An Emerging Role for Enhancer RNAs in Brain Disorders.

Noncoding DNA undergoes widespread context-dependent transcription to produce noncoding RNAs. In recent decades, tremendous advances in genomics and transcriptomics have revealed important regulatory roles for noncoding DNA elements and the RNAs that they produce. Enhancers are one such element that are well-established drivers of gene expression changes in response to a variety of factors such as external stimuli, cellular responses, developmental cues, and disease states. They are known to act at long distances, interact with multiple target gene loci simultaneously, synergize with other enhancers, and associate with dynamic chromatin architectures to form a complex regulatory network. Recent advances in enhancer biology have revealed that upon activation, enhancers transcribe long noncoding RNAs, known as enhancer RNAs (eRNAs), that have been shown to play important roles in enhancer-mediated gene regulation and chromatin-modifying activities. In the brain, enhancer dysregulation and eRNA transcription has been reported in numerous disorders from acute injuries to chronic neurodegeneration. Because this is an emerging area, a comprehensive understanding of eRNA function has not yet been achieved in brain disorders; however, the findings to date have illuminated a role for eRNAs in activity-driven gene expression and phenotypic outcomes. In this review, we highlight the breadth of the current literature on eRNA biology in brain health and disease and discuss the challenges as well as focus areas and strategies for future in-depth research on eRNAs in brain health and disease.

Sex-based eRNA expression and function in ischemic stroke.

Enhancer-derived RNAs (eRNAs) are a new class of long noncoding RNA that have roles in modulating enhancer-mediated gene transcription, which ultimately influences phenotypic outcomes. We recently published the first study mapping genome-wide eRNA expression in the male mouse cortex during ischemic stroke and identified 77 eRNAs that were significantly altered following a 1 h middle cerebral artery occlusion (MCAO) and 6 h of reperfusion, as compared to sham controls. Knockdown of one such stroke-induced eRNA - eRNA_06347 - resulted in significantly larger infarcts, demonstrating a role for eRNA_06347 in modulating the post-stroke pathophysiology in males. In the current study, we applied quantitative real-time PCR to evaluate whether the 77 eRNAs identified in the male cortex also show altered expression in the post-stroke female cortex. Using age-matched and time-matched female mice, we found that only a subset of the 77 eRNAs were detected in the post-stroke female cortex. Of these, only a small fraction showed similar temporal expression characteristics as males, including eRNA_06347 which was highly induced in both sexes. Knockdown of eRNA_06347 in the female cortex resulted in significantly increased infarct volumes that were closely matched to those in males, indicating that eRNA_06347 modulates the post-stroke pathophysiology similarly in males and females. This suggests a common underlying role for eRNA_06347 in the two sexes. Overall, this is the first study to evaluate eRNA expression and perturbation in the female cortex during stroke, and present a comparative analysis between males and females. Our findings show that eRNAs have sex-dependent and sex-independent expression patterns that may be of significance to the pathophysiological responses to stroke in the two sexes.

Modulation of Brain Pathology by Enhancer RNAs in Cerebral Ischemia.

Recent studies have reported widespread stimulus-dependent transcription of mammalian enhancers into noncoding enhancer RNAs (eRNAs), some of which have central roles in the enhancer-mediated induction of target genes and modulation of phenotypic outcomes during development and disease. In cerebral ischemia, the expression and functions of eRNAs are virtually unknown. Here, we applied genome-wide H3K27ac ChIP-seq and genome-wide RNA-seq to identify enhancer elements and stroke-induced eRNAs, respectively, in the mouse cerebral cortex during transient focal ischemia. Following a 1-h middle cerebral artery occlusion (MCAO) and 6 h of reperfusion, we identified 77 eRNAs that were significantly upregulated in stroke as compared to sham, of which 55 were exclusively expressed in stroke. The knockdown of two stroke-induced eRNAs in the mouse brain resulted in significantly larger infarct volumes as compared to controls, suggesting that these eRNAs are involved in the post-stroke neuroprotective response. A preliminary comparison of eRNA expression in the male versus female cortices revealed sex-dependent patterns that may underlie the physiological differences in response to stroke between the two sexes. Together, this study is the first to illuminate the eRNA landscape in the post-stroke cortex and demonstrate the significance of an eRNA in modulating post-stroke cortical brain damage.

