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. 2009 Jan 6;106(1):316-21.
doi: 10.1073/pnas.0806518106. Epub 2008 Dec 30.

Synaptic Activity-Responsive Element in the Arc/Arg3.1 Promoter Essential for Synapse-To-Nucleus Signaling in Activated Neurons

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Free PMC article

Synaptic Activity-Responsive Element in the Arc/Arg3.1 Promoter Essential for Synapse-To-Nucleus Signaling in Activated Neurons

Takashi Kawashima et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The neuronal immediate early gene Arc/Arg-3.1 is widely used as one of the most reliable molecular markers for intense synaptic activity in vivo. However, the cis-acting elements responsible for such stringent activity dependence have not been firmly identified. Here we combined luciferase reporter assays in cultured cortical neurons and comparative genome mapping to identify the critical synaptic activity-responsive elements (SARE) of the Arc/Arg-3.1 gene. A major SARE was found as a unique approximately 100-bp element located at >5 kb upstream of the Arc/Arg-3.1 transcription initiation site in the mouse genome. This single element, when positioned immediately upstream of a minimal promoter, was necessary and sufficient to replicate crucial properties of endogenous Arc/Arg-3.1's transcriptional regulation, including rapid onset of transcription triggered by synaptic activity and low basal expression during synaptic inactivity. We identified the major determinants of SARE as a unique cluster of neuronal activity-dependent cis-regulatory elements consisting of closely localized binding sites for CREB, MEF2, and SRF. Consistently, a SARE reporter could readily trace and mark an ensemble of cells that have experienced intense activity in the recent past in vivo. Taken together, our work uncovers a novel transcriptional mechanism by which a critical 100-bp element, SARE, mediates a predominant component of the synapse-to-nucleus signaling in ensembles of Arc/Arg-3.1-positive activated neurons.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Multiple regulatory elements in the mouse Arc/Arg-3.1 promoter. (A) Time course of endogenous Arc/Arg-3.1 protein induction by 4AP/BIC stimulation. Cultured cortical neurons pretreated with TTX were stimulated with 4AP/BIC for indicated hours. (Scale bar, 100 μm.) (B) Evolutionarily conserved genomic regions in the upstream of the Arc/Arg-3.1 gene. Regions conserved between mice and humans are shown as white boxes. A destabilized luciferase (Luc-PEST) gene was used as a reporter. (C) Transcriptional regulatory activities of Arc1000–5000. Cultured cortical neurons were stimulated with 4AP/BIC for 4 h. Luciferase activities are normalized relative to the activity of Arc1000 under the TTX treatment. This normalization applies to all luciferase assay data in this study. Statistical analyses were performed separately for the TTX and 4AP/BIC data sets. n = 5 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the TTX or the 4AP/BIC value of Arc1000 (1-way ANOVA with Tukey's test). (D) Strong activation ability of Arc7000. The y axis for the basal levels were expanded and shown in the inset for clarity. n = 7 independent experiments. **, P < 0.01; ns, not significant (paired t test) compared with the 4AP/BIC or the TTX value of Arc4000. (E) Arc7000-driven luciferase correlates with endogenous Arc induction. Left, coexpression of luciferase and endogenous Arc protein after stimulation. (Scale bar, 50 μm.) Right, simultaneous quantification of luciferase activities and endogenous Arc protein levels in the same samples. Cell lysates were prepared from neurons with no treatment (normal medium), with TTX, or with 4AP/BIC for 4 h. Arc protein levels were quantified by Western blotting and plotted against the reporter luciferase activities. Duplicated samples were analyzed and shown.
Fig. 2.
Fig. 2.
The distal conserved region is crucial for the Arc7000 promoter activity. (A) Arc7000 deletion mutants. The genomic locus framed by a dashed box in Fig. 1B is expanded and shown. (B) Presence of a potent enhancer element between Arc7000-del no. 1 and Arc7000-del no. 2. n = 6 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the TTX or the 4AP/BIC value of Arc7000-del no. 1 (1-way ANOVA with Tukey's test). (C and D) Rapid and transient induction of luciferase luminescence by Arc7000 after high-frequency electrical stimulation. Hippocampal neurons transfected with a click beetle luciferase (ELuc) vector was electrically stimulated at 0 h (triangle), and luminescence in the soma was monitored. Representative images were shown in C. n = 9 neurons. (Scale bar, 50 μm.)
Fig. 3.
Fig. 3.
