and TSK SP-NPR HPLC columns. MRGBP-associating pro- teins in the peak fraction from TSK SP-NPR HPLC were applied to a Superose 6 column (3.2/30, Amersham Biosciences) in 20 mM Hepes- NaOH (pH 7.9), 0.1 M KCl, 0.5 mM EDTA, 1 mM DTT, and 5% (v/v) glycerol. The column was eluted at 20 l/min, and 50-l fractions were collected. Proteins present in fractions were analyzed by 11% Tris- Tricine SDS-polyacrylamide gel electrophoresis, and proteins were vi- sualized by Coomassie Blue staining. The bands marked with brackets were excised, and proteins present in gel slices were identified by mass spectrometry as described under “Experimental Procedures.” The posi- tions and relative molecular masses in kilodaltons of protein size mark- ers are indicated on the left. Catalase and chymotrypsinogen standards eluted from Superose 6 column in peaks centered at fractions 32 and 41, respectively.

Article Snippet: Anti-TIP60 antibodies were from Upstate-Cell Signaling Solutions.

Techniques: Filtration, Chromatography, Polyacrylamide Gel Electrophoresis, Staining, Mass Spectrometry