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.

No abstract

Small interfering RNAs (siRNAs) directed to gene promoters can silence genes at the transcriptional level. siRNA-directed transcriptional silencing (RdTS) was first described in plants and yeasts and more recently in mammalian cells. RdTS has been associated with the induction of epigenetic changes and the formation of complexes containing RNA interference and chromatin-remodelling factors. Here, we show that a promoter-targeted siRNA inhibits transcription of the c-myc gene. Transcriptional silencing of c-myc did not involve changes of known epigenetic marks. Instead, the c-myc promoter-targeted siRNA interfered with transcription initiation blocking the assembly of the pre-initiation complex. Transcriptional interference depended on Argonaute 2 and a noncoding promoter-associated RNA initiated upstream and overlapping the transcription start site. Silencing of c-myc led to growth arrest, reduced clonogenic potential and senescence of c-myc over-expressing prostate cancer cells with minimal effect on normal cells. RNA-directed transcriptional interference may be a natural mechanism of transcriptional control and siRNAs targeting noncoding RNAs participating in this regulatory pathway could be valuable tools to control expression of deregulated genes in human diseases.

Apolipoprotein A-1 (APOA1) is the major protein component of high-density lipoprotein (HDL) in plasma. We have identified an endogenously expressed long non-coding natural antisense transcript, APOA1-AS, which acts as a negative transcriptional regulator of APOA1, both in vitro and in vivo. Inhibition of APOA1-AS in cultured cells resulted in the increased expression of APOA1, and two neighboring genes in the APO cluster. Chromatin immunoprecipitation (ChIP) analyses of a ~50Kb chromatin region flanking the APOA1 gene demonstrated that APOA1-AS can modulate distinct histone methylation patterns that mark active and/or inactive gene expression, through the recruitment of histone-modifying enzymes. Targeting APOA1-AS using short antisense oligonucleotides also enhanced APOA1 expression in both human and monkey liver cells, and induced an increase in hepatic RNA and protein expression in African green monkeys. The results presented here further highlight the significant local modulatory effects of long non-coding antisense RNAs, and demonstrate the therapeutic potential of manipulating the expression of these transcripts both in vitro and in vivo.

The underlying genetic variations of late onset Alzheimer’s Disease (LOAD) cases remain largely unknown. A combination of genetic variations with variable penetrance and lifetime epigenetic factors may converge on transcriptomics alterations that drive LOAD pathological process. Transcriptome profiling using deep sequencing technology offers insight into common altered pathways regardless of underpinning genetic or epigenetic factors and thus represents an ideal tool to investigate molecular mechanisms related to the pathophysiology of LOAD. We performed directional RNA sequencing on high quality RNA samples extracted from hippocampi of LOAD and age-matched controls. We further validated our data using qRT-PCR on a larger set of post-mortem brain tissues, confirming downregulation of the gene encoding substance P (TAC1) and upregulation of the gene encoding the plasminogen activator inhibitor-1 (SERPINE1). Pathway analysis indicates dysregulation in neural communication, cerebral vasculature and Amyloid-β clearance. Beside protein coding genes, we identified several annotated and non-annotated long noncoding RNAs that are differentially expressed in LOAD brain tissues, three of them are activity-dependent regulated and one is induced by Aβ1-42 exposure of human neural cells. Our data provide a comprehensive list of transcriptomics alterations in LOAD hippocampi and warrant holistic approach including both coding and non-coding RNAs in functional studies aimed to understand the pathophysiology of LOAD.