The analysis showed that SARE clusters were most abundant in intergenic and intronic regions (Fig. 1), potential areas for gene expression control. The list of SARE-containing genes showed genes with central roles in the nervous system such as NMDA (Grin2a, essential for excitatory synapses), Robo2 (with major functions in axon guidance) and Cutl/Cux2 (determinant for cerebral cortex layer II-IV) (examples in Table 1). Classification of genes containing SARE sequences at the GO categories using Toppfun application (http://toppgene.cchmc.org/“ToppFun”) indicated that the processes potentially affected by SARE regulation are clearly related to the nervous system (Table S2). This analysis yielded several enriched GO categories, out of 112 significantly enriched GO biological processes, 21 (18,75%) of them related to neural functions (Table 2). All of these categories are specifically related to nervous system development and maintenance, and many showed significant greater enrichment than other categories (Table 2 and Table S2). In accordance with our hypothesis, this prevalence of neural functions supports potentially important, selective action of SARE-mediated mechanisms in the nervous system. Next, SARE containing genes were grouped into two main categories representing potential distal and proximal regulatory sequences: intergenic; and intragenic, cds, promoter and utr regions (Table S2) and GO analysis was performed separately with these two groups. Both groups showed similar enrichment in neural functions; therefore it did not favor proximal or distal regulatory regions as more relevant to plasticity.

The analysis of genes containing the SARE cluster appeared an appropriate approach for identification of mechanisms of homeostasis, plasticity and activity-dependent remodeling in the nervous system. This study disclosed a large number of genes known to participate in plasticity and synaptogenesis (examples in Table 1); Homer1 is an example in this category. Homer genes encode scaffolding proteins that bind Ca²⁺ signaling proteins and target them to their correct subcellular localization [9], [10]; they are essential for dynamic regulation of the synapse, synaptic plasticity, and spatial learning [11], [12]. Coincident expression of experience-triggered Homer and Arc proteins is found in hippocampal and cortical neurons [13], which supports simultaneous activation, as predicted by our analysis. We also identified axonal guidance molecules (Table 1 and Table S1), including PlxnA4 and its ligand Sema6A [14] as molecules potentially regulated by SARE. The semaphorin and plexin receptor families, together with neuropilins, are crucial during nervous system development and homeostasis, and mark the pathway for axon growth [14]. These proteins also control synaptogenesis, axon pruning, the density and maturation of dendritic spines and are implicated in a number of developmental, psychiatric and neurodegenerative disorders [15]. As for the axon guidance cues, we found a number of genes that encode cytoskeletal remodeling molecules at the synapse (Table 1). For example, ankyrins link integral membrane proteins to the underlying spectrin-actin cytoskeleton; they have key roles in activities such as cell motility, activation, proliferation, contact, and maintenance of specialized membrane domains. They might be involved in bipolar disorder and other mental alterations [16].

Less anticipated were SARE-containing genes not previously implicated in plasticity or structural maintenance of the synapse; in this category, we found neuronal subtype-specific TF such as Cux1 and Cux2 [17], Zic2 [18] and Sox6 [19], [20] (Table 1 and Table S1). Cux TF expression is restricted to neurons of layers II-III and IV of the cerebral cortex. During development, Cux regulate dendritic branching, spine morphogenesis and synapse maturation [17]. Cux expression is maintained through adulthood, but nothing is yet known of their function in mature neurons. Whereas Cux functions could be associated with plasticity at the postsynaptic site [17], Zic2 might act on the presynaptic terminal, as it is associated with axon development in retinal ganglion cells; Sox6, in turn, is described as essential for neuronal differentiation [19], [20]. These observations suggest that activity-dependent mechanisms act on pathways specific to neuronal subtypes.

