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Department of Biomolecular Sciences,1 Department of Anatomy and Physiology,2 Department of Ophthalmology, Saga Medical School, Saga 849-8501,5 Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan,3 Mammalian Genetics Unit, Medical Research Council, Harwell, Didcot, Oxon 0X11 0RD, United Kingdom4
Received 5 May 2003/ Returned for modification 10 July 2003/ Accepted 2 October 2003
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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U2af1-rs1 is a mouse imprinted gene and is transcribed exclusively from the paternal allele in embryonic and neonatal mice (16, 17). This gene is intronless and located within the first intron of Murr1. It is transcribed in the direction opposite of that of the host gene in the proximal region A3.2 of mouse chromosome 11 (27). The presence of two maternal copies of this region resulted in prenatal growth retardation, while two paternal copies resulted in fetal overgrowth (6). The U2af1-rs1 gene was supposedly created by retrotransposition during mouse evolution after the divergence of mice and humans, as the human MURR1 gene does not contain the homologue of the U2af1-rs1 gene within its introns (reference 27 and unpublished data).
Imprinted genes are a suitable model system for the study of epigenetic control of gene expression because their allelic expressions are essentially controlled by this mechanism. We initiated the analysis of the Murr1 gene in order to investigate the imprinting mechanisms of the U2af1-rs1 locus. The U2af1-rs1 gene was the only gene reported to be imprinted in this chromosomal region at that time. It was necessary to know if there were other imprinted genes around the U2af1-rs1 locus because clustered imprinted genes are thought to be controlled by a common regulatory mechanism rather than being independently regulated (10, 30). Although Murr1 has been reported to be expressed biallelically in the neonatal mouse (27), we have analyzed Murr1 expression in the tissues of both adult and embryonic mice because allelic expression of some imprinted genes is tissue- and developmental stage specific (7, 15, 24, 38). In this study we have found that the expression of the Murr1 gene shows a moderate bias of expression toward the maternal allele in the adult tissues analyzed so far. Moreover, this gene showed a predominant expression of the maternal allele specifically in adult brain but not in brains of neonatal or embryonic mice. We proposed that this mechanism might come from transcriptional interference by antisense-oriented U2af1-rs1.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice with two paternal copies of proximal chromosome 11, including the Murr1 locus (paternal duplication [PatDp] prox11), were produced by a standard protocol using the mouse reciprocal translocation T(7;11)40Ad and identified by using genetic markers (4).
Allelic expression of the Murr1 and U2af1-rs1 genes.
Total RNAs were isolated with ISOGEN (Nippon Gene) from various tissues of adult F1 progeny obtained by reciprocal crosses between C57BL/6 and PWK mice. Contaminating chromosomal DNA was removed by DNase I treatment. Reverse transcription was conducted with oligo(dT) primers according to the manufacturer's protocol of the RNA PCR kit (Takara). To exclude the problem of heteroduplex formation skewing the results of restriction endonuclease digestion, hot-stop reverse transcription-PCR (RT-PCR) was employed (40). Thirty cycles of PCR with primers MuF1 (5'-TGGAGGGTGGCAAGTCCCTG-3') and UMUR6 (5'-GGTAACACCAGTGGGCAAAG-3') (Fig. 1) were followed by one cycle of amplification with primer UMUR6 labeled with 
-32P. The resulting 686-bp fragment was digested with BspHI and electrophoresed. The intensity of electrophoretic bands was measured by using BAS2000 (Fujifilm). For the analysis of U2af1-rs1 gene, a regular PCR with primers 5'-U2af1rs1 (5'-AGATAACCACGGATACCTGG-3') and 3'-U2af1rs1 (5'-AGTACATAGGCCTGCCCATG-3') was performed. The resulting 236-bp PCR product was digested with MspI and electrophoresed.
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The kinetics of PCR amplification was monitored as the kinetics of enhancement of the fluorescence of SYBR Green (a dye specific for double-stranded DNA) in the reactions. The relative amounts of three mRNAs were obtained by determining the numbers of cycles required to obtain the same levels of fluorescence in each PCR. In order to evaluate the quantification of this assay and to condition the PCRs, pilot experiments were performed by using serially diluted cDNAs of these mRNAs as the templates according to the manufacturer's protocol.