Arc/Arg-3.1 SARE replicates Arc7000 promoter activity. (A) Comparison across multiple mammalian species. Conserved sequences were highlighted in gray. A short stretch of sequences (thick line) consisted of 4 highly conserved boxes (boxes A–D) was termed a synaptic activity responsive element (SARE). (B) Dendrogram showing the divergence of SARE sequences across various mammalian species. The numbers represent the branch length, which indicates the degree of nucleotide differences. Detailed nucleotide information for this analysis is shown in Fig. S3. (C) SARE-ArcMin reporter vector. SARE was fused directly upstream of ArcMin, a TATA-containing sequence around the transcription initiation site of the Arc/Arg-3.1 gene. (D) SARE-ArcMin replicates the activation ability of Arc7000. n = 5 independent experiments. ***, P < 0.001; ns, not significant, compared with the TTX or the 4AP/BIC value of ArcMin (1-way ANOVA with Tukey's test).
Fig. 4.
Fig. 4.
Involvement of coclustered CREB, MEF2, and SRF in the SARE activation. (A) Loss of the SARE activity by mutations in the boxes B, C, and D, but not A, in the context of a minimal CMV promoter (SARE-minCMV). Note that the basal level (TTX) was elevated by a mutation in the box D (inset). WT, wild-type; ***, P < 0.001; ns, not significant, compared with the TTX or the 4AP/BIC value of WT (1-way ANOVA with Tukey's test). (B) Loss of the Arc7000 activity by mutations in the boxes B, C, and D. WT, wild-type; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, compared with the 4AP/BIC value of WT (1-way ANOVA with Tukey's test). (C) EMSA revealed binding of CREB, MEF2, and SRF to the boxes B, C, and D. Top, the matched nucleotides are indicated by dots. The evolutionarily conserved nucleotides are highlighted in gray. The mutated nucleotides are shown with white letters. Bottom, representative results of EMSA. The brain nuclear extracts were added with probes of the boxes B, C, and D. Specific DNA-protein complexes (arrows) were observed, which disappeared by adding excessive amounts of unlabeled competitors (Comp), but not by mutated competitors (Mut Comp). Antibodies against CREB, MEF2, and SRF disrupted or supershifted the complexes, whereas a control antibody (anti-Myc) had no effects. (D) Chromatin immunoprecipitation (ChIP) assays revealed physical binding of CREB, MEF2, and SRF to SARE in the genome. Target genomic sequences were amplified with specific primer sets by qPCR. Left, the genomic region adjacent to the SARE sequence (within 200 bp) was detected in the immunoprecipitates obtained using an anti-CREB antibody, while a negative control sequence 10 kb downstream of SARE was not. The Somatostatin promoter was used as a positive control. Middle, detection of SARE in the immunoprecipitates obtained using an anti-MEF2 antibody. The Nur77 promoter was used as a positive control. Right, detection of SARE in the immunoprecipitates obtained using an anti-SRF antibody. The β-actin promoter was used as a positive control.
Fig. 5.
Fig. 5.
Lentivirus-based genomic integration of the SARE reporter. (A) Design of lentiviral vectors. Top, the SARE reporter vector encodes an inducible reporter GFP (d2EGFP) under the control of SARE-ArcMin and a constitutively expressed infection marker RFP (TurboFP635) under the control of a pgk promoter. Bottom, a control vector lacking SARE. (B) Hippocampal neurons infected with the SARE reporter lentivirus were stimulated with BDNF for 6 h at 14 days postinfection. (Scale bar, 50 μm.) (C) GFP live-cell imaging of SARE-virus-infected neurons stimulated with high-frequency electrical pulses. Hippocampal neurons infected with the SARE reporter lentivirus were stimulated with field electrical pulses at time 0 h (triangle, 100 pulses at 100 Hz, 9 times), and GFP fluorescence was monitored. Gray lines, traces of individual neurons. Filled squares, the average trace of all neurons examined (n = 59). Open circles, the average trace of highly reactive neurons (top 10% of all neurons sorted by the F/F0 value at the time of 8 h, n = 6). (D) No GFP fluorescence changes were observed in control-virus-infected neurons. Filled squares, the average trace of all neurons examined (n = 18).
Fig. 6.
Fig. 6.
Visualization of activated neurons in vivo by the SARE viral vector. (A) Manipulation of neuronal activity in the mouse visual cortex. The virus-infected mice were sensory-deprived on 1 eye by suture, dark reared for 2–3 days, and exposed to light on the intact open eye. (B) Endogenous Arc/Arg-3.1 immunohistochemistry showing unilateral activation of the visual cortex. (Scale bar, 0.5 mm.) (C) The SARE reporter virus-infected neurons in layers 2/3 of the visual cortex. Activity-reporter GFP signals were detected in the contralateral side, but not in the ipsilateral side. (Scale bar, 20 μm.) (D) The percentages of GFP-positive neurons over RFP-positive neurons in each hemisphere. n = 6 mice for SARE-ArcMin, n = 5 mice for ArcMin. **, P < 0.01; ns, not significant, compared to the ipsilateral side (paired t test).

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