Imprinted expression of the Igf2r gene depends on an intronic CpG island

Abstract

Gametic imprinting is a developmental process that induces parental-specific expression or repression of autosomal and X-chromosome-linked genes1,2. The mouse Igf2r gene (encoding the receptor for insulin- like growth factor type-2) is imprinted and is expressed from the maternal allele after embryonic implantation3. We previously proposed that methylation of region 2, a region rich in cytosine-guanine doublets (a ‘CpG island’) in the second intron of Igf2r, is the imprinting signal that maintains expression of the maternal allele4. Here we use mouse transgenes to test the role of region 2 and the influence of chromosome location on Igf2r imprinting. Yeast artificial chromosome transgenes successfully reproduced the imprinted methylation and expression pattern of the endogenous Igf2r gene; deletion of region 2 from these transgenes caused a loss of imprinting and restored biallelic Igf2r expression. These results define a primary role for region 2 and a negligible role for chromosomal location in Igf2r imprinting; they also show that methylation imprints can maintain allelic expression. Short transgenes containing only region 2 and yeast artificial chromosome transgenes with an inactive Igf2r promoter do not attract parental-specific methylation. All transgenes showing paternal-specific repression of Igf2r produced an antisense RNA whose transcription was dependent on region 2. The production of an antisense RNA by the repressed parental allele is reminiscent of the imprinting of the Igf2/H19 gene pair5 and may indicate that expression competition could play a general role in imprinting.

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Figure 1: Cloning of YACs in S. cerevisiae.
Figure 2: a, YAC DNA for microinjection (20?µl) separated by PFGE was hybridized with Plg cDNA; the 300-kb signal indicates intact YAC molecules (ST; slot).
Figure 3: Parental-specific expression and methylation of YAC-T1/P transgenes.
Figure 4: a and b, expression of the region-2-deleted YAC-T1/PΔR2 transgenes.
Figure 5: Methylation of region 2 on short transgenes.

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Acknowledgements

We thank M. King for maintaining the mouse colony; R. Kurzbauer and G. Schaffner for DNA sequencing and oligonucleotide synthesis; F. Sleutels for mapping the line-c transgene; and M.Sibilia, G. Christofori and A. Berns for critical comments. A.W. particularly thanks A. Schedl for help with the YAC protocol and T. Jenuwein for introduction to and help with transgenic technology. Part of this work was supported by the Austrian Industrial Promotion Fund and a Schrödinger Fellowship to O.W.S.

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  1. Correspondence and requests for materials should be addressed to D.P.B.

    Correspondence to Denise P. Barlow.

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    Wutz, A., Smrzka, O., Schweifer, N. et al. Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature 389, 745–749 (1997). https://doi.org/10.1038/39631

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