To test the relevance of our findings and the predictive capability of our gene set to identify genes up-regulated upon neuronal activation we searched for experimental confirmation. Several studies of gene expression changes induced by neuronal activation have been reported and made useful available contributions. Many of them analyze the effects of the gabaergic inhibitor bicuculline to trigger neuronal excitatory response. We therefore compare the list of SARE containing genes with those of genes which expression was modify in studies analyzing the in vivo effects of infusion of bicuculline into the accessory olfactory bulb [21]; in vitro bicuculline treatment of cortical cells [22]; and hippocampal neuronal cultures [23]. This allowed us to extend our validation to several neuronal types. In all three cases, the comparison revealed a highly significant enrichment between the SARE containing genes and those up-regulated upon neuronal activation, but none or of lower statistical significance, when compared to the list of genes that are down-regulated (Table 3). Several of the SARE genes, such as Homer, Atf3, Klf6 and Bdnf are common to two or the three studies, and may represent a general pan-neuronal response, while unique ones might represent tissue-specific responses. These significant overlapping validate our results with external independent data. We next took the reverse approach and tested the predictive capability of our study by testing the expression of genes picked from our list upon neuronal activation. Cells from E18 mouse cortex were dissociated, neurons were cultivated and neuronal activity triggered using bicuculline [7]. RNA was obtained and transcript expression of twelve SARE containing genes, including Arc, analyzed by using quantitative real time RT-PCR (Q-PCR) (Fig. 2A). Up-regulation of Arc gene demonstrated efficient neuronal activation and, also expected, the levels of the S-isoform of Homer1 were increased [24], [25]. Six more genes showed up-regulation when neurons were activated. Atf3, Impdh2, and Npas4 up-regulation in cortical cells was in agreement with our own analysis of the raw data obtained from gene expression arrays reported by other investigators [22], and further confirmed our comparison with external sources (Table 3). Interestingly, up-regulation of Cux1, Cux2, and PlxnA4, genes not suspected to be regulated by activity, again confirmed the predictive capacity of our study. Four genes, Lmo4, Robo1, Robo2 and Klf6 did not show significant changes. This can be ascribed to the almost certain possibility of a number of false positive in our list, to the fact that other splicing variants might be affected, or to the possibility that subsets of genes may respond differently depending on the stimulus that triggers neuronal response.

Next, the sequence corresponding to two of these SARE sequences were cloned upstream of a minimal promoter into vectors containing luciferase reporters to test their ability to activate transcription in response to neuronal activity. Cells from E18 mouse cortex were transfected with reporter constructs, neuronal activity triggered using bicuculline [7], and luciferase activity compared to control tetrodotoxin (TTX) treated neurons. These experiments demonstrated that these novel identified SARE sequences replicate the promoter activity of the SARE sequence corresponding to the Arc gene and significantly increase transcription upon depolarization (Fig. 2B).

Our analyses thus point to overlooked pathways that might participate in activity-dependent regulatory mechanisms and, by extension, suggests the identification of genes potentially linked to mental diseases caused by plasticity defects [26], [27], [28]. This is the case of genes reported as candidates for autism in which we found SARE sequences, such as, NRXN1 and 2 [29], FOXP1 [30], FOXP2 [30], [31], GRID2 [32], KCNMA1 [33] and others (http://gene.sfari.org) [34] (see Table S1).

Validation of our prediction nonetheless required evaluation of true enrichment of genes involved in cognitive dysfunction. Fragile X syndrome (FXS) is a well-characterized form of autism, caused by loss of function of the Fragile X mental retardation protein (FMRP), which regulates local translation and plasticity at pre- and postsynaptic sites [28], [35]. Based on a recent extensive list of genes targeted by FMRP, from which the authors extract a stringent set of 842 reliable targets [36], we hypothesized that the list of SARE-regulated genes will be enriched in FMRP targets. Comparison of SARE-containing genes (including those containing SARE clusters at intergenic locations) with the stringent list of FMRP targets resulted in 70 genes common to both (8.5% overlap), an enrichment of biological relevance (p=4.3909–13; see Methods) (Table 4). The relationship between the SARE-containing genes and FMRP targets thus strongly supports SARE involvement in activity-dependent regulation. In addition, it suggests that mutations in SARE or SARE-containing genes and pathways can contribute to mental retardation, autism spectrum disorders and other psychiatric diseases.

Table S1

Table S2