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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.


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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.
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f8: 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.

Mentions: To investigate the effect of Arc expression on endogenous Tip60 protein, hippocampal neurons were transiently transfected with Arc-YFP, fixed and stained with an anti-Tip60 antibody. Tip60 antibody staining in neuronal nuclei was mostly homogenous (Fig. 8A), with at most one or two detectable hotspots. However, Arc expression strongly induced the formation of bright endogenous Tip60 puncta in the nucleus (Fig. 8B,C). Figure 8 illustrates that Arc-positive nuclei contained more endogenous Tip60 puncta (∼10 per nucleus) than untransfected controls (0 − 2 per nucleus). Although some of the Tip60 puncta were seen to associate with Arc (Fig. 8B,C, insets), the two proteins did not overlap as closely as when they were both overexpressed (Fig. 2D). Coexpression of Arc-YFP and βSpIVΣ5-CFP in hippocampal neurons also induced formation of extra Tip60 hotspots and the Arc- βSpIVΣ5 complex still associated with Tip60 speckles (Fig. 8D). Our finding that Arc expression modulates the concentration of endogenous Tip60 protein in the nucleus suggests that Arc may be modulating the function of Tip60.


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 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4596143&req=5

f8: 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.
Mentions: To investigate the effect of Arc expression on endogenous Tip60 protein, hippocampal neurons were transiently transfected with Arc-YFP, fixed and stained with an anti-Tip60 antibody. Tip60 antibody staining in neuronal nuclei was mostly homogenous (Fig. 8A), with at most one or two detectable hotspots. However, Arc expression strongly induced the formation of bright endogenous Tip60 puncta in the nucleus (Fig. 8B,C). Figure 8 illustrates that Arc-positive nuclei contained more endogenous Tip60 puncta (∼10 per nucleus) than untransfected controls (0 − 2 per nucleus). Although some of the Tip60 puncta were seen to associate with Arc (Fig. 8B,C, insets), the two proteins did not overlap as closely as when they were both overexpressed (Fig. 2D). Coexpression of Arc-YFP and βSpIVΣ5-CFP in hippocampal neurons also induced formation of extra Tip60 hotspots and the Arc- βSpIVΣ5 complex still associated with Tip60 speckles (Fig. 8D). Our finding that Arc expression modulates the concentration of endogenous Tip60 protein in the nucleus suggests that Arc may be modulating the function of Tip60.

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