Abstract
Here we demonstrate that natural antisense transcripts (NATs), which are abundant in mammalian genomes, can function as repressors of specific genomic loci and that their removal or inhibition by AntagoNAT oligonucleotides leads to transient and reversible upregulation of sense gene expression. As one example, we show that Brain-Derived Neurotrophic Factor (BDNF) is under the control of a conserved noncoding antisense RNA transcript, BDNF-AS, both in vitro and in vivo. BDNF-AS tonically represses BDNF sense RNA transcription by altering chromatin structure at the BDNF locus, which in turn reduces endogenous BDNF protein and function. By providing additional and analogous examples of endogenous mRNA upregulation, we suggest that antisense RNA mediated transcriptional suppression is a common phenomenon. In sum, we demonstrate a novel pharmacological strategy by which endogenous gene expression can be upregulated in a locus-specific manner.
Introduction
Although RNA interference and other technologies have provided useful tools for downregulation of mRNA and subsequently proteins, specific upregulation of individual genes remains challenging. Natural antisense transcripts (NATs) are transcribed from the opposite strand of many protein-coding (sense) genes and overlap in part with sense RNA, promoter region and their regulatory regions12. Here, we demonstrate a potent mechanism by which endogenous NATs suppress transcription of their sense gene counterparts. We show that endogenous gene expression can be upregulated, in a locus-specific manner by the removal or inhibition of the NATs, which are transcribed from most transcriptional units1,3. Our study provides examples of functional ncRNAs that regulate protein output, by altering chromatin structure and we posit that this phenomenon is applicable to many other genomic loci.
Brain-derived Neurotrophic Factor (BDNF) is a member of the "neurotrophin" family of growth factors, essential for neuronal growth, maturation4,5, differentiation and maintenance6. BDNF is also essential for neuronal plasticity and shown to be involved in learning, and memory processes7. The BDNF locus is on chromosome 11 and shows active transcription from both strands, which leads to transcription of a noncoding NATs8. Here, we characterize the regulatory role of this antisense RNA molecule, BDNF-AS that exerts a potent reciprocal and dynamic regulation over the expression of sense BDNF mRNA and protein, both in vitro and in vivo. We also report similar upregulation of Glial-derived Neurotrophic Factor (GDNF) and Ephrin receptor B2 (EphB2), by blocking endogenous NATs. We introduced a novel strategy for upregulation of mRNA expression, using antisense RNA transcript inhibitory molecules, which we term AntagoNATs.
Results
Genomic organization of the human BDNF locus
BDNF mRNA as well as BDNF antisense RNA (BDNF-AS, also annotated as BDNF-OS) displays a complex splicing pattern; however, all variants share a common sense-antisense overlapping region8,9. The transcription start site (TSS) of human BDNF-AS is approximately 200 kb downstream from the BDNF promoter and it is located on the positive strand of chromosome-11. Transcription from this site gives rise to 16–25 splice variant long ncRNAs with 6–8 exons8. Exon-5 of BDNF-AS, which contain 225-nucleotides of full complementarity to BDNF mRNA (overlapping) and exon-4 (non-overlapping) are common between all these variants (Fig. 1a). Nucleotide sequence of human BDNF-AS is provided in supplementary data file 1. BDNF mRNA is transcribed from the negative strand of chromosome-11 and shows 11 alternative splicing patterns and one coding exon. All variants of BDNF mRNA also share the 225-nucleotide overlapping region with the BDNF-AS transcript. Our next generation sequencing (deep sequencing) data confirms expression of BDNF-AS transcript in human brain RNA samples (Fig. 1a inset data). Therefore, BDNF-AS has the potential to form an in vivo RNA-RNA duplex with BDNF mRNA through 225 complementary nucleotides overlap.
