Alternative promoters of Peg3 with maternal specificity

Abstract

Peg3 (paternally expressed gene 3) is an imprinted gene localized within an evolutionarily conserved 500-kb domain in human chromosome 19q13.4 and mouse proximal chromosome 7. In the current study, we have identified three alternative promoters for mouse Peg3 and one alternative promoter for human PEG3. These alternative promoters are localized within the 200-kb upstream region of human and mouse PEG3, which is well conserved and thus predicted to harbor several cis-regulatory elements for the PEG3 domain. In the mouse, two of these alternative promoters drive maternal-specific expression of Peg3 specifically in the hypothalamus of the adult brain, while the remaining third promoter drives bi-allelic expression of Peg3 with a paternal bias only in the neonatal-stage brain. In human, an alternative transcript is also detected at relatively very low levels in adult brain and placenta. Overall, the identification of alternative promoters in both mouse and human models suggests that these alternative promoters may be functionally selected features for the PEG3 imprinted domain during mammalian evolution.

Figure 1. Genomic structures of the wild-type and mutant alleles of Peg3.

(A) Schematic representation of the wild-type alleles of mouse Usp29 and Peg3 loci. Gray and black boxes indicate the exons of Usp29 and Peg3, respectively. Transcriptional direction for each gene is represented by arrows with corresponding colors. (B) Schematic representation of the KO2 mutant allele. The KO2-Neo mutant has a 6.0-kb insertion containing a PGK promoter-driven neomycin resistance gene (NeoR) as selection marker, shown by the red box. NeoR, along with the 4.0-kb promoter region of Peg3 containing first exons of Usp29 and Peg3 are flanked by two LoxP sites as indicated with triangles. Cre-recombinase was used to delete the first exons of Usp29 and Peg3, producing the KO2 mutant. (C) Schematic representation of the DelKO mutant allele. The conditional-ready knockout (CoKO) allele has a 7.1-kb insertion containing a promoterless β-galactosidase gene (β-Gal) as shown by the blue box and human β-actin promoter-driven neomycin resistance gene (NeoR) as shown by the red box. The insertion cassette of the CoKO allele is removed by FLP-mediated recombination, producing the FlipKO allele which has the 6^th exon of Peg3 flanked by two LoxP sites as indicated with triangles. Cre recombinase was used to delete exon 6, producing the DelKO allele for the Peg3 locus.

Figure 2. Alternative 1^st exons and promoters of Peg3.

(A) Map of the mouse Peg3 locus. Gray and black boxes indicate the exons of Usp29 and Peg3, respectively. Transcriptional direction for each gene is represented by arrows with corresponding colors. The solid boxes indicate the position of the exons of Peg3, labeled E1 through E9, followed by ovals to indicate the position of upstream alternative exons. The closed oval represents the shared upstream exon U0, and open ovals represent alternative 1^st exons U1, U2, and U3, respectively. The deleted Peg3 exons corresponding to KO2^(+/−p) and DelKO^(+/−p) mutant alleles are shown using parentheses and dashed lines. The two extended arrows show the anchoring primers used for nested PCR after 5′ RACE: Ex2-R2 and Ex2-R3 for KO2^(+/−p) mutant and its WT counterpart, as shown by scheme I. The two extended arrows underneath it shows the anchoring primers used for nested PCR after 5′ RACE: Ex6-R3 and U0 for DelKO^(+/−p) adult hypothalamus and a WT neonate brain as indicated by the scheme II. A summary of the sequence reads is shown in Table 1. (B) The exon structures of Peg3 alternative transcripts identified from the mouse brain. The E1 - E2 exon structure indicates the transcription by the main promoter of Peg3. The exon U1 is positioned 20-kb upstream of E1, the 1^st exon of Peg3. The alternative transcript starting from the U1 exon connects to U0 (16-kb upstream of E1) and E2 exons while skipping E1. The exon U2 is positioned 26-kb upstream of E1. This alternative transcript starting from the U2 exon connects to U0 and E2 exons while skipping U1 and E1 exons. The exon U3 is positioned 163-kb upstream of E1. The alternative transcript starting from the U3 exon connects to U0 and E2 exons while skipping U2, U1 and E1 exons. Genomic distances are not drawn to scale.

Figure 3. Allele and tissue specificity of the alternative transcripts of Peg3.