In situ hybridization of Murr1 mRNAs in mouse brain. The fresh cuts of brains were fixed immediately in paraformaldehyde after removal and sectioned after being embedded in paraffin. The product of RT-PCR with primers Murr1F (5'-TAGCGCAGAACGCCTTTCAC-3') and Mu3' (5'-ATCCTTGAAGACTTTCATGC-3') (Fig. 1) was cloned into the pT7Blue T-vector (Takara) and used as the template for in vitro transcription of antisense and sense single-stranded RNA probes for Murr1. The antisense and sense RNA probes for U2af1-rs1 were prepared in the same manner from the RT-PCR product with primers U2F (5'-GGAAACACGACTTTCCCACG-3') and U2AluI-R (5'-GCTGTCCTCTTCTCTTCCTC-3'). The digoxigenin-labeled antisense and sense probes were synthesized with T7 RNA polymerase and digoxigenin-UTP according to the manufacturer's protocol (RNA labeling and detection kit; Roche). The transcripts were used as probes at a concentration of 10 ng/µl in hybridization buffer. The hybridized probe was detected with alkaline phosphatase-coupled antibodies to digoxigenin according to the manufacturer's protocol (RNA labeling and detection kit; Roche).
Methylation analyses. The DNA methylation status of the CpG islands (CGIs) was assessed by sodium bisulfite PCR and sequencing assay as described previously (46). Briefly, genomic DNA from the brain of a C57BL/6 mouse was extracted by standard phenol-chloroform extraction methods and treated with sodium bisulfite prior to PCR with each primer set. PCR products were cloned into pT7Blue T-vector, and each cloned PCR product was amplified by colony PCR using a Takara Ex Taq Kit (Takara Shuzo). The amplified products were subjected to sequencing reaction using BigDye (Applied Biosystems) and DYEnamic ET terminator kits (Amersham Pharmacia Biotech) after treatment with exonuclease I and shrimp alkaline phosphatase (both from Amersham Pharmacia Biotech).
Detection and quantification of the primary transcript of the Murr1 and U2af1-rs1 by strand-specific RT-PCR and semiquantitative RT-PCR. DNase I-treated total RNAs (0.25 µg) isolated from the brain, liver, heart, or lung of adult mice using ISOGEN (Nippon Gene) were mixed with 5 pmol of either forward primers 1, 3, 4, and 5 or reverse primer 2 (see Fig. 6) in a final volume of 10 µl; the RNAs were then reverse transcribed for 30 min at 55°C, heat inactivated at 99°C for 5 min, and cooled on ice. Following the reverse transcription and denaturation, 33 PCR cycles with four different primer sets corresponding to the primers for reverse transcription were performed, respectively.
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RESULTS |
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Predominant maternal expression was observed in all regions of adult brain. Maternal allele-predominant expression of the Murr1 gene in the adult brain may suggest a function of the gene in the development of the brain after birth. In order to gain insight into this function, we determined the regions expressing the gene by RNA in situ hybridization on the adult brains of C57BL/6 mice (Fig. 4a, b, c, f, i, and l). The expression of Murr1 was observed in all the regions of the cerebrum, cerebellum, and medulla oblongata. The hippocampus region of the cerebrum had the highest levels of expression. The expression was seen mainly in neuronal cells.
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The CGIs associated with the Murr1 promoter region are not methylated. Three CGIs have been identified in the region spanning from 10 kb upstream to 10 kb downstream of the Murr1 gene (Fig. 1). CGI1 is located approximately 8 kb upstream from the Murr1 gene, and CGI2 spans from the promoter region to the 5' part of exon 1. CGI3 is the region spanning from the promoter to the body of the U2af1-rs1 gene. CGI3 has been reported to be methylated only on the maternal allele and has been thought to be involved in the paternal expression of U2af1-rs1 (17). These three CGIs were analyzed for methylation in the brain genome by a sodium bisulfite sequencing method (data not shown). One can expect that the CGIs 1 and/or 2 may be paternally methylated if differential methylation is involved in the maternal expression of Murr1. The CGI3 was found to be differentially methylated in the brain as reported previously. On the other hand, no methylation was found in the other two CGIs, 1 and 2. Nonmethylation of these CGIs suggests that differential methylation within the genome is not involved in the allelic expression of Murr1, in contrast to many of the imprinted genes.
