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Research ArticleNew Research, Neuronal Excitability

Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation

Caroline L. Wee, Shaun Teo, Nicodemus E. Oey, Graham D. Wright, Hendrika M.A. VanDongen and Antonius M.J. VanDongen
eNeuro 12 November 2014, 1 (1) ENEURO.0019-14.2014; https://doi.org/10.1523/ENEURO.0019-14.2014
Caroline L. Wee
1Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 169857
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Shaun Teo
1Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 169857
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Nicodemus E. Oey
1Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 169857
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Graham D. Wright
2Institute of Medical Biology, a*STAR, Singapore 138648
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Hendrika M.A. VanDongen
1Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 169857
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Antonius M.J. VanDongen
1Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 169857
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  • Figure 1
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    Figure 1

    Segmentation of nuclear substructures. A region of interest containing Arc-YFP puncta in a single neuronal nucleus is segmented to outline the Arc structures using Elements AR. The area of each structure is calculated and the mean and SEM are reported for each nucleus, together with the number of puncta (N).

  • Figure 2.
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    Figure 2.

    Arc, β-Spectrin IV (βSpec), and Tip60 colocalize in the nuclei of hippocampal neurons. Fluorescently tagged versions of each protein were (co-)expressed in cultured hippocampal neurons. See text for details. DNA was labeled using DAPI (blue). Wide-field z-stacks were deconvolved using AutoQuant 3D deconvolution and a representative optical section through the center of the nucleus is shown. Scale bars, 2 μm. Four insets on the right show the structures at higher resolution. Inset scale bars, 0.5 μm.

  • Figure 3.
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    Figure 3.

    Arc, β-Spectrin IV (βSpec), and Tip60 form a tight complex. Fluorescently-tagged versions of Arc, β-Spectrin IV, and Tip60 were coexpressed in hippocampal neurons (A−D) or HEK293 cells (E−H) and imaged as described in Fig. 2. Scale bar, 2 μm. I, Arc, βSpIVΣ5, and Tip60 fused to spectrally distinct GFP isoforms were expressed in hippocampal neurons, either individually, as pairs, or all three together. Neuronal nuclei were outlined using DAPI staining and subnuclear structures (puncta) were segmented as described in the Materials and Methods section. For each nucleus, the mean area and number of puncta was calculated. The scatter plot graphs number of puncta versus mean area for the different experiments. Arc, βSpIVΣ5, and Tip60, when expressed alone, each occupy a distinct region of the plot. Paired and tripartite complexes have properties similar to Tip60 alone.

  • Figure 4.
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    Figure 4.

    Arc localizes the Arc-β-Spectrin-Tip60 complex to the perichromatin region. A, Fluorescently-tagged versions of Arc (green), β-Spectrin (red), and Tip60 (blue) were coexpressed in HEK293 cells and imaged as described in Fig. 3. Because of their strict overlap, Arc-β-Spectrin-Tip60 complexes appear as white puncta. The DAPI signal is shown in magenta. The insets (B−E) show the relationship between individual complexes and chromatin with higher magnification. Segmentation of the DAPI structures is indicated with a magenta line. B shows a complex in the center of an interchromatin domain. C and D show complexes localized to the perichromatin region, while E illustrates a complex surrounded by dense DAPI staining. Analysis of five nuclei indicated that the majority of puncta were localized to the perichromatin region (84%, n = 151), while the puncta were less frequently observed in the center of the interchromatin region (9%, n = 16) or in densely packed chromatin (7%, n = 12). F, Tip60-YFP was expressed in HEK293 cells, which were fixed as stained for DNA by DAPI. Tip60 occupies large parts of the interchromatin domains. Insets (G−J) show individual Tip60 speckles in higher magnification. Not the larger size of Tip60 compared to the tripartite complexes shown in B−E. K, Arc-YFP was expressed in HEK293 cells, which were fixed and stained with DAPI. Many small Arc puncta are seen, which associate with the interface of the IC domains and the dense chromatin strongly labeled with DAPI, indicated with a red line that segments the DAPI structure. Insets (L−M) show the relationship of Arc puncta with chromatin at higher magnification. P, Arc-YFP was expressed in hippocampal neurons (18 DIV), which were fixed and labeled with DAPI. White arrows indicate Arc puncta that localize to the interchromatin -chromatin interface indicated with the red line. Insets (Q−T) show Arc localization in higher magnification.

  • Figure 5.
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    Figure 5.

    Arc and βSpIVΣ5 interact with Tip60. A, Tip60 interacts with Arc. HEK293T cells were transfected with either Arc in pCDNA3.1 and YFP vector, or Arc-pCDNA3.1 and Tip60-YFP. Tip60-YFP was immunoprecipitated with a mouse anti-GFP antibody, and Arc was detected with a rabbit anti-Arc antibody. B, Tip60 interacts with βSpIVΣ5. HEK293T cells were transfected with either βSpIVΣ5-HA and YFP vector or βSpIVΣ5-HA and Tip60-YFP. Tip60-YFP was immunoprecipitated with a mouse anti-GFP antibody, and βSpIVΣ5-HA was detected with a mouse anti-HA antibody. C, Tip60 interacts with both Arc and βSpIVΣ5. HEK293T cells were transfected with Arc, βSpIVΣ5-HA, and either YFP or Tip60-YFP. Tip60-YFP was immunoprecipitated with a mouse anti-GFP antibody, and Arc or βSpIVΣ5-HA was detected with a rabbit anti-Arc or mouse anti-HA antibody respectively. The asterix (*) denotes an unidentified band.

