The 3' portion of the mouse H19 Imprinting-Control Region is required for proper tissue-specific expression of the Igf2 gene.

Genomic imprinting at the H19/Igf2 locus is governed by a cis-acting Imprinting-Control Region (ICR), located 2 kb upstream of the H19 gene. This region possesses an insulator function which is activated on the unmethylated maternal allele through the binding of the CTCF factor. It has been previously reported that paternal transmission of the H19(SilK) deletion, which removes the 3' portion of H19 ICR, leads to the loss of H19 imprinting. Here we show that, in the liver, this reactivation of the paternal H19 gene is concomitant to a dramatic decrease in Igf2 mRNA levels. This deletion alters higher-order chromatin architecture, Igf2 promoter usage and tissue-specific expression. Therefore, when methylated, the 3' portion of the H19 ICR is a bi-functional regulatory element involved not only in H19 imprinting but also in 'formatting' the higher-order chromatin structure for proper tissue-specific expression of both H19 and Igf2 genes.

A conserved imprinting control region at the HYMAI/ZAC domain is implicated in transient neonatal diabetes mellitus.

Transient neonatal diabetes mellitus (TNDM) is associated with intra-uterine growth retardation, dehydration and a lack of insulin. Some TNDM patients exhibit paternal uniparental disomy (UPD) of chromosome 6q24, where at least two imprinted genes, HYMAI and ZAC, have so far been characterized. Here we show that the differentially methylated CpG island that partially overlaps mZac1 and mHymai at the syntenic mouse locus is a likely imprinting control region (ICR) for the approximately 120--200 kb domain. The region is unmethylated in sperm but probably methylated in oocytes, a difference that persists between parental alleles throughout pre- and post-implantation development. We also show that within this ICR, there is a region that exhibits a high degree of homology between mouse and human. Using a reporter expression assay, we demonstrate that this conserved region acts as a strong transcriptional repressor when methylated. Finally, we provide in vivo evidence that in the majority of TNDM patients with a normal karyotype, there is a loss of methylation within the highly homologous region. We propose that this ICR regulates expression of imprinted genes within the domain; epigenetic or genetic mutations of this region probably result in TNDM, possibly by affecting expression of ZAC in the pancreas and/or the pituitary.

Epigenetic reprogramming of the genome--from the germ line to the embryo and back again.

Mammalian parental genomes are not functionally equivalent, and both a maternal and paternal contribution is required for normal development. The differences between the parental genomes are the result of genomic imprinting--a form of gene regulation that results in monoallelic expression of imprinted genes. Cis-regulatory elements at imprinted loci are responsible for directing allele-specific epigenetic marks required for correct gene expression. This cis information must be interpreted at various points in development, including in the germline where existing imprints are erased and reset. Imprints must also be maintained during preimplantation development, when the genome undergoes dramatic global epigenetic changes.

A novel imprinted gene, HYMAI, is located within an imprinted domain on human chromosome 6 containing ZAC.

Transient neonatal diabetes mellitus (TNDM) is a rare disease characterized by intrauterine growth retardation, dehydration, and failure to thrive due to a lack of normal insulin secretion. This disease is associated with paternal uniparental disomy or paternal duplication of chromosome 6, suggesting that the causative gene(s) for TNDM is imprinted. Recently, Gardner et al. (1999, J. Med. Genet. 36: 192-196) proposed that a candidate gene for TNDM lies within chromosome 6q24.1-q24.3. To find human imprinted genes, we performed a database search for EST sequences that mapped to this region, followed by RT-PCR analysis using monochromosomal hybrid cells with a human chromosome 6 of defined parental origin. Here we report the identification of a novel imprinted gene, HYMAI. This gene exhibits differential DNA methylation between the two parental alleles at an adjacent CpG island and is expressed only from the paternal chromosome. A previously characterized imprinted gene, ZAC/LOT1, is located 70 kb downstream of HYMAI and is also expressed only from the paternal allele. In the pancreas, both genes are moderately expressed. HYMAI and ZAC/LOT1 are therefore candidate genes involved in TNDM. Furthermore, the human chromosome 6q24 region is syntenic to mouse chromosome 10 and represents a novel imprinted domain.

Deletion of a silencer element disrupts H19 imprinting independently of a DNA methylation epigenetic switch.

The H19 imprinted gene is silenced when paternally inherited and active only when inherited maternally. This is thought to involve a cis-acting control region upstream of H19 that is responsible for regulating a number of functions including DNA methylation, asynchronous replication of parental chromosomes and an insulator. Here we report on the function of a 1.2 kb upstream element in the mouse, which was previously shown to function as a bi-directional silencer in Drosophila. The cre-loxP-mediated targeted deletion of the 1.2 kb region had no effect on the maternal allele. However, there was loss of silencing of the paternal allele in many endodermal and other tissues. The pattern of expression was very similar to the expression pattern conferred by the enhancer elements downstream of H19. We could not detect an effect on the expression of the neighbouring imprinted Igf2 gene, suggesting that the proposed boundary element insulating this gene from the downstream enhancers was unaffected. Despite derepression of the paternal H19 allele, the deletion surprisingly did not affect the differential DNA methylation of the locus, which displayed an appropriate epigenetic switch in the parental germlines. Furthermore, the characteristic asynchronous pattern of DNA replication at H19 was also not disrupted by the deletion, suggesting that the sequences that mediate this were also intact. The silencer is therefore part of a complex cis-regulatory region upstream of the H19 gene and acts specifically to ensure the repression of the paternal allele, without a predominant effect on the epigenetic switch in the germline.

A silencer element identified in Drosophila is required for imprinting of H19 reporter transgenes in mice.

The H19 gene is subject to genomic imprinting because it is methylated and repressed after paternal inheritance and is unmethylated and expressed after maternal inheritance. We recently identified a 1.1-kb control element in the upstream region of the H19 gene that functions as a cis-acting silencer element in Drosophila. Here we investigate the function of this element in mice. We demonstrate that both H19-lacZ and H19-PLAP reporter transgenes can undergo imprinting with repression and hypermethylation after paternal transmission at many integration sites. However, transgenes that were deleted for the 1.1-kb silencer element showed loss of paternal repression, but they did not show marked changes in the paternal methylation of the remaining upstream region. This study demonstrates that the 1.1-kb control element identified in Drosophila is required to silence paternally transmitted H19 minitransgenes in mice.