The expression levels of Murr1 and U2af1-rs1 change inversely during brain development. In the adult brain, U2af1-rs1 and Murr1 show reciprocal allelic expression. The U2af1-rs1 gene is expressed exclusively from the paternal allele, and the Murr1 gene is expressed predominantly from the maternal allele. This fact suggests that there may be some kinds of interaction, such as transcriptional interference, between allelic expressions of these genes. In order to investigate this possibility, the mRNA levels of these genes were quantified in the brain and liver during development by quantitative real-time RT-PCR (Fig. 5).
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On the other hand, the expression of both genes decreased simultaneously during development in the liver, in which the maternal allele-predominant expression of Murr1 does not occur (Fig. 5D and E). Relative levels of expression of U2af1-rs1 and Murr1 do not change significantly during development (Fig. 5F). The expression of Murr1 is more than 10-fold higher than that of U2af1-rs1 in both embryonic and adult periods.
Transcription from U2af1-rs1 may act through the promoter region of Murr1. Antisense orientation of the U2af1-rs1 gene relative to Murr1 suggests the possibility that transcription originating from U2af1-rs1 promoter overlaps the promoter region of the Murr1 gene and interferes with Murr1 transcription. The 3' end of a mature mRNA is created by the cleavage of the primary transcript. A primary transcript is synthesized beyond the end of the gene, sometimes far more than a distance of 1 kb (29). If this is because of the paternal expression of U2af1-rs1, this interference should occur only on the paternal allele and may cause the reduction of the paternal expression of Murr1. We employed strand-specific and semiquantitative RT-PCR to detect and quantify the transcripts in the region downstream of U2af1-rs1 up to the promoter region of Murr1 (Fig. 6).
In the analysis performed using adult brain RNA, the transcript was detected in the promoter region when primer 1 with sense orientation was used for cDNA synthesis. In contrast, no transcript was detected in this region when primer 2 with antisense orientation was used as the primer for cDNA synthesis (Fig. 6B). This result indicated that the Murr1 promoter region was transcribed in the antisense direction relative to the Murr1 gene. The sequencing of the PCR product revealed that the transcript was expressed exclusively from the paternal allele (Fig. 6D). On the other hand, when liver RNA was used for this analysis, no transcript was detected with either of the primers for cDNA synthesis using PCR with same number of amplification cycles (33 cycles) (Fig. 6B). An antisense transcript was, however, detected in the liver when PCR was done with 36 amplification cycles (data not shown). In the brain, transcripts with antisense orientation relative to Murr1 were also detected at two sites in the first intron downstream of U2af1-rs1. Only the transcript at the site close to U2af1-rs1 was detected in the liver, however. No transcript with antisense orientation was detected at the promoter region of U2af1-rs1 in either the brain or liver (Fig. 6C). These results strongly suggest that transcription from the U2af1-rs1 promoter to the upstream region of the Murr1 gene on the paternal chromosome occurs more abundantly in the brain than in the liver, although we have no direct evidence that these antisense transcripts are parts of one continuous transcript. This finding implies that the transcription of U2af1-rs1 interferes with the expression of the paternal allele of Murr1 in the adult brain. If this is the case, U2af1-rs1 should be expressed in the regions where Murr1 is expressed in the brain. This has been shown to be true by in situ hybridization, in which the expression of U2af1-rs1 observed in the regions was essentially the same as that of Murr1 expression in C57BL/6 brain (Fig. 4e, h, k, and n). We also detected the same antisense transcripts at the Murr1 promoter in lung and kidney cells. Semiquantitative RT-PCR indicated that the primary antisense transcript in the Murr1 promoter region was much more abundant in the adult brain than in the heart, lung, and liver (data not shown).
