The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.
The mouse H19 gene was identified as an abundant hepatic fetal-specific mRNA under the transcriptional control of a trans-acting locus termed raf. The For the majority of cloned eucaryotic genes, their protein products were already known prior to cloning. More recently, differential hybridization screens of cDNA libraries have been used to clone genes with specific tissue and temporal patterns of expression, without regard to their products. The murine H19 gene is such a gene. It was originally identified in a screen of a fetal liver cDNA library to find genes that were coordinately regulated with the a-fetoprotein (AFP) gene (25). The aim of the screen was to find fetal-specific mRNAs under the control of raf, a transacting locus that determines, at least in part, the adult basal level of AFP mRNA (2, 24). The only cDNA that fulfilled these criteria was the H19 cDNA. This gene was expressed at high levels in fetal and neonatal liver, and its 600-fold repression in the adult was under the control of raf. Its coordinate expression with AFP extended to the visceral endoderm of the yolk sac and the fetal gut. However, unlike AFP, H19 mRNA was also found in both fetal skeletal and cardiac muscle at high levels and at reduced levels in all adult muscle, although in these tissues it is not under the influence of raf (2).Sequence analysis of the single-copy H19 gene revealed the presence of multiple small open reading frames (ORFs), none of which spanned more than two of its five exons. The largest of these, called ORF5 (referred to as ORF1 in reference 26), was located entirely within the first exon and could potentially encode a 132-amino-acid protein. This ORF begins 680 bases downstream of the cap site and is preceded by four small ORFs, the largest of which could encode a protein of 27 amino acids. As there was no precedent for such a gene organization, we decided to investigate its possible significance.As a first step, we cloned and sequenced the H19 gene from the human genome, reasoning that a comparison between the murine and human homologs would identify regions that are conserved and therefore important to the function of the gene. The demonstration that the genes shared no ORF led us to investigate the subcellular distribution of H19 mRNA, which indicated that the majority of the mRNA is located in a cytoplasmic particle. * Corresponding author. MATERIALS AND METHODScDNA library construction. A 20-,Ig sample of poly(A)+ RNA from Hep3B human hepatoma cells in 90 ,l of distilled H20 was heat denatured at 65°C for 3 min and then placed at 4°C. To this was added 20 ,ug of oligo(dT) (Boehringer Mannheim Biochemicals), 80 U of RNasin (Promega Biotec), 1.5 mM each dGTP, dATP, dCTP, and dTTP, 50 mM Tris hydrochloride (pH 8.3), 10 mM MgCl2, 70 mM KCI, 10 mM dithiothreitol, and 300 U of reverse transcriptase (Life Sciences, Inc.) to a final reaction volume of 200 ,ul; the mixture was incubated at 42°C for 1 h. This preparation was then mixed with 400 ,u1 of a solution containing 20 mM Tris hydrochloride (pH 7.5), 5...
The imprinted H19 gene, which encodes an untranslated RNA, lies at the end of a cluster of imprinted genes in the mouse. Imprinting of the insulin-2 and insulin-like growth factor 2 genes, which lie about 100 kilobases upstream of H19, can be disrupted by maternal inheritance of a targeted deletion of the H19 gene and its flanking sequence. Animals inheriting the H19 mutation from their mothers are 27% heavier than those inheriting it from their fathers. Paternal inheritance of the disruption has no effect, which presumably reflects the normally silent state of the paternal gene. The somatic overgrowth of heterozygotes for the maternal deletion is attributed to a gain of function of insulin-like growth factor 2, rather than a loss of function of H19.
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