Epigenetic and Phenotypic Consequences of a Truncation Disrupting the Imprinted Domain on Distal Mouse Chromosome 7

Abstract

The distal end of mouse chromosome 7 (Chr 7) contains a large cluster of imprinted genes. In this region two cis-acting imprinting centers, IC1 (H19 DMR) and IC2 (KvDMR1), define proximal and distal subdomains, respectively. To assess the functional independence of IC1 in the context of Chr 7, we developed a recombinase-mediated chromosome truncation strategy in embryonic stem cells and generated a terminal deletion allele, DelTel7, with a breakpoint in between the two subdomains. We obtained germ line transmission of the truncated Chr 7 and viable paternal heterozygotes, confirming the absence of developmentally required paternally expressed genes distal of Ins2. Conversely, maternal transmission of DelTel7 causes a midgestational lethality, consistent with loss of maternally expressed genes in the IC2 subdomain. Expression and DNA methylation analyses on DelTel7 heterozygotes demonstrate the independent imprinting of IC1 in absence of the entire IC2 subdomain. The evolutionarily conserved linkage between the subdomains is therefore not required for IC1 imprinting on Chr 7. Importantly, the developmental phenotype of maternal heterozygotes is rescued fully by a paternally inherited deletion of IC2. Thus, all the imprinted genes located in the region and required for normal development are silenced by an IC2-dependent mechanism on the paternal allele.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Titia de Lange for the cloned array of telomeric repeats, Jörn Walter and Jacquetta Trasler for help with the IC2 bisulfite sequencing protocols, Marisa Bartolomei for sharing results before publication, and Aaron Bogutz for help with blastocyst collections. We thank Wendy Robinson and Meaghan Jones for comments on the manuscript, and Vincenzo Pirrotta for bringing the Hox gene data to our attention.

All animal experiments were performed under certificate A03-0289 from the UBC Animal Care Committee and complied with the national CCAC guidelines to the care and use of experimental animals.

This work was supported in part by the CIHR operating grants MOP-64193 (to L.L.) and FRN-13687 (to A.N.). L.L. is a Michael Smith Foundation for Health Research Scholar and holds a Canada Research Chair.