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DISCUSSION |
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Our preliminary analysis indicates that MURR1, the human homologue of mouse Murr1, is expressed biallelically in the adult brain and contains no imprinted gene in the first intron (Zhang et al., unpublished data). This strongly suggests that the antisense gene, U2af1-rs1, causes the maternal expression of Murr1 in the mouse. The expressions of Murr1 and U2af1-rs1 show a reverse change during the development of the brain (Fig. 5A through C). Murr1 expression decreases during brain development, whereas U2af1-rs1 expression increases. Given the assumption that the mRNA levels reflect the transcriptional activities of these genes, these results indicate that relative transcriptional activity of U2af1-rs1 is increasing compared to that of Murr1 during brain development. On the other hand, analyses by strand-specific RT-PCR suggested that the transcription from the U2af1-rs1 promoter reaches to the promoter region of Murr1 on the paternal allele in adult brain, liver, kidney, and lung to a different extent (Fig. 6B and C and data not shown). A possible hypothesis is that the transcriptional interference of the U2af1-rs1 gene against Murr1 causes maternal allele-preferential or -predominant expression of the Murr1 gene (Fig. 7). In this hypothesis, we assume that the transcription from U2af1-rs1 gene may interfere with Murr1 transcription, possibly by its initiation on the paternal allele, and cause a reduction in Murr1 expression dependent on the balance of the transcriptional activities of both genes. Transcriptional interference has been reported in yeast, where two genes are closely spaced and transcribed in the same direction. The transcription of the downstream gene is affected when the upstream gene is expressed (13). A similar phenomenon has been reported in mammalian cells (14).
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Another possible explanation for the developmental stage-specific and tissue-specific imprinting is the usage of distinct promoters, such as those reported on the human imprinted gene IGF2. Among four promoters of this gene, the expression of the P1 promoter is biallelic, whereas those of the P2 to P4 promoters are paternal allele specific. In human liver, the P1 promoter and the P3 and P4 promoters are used primarily in the adult and fetal stages, respectively. This promoter switch causes fetal stage-specific imprinting of the gene in the human liver (43). We have obtained evidence for a single promoter of the Murr1 gene by 5' rapid amplification of cDNA ends. However, performing other assays, such as S1 mapping or RNase protection, would be needed in order to confirm a single promoter of this gene and to exclude the alternative explanation.
Antisense RNAs have been found previously to be associated with imprinted genes (33). The representative pairs of imprinted gene with antisense RNA are Igf2r/Air, Ube3A/Ube3A-ATS, Kvlqt1/Lit1, and Nesp/Nespas. These antisense RNAs are very long noncoding RNAs (more than 100 kb), and their genes are imprinted oppositely to the partner gene (21, 25, 32, 44). They are suggested to have a role in establishing and/or maintaining imprinting of the partner genes. For example, the antisense RNA, Air, has been shown to function in cis to repress not only the partner, Igf2r, but also two other imprinted genes located upstream of Air (35). Premature disruption of Air transcription resulted in the loss of imprinting of all three genes. The imprinting of the upstream genes cannot be due to the sort of transcriptional interference discussed here. One hypothesis is that the Air RNA functions in cis to cause a repressed chromatin state in the Igf2r-imprinted cluster in a way similar to that of Xist RNA in X-chromosome inactivation (2). Although we cannot rule out the possibility that U2af1-rs1 RNA also harbors such a cis-acting function to form a repressed chromatin state in this locus, the transcriptional interference is more plausible because of the following reasons. First, U2af1-rs1 is expressed paternally in all the tissues, whereas adult brain is the only tissue in which Murr1 is imprinted. In contrast, the pairing of Igf2r and Air shows reciprocal imprinting in many adult tissues (18). Second, we have searched imprinted genes in 500-kb genomic region around Murr1, and no neighboring genes were imprinted (Zhang et al., unpublished data). Third, U2af1-rs1 RNA is a protein-coding mRNA and should diffuse out to the cytoplasm. Harboring cis-acting functions may be difficult in this kind of RNA.
