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 inhibit H19 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 H19 transcription 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) containing Igf2 and H19 following transfer into differentiated tissue culture cells. Contrary to expectations, both H19 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.

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

“…However, from recent work on transgenic mice it follows that hypomethylation of the H19 upstream element is not sufficient to repress Igf2 in cis. Hence, unmethylated YAC transgene constructs containing the Igf2-H19 region, when transfected into differentiated cells, showed expression of both H19 and Igf2 (59). This, together with the finding (34) that the H19 upstream element functions as a silencer in Drosophila (an organism in which methylation is absent [54]) and as a boundary element on the unmethylated maternal chromosome in the mouse, suggests involvement of epigenetic features other than DNA methylation.…”

Insulators are fundamental components of the eukaryotic genomes

“…When present on the maternal chromosome, its insulator function blocks access of the IGF2 promoter to endodermal enhancers, resulting in exclusive H19 expression. When present on the paternal chromosome, the ICR is methylated and impedes establishment of the insulation (Thorvaldsen et al, 1998;Webber and Tilghman, 1998). ICR, then, has two antagonistic roles depending on its parental origin: either it displays an insulator function or it is implicated in the maintenance of a methylated state of the chromatin (Engel et al, 2004) (Figure 4).…”

The Undermethylated State of a CpG Island Region in Igf2 Transgenes Is Dependent on the H19 Enhancers

“…If the differentiated descendants of these early cells downregulate the island demethylation activity and the de novo methylation activity (as their tissue culture counterparts do), a continuous interaction between the enhancers and the island may no longer be necessary. The idea of a narrow window of time during development when the H19 enhancers are required for protection of the Igf2 CpG island region from methylation has its parallel in recent findings that the H19 enhancers may be required to set Igf2 transcriptional activity only around the time when tissue differentiation is initiated: these enhancers are not necessary for Igf2 activity prior to that (e.g., in undifferentiated ES cells) (Sun et al, 1997) or in constructs introduced into fully differentiated cells (Webber and Tilghman, 1998).…”