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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.


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.
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f6: 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.

Mentions: The finding that nuclear Arc colocalizes with and physically binds to Tip60 was novel and therefore prompted a more careful look into the nature of the endogenous protein−protein interactions. To this end, we employed two differing yet complementary super-resolution microscopy approaches, 3D-stimulated emission depletion microscopy (3D-STED) and 3D-stochastic optical reconstruction microscopy (3D-STORM), which push the boundaries of the resolution limit of light microscopy. Upon stimulation of network activity by a combination of 4AP, bicuculline, and forskolin (4AP−Bic−Fors, see Materials and Methods), a subset of neurons expressed endogenous Arc, which we immunostained using an antibody recognizing Arc protein. At resolutions narrowly eclipsing 100 nm, 3D-STED revealed that in these activated neuronal nuclei, the distribution of Tip60 protein highly paralleled that of Arc (Fig. 6, top). The two proteins abutted each other in many distinct conformations (Fig. 6, bottom). In order to definitively delineate this interaction, we proceeded to use 3D-STORM, which has the capability of imaging single molecules, and observed that in support of the results obtained through wide-field and STED microscopy, molecules of Arc and Tip60 do indeed interact in the activated neuronal nucleus with unforeseen clarity (Fig. 7).


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)

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

License
Show All Figures
getmorefigures.php?uid=PMC4596143&req=5

f6: 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.
Mentions: The finding that nuclear Arc colocalizes with and physically binds to Tip60 was novel and therefore prompted a more careful look into the nature of the endogenous protein−protein interactions. To this end, we employed two differing yet complementary super-resolution microscopy approaches, 3D-stimulated emission depletion microscopy (3D-STED) and 3D-stochastic optical reconstruction microscopy (3D-STORM), which push the boundaries of the resolution limit of light microscopy. Upon stimulation of network activity by a combination of 4AP, bicuculline, and forskolin (4AP−Bic−Fors, see Materials and Methods), a subset of neurons expressed endogenous Arc, which we immunostained using an antibody recognizing Arc protein. At resolutions narrowly eclipsing 100 nm, 3D-STED revealed that in these activated neuronal nuclei, the distribution of Tip60 protein highly paralleled that of Arc (Fig. 6, top). The two proteins abutted each other in many distinct conformations (Fig. 6, bottom). In order to definitively delineate this interaction, we proceeded to use 3D-STORM, which has the capability of imaging single molecules, and observed that in support of the results obtained through wide-field and STED microscopy, molecules of Arc and Tip60 do indeed interact in the activated neuronal nucleus with unforeseen clarity (Fig. 7).

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.