Imprinted Expression of Neuronatin from Modified BAC Transgenes Reveals Regulation by Distinct and Distant Enhancers

Abstract

Neuronatin (Nnat) is an imprinted gene that is expressed exclusively from the paternal allele while the maternal allele is silent and methylated. The Nnat locus exhibits some unique features compared with other imprinted domains. Unlike the majority of imprinted genes, which are organised in clusters and coordinately regulated, Nnat does not appear to be closely linked to other imprinted genes. Also unusually, Nnat is located within an 8-kb intron of the Bc10 gene, which generates a biallelically expressed, antisense transcript. A similar organisation is conserved at the human NNAT locus on chromosome 20. Nnat expression is first detected at E8.5 in rhombomeres 3 and 5, and subsequently, expression is widespread within postmitotic neuronal tissues. Using modified BAC transgenes, we show that imprinted expression of Nnat at ectopic sites requires, at most, an 80-kb region around the gene. Furthermore, reporter transgenes reveal distinct and dispersed cis-regulatory elements that direct tissue-specific expression and these are predominantly upstream of the region that confers allele-specific expression.