In contrast to the clustering of many imprinted genes, the Murr1/U2af1-rs1 locus seems to be an isolated imprinted locus. U2af1-rs1 is thought to be a neomorphic gene caused by retrotransposition (27). This gene shows complete imprinted expression, whereas Murr1, containing U2af1-rs1, shows incomplete imprinting, that is, maternal-predominant or -preferential expression. The mouse Bc10/Nnat locus shares some features with the Murr1/U2af1-rs1 locus. Nnat is expressed exclusively from the paternal allele and is associated with a maternally and differentially methylated region, and it resides within the intron of a gene, Bc10, which is transcribed in the opposite direction (20). They have some differences, however. The imprinted gene Nnat contains introns, unlike U2af1-rs1. The host gene, Bc10, is expressed biallelically, although the analysis was done only in embryos. Moreover, a similar arrangement described as "a microimprinted domain" was reported recently for the imprinted genes Nap115 (chromosome 6) and Peg13 (chromosome 15) (36). As is true for the Nnat gene, the Nap115 and Peg13 genes are also expressed exclusively from the paternal allele and located within the introns of other biallelically expressed genes. In order to know whether these loci are isolated imprinted loci, one should analyze neighboring genes on their allelic expression. We are performing this line of analyses with the Murr1/U2af1-rs1 locus, and all the neighboring genes are not imprinted in 500-kb region around Murr1 as mentioned above. The studies on these isolated loci may be valuable for studies on imprinting mechanisms because of their simplicity and their imprinting mechanisms, which possibly differ from those of large imprinted domains.
DNA methylation can serve as a signal to mark the parental alleles. Allele-specific methylation patterns are a common feature of most imprinted genes (9, 12, 37). Such differentially methylated regions (DMRs) are usually located within CGIs, in contrast to the general observation that CGIs are unmethylated. Many of the DMRs have important functions in the allele-specific expression of the imprinted genes (22, 34). Contrary to this general association of DMRs with imprinted genes, the CGIs found in the Murr1 locus are not methylated on both alleles in brain cells, except for the maternally methylated CGI3 spanning from the promoter to the body of theU2af1-rs1 gene. The DMR in CGI3 is considered to be involved in the paternal expression of U2af1-rs1. This observation suggests that the allelic expression of Murr1 does not depend on differential DNA methylation of the genome. Similar results were observed for the Kip2/Lit1 subdomain of mouse chromosome 7F5 (46). There are seven imprinted genes in this region, and no DMR was found in three out of six genes carrying CGIs in the promoter. There was an imprinting control region (ICR) in this subdomain, and results suggested that this ICR governs the imprinted expression of those seven imprinted genes (11). Similarly, an ICR would also probably exist in the Murr1/U2af1-rs1 region. CGI3 is one such candidate. To clarify if this is the case, it would be necessary first to determine the span of the imprinting domain, including Murr1/U2af1-rs1, and then to search for an ICR in this domain.
Moderate preferential expression of the maternal allele is the general expression pattern of the Murr1 gene (Fig. 3A and B). This maternal preference is higher in tissues of BPF1 mice than in those of PBF1 mice. This difference between these F1 mice may be due to the disparity in promoter activities of Murr1 alleles of the mouse strains C57BL/6 and PWK. It can be assumed that the promoter of the C57BL/6 allele may have higher activity than that of the PWK allele. In the PBF1 mouse, in spite of a mechanism to express the maternal allele preferentially, the greater activity of the paternal allele makes the difference between the two alleles small. In BPF1, the C57BL/6 allele is maternal, and this situation enhances the allelic difference. We have, in fact, found some polymorphisms in the promoter region of the Murr1 gene between these two strains, although we have not analyzed whether these sequence variations affect the promoter activity.
The dog Murr1 homologue has recently been reported (42). Exon 2 of the canine MURR1 gene was found to be deleted in both alleles of a certain purebred dog population that suffered from copper toxicosis. Copper toxicosis is characterized by accumulation of copper in the liver and subsequently in the brain and other organs as a result of inefficient biliary excretion of copper. The presence of the mutation suggests that MURR1 is the disease-causing gene in copper toxicosis in dogs. Therefore, it is possible that the mouse Murr1 gene is also involved in copper metabolism. In order to analyze the function of the gene, it would be necessary to produce mice with a deletion of the gene.
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ACKNOWLEDGMENTS |
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This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology in Japan.
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FOOTNOTES |
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, BPF1; 
, PBF1.