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. 2004 Jan;24(1):270-9.
doi: 10.1128/MCB.24.1.270-279.2004.

The mouse Murr1 gene is imprinted in the adult brain, presumably due to transcriptional interference by the antisense-oriented U2af1-rs1 gene

Youdong Wang  1 , Keiichiro Joh, Sadahiko Masuko, Hitomi Yatsuki, Hidenobu Soejima, Akira Nabetani, Colin V Beechey, Satoshi Okinami, Tsunehiro Mukai
Affiliations

The mouse Murr1 gene is imprinted in the adult brain, presumably due to transcriptional interference by the antisense-oriented U2af1-rs1 gene

Youdong Wang et al. Mol Cell Biol. 2004 Jan.

Abstract

The mouse Murr1 gene contains an imprinted gene, U2af1-rs1, in its first intron. U2af1-rs1 shows paternal allele-specific expression and is transcribed in the direction opposite to that of the Murr1 gene. In contrast to a previous report of biallelic expression of Murr1 in neonatal mice, we have found that the maternal allele is expressed predominantly in the adult brain and also preferentially in other adult tissues. This maternal-predominant expression is not observed in embryonic and neonatal brains. In situ hybridization experiments that used the adult brain indicated that Murr1 gene was maternally expressed in neuronal cells in all regions of the brain. We analyzed the developmental change in the expression levels of both Murr1 and U2af1-rs1 in the brain and liver, and we propose that the maternal-predominant expression of Murr1 results from transcriptional interference of the gene by U2af1-rs1 through the Murr1 promoter region.

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Figures

FIG. 1.
FIG. 1.
Structure of the mouse Murr1 gene. (A) The mRNA and coding sequence (CDS, shown as gray box) of Murr1. The arrows indicate the primers employed in the allelic expression analysis (MuF1 and UMUR6) and probe syntheses for Northern blot (MuN and UMUR6) and in situ hybridization (Murr1F and Mu3′). The BspHI site, which is polymorphic between the C57BL/6 and PWK strains, is shown above the mRNA. The site exists only in the C57BL/6 sequence. (B) The Murr1 gene consists of three exons and two introns with a length of about 83 kb. An imprinted gene, U2af1-rs1, is located within the first intron of the Murr1 gene. Three CGIs were detected in the region ranging from 10 kb upstream to 10 kb downstream of Murr1.
FIG. 2.
FIG. 2.
Expression of the Murr1 gene in mouse adult tissues. A premade mouse multiple tissue Northern blot (Clontech Co.) was probed with the PCR fragment synthesized with primers MuN and UMUR6 (Fig. 1). The Murr1 gene is expressed ubiquitously but is most abundant in the heart, liver, and testis.
FIG. 3.
FIG. 3.
Analysis of allelic expression of Murr1 and U2af1-rs1. Total RNAs were prepared from various tissues of adult, newborn, and embryonic F1 progeny obtained from crosses between C57BL/6 and PWK mice. Allelic expression analyses of Murr1 in the tissues of adult mice (A) or in the brains of 13.5-dpc embryonic, newborn, 2-, 4-, 6-, and 12-week-old mice (B). The products of hot-stop RT-PCR were digested with BspHI, which digests only the product from the B6 allele (Fig. 1). The ratio of the intensity of maternal product to paternal product is indicated beneath each lane. Allelic expression analyses of U2af1-rs1 in the tissues of adult mice (C) or in the brains of 13.5-dpc embryonic, newborn, 2-week-old, and 12-week-old mice (D). The products of RT-PCR were digested with MspI, which digests only the product from the B6 allele.
FIG. 4.
FIG. 4.
In situ hybridization of Murr1 and U2af1-rs1 mRNAs in mouse brain. RNA probes of Murr1 and U2af1-rs1 were hybridized on brains of C57BL/6 (B6) and PatDp(prox11) mice. Antisense probes (panels a through n) and sense probes as negative control (panels o through q) were used. Shown are coronal sections through the forebrain (a) and the cerebellum (b) of C57BL/6 mice hybridized with Murr1 probe; also shown are magnified images of the frontal cortex (c through e), hippocampus (f through h), thalamus (i through k), and cerebellar cortex (l through q) hybridized with the Murr1 probe on C57BL/6 brain (panels c, f, i, l, and o), Murr1 probe on PatDp(prox11) brain (panels d, g, j, m, and p), and U2af1-rs1 probe on C57BL/6 brain (panels e, h, k, n, and q) are shown. FrC, frontal cortex; Hi, hippocampus; Thal, thalamus; Hypoth, hypothalamus; CA1 to -3, fields CA1 to CA3 of Ammon's horn; DG, dentate gyrus; ParC, parietal cortex; PirC, piriform cortex; CPu, caudate putamen; Amy, amygdala; ic, internal capsule; CbC, cerebellar cortex; 3V, third ventricle. Scale bars, 100 μm.
FIG. 5.
FIG. 5.
Change of the expression of Murr1 and U2af1-rs1 during development in the brain and liver. The expression of Murr1 and U2af1-rs1 was analyzed in the brains and the livers of BPF1 and PBF1 mice during development by quantitative real-time PCR. All the analyses were performed twice for each of the RNA samples. The relative amounts of Murr1 and U2af1-rs1 mRNAs were calculated by normalizing their values with the housekeeping gene Gapdh. Shown are expression levels of Murr1/Gapdh (A), U2af1-rs1/Gapdh (B), and Murr1/U2afi-rs1 (C) in the brains; also shown are expression levels of Murr1/Gapdh (D), U2af1-rs1/Gapdh (E), and Murr1/U2afi-rs1 (F) in the livers. E, 13.5-dpc embryo; N, newborn; 2W, 2-week-old mice; 12W, 12-week-old mice; □, BPF1; ▪, PBF1.
FIG. 6.
FIG. 6.
Strand-specific RT-PCR of the Murr1 gene. (A) Indicated are the locations of the primers for RT (1, 2, 3, 4 and 5) and the regions amplified by PCR in the Murr1/U2af1-rs1 region. (B) RT-PCR in the 300-bp region upstream of Murr1 with the total RNAs of adult brain and liver of an adult C57BL/6 mouse. A transcript with antisense orientation relative to Murr1 was detected in the brain but not in the liver. (C) Transcripts with antisense orientation were also investigated in the intronic region downstream and upstream of U2af1-rs1 with the total RNAs of adult brain and liver. A transcript near the exon 1 of Murr1 was detected in the brain but not in the liver, while the transcript near U2af1-rs1 was detected in both the brain and the liver. The antisense primary transcript was not detected in the region upstream of the U2af1-rs1 gene. All PCRs were performed with 33 amplification cycles. (D) The sequence analysis of the PCR products in the 300-bp region upstream of Murr1. The genomic DNA and the total RNAs were obtained from the brains of the adult F1 progeny from the crosses of B6 and PWK mice. The polymorphic bases are boxed in the sequences.
FIG. 7.
FIG. 7.
The expression-interference model between the Murr1 and U2af1-rs1 genes. The antisense transcription from the U2af1-rs1 gene (starting from the slashed square) is hypothesized to interfere with the Murr1 transcription (starting from the blank square) on the paternal allele and to cause the maternal allele-preferential or -predominant expression in the adult brain.
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