Long Noncoding RNAs in the Pathophysiology of Ischemic Stroke.

Ischemic stroke is an acute brain injury with high mortality and disability rates worldwide. The pathophysiological effects of ischemic stroke are driven by a multitude of complex molecular and cellular interactions that ultimately result in brain damage and neurological dysfunction. The Human Genome Project revealed that the vast majority of the human genome (and mammalian genome in general) is transcribed into noncoding RNAs. These RNAs have several important roles in the molecular biology of the cell. Of these, the long noncoding RNAs are gaining particular importance in stroke biology. High-throughput analysis of gene expression using methodologies such as RNA-seq and microarrays have identified a number of aberrantly expressed lncRNAs in the post-stroke brain and blood in experimental models as well as in clinical samples. These expression changes exhibited distinct temporal and cell-type-dependent patterns. Many of these lncRNAs were shown to modulate molecular pathways that resulted in deleterious as well as neuroprotective outcomes in the post-stroke brain. In this review, we consolidate the latest data from the literature that elucidate the roles and functions of lncRNAs in ischemic stroke. We also summarize clinical studies identifying differential lncRNA expression changes between stroke patients and healthy individuals, and genetic variations in lncRNA loci that are correlated with an increased risk of stroke development.

Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

Discovery of novel stroke-responsive lncRNAs in the mouse cortex using genome-wide RNA-seq.

Long noncoding RNAs (lncRNAs) play major roles in regulating gene expression in mammals, but are poorly understood in ischemic stroke. Using a mouse model of transient focal ischemia, we applied RNA-seq to evaluate for the first time the unbiased, genome-wide expression of lncRNAs as a function of reperfusion time in the cerebral cortex. Focal ischemia was induced in adult male C57BL/6 mice followed by reperfusion for 6, 12 or 24h. Total RNA from ipsilateral cortices was used for Illumina sequencing and reads were mapped to the mouse reference genome (GRCm38). Annotated and novel transcript isoforms were identified and differential expression between the groups was estimated. We observed that the baseline expression of lncRNAs in the healthy cortex was low, but many were highly altered after stroke. Very few of these altered lncRNAs were previously annotated. A total of 259 lncRNA isoforms at 6h, 378 isoforms at 12h, and 217 isoforms at 24h of reperfusion were differentially expressed versus sham controls. Of these, 213, 322 and 171 isoforms at 6, 12 and 24h of reperfusion, respectively, were novel lncRNAs. Reperfusion time-point-specific analyses revealed that the lncRNAs reached peak expression levels at 6h of reperfusion. Positional analysis of ischemia-responsive lncRNAs with respect to ischemia-responsive protein-coding genes identified potential gene-regulatory relationships. Overall, this work shows that transient focal ischemia induces widespread changes in the expression of lncRNAs in the mouse cortex with distinct reperfusion time-point-dependent expression characteristics that may underlie progression of the ischemic pathophysiology. The detection of hundreds of novel ischemia-responsive lncRNAs marks the discovery of new disease-related genomic regions in the adult cortex and may help identify novel opportunities for therapeutic targeting.

Mutual induction of transcription factor PPARγ and microRNAs miR-145 and miR-329.

