A subset of genes in mammals are subject to genomic imprinting. The mouse H19 gene, for example, is active only when maternally inherited and the neighboring Igf2 gene is paternally expressed. This imprinted expression pattern is regulated by the imprinting control region (ICR) upstream of the H19 gene. A maternally inherited H19 ICR inhibits Igf2 gene activation by the downstream enhancer due to its insulator function while it suppresses H19 gene transcription by promoter DNA methylation when paternally inherited. These parent-of-origin specific functions depend on the allele-specific methylation of the ICR DNA, which is established during gametogenesis. Therefore, the ICR may also function as a landmark for epigenetic modifications. To examine whether the ICR confers these activities autonomously, we introduced a 2.9-kbp ICR-containing DNA fragment into a human ␤-globin yeast artificial chromosome at the 3 end of the locus control region and established transgenic mouse lines. Expression of all of the ␤-like globin genes was higher when the transgene was paternally inherited. In accord with this result, transgenic ICR DNA from nucleated erythrocytes was more heavily methylated when paternally transmitted. Chromatin immunoprecipitation assays confirmed that CCCTC binding factor is preferentially recruited to the maternal transgenic ICR in vivo. Surprisingly however, the parent-of-origin specific methylation pattern was not observed in germ cell DNA in testis, demonstrating that methylation was established after fertilization. Thus, the ICR autonomously recapitulated imprinting within the normally nonimprinted transgenic ␤-globin gene locus, but the temporal establishment of imprinting methylation differs from that at the endogenous Igf2͞H19 locus.

The imprinted expression of the mouse Igf2/H19 locus is governed by the differential methylation of the imprinting control region (ICR), which is established initially in germ cells and subsequently maintained in somatic cells, depending on its parental origin. By grafting a 2.9-kbp H19 ICR fragment into a human ␤-globin yeast artificial chromosome in transgenic mice, we previously showed that the ICR could recapitulate imprinted methylation and expression at a heterologous locus, suggesting that the H19 ICR in the ␤-globin locus contained sufficient information to maintain the methylation mark (K. Tanimoto Curiously, however, the transgenic H19 ICR was not methylated in sperm, which was distinct from that seen in the endogenous locus. Here, we reevaluated the ability of the H19 ICR to mark the parental origin using more rigid criteria. In the testis, the methylation levels of the solitary 2.9-kbp transgenic ICR fragment varied significantly between six transgenic mouse lines. However, in somatic cells, the paternally inherited ICR fragment exhibited consistently higher methylation levels at five out of six randomly integrated sites in the mouse genome. These results clearly demonstrated that the H19 ICR could acquire parent-of-origin-dependent methylation after fertilization independently of the chromosomal integration site or the prerequisite methylation acquisition in male germ cells.

Enhancer elements modulate promoter activity over vast chromosomal distances, and mechanisms that ensure restrictive interactions between promoters and enhancers are critical for proper control of gene expression. The human ␤-globin locus control region (LCR) activates expression of five genes in erythroid cells, including the proximal embryonic -and the distal adult ␤-globin genes. To test for possible distance sensitivity of the genes to the LCR, we extended the distance between the LCR and genes by 2.3 kbp within the context of a yeast artificial chromosome, followed by the generation of transgenic mice (TgM). In these TgM lines, -globin gene expression decreased by 90%, while the more distantly located ␥-or ␤-globin genes were not affected. Remarkably, introduction of a consensus EKLF binding site into the -globin promoter rendered its expression distance insensitive; when tested in an EKLF-null genetic background, expression of the mutant -globin gene was severely compromised. Thus, the -globin gene differs in its distance sensitivity to the LCR from the other ␤-like globin genes, which is, at least in part, determined by the transcription factor EKLF.Proper temporal and spatial expression of genetic information is tightly regulated by DNA cis elements such as promoters, enhancers, and insulators. Specific combinations of enhancers and promoters often act synergistically to finely tune gene expression. In contrast, inappropriate communication between an enhancer and promoter can disturb their in vivo expression patterns. Enhancers were originally defined by their ability to activate transcription of cis-linked genes over considerable distances in an orientation-independent manner. Increasing evidence suggests that enhancers can physically and functionally interact with promoters over long distances, exceeding several hundreds of kilobase pairs in cis, or even with promoters located on different chromosomes in trans (reviewed in references 5 and 13). This suggests the presence of molecular mechanisms that allow specific enhancer-promoter interactions to take place while protecting promoters from being inappropriately activated by enhancer elements that might be located in neighboring gene loci or even on different chromosomes.The human ␤-globin genes are organized within a 70-kbp span of chromosome 11, with the embryonic ε-globin gene located most 5Ј, followed by the two fetal ␥-globin genes (G␥ and A␥), while the adult ␦-and ␤-globin genes are at the 3Ј end of the locus (Fig. 1A). Expression of all the ␤-like globin genes, in primitive (embryonic) as well as definitive (fetal and adult) erythroid cells, requires the activity of the locus control region (LCR)/enhancer, which consists of five DNase I hypersensitive sites (HSs) and is located 6 or 48 kbp 5Ј to the transcription initiation sites of the ε-and ␤-globin genes, respectively (19, 27).How distal enhancer elements and LCRs activate gene expression has long been a subject of intense debate, and numerous models have been proposed. Among them, two models...