Gene Isoform Specificity through Enhancer-Associated Antisense Transcription

Courtney S. Onodera,Jason G. Underwood,David Greenberg,Sofie R. Salama,Frank Jacobs,Sol Katzman,David Haussler,Charalampos Babis Spilianakis

doi:10.1371/journal.pone.0043511

Courtney S. Onodera, Jason G. Underwood + Show 6 more

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https://doi.org/10.1371/journal.pone.0043511

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Abstract

Enhancers and antisense RNAs play key roles in transcriptional regulation through differing mechanisms. Recent studies have demonstrated that enhancers are often associated with non-coding RNAs (ncRNAs), yet the functional role of these enhancer:ncRNA associations is unclear. Using RNA-Sequencing to interrogate the transcriptomes of undifferentiated mouse embryonic stem cells (mESCs) and their derived neural precursor cells (NPs), we identified two novel enhancer-associated antisense transcripts that appear to control isoform-specific expression of their overlapping protein-coding genes. In each case, an enhancer internal to a protein-coding gene drives an antisense RNA in mESCs but not in NPs. Expression of the antisense RNA is correlated with expression of a shorter isoform of the associated sense gene that is not present when the antisense RNA is not expressed. We demonstrate that expression of the antisense transcripts as well as expression of the short sense isoforms correlates with enhancer activity at these two loci. Further, overexpression and knockdown experiments suggest the antisense transcripts regulate expression of their associated sense genes via cis-acting mechanisms. Interestingly, the protein-coding genes involved in these two examples, Zmynd8 and Brd1, share many functional domains, yet their antisense ncRNAs show no homology to each other and are not present in non-murine mammalian lineages, such as the primate lineage. The lack of homology in the antisense ncRNAs indicates they have evolved independently of each other and suggests that this mode of lineage-specific transcriptional regulation may be more widespread in other cell types and organisms. Our findings present a new view of enhancer action wherein enhancers may direct isoform-specific expression of genes through ncRNA intermediates.

Highlights

IntroductionMany studies have revealed that antisense transcription (transcription from the opposite strand of a protein-coding or sense gene) is widespread throughout the genome
Many studies have revealed that antisense transcription is widespread throughout the genome
For the remainder of our analyses, we grouped reads from the same cell/compartment type together to simplify our libraries to 4 main types, UnNuc, UnCyt, NPNuc, and NPCyt, corresponding to undifferentiated nuclear, undifferentiated cytoplasmic, neural precursor cells (NPs) nuclear, and NP cytoplasmic, respectively

Summary

Introduction

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 [1]). 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) [2] This correlated expression suggests that the antisense transcripts in S/. 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

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