Activity-dependent human brain coding/noncoding gene regulatory networks

Abstract

While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The brain-derived neurotrophic factor (BDNF) gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long noncoding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and noncoding human transcription using a custom lncRNA microarray identified 1288 differentially expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional 8 were adjacent to or overlapping with differentially expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, composing an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF-BDNFOS co-expression that was not achieved with a single depolarization. RNAi-mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-messenger RNA pairs validated the effect of chronic neuronal activity on the transcriptome and implied various lncRNA regulatory mechanisms. lncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity.

Figure 1

Reciprocal pattern of BDNF and BDNFOS gene expression in electrically active human neocortex. (A) Summary of human epilepsy patients showing the ratios of electrical discharges, regions of neocortex sampled for each, and histopathology. All tissue sampled for gene expression changes had a normal histological structure, even in the presence of nearby structural abnormalities. (B) Long-term brain-surface recordings obtained prior to tissue resection were used to differentiate electrode locations with high- and low-spiking interictal activities for each patient. (C) While both the activity-dependent immediate early gene EGR1 and BDNF are constitutively upregulated in high-spiking cortex, BDNFOS was consistently downregulated in the same samples. The downregulation was significant (P = 0.016, Wilcoxon’s test; BDNFOS fold-change < −1.1, 95% C.I.). Bars represent average values for all seven patients shown with the color of the circles corresponding to the patients shown in A.

Figure 2

Downregulation of BDNFOS induces BDNF and LIN7C expression in SH-SY5Y cells. (A) The BDNFOS/BDNF gene locus shows antisense overlap between BDNF and BDNFOS (UCSC Genome Browser) (Kent 2002). Three siRNAs were generated from a non-overlapping BDNFOS exon (S1, S2, S3). (B) Downregulation of BDNFOS lncRNA using each of these siRNAs produced a corresponding increase in BDNF and, to a lesser extent, in LIN7C mRNA levels at 24 and 48 hr. Expression level changes are relative to a mock-electroporation negative control. Standard error bars are displayed. No further BDNFOS knockdown or BDNF rescue persisted at the 48-hr time point for s1 and s3 (data not shown).

Figure 3

Genome-wide analysis of human cortex reveals activity-dependent coding–noncoding gene pairs and stand-alone lncRNAs. (A) This experimental design of paired high- and low-spiking brain samples from the seven patients shown in Figure 1A was used to interrogate both coding and noncoding gene transcription as a function of brain activity. A flip-dye, quadruplicate microarray design was used with both a genome-wide coding array and a novel custom lncRNA array encompassing 5586 lncRNA genes with seven probes per gene. Based on a rigorous statistical cutoff, a total of 4044 protein-coding and 1288 lncRNA genes were identified for these seven patients (>1.4-fold; FDR <5% for each probe). lncRNA genes were further subdivided based on known cis-antisense partners of protein-coding genes, lncRNAs located <10 kb from any known gene, or stand-alone lncRNAs >10 kb from any known gene. Due to gene chains (Engström et al. 2006), some lncRNAs belonged simultaneously to the first two of these three categories. (B) Pairs of differentially expressed protein-coding and lncRNA genes that were either in an antisense overlap or <10-kb neighbors are shown together with stand-alone lncRNAs. Many of the affected protein-coding genes have known functions in synaptic plasticity. The green arrows to the left of the gene pairs have been validated by qPCR. *BDNFOS-BDNF was discovered by targeted qPCR and did not meet statistical significance on the microarrays.

Figure 4

Parallel patterns of activity-dependent coding and noncoding genes. As a means to identify activity-dependent lncRNAs with potential roles in synaptic plasticity, we probed the expression patterns of 13 known activity-dependent coding genes against the entire data set of lncRNAs for parallel patterns of expression. This figure shows all significant relationships between these 13 genes and 26 lncRNAs identified using an R > 0.90 cutoff. Each line represents a significant correlation and the proximity of the genes is directly proportional to this significance. The length of each line is inversely proportional to the correlation coefficient that is based on the average of correlations from probes above the 0.90 cutoff. The width of each line is directly proportional to the number of probes above the 0.90 cutoff. Coding genes are shown in blue while lncRNAs are pink. This figure was prepared using Cytoscape (http://www.cytoscape.org).

Figure 5

Repeated depolarization in vitro can replicate patterns of coding–noncoding gene transcription. (A) While transient CREB phosphorylation (top and middle) is induced with a single depolarization of Sy5Ycells with 100 mM KCl producing downregulation of BDNF and BDNFOS (bottom), (B) repeated depolarization produces more sustained CREB activation (top), accompanied by a clear oscillation of the pCREB:CREB ratio with pCREB maximum peaks clearly following each depolarization treatment (middle), and results in a reciprocal pattern of BDNF/BDNFOS transcription at 24 and 48 hr (shown by the brace) (bottom). *Reciprocal expression with upregulation of BDNF at 24 hr (P = 0.027) and at 48 hr (P = 0.019) and downregulation of BDNFOS at 24 ht (P = 0.004) and at 48 hr (P = 0.013) were observed. (A and B, middle panels) Quantification of triplicate Western blots. (A and B, bottom panels) Triplicate Taqman qRT-PCR results for EGR1 as a positive control for induced activity-dependent transcription, together with BDNF and BDNFOS at each time point. (C) The expression of three cis-encoded lncRNA–mRNA pairs and one known functional lncRNA (NEAT1) was examined in the same SY5Y repeated depolarization time course as in B, showing multiple distinct patterns of expression of both coding–noncoding pairs and the stand-alone lncRNA.