Transcriptome-wide identification of novel imprinted genes in neonatal mouse brain - PubMed (original) (raw)

Transcriptome-wide identification of novel imprinted genes in neonatal mouse brain

Xu Wang et al. PLoS One. 2008.

Abstract

Imprinted genes display differential allelic expression in a manner that depends on the sex of the transmitting parent. The degree of imprinting is often tissue-specific and/or developmental stage-specific, and may be altered in some diseases including cancer. Here we applied Illumina/Solexa sequencing of the transcriptomes of reciprocal F1 mouse neonatal brains and identified 26 genes with parent-of-origin dependent differential allelic expression. Allele-specific Pyrosequencing verified 17 of them, including three novel imprinted genes. The known and novel imprinted genes all are found in proximity to previously reported differentially methylated regions (DMRs). Ten genes known to be imprinted in placenta had sufficient expression levels to attain a read depth that provided statistical power to detect imprinting, and yet all were consistent with non-imprinting in our transcript count data for neonatal brain. Three closely linked and reciprocally imprinted gene pairs were also discovered, and their pattern of expression suggests transcriptional interference. Despite the coverage of more than 5000 genes, this scan only identified three novel imprinted refseq genes in neonatal brain, suggesting that this tissue is nearly exhaustively characterized. This approach has the potential to yield an complete catalog of imprinted genes after application to multiple tissues and developmental stages, shedding light on the mechanism, bioinformatic prediction, and evolution of imprinted genes and diseases associated with genomic imprinting.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Alignment of Illumina sequence reads for Igf2 transcript.

The top panel is the summary window or all 1,253 cDNA reads that mapped to the 4,038 bp Igf2 transcript (NM_010514). The blue arrows represent the sense reads and the red arrows represent antisense reads. From the figure, most of the reads were aligned to the 1 kb region near the 3′-end of the transcript. The bottom left panel gives the Illumina read names, and the bottom right gives the sequence alignment. Sense reads are printed in black font and the antisense reads are in red font. There are many overlapping 32-bp reads aligned uniquely to the transcript, with a quality score for each nucleotide. There is a SNP (A/G) located in the middle. By directly counting the number of reference and alternative nucleotides at the SNP, we were able to quantify the relative expression level of the two parental alleles.

Figure 2

Figure 2. Verification for known imprinted gene Nnat (also known as Peg5).

(A) Allele counts for Perlegen SNP NES08901860, NES08901861, NES08901863 and NES08901864. The blue bars (from left to right) represent the Illumina read counts from the paternal allele in PWD x AKR and AKR x PWD F1s respectively (maternal genotype listed first). The red bars represent the maternal allele Illumina read counts. (B) Sanger sequencing verification for Perlegen SNP NES08901861. We discovered an adjacent SNP position before NES08901861. The target sequence is GCCCT(AC/GA)ATCT. (C), Pyrosequencing verification for Perlegen SNP NES08901861. The target sequence is GCCCT(AC/GA)ATCT.

Figure 3

Figure 3. Verification for the known imprinted gene Gtl2.

(A) Allele counts for the 4 new SNPs discovered by assembling the Solexa reads. The blue bars (from left to right) stand for the counts from the paternal allele in PWD x AKR and AKR x PWD F1s respectively. The red bars represent the maternal allele counts. Four novel SNPs were discovered in one Gtl2 transcript (XR_035484), consistent with monoallelic expression from the maternal allele in both reciprocal crosses and confirmed by Pyrosequencing. Another splicing variant of Gtl2, NM_144513, previously was found by us to be imprinted using a custom Agilent allele-specific microarray (unpublished), with an 1,847-fold difference in probe intensity in PWD x AKR cross and 793-fold in the reciprocal cross. A Perlegen SNP (NES17649478) in NM_144513 but not XR_035484 was verified by Pyrosequencing. We conclude that both XR_035484 and NM_144513 are imprinted in the neonatal brain. (B) Pyrosequencing verification for novel SNP1 in Gtl2. The target sequence is TGT(A/G)GAGGGA. (C) Pyrosequencing verification for Perlegen SNP NES17649478. The target sequence is GA(A/G)GATAG.

Figure 4

Figure 4. Sense-antisense gene pairs covered by the Illumina sequence data.

Gene structures of the three gene pairs showing nested structures. The blue shading represents the paternal allele and the pink shading indicates for the maternal allele. Boxes with dashed lines indicate no expression. The arrows represent the direction of transcription. The sum of the heights of the two parental exons for each gene is in proportion to the expression level, which is quantified by the total counts of the perfect-match Illumina reads. The relative heights of the exons for the paternal and maternal allele within the same gene represent the relative expression level of the two parental alleles. The short vertical lines under the exons indicate the SNP positions, and the total counts of the two reciprocal crosses for the maternal and paternal allele are labeled.

Figure 5

Figure 5. Chromosomal scans of imprinting status.

(A) Imprinting status for chromosome 2. (B) Imprinting status for chromosome 7. Each plot contains unique Entrez genes covered by SNP-containing Illumina reads with counts no less than 4 in both reciprocal crosses. The height of each bar is the difference of the AKR percentage in the two reciprocal crosses (p1-p2), representing the intensity of imprinting. The color stands for the direction of imprinting, blue for paternal expression and red for maternal expression. The intensity of the color represents the significance, grey for not significant (_q_-value ≥0.10), lighter blue and pink for marginally significant (0.05≤ _q_-value <0.10), darker blue and red for significant (_q_-value <0.05). The gene name is indicated if | _p1_-p2| ≥0.3.

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