MicroRNAs (miRNAs) are small non-coding RNAs that are known to control mRNA translation. Most miRNAs are transcribed from specific genes with well-defined promoters located throughout the genome. The mechanisms that control miRNA expression under normal and pathological conditions are not yet understood clearly. Peroxisome proliferator-activated receptor (PPAR) γ is a ligand-activated transcription factor that is extensively distributed in the CNS. PPARγ activation induces neuroprotection by modulating genes that contain peroxisome proliferator response elements (PPREs) in their promoters. We presently evaluated if PPARγ modulates miRNA expression. When adult rats were treated with PPARγ agonist rosiglitazone, expression of 28 miRNAs altered significantly (12 up- and 16 down-regulated; 3-119 fold) in the cerebral cortex compared to vehicle-treated controls. In silico analysis showed 1-5 PPREs in the putative promoter regions (within 1 Kb upstream of the transcription start site) of these miRNA genes. Cotransfection with a PPARγ constitutively expressing vector significantly induced the miR-145 and miR-329 promoter vectors (each have four PPREs), which was curtailed by point mutations of PPREs in their promoters. Interestingly, the PPARγ promoter has binding sites for both these miRNAs and transfection with miR-329 mimic and miR-145 mimic induced the PPARγ expression. Thus, these studies show a cyclical induction of miRNAs and PPARγ, indicating that the pleiotropic beneficial effects of PPARγ agonists might be modulated in part by miRNAs and their down-stream mRNAs. We proposed that promoters of many microRNAs contain the binding sites for the transcription factor PPARγ. Activation of PPARγ modulates the expression of these microRNAs. Two such PPARγ-responsive microRNAs (miR-145 and miR-329) bind to PPARγ promoter to induce its expression. This indicates the presence of a feedback loop by which transcription factors and microRNAs can modulate each other.

Expression of transcribed ultraconserved regions of genome in rat cerebral cortex.

Emerging evidence indicates that 481 regions of the genome (>200 bp) that actively transcribe noncoding RNAs shows 100% homology between humans, rats and mice. These transcribed ultraconserved regions (T-UCRs) are thought to control the essential regulatory functions basic for life in rodents and mammals. Using microarray analysis, we presently show that 107 T-UCRs are actively expressed in adult rat cerebral cortex. They are grouped into intragenic (61) and intergenic (46) based on their genic location. Interestingly, 10 T-UCRs are expressed at unusually high levels in cerebral cortex. Additionally, many T-UCRs also showed cogenic expression. We further analyzed the correlation of intragenic T-UCRs with their host protein coding genes. Surprisingly, most of the expressed intragenic T-UCRs (54 out of 61) displayed a negative correlation with their host gene expression. T-UCRs are thought to control the splicing and transcription of the protein-coding genes that host them and flank them. Bioinformatics analysis indicated that the protein products of majority of these genes are nuclear in localization, share protein domains and are involved in the regulation of diverse biological and molecular functions including metabolism, development, cell cycle, binding and transcription factor regulation. In conclusion, this is the first study to shows that many T-UCRs are expressed in rodent brain and they might play a role in physiological brain functions.

MicroRNA miR-324-3p induces promoter-mediated expression of RelA gene.

MicroRNAs (miRNAs) are known to repress translation by binding to the 3'UTRs of mRNAs. Using bioinformatics, we recently reported that several miRNAs also have target sites in DNA particularly in the promoters of the protein-coding genes. To understand the functional significance of this phenomenon, we tested the effects of miR-324-3p binding to RelA promoter. In PC12 cells, co-transfection with premiR-324-3p induced a RelA promoter plasmid in a dose-dependent manner and this effect was lost when the miR-324-3p binding site in the promoter was mutated. PremiR-324-3p transfection also significantly induced the endogenous RelA mRNA and protein expression in PC12 cells. Furthermore, transfection with premiR-324-3p increased the levels of cleaved caspase-3 which is a marker of apoptosis. Importantly, the miR-324-3p effects were Ago2 mediated as Ago2 knockdown prevented RelA expression and cleavage of caspase-3. Thus, our studies show that miRNA-mediated transcriptional activation can be seen in PC12 cells which are neural in origin.

Increased binding of stroke-induced long non-coding RNAs to the transcriptional corepressors Sin3A and coREST.