(A) A schematic of the mouse Peg3 locus with RT-PCR primer combinations. Gray and black boxes indicate the exons of Usp29 and Peg3, respectively. The transcriptional direction for each gene is represented with arrows and corresponding colors above the map. The arrows below the map indicate the directionality of primers: Peg3-RT-Ex6-R3 was coupled with U0, U1, U2, and U3 specific primers to amplify the respective alternative exons. The DelKO^(+/−p) mutant has a deletion on exon 6 of the Peg3 paternal allele, enabling the detection of its maternal allele expression. Similarly, the DelKO^(−m/+) mutant has a deletion on exon 6 of the Peg3 maternal allele, enabling the detection of Peg3 paternal allele expression. (B) Allele, tissue, and stage specificity of upstream alternative promoters of Peg3. The left RT-PCR panel shows the maternal expression patterns of the identified alternative promoters using total RNA isolated from tissues of DelKO^(+/−p) mice: OB (olfactory bulb), MB (midbrain), HT (hypothalamus), CB (cerebellum), TM (thymus), HR (heart), LG (lung), LV (liver), KD (kidney), FT (fat), TT (testis), OV (ovary), and NB (neonate head). The right RT-PCR panel shows the paternal expression patterns using total RNA isolated from tissues of DelKO^(−m/+) mice: OB (olfactory bulb), MB (midbrain), HT (hypothalamus), CB (cerebellum), and NB (neonate head). The U3-Ex6R3, U2-Ex6R3, and U1-Ex6R3 primer combinations amplified the upstream alternative 1^st exons of Peg3, whereas U0-Ex6R3 primer combination amplified a shared upstream exon of Peg3 to show the expression profile preferred by each upstream exon. The combination of exon 1 and exon 2 primers was used to explore the expression pattern of Usp29. Equal amounts of total RNA were used for RT-PCR, which were further normalized and visualized by β-actin expression levels. We repeated this analysis using three independently derived replicates.

Figure 4. DNA methylation analysis of alternative promoters of Peg3.

(A) DNA methylation analysis of the E1, U1, U2, and U3 promoter regions. Genomic DNA was purified from the different parts of the brain (olfactory bulb, hypothalamus, midbrain, cerebellum) and kidney of a two-month-old female mouse, and then used for bisulfite conversion. The bisulfite-converted DNAs were subsequently amplified with PCR using specific primer sets designed for each promoter region (Table S1). The amplified PCR products were analyzed by COBRA. The restriction enzymes used for each digestion is shown on the left side of the panel, while the right side of the panel indicates the promoter regions under investigation. The red lines indicate methylated DNA whereas the blue lines indicate unmethylated DNA. The methylation levels of U2 and E1 promoter regions were calculated using the ImageJ software for three independent trials. (B) Quantitative methylation analysis of U1 and U3 promoter regions. The bisulfite-converted DNAs were amplified with PCR and used for NGS-based deep sequencing to obtain the methylation level for each adult mouse tissue. A single red box and a blue box indicate a single read for methylated and an unmethylated CpG site, respectively. Each column in the methylation array represents a single CpG site for the respective tissue sample. The U3 and U1 promoter regions represent DNA methylation changes observed from four and six CpG sites, respectively. The overall percentage of methylation is indicated at the bottom of each tissue for comparison. The number of reads are represented by the (n) underneath each tissue sample.

Figure 5. Upstream alternative promoter of human PEG3.

(A) A schematic of the human PEG3 locus with RT-PCR primer combinations. Gray and black boxes indicate the exons of MIMT1 and PEG3, respectively. Transcriptional direction for each gene is represented using arrows with corresponding colors. The black arrow indicates the directionality of Ex2-R1, the primer used for cDNA synthesis. The open oval represents the alternative 1^st exon U1. (B) 5′ RACE analysis for upstream exons of PEG3. The Ex2-R2 indicates the anchoring primer used for nested PCR after 5′ RACE. This primer was coupled with U1 and E1 specific primers to amplify the respective exons. The percentage of transcripts preferred by the adult human brain was calculated by counting the sequences specific for U1 and E1. (C) RT-PCR analysis of upstream alternative exons in human tissues. The RT-PCR panel shows the expression patterns of PEG3 using cDNA from AB (adult brain), NB (neonate brain), HT (adult heart), LV (adult liver), KD (adult kidney) and PC (adult placenta). U1-Ex2R2 and E1-Ex2R2 primer combinations amplified the upstream exons of PEG3 to show the expression profile preferred by each 1^st exon. The Ex1-Ex2 primer combination amplified the expression pattern of MIMT1 in the AB and NB. Equal amounts of total RNA were used for RT-PCR, which were normalized by β-actin expression levels.

Figure 6. Evolutionary conservation of mammalian Peg3 domains.

The 500-kb genomic interval of the Peg3 imprinted domain is represented using the mouse region as a representative locus. The blue and red colors indicate paternally expressed (APeg3, Peg3, Usp29, Zfp264) and maternally expressed genes (Zim1, Zim2, Zim3), respectively. Transcriptional direction for each gene is represented using arrows with corresponding colors. The evolutionarily conserved regions (ECRs) are shown using green lines. The upstream alternative promoters for Peg3 in mouse chromosome 7 are shown using three black arrows. The upstream alternative promoter for PEG3 in human chromosome 19 is shown using a black arrow. The dotted box indicates the genomic region responsible for stillbirth in cows (64, 325, 122-64, 431, 506). The evolutionary conservation of the mammalian Peg3 domains is indicated by the mammal conservation plot, which is represented in green.