Uncoupling antisense-mediated silencing and DNA methylation in the imprinted Gnas cluster

There is increasing evidence that non-coding macrornas are major elements for silencing imprinted genes,but their mechanism of action is poorly understood. within the imprinted gnas cluster on mouse chromosome 2,nespas is a paternally expressed macrorna that arises from an imprinting control region and runs antisense to nesp,a paternally repressed protein coding transcript. here we report a knock-in mouse allele that behaves as a nespas hypomorph. the hypomorph mediates down-regulation of nesp in cis through chromatin modification at the nesp promoter but in the absence of somatic dna methylation. notably there is reduced demethylation of h3k4me3,sufficient for down-regulation of nesp,but insufficient for dna methylation; in addition,there is depletion of the h3k36me3 mark permissive for dna methylation. we propose an order of events for the regulation of a somatic imprint on the wild-type allele whereby nespas modulates demethylation of h3k4me3 resulting in repression of nesp followed by dna methylation. this study demonstrates that a non-coding antisense transcript or its transcription is associated with silencing an overlapping protein-coding gene by a mechanism independent of dna methylation. these results have broad implications for understanding the hierarchy of events in epigenetic silencing by macrornas. © 2011 williamson et al.