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Differential expression and imprinting status of Ins1 and Ins2 genes in extraembryonic tissues of laboratory mice
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L. Deltoura, J. Vandammea, Y. Jouvenota,1, B. Duvillie´b, K. Kelemenc, P. Schaerlyd, J. Jamia, A. Paldid,*
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De´partement Ge´ne´tique, De´veloppement et Pathologie Mole´culaire, Institut Cochin, Inserm U567, CNRS UMR 8104, Universite´ Rene´ Descartes, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France b Unite´ 457 INSERM, Hopital Robert Debre´, 48, Bd Se´rurier, 75019 Paris, France c Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, CO 80262, USA d Institut Jacques Monod and EPHE, FRANCE 2, place Jussieu, 75005 Paris, France
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Received 25 February 2004; accepted 20 April 2004
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Abstract
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There are two functional insulin genes in the mouse genome. The Ins2 gene is imprinted and expressed monoallelically from the paternal allele in the yolk sac. In the present study we have re-examined the imprinting status of Ins1. We found that Ins1 is not expressed in the yolk sac of several laboratory mouse strains. The asynchrony of replication at the wild type locus was significantly lower than at imprinted loci and was more similar to non-imprinted loci. Finally, we have taken the advantage of the Ins1neo allele created by homologous recombination to examine the allelic usage at this locus. We observed that the neo gene inserted at the Ins1 locus was expressed from both the paternally and the maternally transmitted allele. Therefore, the Ins1 gene does not share any of the basic properties of imprinted genes. On the basis of these data, we concluded that Ins1 locus is unlikely to be imprinted in common laboratory mice. q 2004 Published by Elsevier B.V.
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Keywords: Insulin; Yolk sac; Monoallelic
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Mice, rat, xenopus and some fishes, have two non-allelic Insulin genes. These genes are both functional and the two proteins are synthetized in the pancreas in a 1:2 ratio for Ins1 and Ins2, respectively (Deltour et al., 1993). The Ins1 gene arose by retrotransposition of a partially processed Ins2 transcript and is highly similar to it but lacks the second intron. Ins1 maps to the telomeric region of the mouse chromosome 19 (Davies et al., 1994). The single Ins gene found in human, pork, chicken and other species has a structure similar to that of the mouse Ins2 gene, i.e. two introns and is considered as the ancestral Ins gene (Wentworth et al., 1986). The ancestral Ins gene is located
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* Corresponding author. Present address: Genethon, 1bis rue de l’Internationale, BP60 91002 Evry Ce´dex, France. Tel.: C33 169 47 12 75; fax: C33 169 47 28 38. E-mail address:
[email protected] (A. Paldi). 1 Present address: Sangamo Biosciences Inc., 501 Canal Blvd, suite A100, Richmond, CA 94804, USA.
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1567-133X/$ - see front matter q 2004 Published by Elsevier B.V. doi:10.1016/j.modgep.2004.04.013
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in an evolutionary conserved imprinted chromosome domain, on the chromosome 7 in mouse and 11p15 in human. Although paralogous, these two mouse Ins genes display a differential pattern of expression in embryos. We showed that during development Ins2 expression is first detected in E8.5 days embryos while Ins1 expression was first detected one day later. We also discovered that Ins2 transcripts but not Ins1 RNA was expressed in the brain (Deltour et al., 1993). Ins2 gene expression has also been shown to be monoallelic in the yolk sac (Giddings et al., 1994; Deltour et al., 1995). We have found that monoallelic paternal expression of Ins2 in the yolk sac depends on the developmental stage. Before E13.5, both alleles are expressed but the maternal allele gradually becomes silenced and after E14.5 only the paternal allele is expressed (Deltour et al., 1995). Although, Giddings et al found that Ins1 was also imprinted in yolk sac, with transcripts of only the paternal
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allele detected in late gestation (Giddings et al., 1994), we did not find any Ins1 expression in this tissue on day 14.5 (Deltour et al., 1995). Because these previous studies were done at different embryonic stages, we reexamined the imprinting status of the Ins1 in late gestation of laboratory mouse embryos. The strains we choose are commonly used in any laboratory and therefore represent universal wild type control. Yolk sacs from 129, Blsw, C57Bl6 and B6CBAF1 laboratory mouse strains were collected at E17.5, RNAs extracted and RT-PCR performed. We used a common set of primers for Ins1 and Ins2 genes that coamplifie both transcripts with the same efficiency (Deltour et al., 1992, 1995). The RT-PCR products were digested by Msp1 and the diagnostic fragments were detected on Southern-blot with an oligonucleotide probe common for Ins1 and Ins2. Fig. 1A shows the result of a semi-quantitative amplification of one individual E17.5 yolk sac from each strain. The same amount of pancreatic RNA was also amplified and, as expected, both transcripts were detected. On the opposite, only Ins2 transcripts were visible on each yolk sac sample. To be absolutely certain of the lack of Ins1 RNA in the samples, we increased the number of amplification cycles up to 40 and we used pooled yolk sacs RNAs. Fig. 1B shows
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Fig. 1. Southern blot analysis of the RT-PCR products of Ins1 and Ins2 in E17 yolk sac of B6CBAF1, C57Bl, Blsw, and 129 mouse foetuses. (A) Analysis of RNAs extracted from individual yolk sacs after 30 cycles of PCR amplification. (B) RT-PCR analysis of Ins1 and Ins2 transcripts in RNAs extracted from pools of five yolk sacs after 40 cycles of amplification. P, pancreas; M, molecular DNA marker.
