High-resolution analysis of parent-of-origin allelic expression in the mouse brain

Abstract

Genomic imprinting results in preferential expression of the paternal or maternal allele of certain genes. We have performed a genome-wide characterization of imprinting in the mouse embryonic and adult brain. This approach uncovered parent-of-origin allelic effects of more than 1300 loci. We identified parental bias in the expression of individual genes and of specific transcript isoforms, with differences between brain regions. Many imprinted genes are expressed in neural systems associated with feeding and motivated behaviors, and parental biases preferentially target genetic pathways governing metabolism and cell adhesion. We observed a preferential maternal contribution to gene expression in the developing brain and a major paternal contribution in the adult brain. Thus, parental expression bias emerges as a major mode of epigenetic regulation in the brain.

Fig. 1

A map of imprinted gene expression in the adult CNS identifies distinctive hot- and cold-spots. Presence (colored squares) versus absence (dark grey squares) of imprinted gene expression was mapped in a representative subset of brain regions (full map in Fig. S1). Randomly selected biallelic control genes are indicated by green squares. The heatmap was assigned for each brain region according to the number of standard deviations from the mean for the number of imprinted genes expressed (Cooler to Warmer (standard deviations): <−2, <−1.5, <−1, >+1, >+1.5, >+2).

Fig. 2

Identification of loci exhibiting parent-of-origin allelic effects in the embryonic and adult CNS using Illumina sequencing. (A) Plots of the number of SNP sites exhibiting parental expression bias identified by sequencing (black) compared to chance expectations (orange) at various χ2 P value cutoffs. Green values indicate number of imprinted SNPs detected at P<0.05. (B) Scatterplot of the -log (P) of the two-tailed χ2 probability (P) for individual SNP sites for the F1i versus the F1r cross (POA shown). SNP sites identified by P<0.05 cutoff in each cross are indicated by red and blue dots. (C) Numbers of known and uncovered genes associated with parental allelic effects.

Fig. 3

Features of imprinted gene clusters associated with neurological disorders and diseases. (A) UCSC browser tracks indicate all reads aligning uniquely to the genome (Expression, black) and the percentage of transcriptome aligned reads at SNP sites that were assigned to the paternal (blue, positive) versus maternal allele (red, negative). Analysis of 1810044A24Rik isoforms (1: uc007wbn.1, 2: uc007wbl.1 (uc007wbm.1 not shown), 3: uc007wbp.1) revealed mixed isoform specific imprinting at this locus. A paternally expressed isoform (uc007wbn.1) was identified by SNPs located in the unique 3’ exon and UTR. (B) Bar graphs indicate parental bias for SNP sites located in a 1810044A24Rik shared exon (SNP_ID: uc007wbl.1_1339), the 3’UTR of the uc007wbn.1 isoform (SNP_ID: uc007wbn.1_4207), and Eif2c2 (SNP_ID: uc007wbu.1_1944). Sequenom analysis validated maternal expression of Eif2c2 (E15 brain). Highlighted regions in browser tracks (A; red: Eif2c2; blue: 1810044A24Rik) indicate regions detailed in bar graphs. χ2 analysis (***P<0.001; **P< 0.01). (C) A large region of paternally-biased transcription was uncovered in the PWS-AS gene cluster between the SNRPN/SNURF locus and Ndn. (D) Sequenom validation of paternally-biased expression by DOKist4 in the PWS-AS cluster (SNP_ID: uc009hex.1_3057).

Fig. 4

Parent-of-origin allelic effects influence gene expression in a developmental and region specific manner in the CNS. (A) Comparison of the total numbers of UCSC annotated genes with parent-of-origin allelic effects in the E15 brain, adult POA and mPFC. Red and blue bars indicate MEGs and PEGs identified in each sample, respectively. (B) Pie charts comparing the relative numbers of PEGs and MEGs identified in the E15 brain, mPFC, and POA. (C) Spatiotemporal regulation of imprinting at the H19-Igf2 locus revealed by UCSC Browser tracks of raw expression data (black) and parental expression bias (blue: paternal, red: maternal) at identified SNP sites in H19 and Igf2. (D) Igf2 allele-specific expression inversion confirmed by Illumina and Sequenom (SNP_ID: uc009kod.1_2313). Raw expression tracks of reads uniquely aligning to the genome are shown below in black. χ2 analysis (***P<0.001; **P< 0.01; *P< 0.05).

Fig. 5

Characterization of complex genes associated with parental allelic effects in the CNS transcriptome. (A) Numbers of previously known and newly uncovered consensus, complex, or single SNP imprinted genes. (B) Proportions of complex genes with parent-of-origin allelic effects localized to one or more exons (Exon), the 3’UTR (last exon), or 3’UTR+Exons, or other outcomes (ie. disagreements between SNPs in the same exon or 3’UTR). Exons or 3’ UTRs with more than one SNP for evidence are indicated separately (MultiSNP). (C) UCSC browser tracks at the cdh15 locus indicate preferential expression of the paternal allele (paternal allele expression bias: blue, POA data shown). Illumina and Sequenom analysis confirmed preferential expression of the paternal allele for cdh15 (SNP_ID: uc009ntv.1_2522). (D) Complex spatiotemporal and isoform-specific imprinting at the Inpp5f locus. A significant maternal bias was observed specifically in the region of Inpp5f_v1 that overlaps with mKIAA0966 in adult POA. Highlighted SNP sites of particular interest are statistically significant in both crosses by χ2 analysis (***P<0.001; **P< 0.01; *P< 0.05).