Identification of mouse Bdnf-AS transcript
Although several human EST’s have been reported to have the potential of forming sense-antisense pairs with BDNF transcripts8, the mouse antisense transcript was not previously identified and thus BDNF-AS was erroneously reported as a primate-specific transcript by others9. Using 5’ and 3’ Rapid amplification of cDNA ends (RACE) experiments; we identified the mouse Bdnf-AS transcript (Fig. 1b). Based on RACE data we designed primers and probes for detection of mouse Bdnf-AS by real-time PCR (RT-PCR) experiments. RACE experiments followed by sequencing and RT-PCR indicate the existence of a conserved noncoding antisense transcript to the mouse Bdnf mRNA. The mouse Bdnf-AS transcript has two splice variants with 1–2 exons and 934-nucleotide complementarity to Bdnf mRNA (Fig. 1b). Nucleotide sequence of mouse Bdnf-AS is provided in Supplementary data file-2. Although the architectures of BDNF-AS in human and mouse are not entirely similar, the 225 bp overlapping region is almost identical between these two species (90% homology across the two species), suggesting the presence of an evolutionarily conserved functional binding domain.
Expression analysis of BDNF and BDNF-AS
We assessed expression of BDNF and BDNF-AS transcripts in various human (adult and embryonic), as well as monkey and mouse tissues, by RT-PCR and RNA fluorescence in situ hybridization (FISH). We also measured the absolute expression of BDNF and BDNF-AS transcript by generating standard curves, using DNA vectors containing cDNA of each transcript (Supplementary Fig. 1). BDNF mRNA levels are generally 10–100 fold higher than BDNF-AS transcript, except in testis, kidney and heart, which contain equal or higher levels of BDNF-AS. Both transcripts are expressed in brain, muscle and embryonic tissues (Supplementary Fig. 2). BDNF mRNA levels were relatively low in all post-natal tissues examined except in brain, bladder, heart and skeletal muscle (Supplementary Fig. 3). We examined the expression pattern of sense and antisense transcripts in rhesus monkey (Supplementary Fig. 4) and mouse tissues by RT-PCR (Supplementary Fig. 5) and RNA FISH (Supplementary Fig. 6). Both transcripts are co-expressed in many tissues, which suggest BDNF-AS potential for regulation of BDNF mRNA.
Knockdown of BDNF-AS increases BDNF in vitro
Transfection of several human and mouse cell lines including HEK293T cells with three independent siRNA molecules that targeted to non-overlapping regions of the BDNF-AS transcript, shown by asterisks in Figure 1b, resulted in over 85% knockdown of BDNF-AS transcript, accompanied by 2 to 6 fold upregulation of the BDNF transcript (Fig. 2a). Sequence information of these siRNAs as well as scrambled controls, AntagoNATs and other oligonucleotides are listed in supplementary Table S1. The upregulation of BDNF was not related to the choice of endogenous controls (Supplementary Fig. 7). BDNF-AS is a very low abundance transcript and therefore, in order to reliably detect the transcript, avoiding false signals from genomic DNA contamination and as control for RT-PCR reactions, we included no reverse transcriptase (NRT) and no template (NTC) controls in our experiments. Additionally, we tested the final PCR products for each set of primers and probe on a gel to ensure that only one product was amplified (Supplementary Fig. 8).
To monitor the sequential events after administration of BDNF-AS-targeted siRNA, we performed a time-course study (Fig. 2b) in which we collected HEK-293 cells and assessed the (endogenous) expression of both BDNF and BDNF-AS transcripts at several time points after treatment. We observed downregulation of BDNF-AS at 6 h with maximum efficacy between 24–48 h and it lasting up to 72 h (Fig. 2b). Upregulation of BDNF mRNA only started at 18 h and reached the maximum at 48 h then decreased by 72 h, the expression returning to pre-treatment levels by 96 h. These data show that the observed increase of BDNF commences sequentially after reduction of the BDNF-AS transcript and displays full reversibility.
Moreover, we examined BDNF protein levels following treatment of HEK-293 cells with two active BDNF-AS siRNAs, control siRNA or scrambled siRNAs by ELISA (Fig. 2c). We measured BDNF protein level by western blot following transfection of cells with BDNF-AS siRNA-1 or control siRNA (Fig. 2d). Both ELISA (Fig. 2c) and western blotting (Fig 2d) experiments demonstrated that siRNA targeting BDNF-AS significantly increased the expression of BDNF protein. We observed that the magnitude of BDNF protein upregulation (~2 fold) was somewhat lower than with mRNA upregulation (2–6 fold); this suggests that protein upregulation kinetics lags that of mRNA in these cells or possibly an involvement of post-transcriptional regulatory mechanisms aside from antisense RNA to control BDNF protein output, including, for example, microRNA regulation10–12 (Supplementary Fig. 9).
