Summary Enhancer RNAs (eRNAs) are a class of long noncoding RNAs (lncRNA) expressed from active enhancers, whose function and action mechanism are yet to be firmly established. Here we show that eRNAs facilitate the transition of paused RNA Polymerase II (RNAPII) into productive elongation by acting as a decoy for the negative elongation factor (NELF) complex upon induction of immediate early genes (IEGs) in neurons. eRNAs are synthesized prior to the culmination of target gene transcription and interact with the NELF complex. Knockdown of eRNAs expressed at neuronal enhancers impairs transient release of NELF from the specific target promoters during transcriptional activation, coinciding with a decrease in target mRNA induction. The enhancer-promoter interaction was unaffected by eRNA knockdown. Instead chromatin looping might enable eRNAs to act locally at a specific promoter. Our findings highlight the spatiotemporally regulated action mechanism of eRNAs during early transcriptional elongation.

The c-fos gene is induced by a broad range of stimuli, and has been commonly used as a reliable marker for neural activity. Its induction mechanism and available reporter mouse lines are exclusively based on the c-fos promoter activity. Here, we demonstrate that multiple enhancers surrounding the c-fos gene are critical for ensuring robust c-fos response to various stimuli. Membrane depolarization, brain-derived neurotrophic factor (BDNF), and Forskolin activate distinct subsets of the enhancers to induce c-fos transcription in neurons, suggesting that stimulus-specific combinatorial activation of multiple enhancers underlies the broad inducibility of the c-fos gene. Accordingly, the functional requirement of key transcription factors varies depending on the type of stimulation. Combinatorial enhancer activation also occurs in the brain. Providing a comprehensive picture of the c-fos induction mechanism beyond the minimal promoter, our study should help in understanding the physiological nature of c-fos induction in relation to neural activity and plasticity.

Summary Homeostatic scaling allows neurons to maintain stable activity patterns by globally altering their synaptic strength in response to changing activity levels. Suppression of activity by blocking action potentials increases synaptic strength through an upregulation of surface AMPA receptors. Although this synaptic up-scaling was shown to require transcription, the molecular nature of the intrinsic transcription program underlying this process and its functional significance have been unclear. Using RNA-seq, we identified 73 genes that were specifically upregulated in response to activity suppression. In particular, Neuronal pentraxin-1 (Nptx1) increased within 6 h of activity blockade, and knockdown of this gene blocked the increase in synaptic strength. Notably, Nptx1 induction is mediated by calcium influx through the T-type Voltage-Gated Calcium Channel, as well as two transcription factors, SRF and ELK1. Taken together, these results uncover a transcriptional program that specifically operates when neuronal activity is suppressed, to globally coordinate the increase in synaptic strength.

Synapse formation is the key step in the development of neuronal networks. Precise synaptic connections between nerve cells in the brain provide the basis of perception, learning, memory, and cognition. Recently, we have shown that the trans-synaptic interaction of postsynaptic glutamate receptor (GluR) d2 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum (Uemura et al. 2010). This finding raises a question whether GluRd1 regulates synapse formation by trans-synaptic interaction with NRXNs through Cbln subtypes in the forebrain since GluRd1 shares 56% amino acid identity with GluRd2 (Yamazaki et al. 1992;Araki et al. 1993;Lomeli et al. 1993). Previous studies showed that GluRd1 expressed in human embryonic kidney (HEK) 293T cells induced presynaptic differentiation of cultured cerebellar granule cells AbstractGlutamate receptor (GluR) d1 is widely expressed in the developing forebrain, whereas GluRd2 is selectively expressed in cerebellar Purkinje cells. Recently, we found that trans-synaptic interaction of postsynaptic GluRd2 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum. Thus, a question arises whether GluRd1 regulates synapse formation in the forebrain. In this study, we showed that the N-terminal domain of GluRd1 induced inhibitory presynaptic differentiation of some populations of cultured cortical neurons. When Cbln1 or Cbln2 was added to cultures, GluRd1 expressed in HEK293T cells induced preferentially inhibitory presynaptic differentiation of cultured cortical neurons. The synaptogenic activity of GluRd1 was suppressed by the addition of the extracellular domain of NRXN1a or NRXN1b containing splice segment 4. Cbln subtypes directly bound to the N-terminal domain of GluRd1. The synaptogenic activity of GluRd1 in the presence of Cbln subtypes correlated well with their binding affinities. When transfected to cortical neurons, GluRd1 stimulated inhibitory synapse formation in the presence of Cbln1 or Cbln2. These results together with differential interactions of Cbln subtypes with NRXN variants suggest that GluRd1 induces preferentially inhibitory presynaptic differentiation of cortical neurons by interacting with NRXNs containing splice segment 4 through Cbln subtypes.

The prolonged effects of N-methyl-D-aspartate (NMDA) receptor activation on the proliferation and differentiation of hippocampal neural progenitor cells (NPCs) were studied. Under conditions of mitogen-mediated proliferation, a single NMDA pulse (5 μM) increased the fraction of 5-bromo-2-deoxyuridine (BrdU)-positive (BrdU+) cells after a delay of 72 hours. Similarly, a single systemic injection of NMDA (100 mg/kg) increased the number of BrdU+ cells in the dentate gyrus (DG) after 28 days, but not after 3 days. NMDA receptor activation induced an immediate influx of Ca2+ into the NPCs and the NPCs expressed and released vascular endothelial growth factor (VEGF) in an NMDA receptor-dependent manner within 72 hours. With repetitive stimulation at the same dose, NMDA stimulated the acquisition of a neuronal phenotype accompanied by an increase in the expression of proneural basic helix-loop-helix (bHLH) factors. Together these findings suggest that neurogenesis in the developing brain is likely to be both directly and indirectly regulated by complex interactions between Ca2+ influx and excitation-releasable cytokines, even at mild levels of excitation. In addition, our results are the first to show that stimulation of NPCs may lead to either proliferation or neuronal differentiation, depending on the level of NMDA receptor activation.