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Molecular and Cellular Biology, April 2002, p. 2450-2462, Vol. 22, No. 8
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.8.2450-2462.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Analysis of Sequence Upstream of the Endogenous H19 Gene Reveals Elements Both Essential and Dispensable for Imprinting

Joanne L. Thorvaldsen, Mellissa R. W. Mann, Okechukwu Nwoko, Kristen L. Duran, and Marisa S. Bartolomei*

Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received 10 October 2001/ Returned for modification 14 November 2001/ Accepted 12 January 2002

Imprinting of the linked and oppositely expressed mouse H19 and Igf2 genes requires a 2-kb differentially methylated domain (DMD) that is located 2 kb upstream of H19. This element is postulated to function as a methylation-sensitive insulator. Here we test whether an additional sequence 5' of H19 is required for H19 and Igf2 imprinting. Because repetitive elements have been suggested to be important for genomic imprinting, the requirement of a G-rich repetitive element that is located immediately 3' to the DMD was first tested in two targeted deletions: a 2.9-kb deletion ({Delta}DMD{Delta}G) that removes the DMD and G-rich repeat and a 1.3-kb deletion ({Delta}G) removing only the latter. There are also four 21-bp GC-rich repetitive elements within the DMD that bind the insulator-associated CTCF (CCCTC-binding factor) protein and are implicated in mediating methylation-sensitive insulator activity. As three of the four repeats of the 2-kb DMD were deleted in the initial 1.6-kb {Delta}DMD allele, we analyzed a 3.8-kb targeted allele ({Delta}3.8kb-5'H19), which deletes the entire DMD, to test the function of the fourth repeat. Comparative analysis of the 5' deletion alleles reveals that (i) the G-rich repeat element is dispensable for imprinting, (ii) the {Delta}DMD and {Delta}DMD{Delta}G alleles exhibit slightly more methylation upon paternal transmission, (iii) removal of the 5' CTCF site does not further perturb H19 and Igf2 imprinting, suggesting that one CTCF-binding site is insufficient to generate insulator activity in vivo, (iv) the DMD sequence is required for full activation of H19 and Igf2, and (v) deletion of the DMD disrupts H19 and Igf2 expression in a tissue-specific manner.

* Corresponding author. Mailing address: Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. Phone: (215) 898-9063. Fax: (215) 573-6434. E-mail: bartolom{at}mail.med.upenn.edu.

Molecular and Cellular Biology, April 2002, p. 2450-2462, Vol. 22, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.8.2450-2462.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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