BDNF-AS did not alter BDNF sense RNA stability
In order to examine the effects of BDNF-AS transcript on the stability of BDNF sense transcript, we depleted BDNF-AS with siRNA then treated cells with α-amanitin. We found that the baseline half-life for BDNF-AS was 15.3 h, nearly 3 h longer than that of the BDNF sense transcript (Supplementary Fig. 10). There was no significant change in BDNF sense RNA stability after reduction of BDNF-AS transcript, suggesting that unlike some other highly abundant antisense transcripts13, BDNF-AS does not alter BDNF sense mRNA stability. Recently it has been suggested that some NATs can produce endogenous siRNAs from the overlapping region between sense and antisense RNAs. Indeed Watanabe et al. published a list of endogenous siRNAs from mouse oocytes14. We examined this list but we were not able to find any endogenous siRNAs originating from the Bdnf locus.
Targeting of BDNF-AS by AntagoNATs
We introduce the term AntagoNAT here to describe single-stranded oligonucleotide molecules that inhibit sense-antisense interactions (with different modifications, see supplementary methods). We hypothesized that use of AntagoNATs should have a similar outcome on BDNF-AS as that observed with BDNF-AS siRNA and designed gapmer single–stranded oligonucleotides, 14 nucleotides in length, with 2’O-Methyl RNA and/or locked nucleic acid (LNA) modifications. Using this strategy, we tiled the entire overlapping region between human BDNF-AS and BDNF transcripts and identified several efficacious AntagoNATs capable of upregulating of BDNF mRNA. hBDNF-AntagoNAT1 and hBDNF-AntagoNAT4, targeting the first part of the overlapping region, produced the largest response (Supplementary Fig. 11). Our data suggests that blockage of BDNF antisense RNA, by single-stranded AntagoNATs, is sufficient in causing an increase in BDNF mRNA.
We then designed single-stranded gapmer LNA-modified15 DNA oligonucleotides (AntagoNATs) 16-nucleotides in length with phosphorothioate backbone, complementary to mouse Bdnf-AS. Two AntagoNATs (mBdnf-AntagoNAT3 and mBdnf-AntagoNAT9) consistently showed a statistically significant increase in Bdnf mRNA levels in mouse N2a cells (Supplementary Fig. 12). In order to establish an optimal dosage for further in vitro studies, we performed concentration-response experiments with 11 different concentrations (1:3 serial dilutions ranging from 300 nM to 5 pM) of mBdnf-AntagoNAT9, measuring fold changes in Bdnf mRNA levels (Fig. 2e). A concentration-dependent increase in Bdnf mRNA levels at 1–300 nM with EC50 of 6.6 nM was determined.
Knockdown of GDNF-AS increases GDNF mRNA in vitro
We selected another low abundance noncoding antisense RNAs that is transcribed from opposite strand of GDNF at chromosome 5 and designed several AntagoNATs targeting the GDNF antisense transcript. We found two AntagoNATs that significantly increase the GDNF mRNA by 3–4 fold (Fig. 2f). Additionally, we show AntagoNAT-mediated upregulation of EphB2, a member of a different gene family (Supplementary Fig. 13). These results suggest that antisense RNA mediated transcriptional suppression is a widespread phenomenon in the mammalian genome.
Bdnf upregulation increases neuronal outgrowth
Consistent with many previous reports that indicate stimulatory effects of Bdnf on neuronal outgrowth and adult neurogenesis16–17, we found that an increase in the endogenous Bdnf level due to the knockdown of Bdnf-AS transcript resulted in increased neuronal cell number and in neurite outgrowth and maturation at 3 and 7 days post-plating in neurospheres (Fig. 3 a–d). These data suggest that the upregulation of endogenous Bdnf, due to inhibition of antisense RNA, induces neuronal differentiation in neuronal progenitor cells and might cause a mature phenotype in nascent neurons.
