The Absence of Enhancer Competition betweenIgf2 and H19 following Transfer into Differentiated Cells

ABSTRACT

H19 and Igf2 are reciprocally imprinted genes that lie 90 kb apart on mouse chromosome 7. The two genes are coexpressed during development, with the H19 gene expressed exclusively from the maternal chromosome and Igf2 from the paternal chromosome. It has been proposed that their reciprocal imprinting is governed by a competition between the genes for a common set of enhancers. The competition on the paternal chromosome is influenced by extensive allele-specific methylation of the H19 gene and its 5′ flank, which acts to inhibitH19 transcription and thus indirectly leads to the activation of the Igf2 gene. In contrast, no allele-specific methylation has been detected on the maternal chromosome, and the basis for the preference for H19transcription on that chromosome is unresolved. In this investigation, the mechanism controlling the silencing of the Igf2 gene on the maternal chromosome was explored by studying the transcriptional activity of a yeast artificial chromosome (YAC) containingIgf2 and H19 following transfer into differentiated tissue culture cells. Contrary to expectations, bothH19 and Igf2 were expressed from a single integrated copy of the YAC. Furthermore, Igf2 expression appeared to be independent of the H19 locus, based on deletions of the H19 gene promoter and its enhancers. These results suggest that an active process is responsible for the transcriptional bias toward H19 on the maternal chromosome and that the hypomethylated state of this chromosome cannot be viewed as a “default” state. Moreover, the active process is not reproduced in a differentiated cell and may require passage through the female germ line.

ACKNOWLEDGMENTS

We thank the members of the Rose, Broach, and Waters labs for advice and reagents; William Strauss for advice on YAC lipofection; and members of the Tilghman lab, especially Laurie Jo Kurihara and Lisa Sandell, for continued interest and support. We thank Sharon Zemel, who identified the Igf2/H19 YAC.

This work was supported by a grant from the National Institute of General Medical Sciences. S.M.T. is an Investigator of the Howard Hughes Medical Institute.