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Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation(1,2,3).

Wee CL, Teo S, Oey NE, Wright GD, VanDongen HM, VanDongen AM - eNeuro (2014)

Bottom Line: Although Arc protein is found at synapses, it also localizes to the neuronal nucleus, where its function is less understood.Neuronal activity-induced expression of Arc (1) increases endogenous nuclear Tip60 puncta, (2) recruits Tip60 to PML bodies, and (3) increases histone acetylation of Tip60 substrate H4K12, a learning-induced chromatin modification.These mechanisms point to an epigenetic role for Arc in regulating memory consolidation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School , Singapore 169857.

ABSTRACT
Arc is an immediate-early gene whose genetic ablation selectively abrogates long-term memory, indicating a critical role in memory consolidation. Although Arc protein is found at synapses, it also localizes to the neuronal nucleus, where its function is less understood. Nuclear Arc forms a complex with the β-spectrin isoform βSpIVΣ5 and associates with PML bodies, sites of epigenetic regulation of gene expression. We report here a novel interaction between Arc and Tip60, a histone-acetyltransferase and subunit of a chromatin-remodelling complex, using biochemistry and super-resolution microscopy in primary rat hippocampal neurons. Arc and βSpIVΣ5 are recruited to nuclear Tip60 speckles, and the three proteins form a tight complex that localizes to nuclear perichromatin regions, sites of transcriptional activity. Neuronal activity-induced expression of Arc (1) increases endogenous nuclear Tip60 puncta, (2) recruits Tip60 to PML bodies, and (3) increases histone acetylation of Tip60 substrate H4K12, a learning-induced chromatin modification. These mechanisms point to an epigenetic role for Arc in regulating memory consolidation.

No MeSH data available.


Related in: MedlinePlus

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.
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f10: 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.

Mentions: Arc-YFP was expressed in cortical neurons for 1 d, after which the cells were fixed and stained using an antibody specific for acetylated lysine 12 of histone H4 (H4K12Ac). We have performed additional experiments comparing the effect of Arc-YFP and YFP overexpression on H4K12 acetylation following treatment with forskolin, which rescues Arc translation (Bloomer et al., 2008), and the pharmacological combination of 4AP−bicuculine−forskolin, which induces endogenous Arc expression. Overexpression of YFP did not affect H4K12 acetylation, whereas Arc-YFP significantly increased H4K12 acetylation (Fig. 10B). Interestingly, the increase was much higher after network stimulation than with forskolin treatment only. In addition, these experiments revealed that endogenous Arc expression levels correlated strongly with H4K12Ac levels. The relationship between endogenous Arc expression and H4K12Ac levels was investigated by analyzing the 95% of the neurons that were not transfected. Segmented nuclei were ranked according to endogenous Arc expression level and analyzed for H4K12 acetylation (Fig. 10C). The increasing trend displayed by the Arc-sorted H4K12Ac levels indicates a positive correlation between endogenous Arc levels and H4K12 acetylation status.


Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation(1,2,3).

Wee CL, Teo S, Oey NE, Wright GD, VanDongen HM, VanDongen AM - eNeuro (2014)

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.
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f10: 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.
Mentions: Arc-YFP was expressed in cortical neurons for 1 d, after which the cells were fixed and stained using an antibody specific for acetylated lysine 12 of histone H4 (H4K12Ac). We have performed additional experiments comparing the effect of Arc-YFP and YFP overexpression on H4K12 acetylation following treatment with forskolin, which rescues Arc translation (Bloomer et al., 2008), and the pharmacological combination of 4AP−bicuculine−forskolin, which induces endogenous Arc expression. Overexpression of YFP did not affect H4K12 acetylation, whereas Arc-YFP significantly increased H4K12 acetylation (Fig. 10B). Interestingly, the increase was much higher after network stimulation than with forskolin treatment only. In addition, these experiments revealed that endogenous Arc expression levels correlated strongly with H4K12Ac levels. The relationship between endogenous Arc expression and H4K12Ac levels was investigated by analyzing the 95% of the neurons that were not transfected. Segmented nuclei were ranked according to endogenous Arc expression level and analyzed for H4K12 acetylation (Fig. 10C). The increasing trend displayed by the Arc-sorted H4K12Ac levels indicates a positive correlation between endogenous Arc levels and H4K12 acetylation status.

Bottom Line: Although Arc protein is found at synapses, it also localizes to the neuronal nucleus, where its function is less understood.Neuronal activity-induced expression of Arc (1) increases endogenous nuclear Tip60 puncta, (2) recruits Tip60 to PML bodies, and (3) increases histone acetylation of Tip60 substrate H4K12, a learning-induced chromatin modification.These mechanisms point to an epigenetic role for Arc in regulating memory consolidation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School , Singapore 169857.

ABSTRACT
Arc is an immediate-early gene whose genetic ablation selectively abrogates long-term memory, indicating a critical role in memory consolidation. Although Arc protein is found at synapses, it also localizes to the neuronal nucleus, where its function is less understood. Nuclear Arc forms a complex with the β-spectrin isoform βSpIVΣ5 and associates with PML bodies, sites of epigenetic regulation of gene expression. We report here a novel interaction between Arc and Tip60, a histone-acetyltransferase and subunit of a chromatin-remodelling complex, using biochemistry and super-resolution microscopy in primary rat hippocampal neurons. Arc and βSpIVΣ5 are recruited to nuclear Tip60 speckles, and the three proteins form a tight complex that localizes to nuclear perichromatin regions, sites of transcriptional activity. Neuronal activity-induced expression of Arc (1) increases endogenous nuclear Tip60 puncta, (2) recruits Tip60 to PML bodies, and (3) increases histone acetylation of Tip60 substrate H4K12, a learning-induced chromatin modification. These mechanisms point to an epigenetic role for Arc in regulating memory consolidation.

No MeSH data available.


Related in: MedlinePlus