  • Figure 6.
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    Figure 6.

    3D stimulated emission depletion microscopy shows association of endogenous Arc and Tip60. The top left panel shows the distribution of endogenous Arc protein (red) and endogenous Tip60 protein (green) in a representative z-plane of a hippocampal neuronal nucleus following network activation by a 4 h treatment with 4AP-bicuculline-forskolin (see Materials and Methods). In the top right panel, Tip60 structures have been segmented and are shown with a green outline to highlight their relationship with Arc. Scale bar, 1 μm. The bottom panels show examples of the rich variety of association patterns formed by Arc and Tip60 puncta, with many unique conformations. Scale bar, 300 nm.

  • Figure 7
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    Figure 7

    Dual-color super-resolution microscopy of Arc-mEOS2 and endogenous Tip60. A representative z-plane of a hippocampal neuronal nucleus after 4 h of network activation by 4AP-bicuculline-forskolin treatment (see Materials and Methods), showing the single-molecule distribution Arc-mEOS2 (green) imaged using 3D PALM and endogenous Tip60 (red) imaged using dSTORM. Cyan-colored squares outline distinct areas of association between Arc and Tip60 proteins, which were consistently found across reconstructed z-sections. Scale bar, 2 μm. The bottom panel shows detailed images at higher magnification. Scale bar, 200 nm.

  • Figure 8.
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    Figure 8.

    Arc increases nuclear Tip60 puncta. A−D, DIV18 hippocampal neurons were transfected with Arc-YFP or YFP as a control, and imaged the next day. Arc-YFP expression was stimulated for 4 h with 50 μM forskolin or DMSO as a control, fixed and stained for endogenous Tip60 (red). Comparing Arc-negative (A) with Arc-positive (B, C) neurons, it was found that overexpression of Arc-YFP induced the formation of endogenous Tip60 nuclear puncta, which associate with Arc-YFP puncta (B, C, insets). 58 ± 4% (n = 24) of endogenous Tip60 puncta were associated with exogenous Arc-YFP spots. Overexpression of YFP alone did not induce the formation of Tip60 hotspots (D). E, DIV18 hippocampal neurons were cotransfected with Arc-YFP (green) and βSpIVΣ5-CFP (blue), treated for 4 h with forskolin, fixed and stained for endogenous Tip60 (red). Overexpression of both Arc and βSpIVΣ5 similarly induced the formation of endogenous Tip60 nuclear puncta, which associated with the Arc- βSpIVΣ5 complex. Scale bars, 2 μm; insets, 0.5 μm. The * indicates immunostaining of endogenous protein.

  • Figure 9.
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    Figure 9.

    Arc recruits Tip60 to PML bodies. A, Tip60-YFP (green) and PML-mCherry (red) were coexpressed in 18 DIV hippocampal neurons, which were fixed and stained for DNA by DAPI (blue). Although Tip60 speckles are seen on close proximity of PML bodies in the interchromatin domains, they do not overlap. B, When Arc-YFP (green), Tip60-mCherry (blue) and PML-CFP (red) were coexpressed, they were found to tightly overlap, indicated by a puncta containing a preponderance of white pixels in the merged image (far right panel). C, D, When the same experiment was performed in HEK293 cells, Tip60 and PML occupied non-overlapping regions of the interchromatin space (C), while inclusion of Arc resulted in puncta in which all three proteins tightly colocalized (D). E, A nucleus of a representative HEK293 cell expressing Arc (blue), Tip60 (green), and PML (red), showing that in structures containing moderate to high Arc (white arrows 2 − 4) Tip60 is recruited to PML bodies, which does not occur when Arc is low (white arrow 1). The bottom insets are enlarged views of PML bodies 1–4 as viewed through structured illumination microscopy, showing that Tip60 puncta heavily populate and permeate the porous PML bodies more efficiently when Arc is present. Scale bars: A−D, 2 μm; E, 500 nm.

