A 5′ Differentially Methylated Sequence and the 3′-Flanking Region Are Necessary forH19Transgene Imprinting

Abstract

The mouse H19 gene is expressed exclusively from the maternal allele. The imprinted expression of the endogenous gene can be recapitulated in mice by using a 14-kb transgene encompassing 4 kb of 5'-flanking sequence, 8 kb of 3'-flanking sequence, which includes the two endoderm-specific enhancers, and an internally deleted structural gene. We have generated multiple transgenic lines with this 14-kb transgene and found that high-copy-number transgenes most closely follow the imprinted expression of the endogenous gene. To determine which sequences are important for imprinted expression, deletions were introduced into the transgene. Deletion of the 5' region, where a differentially methylated sequence proposed to be important in determining parental-specific expression is located, resulted in transgenes that were expressed and hypomethylated, regardless of parental origin. A 6-kb transgene, which contains most of the differentially methylated sequence but lacks the 8-kb 3' region, was not expressed and also not methylated. These results indicate that expression of either the H19 transgene or a 3' DNA sequence is key to establishing the differential methylation pattern observed at the endogenous locus. Finally, methylation analysis of transgenic sperm DNA from the lines that are not imprinted reveals that the transgenes are not capable of establishing and maintaining the paternal methylation pattern observed for imprinted transgenes and the endogenous paternal allele. Thus, the imprinting of the H19 gene requires a complex set of elements including the region of differential methylation and the 3'-flanking sequence.

Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2

“…These enhancers are then free to drive the expression of Igf2. Thus, although the DMD is not required for transgenic H19 expression (Pfeifer et al 1996;Elson and Bartolomei 1997), possibly because of the absence of the competing Igf2 gene, it appears to be essential for the optimal and exclusive expression of H19 at the endogenous locus. Second, the maternally derived DMD may form a unique chromatin configuration that promotes transcriptional activity.…”

“…Absence of this region in a normally imprinted mouse transgene results in parental-independent expression and hypomethylation of the derivative transgene (Elson and Bartolomei 1997), indicating that the DMD is crucial for suppression of a paternally transmitted transgene. This element also has unique properties in Drosophila where it acts as a silencer, despite the absence of DNA methylation in the Drosophila genome (Lyko et al 1997).…”

“…This idea is supported by H19 transgenic experiments in which constructs harboring 4 kb of upstream flanking sequence, an internally deleted H19 structural gene and the 3Ј endodermal enhancers are imprinted similarly to the endogenous gene (Bartolomei et al 1993;Pfeifer et al 1996; Elson and Bartolomei 1997). We have proposed that this autonomous regulation is governed by paternal-specific DNA methylation present at the endogenous and transgenic loci (Bartolomei et al 1993).…”

“…A 2-kb region located from −2 to −4 kb relative to the start of transcription is methylated exclusively on the paternal allele throughout development, suggesting that this region is crucial to determining the imprinted expression of H19 (Tremblay et al 1995(Tremblay et al , 1997. When this region was deleted from the original imprinted H19 transgene, the new transgenes were expressed and hypomethylated regardless of parental origin (Elson and Bartolomei 1997).…”

Parental Allele-Specific Chromatin Configuration in a Boundary–Imprinting-Control Element Upstream of the Mouse H19 Gene

“…Detailed methylation studies have pinpointed a core region, localized at approximately 2 to 4 kb upstream of the gene, in which CpG dinucleotides are paternally methylated throughout development (38,52,53,57). Furthermore, a study with transgenic mice has suggested that the H19 upstream region is necessary for the imprinted expression of H19 (13). A targeted deletion of the endogenous H19 gene that included 10 kb of upstream sequences resulted in biallelic expression of the 5Ј neighboring insulin-like growth factor 2 (Igf2) and insulin 2 (Ins2) genes.…”