Many studies have revealed that antisense transcription (transcription from the opposite strand of a protein-coding or sense gene) is widespread throughout the genome. This finding was first made possible through large-scale cDNA and EST sequencing efforts and has sparked interest in uncovering functional roles for these antisense transcripts, often termed Natural Antisense Transcripts, or NATs (reviewed in 
). In mouse 
and in human 
, thousands of sense/antisense (S/AS) pairs of transcripts have been revealed in this manner. Additionally, through subsequent microarray analysis, many S/AS pairs have been found to show correlated expression, with concordant expression (positive expression correlation) more common than reciprocal expression (negative expression correlation) 
. This correlated expression suggests that the antisense transcripts in S/AS pairs may often be involved in regulation of their sense partners.
Indeed, antisense transcripts have been demonstrated to exhibit regulatory activity in several instances. Transcriptional interference represents one known mechanism. In this phenomenon, the act of transcription from one strand prevents the initiation or elongation of transcription from the opposite strand through steric hindrance (reviewed in 
). Quite recently, Morrissy et al. used microarrays to show that expression of antisense transcripts strongly correlates with alternative splicing of sense targets 
. The authors propose that antisense transcripts may regulate alternative splicing of their sense targets through base-pairing of the complementary regions, resulting in splice site masking or the production of endogenous siRNAs, or through transcriptional interference.
Of course, gene regulation also occurs at the DNA level, particularly through enhancers. Enhancers have traditionally been understood to be cis
-acting DNA elements which act as binding sites for transcription factors or activator proteins that in turn recruit or stabilize RNA polymerase II transcription at specific promoters (reviewed in 
). Interestingly, recent work has demonstrated widespread transcription at enhancers 
. While it has long been known that enhancers can occur in introns of genes, the finding that extragenic enhancers are often transcribed raises questions regarding the functions of these enhancer-associated transcripts. Recently, short, non-polyadenylated RNA pol II transcripts arising bidirectionally from active enhancers have been reported 
, but functional roles for these transcripts, termed “eRNAs,” have not been identified. Indeed, as bidirectional transcription has also been demonstrated at promoters 
, it is not unreasonable to postulate these eRNAs could largely be transcriptional noise. Among the few enhancer-associated RNAs to be characterized at a functional level, the polyadenylated ncRNA Evf-2 is transcribed from an enhancer and interacts with this same enhancer to drive transcription of homeodomain proteins DLX5 and DLX6 
. Evf-2 currently represents only an isolated example of an enhancer-associated RNA, yet this finding suggests that enhancers and ncRNAs may cooperate on a larger scale to achieve finely-tuned gene regulation.
In this study we functionally characterize two specific novel enhancer-associated transcripts discovered through RNA-Sequencing (RNA-Seq) of mouse embryonic stem cells (mESCs) and their derived neural precursors (NPs). Each of these RNAs originates within and is expressed antisense to a known protein-coding gene. Further, the expression of each antisense RNA is correlated with an active enhancer, located by P300 binding. Finally, in each case the antisense transcript is expressed concomitantly with the sense transcript, but a shorter isoform of the sense gene appears to be preferentially expressed when the antisense transcript is also expressed. From these findings we propose a model in which gene isoform specificity may be achieved through enhancer-associated antisense RNAs. This model challenges the long-held view that enhancers act strictly on promoters of protein-coding genes to accomplish gene regulation.