Antisense Activity across the Nesp Promoter is Required for Nespas-Mediated Silencing in the Imprinted Gnas Cluster

Abstract

Macro long non-coding RNAs (lncRNAs) play major roles in gene silencing in inprinted gene clusters. Within the imprinted <i>Gnas</i> cluster, the paternally expressed <i>Nespas</i> lncRNA downregulates its sense counterpart <i>Nesp</i>. To explore the mechanism of action of <i>Nespas</i>, we generated two new knock-in alleles to truncate <i>Nespas</i> upstream and downstream of the <i>Nesp</i> promoter. We show that <i>Nespas</i> is essential for methylation of the <i>Nesp</i> differentially methylated region (DMR), but higher levels of <i>Nespas</i> are required for methylation than are needed for downregulation of <i>Nesp</i>. Although <i>Nespas</i> is transcribed for over 27 kb, only <i>Nespas</i> transcript/transcription across a 2.6 kb region that includes the <i>Nesp</i> promoter is necessary for methylation of the <i>Nesp</i> DMR. In both mutants, the levels of <i>Nespas</i> were extraordinarily high, due at least in part to increased stability, an effect not seen with other imprinted lncRNAs. However, even when levels were greatly raised, <i>Nespas</i> remained exclusively <i>cis</i>-acting. We propose <i>Nespas</i> regulates <i>Nesp</i> methylation and expression to ensure appropriate levels of expression of the protein coding transcripts <i>Gnasxl</i> and <i>Gnas</i> on the paternal chromosome. Thus, <i>Nespas</i> mediates paternal gene expression over the entire <i>Gnas</i> cluster via a single gene, <i>Nesp</i>.