Abstract
Neurotrophin-regulated gene expression is believed to play a key role in long-term changes in synaptic structure and the formation of dendritic spines. Brain-derived neurotrophic factor (BDNF) has been shown to induce increases in dendritic spine formation, and this process is thought to function in part by stimulating CREB-dependent transcriptional changes. To identify CREB-regulated genes linked to BDNF-induced synaptogenesis, we profiled transcriptional occupancy of CREB in hippocampal neurons. Interestingly, de novo motif analysis of hippocampal ChIP-Seq data identified a non-canonical CRE motif (TGGCG) that was enriched at CREB target regions and conferred CREB-responsiveness. Because cytoskeletal remodeling is an essential element of the formation of dendritic spines, within our screens we focused our attention on genes previously identified as inhibitors of RhoA GTPase. Bioinformatic analyses identified dozens of candidate CREB target genes known to regulate synaptic architecture and function. We showed that two of these, the RhoA inhibitors Par6C (Pard6A) and Rnd3 (RhoE), are BDNF-induced CREB-regulated genes. Interestingly, CREB occupied a cluster of non-canonical CRE motifs in the Rnd3 promoter region. Lastly, we show that BDNF-stimulated synaptogenesis requires the expression of Par6C and Rnd3, and that overexpression of either protein is sufficient to increase synaptogenesis. Thus, we propose that BDNF can regulate formation of functional synapses by increasing the expression of the RhoA inhibitors, Par6C and Rnd3. This study shows that genome-wide analyses of CREB target genes can facilitate the discovery of new regulators of synaptogenesis.
Highlights
Most excitatory synapses in the mammalian brain are found on small, actin-rich protrusions of the dendritic membrane known as dendritic spines [1,2,3,4]
To identify CREB target sites in synaptically active neurons, we performed high-throughput sequencing of CREB chromatin immunoprecipitation (ChIP) material derived from mature cultured hippocampal neurons (Table S2)
CREB ChIP-Seq peaks were markedly enriched at the 59 end of known genes (Figure 1A, 1B, and 1D) and 29% were within 1 kb of an annotated RefSeq transcriptional start site (Table S2)
Summary
Most excitatory synapses in the mammalian brain are found on small, actin-rich protrusions of the dendritic membrane known as dendritic spines [1,2,3,4]. Long-term changes in spine morphogenesis often depend on de novo gene expression [26,27]. Activation of CREBdependent transcription has been linked to de novo and developmental synaptogenesis [25,28,29] Neurotrophic factors, such as brain derived neurotrophic factor (BDNF), are both activators of CREB-dependent transcription and regulators of synaptogenesis [30,31,32,33,34,35,36,37,38,39]. The effect of increased miR132 expression is implicated in regulation of the actin cytoskeleton, and it promotes changes in synaptic connectivity and stimulates dendritic spine formation [20,25,40]. We sought to identify additional CREB-regulated genes that contribute to BDNF-mediated synapse formation
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