Knockdown of Bdnf-AS increases Bdnf in vivo
We utilized osmotic mini-pumps for intracerebroventricular (ICV) delivery of mBdnf-AntagoNAT9 to C57BL/6 mice. We selected mBdnf-AntagoNAT9, which is targeting a non-overlapping region of mouse Bdnf-AS, over other active AntagoNATs, based on its high efficacy to increase in Bdnf mRNA in vitro. After 28 days of continuous AntagoNAT infusion, Bdnf mRNA levels were increased across forebrain regions adjacent to the third ventricle in mice treated with mBdnf-AntagoNAT9 as compared to levels unaltered by an inert control oligonucleotide (Fig. 4a,b). Bdnf and Bdnf-AS transcripts were unaltered in the hypothalamus, a structure that is not immediately adjacent to the third ventricle (Fig. 4c). Moreover, we find that AntagoNAT-mediated blockade of Bdnf-AS results in increased Bdnf protein levels (Fig 4 d,e). These findings correspond with the in vitro data described above and indicate that the blockade of Bdnf-AS results in the increase of Bdnf mRNA and protein expression in vivo.
We injected BrdU in the mice treated with mBdnf-AntagoNAT9 in the first week of the study for 5 days. After 28 days of continuous AntagoNAT infusion, we performed histological examination of brain tissues and quantified neuronal proliferation and survival using Ki67 and BrdU markers, respectively. In mice treated with mBdnf-AntagoNAT9, we observed an increase in Ki67 positive (proliferating) cells as compared to control treated mice (Fig 5a,b). We quantified the number of Ki67 positive cells and found a significant increase in cell proliferation in mice treated with mBdnf-AntagoNAT9 compared to control oligonucleotide (Fig. 5c). In mice treated with mBdnf-AntagoNAT9, there was a significant increase in BrdU incorporation (surviving cells) as compared to the control oligonucleotide- treated mice (Fig. 5d). There were no differences in hippocampal volume between control and mBdnf-AntagoNAT9 treated mice (Fig. 5e). These findings demonstrate that Bdnf-AS regulates Bdnf levels in vivo.
BDNF-AS induces repressive chromatin marks
We measured the association of repressive (H3K9met3, H3K27met3) and active (H3K4met3, H3K36met3) chromatin marks to the BDNF genomic locus (Fig. 6a). Treating with siRNA, intracellular BDNF-AS was depleted and chromatin immunoprecipitation (ChIP) assays were performed. DNA was extracted and analyzed using 16 primer sets matching regions along the entire BDNF locus including the sense-antisense (S-AS) overlapping and BDNF promoter region (Fig-6a). Primers were designed to span the entire BDNF locus, at 20 kb increments, covering more than 270 kb and extending through to neighboring genes LIN7C and KIF18A. Initially, we performed RT-PCR on RNA samples and found that the knockdown of BDNF-AS transcript increases BDNF mRNA without any effects on neurotrophic tyrosine kinase, receptor, type 2 (TrkB) or on neighboring genes (LIN7C and KIF18A) in both directions (Supplementary Fig. 14). Next, we studied the immunoprecipitated DNA, using individual primer sets using RT-PCR and found that the siRNA-mediated depletion of the BDNF-AS transcript causes a significant reduction in H3K27met3 association in both the sense-antisense overlapping region and in the upstream BDNF promoter region (Fig. 6b). We found similar reduction in H3K27met3 binding to the promoter of the mouse Bdnf gene upon treatment of N2a ells with mBdnf-AntagoNAT9 (Supplementary Fig. 15). Furthermore, we found that alterations in repressive marks were specific to H3K27met3 as we were not able to detect significant changes in H3K9met3, H3K4met3 and H3K36met3 (Supplementary Fig. 16). These data suggest that BDNF-AS might play a role in the guidance, introduction and maintenance of H3K27met3 at the BDNF locus.
We observed that the ablation of Ezh2 activity using two different siRNAs phenocopied the BDNF-AS knockdown effect and caused the upregulation of BDNF mRNA (Fig. 6c). A ChIP assay was performed using the Ezh2 antibody. ChIP data revealed a significant reduction in Ezh2 binding at the BDNF promoter, upon depletion of BDNF-AS by siRNA (Fig. 6d). However, not all 16 primer sets gave detectable PCR signals, which could be attributed to the lack of direct Ezh2-chromatin binding. Given the reduction of binding of Ezh2 and the loss of the H3K27met3 at the BDNF promoter when BDNF-AS is knocked down, we conclude that BDNF-AS represses chromatin by recruiting Polycomb Repressive Complex 2 (PRC2) to the BDNF promoter region. Removal or inhibition of BDNF-AS could lead to the locus-specific upregulation of BDNF mRNA and protein.