LncRNAs (long non-coding RNAs) are thought to play a significant role in cellular homeostasis during development and disease by interacting with CMPs (chromatin-modifying proteins). We recently showed that following transient focal ischemia, the expression of many lncRNAs was altered significantly in rat brain. We currently analyzed whether focal ischemia also alters the association of lncRNAs with the CMPs Sin3A and coREST (corepressors of the RE-1 silencing transcription factor). RIP (RNA immunoprecipitation) combined with lncRNA microarray analysis showed that 177 of the 2497 lncRNAs expressed in rat cerebral cortex showed significantly increased binding to either Sin3A or coREST following ischemia compared with sham. Of these, 26 lncRNAs enriched with Sin3A and 11 lncRNAs enriched with coREST were also up-regulated in their expressions after ischemia. A majority of the lncRNAs enriched with these CMPs were intergenic in origin. Evaluation of the expression profiles of corresponding protein-coding genes showed that their expression levels correlate with those of the lncRNAs with which they shared a common locus. This is the first study to show that stroke-induced lncRNAs might associate with CMPs to modulate the post-ischemic epigenetic landscape.

3S: shotgun secondary structure determination of long non-coding RNAs.

Long non-coding RNAs (lncRNAs) have emerged as an important class of RNAs playing key roles in development, disease and epigenetics. Knowledge of lncRNA structure may be critical in understanding function for many lncRNA systems. Due to the enormous number of possible folds for these sequences, secondary structure determination presents a significant challenge, both experimentally and computationally. Here, we present a new strategy capable of determining the RNA secondary structure in the wet lab without significant reliance on computational predictions. First, we chemically probe the entire lncRNA. Next, using a shotgun approach, we divide the RNA into overlapping fragments and probe these fragments. We then compare probing profiles of fragments with the profiles of the full RNA and identify similarities. Sequence regions with profiles that are similar in the fragment and full-length transcript possess only base pairing partners within the fragment. Thus, by experimentally folding smaller and smaller fragments of the full RNA and probing these chemically, we are able to isolate modular sub-domains, dramatically reducing the number of possible folds. The method also eliminates the possibility of pseudoknots within a modular sub-domain. The 3S technique is ideally suited for lncRNAs because it is designed for long RNA sequences. The 3S-determined secondary structure of a specific lncRNA in one species (e.g., human) enables searches for instances of the same lncRNA in other species.

MicroRNA miR-29c down-regulation leading to de-repression of its target DNA methyltransferase 3a promotes ischemic brain damage.

Recent studies showed that stroke extensively alters cerebral microRNA (miRNA) expression profiles and several miRNAs play a role in mediating ischemic pathophysiology. We currently evaluated the significance of miR-29c, a highly expressed miRNA in rodent brain that was significantly down-regulated after focal ischemia in adult rats as well as after oxygen-glucose deprivation in PC12 cells. Bioinformatics indicated that DNA methyltransferase 3a (DNMT3a) is a major target of miR-29c and co-transfection with premiR-29c prevented DNMT3a 3'UTR vector expression. In PC12 cells, treatment with premiR-29c prevented OGD-induced cell death (by 58 ± 6%; p<0.05). Furthermore, treatment with antagomiR-29c resulted in a 46 ± 5% cell death in PC12 cells. When rats were treated with premiR-29c and subjected to transient focal ischemia, post-ischemic miR-29c levels were restored and the infarct volume decreased significantly (by 34 ± 6%; p<0.05) compared to control premiR treated group. DNMT3a siRNA treatment also significantly curtailed the post-OGD cell death in PC12 cells (by 54 ± 6%; p<0.05) and decreased the post-ischemic infarct volume in rats (by 30 ± 5%; p<0.05) compared to respective control siRNA treated groups. The miR-29c gene promoter showed specific binding sites for the transcription factor REST and the miR-29c promoter vector expression was curtailed when cotransfected with a REST expressing plasmid. Furthermore, treatment with REST siRNA prevented the post-ischemic miR-29c down-regulation and DNMT3a induction in PC12 cells and curtailed ischemic cell death (by 64 ± 9%; p<0.05) compared to control siRNA treatment. These studies suggest that miR-29c is a pro-survival miRNA and its down-regulation is a promoter of ischemic brain damage by acting through its target DNMT3a. Furthermore, REST is an upstream transcriptional controller of miR-29c and curtailing REST induction prevents miR-29c down-regulation and ischemic neuronal death.

