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 human ␤-globin locus contains five developmentally regulated ␤-type globin genes. All five genes depend on the locus control region (LCR), located at the 5 end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human ␤-globin yeast artificial chromosome transgenic mice and observed reduced expression of all the ␤-type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancerblocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the -globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the ␤-globin gene.During the normal process of mammalian development, proper temporal and spatial expression of genetic information must be achieved. To this end, gene expression must be tightly regulated through cis-DNA elements: promoters, enhancers, and silencers. After the human genome had been completely sequenced, it became clear that the average size of intergenic regions is roughly 100 kbp. However, substantial evidence argues that enhancers can modulate promoter activity from very long distances, exceeding 100 kbp (19,20). In such a circumstance, one can envision an activity that protects a locus from neighboring gene regulatory elements to prevent improper gene regulation. Indeed, such an activity has been described: DNA insulators were first identified in Drosophila melanogaster, and similar activities were subsequently found in mammals.Insulators can protect a locus by two distinguishable means; one is a chromatin barrier function, while a second is referred to as enhancer-blocking activity. The most extensively characterized vertebrate insulator was originally identified in the chicken ␤-globin LCR, which consists of four DNase I-hypersensitive (HS) sites; the 5Ј-most is HS4. After stable transformation of K562 cells, a 1.2-kbp DNA fragment containing HS4 was found to interfere with enhancer/promoter interactions, but only when it was placed between them (10). Further analysis revealed two separable categories of activity within this 1.2-kbp DNA fragment, one of which protects stably integrated transgenes from silencing after long-term cell culture (28). This property of an insulator is referred to as its chromatin barrier activity.Chung et al. developed an enhancer-blocking assay to refine the position of a 250...

The human ␤-like globin genes (5--G␥-A␥-␦-␤-3) are temporally expressed in sequential order from the 5 to 3 end of the locus, but the nonadult -and ␥-globin genes are autonomously silenced in adult erythroid cells. Two cis elements have been proposed to regulate definitive erythroid ␥-globin repression: the DR (direct repeat) and CCTTG elements. Since these two elements partially overlap, and since a well-characterized HPFH point mutation maps to an overlapping nucleotide, it is not clear if both or only one of the two participate in ␥-globin silencing. To evaluate the contribution of these hypothetical silencers to ␥-globin regulation, we generated point mutations that individually disrupted either the single DR or all four CCTTG elements. These two were separately incorporated into human ␤-globin yeast artificial chromosomes, which were then used to generate ␥-globin mutant transgenic mice. While DR element mutation led to a dramatic increase in A␥-globin expression only during definitive erythropoiesis, the CCTTG mutation did not affect adult stage transcription. These results demonstrate that the DR sequence element autonomously mediates definitive stage-specific ␥-globin gene silencing.