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Table 1 Analysis of replication synchrony at the Ins1, Ins2–Igf2, H19 and Igf2r loci using FISH analysis
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that even at this very high number of cycles, no competition for the common primers was observed in the pancreas sample since both transcripts were coamplified and no Ins1 transcripts, even residuals, were amplified in any of the yolk sac samples. The amplification of Ins2 but not Ins1 with the common set of primers also confirmed the lack of contaminating DNA in our samples. Although silent in the yolk sac, the Ins1 locus might display other characteristics of imprinting. The two parental homologs of imprinted regions usually replicate asynchronously (Kitsberg et al., 1993; Knoll et al., 1994). This feature of imprinted genes is independent of whether the gene is expressed or not. Therefore, we have examined the replication timing of the Ins1 locus in normal embryonal fibroblasts using a fluorescent in situ hybridisation (FISH) method that provides a sensitive measure of allelic replication asynchrony (Selig et al., 1992). Before replication the two alleles of a gene are detected as two independent spots in an interphase nucleus. After replication two doublets are seen. If only one but not the second allele is replicated, a double and a single signal is observed. A 20 kb genomic probe containing the whole Ins1 gene was hybridised on a non-synchronised fibroblast population and the frequency of single/double signals was detected in interphase nuclei (Table 1). Probes detecting known imprinted loci (Ins2–Igf2, H19 and Igf2r) were included as control. As shown in Table 1, the Ins1 gene probe detected only 21% of single/double (S/D) nuclei in non-synchronised fibroblast population, by contrast to more than 30% S/D nuclei for imprinted genes in the same cells. The replication asynchrony at the Ins1 locus was significantly lower than all of the imprinted loci tested (P!0.001 for Igf2/Ins2 and H19 and P!0.01 for Igf2r) and remains within the limits reported for loci considered as non-imprinted (Kitsberg et al., 1993; Knoll et al., 1994). Finally, a reporter gene inserted in an imprinted region of the genome or replacing an imprinted gene usually becomes imprinted by positional effect. This was the case of the pmc-neo cassette inserted at the Ins2 locus by homologous recombination as part of a larger replacement construct (Duvillie´ et al., 1998). By analogy to Ins2, if the region
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On the upper part a typical image of nuclei with two single dots, one single, one double and two double dots are shown (left to right). The total number of nuclei scored with the given pattern and the corresponding proportion in the population is given for each locus examined.
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containing the Ins1 locus was imprinted, one would expect that the same reporter gene cassette inserted at this locus also becomes imprinted. Therefore, the expression of the neo gene was analysed in mutant Ins1neo/C mice (Duvillie´ et al., 1997). E17.5 yolk sacs of Ins1neo/C foetuses that inherited the targeted allele either maternally or paternally were dissected and the expression of neo, Ins1 and Ins2 were examined by RT-PCR. As shown in Fig. 2, the neo gene was expressed independently of its parental origin. Again, the expression of the Ins1 gene was undetectable in the yolk sac. This observation shows that the pmc-neo cassette inserted at the Ins1 locus is expressed independently of the parental origin. All these experiments show that the Ins1 gene does not have any of the basic properties of imprinted genes. The two non-allelic Ins genes are the only known examples in the mouse genome that code for exactly the same protein product, the well-known glucose uptake regulating hormone. The simple fact that these two genes are regulated differently is important by itself (Deltour et al., 1993). In addition, current theories about the biological significance of genomic imprinting are based on the growth regulating function of imprinted genes (review in Pa`ldi, 2003). Since the two Ins genes code exactly the same peptide hormone, the fact that one of them is not imprinted is unexpected on the basis of the current view and calls for further reflection on the biological role of imprinting. In addition, the present study illustrates well the paradoxical difficulty to prove that a gene is not subject to genomic imprinting.