  • Figure 10
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    Figure 10

    Arc expression increases H4K12Ac levels. Cortical neurons (21 DIV) were transfected with Arc-YFP or YFP as a control. The next day, the cultures were treated with either forskolin or the combination of 4AP−bicuculline−forskolin (see Materials and Methods for details). After 4 h of treatment, the neurons were fixed and stained for both H4K12Ac and endogenous Arc, using a red and far-red secondary antibody, respectively. DNA was labeled using DAPI. Scale bars, 10 μm. A, Representative images for the four experimental conditions. Each field contains a transfected neuron (YFP or Arc-YFP) surrounded by untransfected controls. The top row shows the YFP or Arc-YFP signal, and the bottom row represents the H4K12Ac signal. The transfected neuron is indicated with a white arrow. Nuclear outlines are shown as thin blue lines. B, H4K12Ac levels (mean fluorescence intensity per nucleus) were determined for both transfected neurons (YFP or Arc-YFP) and untransfected controls (Con) for both treatment conditions for 30 fields of view containing at least 1 − 4 transfected neurons and 30 − 90 untransfected controls. The bar graph shows the average H4K12Ac levels, normalized using the mean of the untransfected neurons, with error bars indicating SEMs. YFP overexpression did not significantly alter H4K12Ac levels in either condition: p values were 0.07 and 0.12 for forskolin and 4AP−Bic−Fors, respectively. Arc-YFP overexpression increased H4K12 acetylation levels for both treatment scenarios, with high statistical significance (p = 3*10−5 for forskolin, p = 5*10−11 for 4AP−Bic−Fors), although the increase was much larger following network activation by 4AP−Bic−Fors (89%) than with forskolin treatment only (10%). ***p < 0.0001; n.s., not significant. C, The relationship between endogenous Arc expression and H4K12Ac levels was investigated by analyzing the neurons that were not transfected. The inset shows DAPI, endogenous Arc, and H4K12Ac levels for 15 untransfected neuronal nuclei. Five neurons (solid white arrows) strongly expressed endogenous Arc and the same five neurons also displayed high levels of H4K12 acetylation. Eleven neurons (thin gray arrows) had barely detectable Arc levels, and H4K12Ac staining was faint as well. Nuclei are outlined by a thin blue line. The graph was generated by sorting 816 nuclei by their endogenous Arc levels and plotting H4K12Ac levels versus the sortation index, from low to high Arc levels. The solid black line is a moving average of 50 H4K12Ac values. Scale bars, 10 μm.

  • Figure 11
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    Figure 11

    A Tip60 mutant lacking acetyltransferase activity decreases H4K12 acetylation. Hippocampal neurons (21 DIV) were transfected with a catalytically inactive double mutant of Tip60 (Q377E/G380E, abbreviated as Tip60dm). A, B, Representative pair of neuronal nuclei positive (A) and negative (B) for Tip60dm, showing that upon Tip60dm overexpression, the overall staining of H4K12Ac per nucleus is decreased. Scale bars, 2 μm. C, Distributions of nuclear H4K12Ac staining in neurons expressing Tip60dm versus negative control neurons. Each horizontal line represents the average H4K12Ac intensity of a nucleus. All averages were normalized to the mean of the population of Tip60dm-negative neurons. Circles to the left of the distributions indicate the mean and SEM of each population: mean ± SEM (N) is 1.00 ± 0.02 (n = 408) for Tip60dm-negative and 0.62 ± 0.06 (n = 27) for Tip60dm-positive neurons. p = 4*10−9.

  • Figure 12.
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    Figure 12.

    Arc associates with H4K12Ac. A−E, A representative hippocampal neuronal nucleus showing H4K12Ac staining (A) and Arc-YFP puncta (B). The same image with increasing the brightness threshold to show only the brightest 50th percentile H4K12ac shows clear H4K12Ac hotspots (C), which is overlaid with localized Arc-YFP puncta (D; scale bar, 1 μm). Insets below show association of Arc puncta with H4K12ac hotspots (E; scale bar, 200 nm). Hippocampal cultures were treated with 4AP, bicuculline, and forskolin for 4 h to induce Arc expression. F, A representative image of a hippocampal neuron that has been transfected with Arc-YFP, showing Arc-YFP puncta (green), imaged with structured illumination microscopy to observe the localization of H4K12Ac (red) in higher resolution. The increased resolution afforded by SIM did not substantially change the Arc puncta. However, the H4K12Ac staining, which appeared fairly homogenous in A, can now be seen to consist of many tiny structures, the size of which is just below the resolution of the conventional light microscope. They are distributed fairly uniformly over the nucleus, although local concentrations can be observed, which would correspond to the hotspots in C. Some of these clusters of H4K12Ac seemed to associate with Arc puncta. Scale bar, 1.7 μm. G shows six Arc puncta at higher magnification. The top row shows an Arc puncta associating with dense H4K12Ac clusters, while the bottom row shows a few bright H4K12Ac structures surrounding Arc puncta. Scale bar, 500 nm.

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Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation
Caroline L. Wee, Shaun Teo, Nicodemus E. Oey, Graham D. Wright, Hendrika M.A. VanDongen, Antonius M.J. VanDongen
eNeuro 12 November 2014, 1 (1) ENEURO.0019-14.2014; DOI: 10.1523/ENEURO.0019-14.2014

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Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation
Caroline L. Wee, Shaun Teo, Nicodemus E. Oey, Graham D. Wright, Hendrika M.A. VanDongen, Antonius M.J. VanDongen
eNeuro 12 November 2014, 1 (1) ENEURO.0019-14.2014; DOI: 10.1523/ENEURO.0019-14.2014
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Keywords

  • Chromatin Modification
  • epigenetics
  • Histone Acetylation
  • immediate early gene
  • synaptic plasticity

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