Effect of focal ischemia on long noncoding RNAs.

BACKGROUND AND PURPOSE: Long noncoding RNAs (lncRNAs) play a significant role in cellular physiology. We evaluated the effect of focal ischemia on the expression of 8314 lncRNAs in rat cerebral cortex using microarrays. METHODS: Ischemia was induced by transient middle cerebral artery occlusion. Genomic and transcriptomic correlates of the stroke-responsive lncRNAs and the transcription factor binding sties in their promoters were evaluated with bioinformatics. RESULTS: Three hundred fifty-nine lncRNAs were upregulated (>2-fold) and 84 were downregulated (<0.5-fold) at 3 hours to 12 hours of reperfusion after middle cerebral artery occlusion compared with sham. Sixty-two stroke-responsive lncRNAs showed >90% sequence homology with exons of protein-coding genes. Promoters of stroke-responsive lncRNA genes and their homologous protein-coding genes showed highly overlapping transcription factor binding sites. Despite presence of open reading frames, lncRNAs did not form any product when subjected to in vitro translation. CONCLUSIONS: Stroke significantly alters cerebral lncRNA expression profiles.

Altered expression of PIWI RNA in the rat brain after transient focal ischemia.

BACKGROUND AND PURPOSE: The PIWI-interacting RNA (piRNA) is the most predominant RNA species in eukaryotes. The piRNA are a class of noncoding RNAs that bind and degrade the RNA formed by the transposons to control the transposon-induced gene mutations. The role of piRNA after focal ischemia is not yet evaluated. METHODS: We profiled 39 727 piRNAs in the cerebral cortex of adult rats subjected to transient focal ischemia using microarrays. The RT targets of stroke-responsive piRNAs were identified with bioinformatics. To understand how piRNAs are controlled, we analyzed the transcription factor binding sites in the putative promoters of 10 representative stroke-responsive piRNAs. RESULTS: In the ipsilateral cortex of ischemic rats, 105 piRNAs showed altered expression (54 up- and 51 downregulated; >2.5-fold) compared with shams. Twenty-five of those showed a >5-fold change. A bioinformatics search showed that the transposon targets of the highly stroke-responsive piRNAs are distributed among the 20 autosomal chromosomes and there is a redundancy in the targets between the piRNAs. Furthermore, the transposon targets were observed to be highly repetitious for each piRNA across the chromosome length. Of the 159 transcription factors observed to have binding sites in the piRNA gene promoters, 59% belonged to 20 major families indicating that transcription factors control stroke-responsive piRNAs in a redundant manner. CONCLUSIONS: The present study is the first to show that many piRNAs are expressed in adult rodent brain and several of them respond to focal ischemia.

Ischemic pre-conditioning alters cerebral microRNAs that are upstream to neuroprotective signaling pathways.

Cerebral gene expression is known to be significantly influenced by a sublethal ischemic event (pre-conditioning; PC) that induces tolerance to future damaging ischemic events. Small non-coding RNAs known as microRNAs (miRNAs) were recently shown to control the mRNA translation. We currently profiled cerebral miRNAs in the cerebral cortex of rats subjected to PC. The miRNAome reacted quickly and by 6 h following PC, levels of 51 miRNAs were altered (26 up- and 25 down-regulated; > 1.5-fold change). Twenty of these stayed at the altered level even at 3 days after PC. At least nine miRNAs showed > 5-fold change at one or more time points between 6 h to 3 days after PC compared with sham. Bioinformatics analysis showed 2007 common targets of the miRNAs that were up-regulated and 459 common targets of the miRNAs that were down-regulated after PC. Pathways analysis showed that MAP-kinase and Mammalian target of rapamycin (mTOR) signaling are the top two Kyoto Encyclopedia of Genes and Genomes pathways targeted by the up-regulated miRNAs, and Wnt and GnRH signaling are the top two Kyoto Encyclopedia of Genes and Genomes pathways targeted by the down-regulated miRNAs after PC. We hypothesize that alterations in miRNAs and their down-stream mRNAs of signaling pathways might play a role in the induction of ischemic tolerance.