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Fig. 2. RT-PCR analysis of Ins1 and Ins2 and neo transcripts in the yolk sac of heterozygous E17.5 mouse yolk sacs carrying the pmc-neo gene inserted on the paternal (P) or the maternal (M) chromosome. Ins2 and neo but not Ins1 transcripts were detected. The control wt embryo shows both Ins1 and Ins2 expression.
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Yolk sacs were collected from laboratory mouse strains 129, Blsw, C57Bl6 and B6CBAF1. Mice with the Ins1 gene replaced by the pmc-neo reporter gene by targeted mutation were described earlier (Duvillie´ et al., 1997). Normal foetuses were obtained after mating the females to males of the same genotype. Heterozygous mutant foetuses with
Davies, P., Poirier, C., Deltour, L., Montagutelli, X., 1994. Genetic reassignment of the insulin-1 (Ins1) gene to distal mouse chromosome 19. Genomics 21, 665–667. Deltour, L., Jami, J., Bucchini, D., 1992. Discrimination of homologous mRNAs by polymerase chain reaction. Methods Mol. Cell. Biol. 3, 35–38. Deltour, L., Leduque, P., Blume, N., Madsen, O., Dubois, P., Jami, J., 1993. Differential expression of the two nonallelic proinsulin genes
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The authors are grateful to Lucianne Lamotte for assistance and Takuya Imamura for helpful discussions and critical reading of the manuscript.
1. Materials and methods
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the mutant allele transmitted either by the father or by the mother were obtained by mating homozygous mutant animals with wild type mice (C57BlxCBA/J F1). Individual visceral yolk sacs were dissected out on day 17.5 of development. The tissues were stored at K80 8C individually. Wild type embryos at the same developmental stage and adult pancreas were used as control to expression studies. The RNAs were extracted from individual tissues using a kit (Rnaxel, Eurobio). Ten micrograms of RNAs were treated by RNase–free DNase to eliminate residual genomic DNA. Neo, Ins1 and Ins2 transcripts were amplified by RTPCR on 250 ng of RNAs as described earlier (Deltour et al., 1992, 1995). The lack of genomic DNA contamination was systematically controlled by PCR without reverse transcription (not shown). Embryonic fibroblasts were obtained from E12.5 wild type embryos. The cells were grown in DMEM medium. Rapidly growing cells were incubated in 100 mg/ml BrdU for 30 min in order to label nuclei in S-phase. Then the cells were treated with a hypotonic solution and fixed in cold ethanol/acetic acid. Fluorescent in situ hybridisation (FISH) using DIG-labelled genomic probes was performed according to the standard protocol. We used a 22 kb genomic probe for Ins1 (Duvillie´ et al., 1997), a 40 kb cosmid probe for H19, a cosmid probe for Igf2r (kindly provided by D. Barlow) and a plasmid covering the Ins2–Igf2 intergenic region (kindly provided by W. Reik). The probes were detected with FITC-conjugated anti-DIG sheep antibody (Roche) and FITC-conjugated anti-sheep secondary antibody (Vector Laboratories). Incorporated BrdU was detected with a biotinylated monoclonal mouse anti-BrdU antibody (Zymed Laboratories) and Texas Red Avidin DCS (Vector Laboratories). The assay was performed three times and the difference between the replication of Ins1 and all the other genes was always statistically significant. Therefore, the results were merged. The statistical significance was calculated using the standard c2 test on the basis of the merged numbers.
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Kitsberg, D., Selig, S., Brandeis, M.I.S., Keshet, I., Driscoll, D.J., Nicholls, R.D., Cedar, H., 1993. Allele-specific replication timing of imprinted gene regions. Nature 364, 459–463. Knoll, J., Cheng, S., Lalande, M., 1994. Allele specificity of DNA replication timing in the Angelman/Prader-Willi syndrome imprinted chromosomal region. Nat. Genet. 6, 41–46. Pa`ldi, A., 2003. Genomic imprinting: could the chromatin structure be the driving force?. Curr. Top. Dev. Biol. 53, 115–138. Selig, S., Okumura, K., Ward, D.C., Cedar, H., 1992. Delineation of replication time zones by fluorescence in situ hybridization. Eur. Mol. Biol. Org. J. 11, 1217–1225. Wentworth, B.M., Schaeffer, I.M., Villa-Komaroff, L., Chirgwin, J.M., 1986. Characterization of the two non-allelic genes encoding mouse preproinsulin. J. Mol. Evol. 23, 305–312.
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