A Survey for Novel Imprinted Genes in the Mouse Placenta by mRNA-seq

Abstract

Many questions about the regulation, functional specialization, computational prediction, and evolution of genomic imprinting would be better addressed by having an exhaustive genome-wide catalog of genes that display parent-of-origin differential expression. As a first-pass scan for novel imprinted genes, we performed mRNA-seq experiments on embryonic day 17.5 (E17.5) mouse placenta cDNA samples from reciprocal cross F 1 progeny of AKR and PWD mouse strains and quantified the allele-specific expression and the degree of parent-of-origin allelic imbalance. We confirmed the imprinting status of 23 known imprinted genes in the placenta and found that 12 genes reported previously to be imprinted in other tissues are also imprinted in mouse placenta. Through a wellreplicated design using an orthogonal allelic-expression technology, we verified 5 novel imprinted genes that were not previously known to be imprinted in mouse (Pde10, Phf17, Phactr2, Zfp64, and Htra3). Our data suggest that most of the strongly imprinted genes have already been identified, at least in the placenta, and that evidence supports perhaps 100 additional weakly imprinted genes. Despite previous appearance that the placenta tends to display an excess of maternally expressed imprinted genes, with the addition of our validated set of placenta-imprinted genes, this maternal bias has disappeared.

Using next-generation RNA sequencing to identify imprinted genes

“…To distinguish between RMAE and parent-of-origin monoallelic expression, adequate sample size is needed to capture representative individuals expressing an identified paternal/maternal allele in the two reciprocal F1 crosses (Figure 1c). Therefore, validation of novel candidate imprinted genes should be performed across a panel of informative individuals to exclude the possibility of RMAE (Wang et al, 2011(Wang et al, , 2013b. Consistent parent-of-origin allelic expression in both reciprocal crosses is needed to confirm a newly identified imprinted gene (Figure 1d).…”

“…This method requires the tracking of all SNP alleles in each read and is not applicable when a single read contains the reference allele for one SNP and the alternative allele for another SNP. The best way that we have found to remove the alignment bias is to align the reads to both the reference genome and a pseudogenome, constructed by substituting the reference allele with the alternative allele in all transcribed regions, and take the average counts from the reference and pseudogenome (Wang et al, 2011(Wang et al, , 2013b. With this approach, the alignment bias could be reduced to o1% (Figure 4b).…”

“…To search for novel imprinted genes genome-wide in an unbiased way, we and others have applied next-generation RNA sequencing (RNA-seq) and quantitatively measured the allele-specific expression in RNA samples from reciprocal crosses of inbred mouse strains. This was accomplished by directly counting the number reference/alternative allelecontaining reads at polymorphic SNP positions in the parental genome (Babak et al, 2008;Wang et al, 2008Wang et al, , 2011Gregg et al, 2010a, b;DeVeale et al, 2012;Okae et al, 2012). This approach has been successful in identifying a number of additional imprinted genes in mice.…”

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

“…Next generation sequencing (NGS) technologies, including genome-wide DNA sequencing (DNA-seq) and transcriptome-wide RNA sequencing (RNA-seq), have been increasingly utilized for in-depth analysis and detection of novel imprinted genes in both humans and mice [15–17]. While high throughput, such studies require completion of genome sequencing and annotation, intensive bioinformatics, and careful independent validation (e.g., Sanger sequencing) to reduce false positives [7,11,18].…”

Imprinting meets genomics: new insights and new challenges

“…In mammals, the role of imprinted genes in regulating nutrient flow from the mother to the fetus via the placenta is well established [18]. In plants evidence is still lacking, although mutations in several imprinted genes affect the transition from endosperm cell division to cellularization, a developmental step that determines final endosperm and seed size.…”