Sequence-based bioinformatic prediction and QUASEP identify genomic imprinting of the KCNK9 potassium channel gene in mouse and human

Abstract

Genomic imprinting is the epigenetic marking of gene subsets resulting in monoallelic or predominant expression of one of the two parental alleles according to their parental origin. We describe the systematic experimental verification of a prioritized 16 candidate imprinted gene set predicted by sequence-based bioinformatic analyses. We used Quantification of Allele-Specific Expression by Pyrosequencing (QUASEP) and discovered maternal-specific imprinted expression of the Kcnk9 gene as well as strain-dependent preferential expression of the Rarres1 gene in E11.5 (C57BL/6 x Cast/Ei)F1 and informative (C57BL/6 x Cast/Ei) x C57BL/6 backcross mouse embryos. For the remaining 14 candidate imprinted genes, we observed biallelic expression. In adult mouse tissues, we found that Kcnk9 expression was restricted to the brain and also was maternal-specific. QUASEP analysis of informative human fetal brain samples further demonstrated maternal-specific imprinted expression of the human KCNK9 orthologue. The CpG islands associated with the mouse and human Kcnk9/KCNK9 genes were not differentially methylated, but strongly hypomethylated. Thus, we speculate that mouse Kcnk9 imprinting may be regulated by the maternal germline differentially methylated region in Peg13, an imprinted non-coding RNA gene in close proximity to Kcnk9 on distal mouse chromosome 15. Our data have major implications for the proposed role of Kcnk9 in neurodevelopment, apoptosis and tumourigenesis, as well as for the efficiency of sequence-based bioinformatic predictions of novel imprinted genes.

Chromosome-Wide Analysis of Parental Allele-Specific Chromatin and DNA Methylation

“…The Peg13 DMR also had a very clear CTCF peak in the paternal allele. Kcnk9 is maternally expressed in 11.5-dpc embryos and in adult brain (79), and Trappc9 exhibits imprinted expression in the neonatal brain (101). Kcnk9 and Trappc9 were not differentially expressed in MatDup.dist7 versus PatDup.dist7 MEFs, and this was in agreement with the absence of an allele-specific chromatin pattern.…”

“…Recently, large-scale studies aimed at detecting novel imprinted genes (73). The predictions were based on RNA expression differences (1,28,57,83,101), DNA sequence-based computational predictions (50,51,79), DNA methylation differences (35,89), and epigenetic signature (8,18,54,56,103). The chromatin-based studies successfully predicted and identified novel imprinted genes but with variable specificity.…”

“…1A to C): the paternally methylated H19-Igf2 imprinting control region (ICR) (23,67,96,97), the maternally methylated KvDMR1 (19,25,105), the maternally methylated Inpp5f_v2 DMR (104) in distal Chr7, the maternally methylated Slc38a4, and Peg13 DMRs in distal Chr15 (79,89).…”

“…The duplicated chromosomes in MatDup.dist7 and PatDup-.dist7 MEFs additionally harbor the paternally expressed Ampd3 (83), Inpp5f_v2, and Inpp5f_v3 (12,104) on Chr7, the maternally expressed Kcnk9 (79) and Trappc9 (101), and the paternally expressed Peg13 (89) and Slc38a4 (57,89) imprinted genes on distal Chr15.…”

Successful Computational Prediction of Novel Imprinted Genes from Epigenomic Features

“…Recently, computational prediction of imprinted genes using DNA sequence features alone has been used for both the mouse and the human genomes. Data from these studies resulted in experimental validation of three imprinted genes in the mouse genome, and two candidate genes from analysis of the human genome may also be imprinted (26,36,37,50). However, data from reciprocal crosses are not available for the two genes reported to be imprinted in humans, which is essential to distinguish imprinted genes from expression quantitative trait loci (eQTLs).…”