Discussion
The number of ncRNAs in eukaryotic genomes have been shown to increase as a function of developmental complexity18,19 and there is, for example, a great deal of diversity in ncRNAs expressed in the nervous system20,21. Over the past few years, we and others have reported on functional NATs and showed their potential involvement in human disorders, including Alzheimer’s disease13, Parkinson’s disease22 and Fragile X syndrome23. Moreover, previously we showed that upregulation of CD97 sense gene can be attained by knockdown of its antisense RNA transcript1. Upregulation of progesterone receptor (PR), and other endogenous transcripts was reported following targeting of promoter-derived noncoding RNAs24,25. Transcriptional activation of p21 gene26 and Oct4 promoter27 were reported following NATs depletion. Antisense RNA-induced chromatin remodeling seems to be a feasible and dynamic mode of action for many low-copy number NATs2,28. If so, antisense RNA might predominantly exert local effects to maintain or modify chromatin structure, ultimately activating or suppressing sense gene expression.
PCR2 is a protein complex that consists of four core subunits: Eed, Suz12, RbAp48 and the catalytic Ezh2, that catalyzes the trimethylation of histone H3-lysine 27 (H3K27met3)32. Recent studies provide evidence for direct RNA-potein interaction between Ezh2 and many ncRNA transcripts29. Other studies of X inactivation30 and HOX gene cluster31 show RNA transcripts to be involved in the PRC2-mediated induction of H3K27met3, repressive chromatin marks. PRC2 transcriptome profiling has identified over 9,000 PRC2-interacting RNAs in embryonic stem cells, many of them categorized as antisense RNA transcripts29. Epigenetic silencing of p15 and DM1 genes were reported to involve heterochromatin formation by its antisense RNA33,34. The traditional binary division of chromatin into hetero- or eu-chromatin categories might not be complete as recent work has shown that there are five principal chromatin types49 that are more dynamic and flexible than originally believed. Likely applicable to a large number of gene loci, NATs can be manipulated in order to obtain a locus-specific alteration in chromatin modification. As examples, we show here that cleavage (by siRNA) or inhibition (by AntagoNATs) of the antisense transcripts of BDNF, GDNF and EphB2 genes leads to the upregulation of corresponding mRNAs.
Neurotrophins belong to a class of secreted growth factors that enhance the survival, development, differentiation and function of neurons and BDNF is an important molecular mediator of synaptic plasticity35–37. BDNF is suggested to synchronize neuronal and glial maturation38, participate in axonal and dendritic differentiation39 and protect and enhance neuronal cell survival40,41. Neurotrophin expression levels are impaired in neurodegenerative42–45 and in psychiatric and neurodevelopmental disorders46–48. The upregulation of neurotrophins is believed to have beneficial effects on several neurological disorders. AntagoNATs can potentially be used as a therapeutic strategy to inhibit BDNF-AS and consequently enhance neuronal proliferation and survival in a variety of disease states. It cannot be excluded that the herein described approach to upregulate the synthesis of endogenous BDNF molecules, presumed to contain natural modifications and to represent all known splice forms, will prove to be distinct, and perhaps superior, to administrating synthetic BDNF molecules.
Many low abundance noncoding antisense RNAs, which are transcribed on the opposite strands of genomic loci, endogenously suppress corresponding sense gene expression; inhibition or removal of antisense transcript leads to locus-specific upregulation of sense mRNA, protein and function.
Supplementary Material
Acknowledgements
Dr. Qing-Rong Liu from the National Institute of Drug Abuse kindly provided us constructs that contain three splice variants of the human BDNF-AS transcript. We thank Drs. Carlos Coito, Philip Frost, Jane Hsiao and Olga Khorkova at OPKO-CURNA for helpful discussions. The National Institutes of Health (5R01NS063974 and 5RC2AG036596) funded this work.
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