Angiotensin (AT) II, the bioactive octapeptide in the renin-angiotensin system that plays a key role in cardiovascular homeostasis, exerts its multiple effects through the different types of AT receptors, AT1a, AT1b, and AT2. Previously, we showed chronic hypotension in angiotensinogen (the precursor of AT)-deficient mice and a dramatic increase in renin mRNA levels in its kidney, but it remains unclear which types of AT receptors regulate the blood pressure and renin gene expression. In order to elucidate the physiological roles of AT1a receptor, we generated mutant mice with a targeted replacement of the AT1a receptor loci by the lacZ gene. In the heterozygous mutant mice, the strong lacZ staining was found in the glomerulus and juxtaglomerular apparatus of the renal cortex, which coincided with that of the signals detected by in situ hybridization. Chronic hypotension was observed in the heterozygous and homozygous mutant mice, with 10 and 22 mm Hg lower systolic blood pressure, respectively, than that of wild-type littermates. Both levels of renin mRNA in the kidney and plasma renin activity were markedly increased only in the homozygous mutant mice. These results demonstrated that an AT1a-mediated signal transduction pathway is, at least in part, involved in the regulation of blood pressure and renin gene expression.

The mechanism underlying enhancer-blocking by insulators is unclear. We explored the activity of human ␤-globin HS5, the orthologue of the CTCF-dependent chicken HS4 insulator. An extra copy of HS5 placed between the ␤-globin locus control region (LCR) and downstream genes on a transgene fulfills the classic predictions for an enhancer-blocker. Ectopic HS5 does not perturb the LCR but blocks gene activation by interfering with RNA pol II, activator and coactivator recruitment, and epigenetic modification at the downstream ␤-globin gene. Underlying these effects, ectopic HS5 disrupts chromatin loop formation between ␤-globin and the LCR, and instead forms a new loop with endogenous HS5 that topologically isolates the LCR. Both enhancer-blocking and insulatorloop formation depend on an intact CTCF site in ectopic HS5 and are sensitive to knock-down of the CTCF protein by siRNA. Thus, intrinsic looping activity of CTCF sites can nullify LCR function.beta-globin genes ͉ insulator ͉ locus control region ͉ epigenetics ͉ transcription regulation C hromatin insulators are thought to establish domains within which proper enhancer-gene interactions occur: these domains can be visualized in Drosophila cells where a protein complex including Su(Hw) forms loops that tether gypsy insulators to the nuclear lamina (1, 2). Insulators can also interfere with enhancer-gene interaction when placed between the two elements, but the molecular mechanisms underlying enhancerblocking are not well understood.In vertebrates, the protein CTCF is associated with enhancerblocking (3). On maternal chromosomes, the CTCF-dependent imprinting control region (ICR) in the Igf2/H19 locus is thought to form a small loop with a second site, DMR1, upstream of Igf2 that includes Igf2 and restricts access to the gene by enhancers it shares with H19 (4, 5). Because detection of CTCF at DMR1 is dependent on its interaction at ICR, it is not clear whether CTCF or other proteins interact at DMR1 and participate in the long-range interactions. Other data indicate the ICR directly contacts Igf2 and the enhancers (6), suggesting that a complex series of chromosomal interactions may be involved in enhancerblocking at this locus. CTCF also mediates the enhancerblocking activity of the chicken ␤-globin insulator, 5ЈHS4, which forms the upstream border of the globin locus (7). Although a prediction (8), enhancer-blocking through loop formation by interacting CTCF insulator sites has not been demonstrated.Locus control regions (LCRs) are complex enhancers that activate genes over long distances through their ability to establish close contacts with target promoters (9). For example, the ␤-globin LCR and active globin genes come into proximity to form a chromatin loop in erythroid cells (10, 11), an interaction that requires the erythroid factors GATA-1 and erythroid Kruppel-like factor (EKLF) (12, 13). The human ␤-globin LCR is composed of four DNase I hypersensitive sites, HS1 to HS4, far upstream of the structural genes. HS5, 3-Kb upstream of HS4, is more widely ...

The TR2 and TR4 orphan nuclear receptors comprise the DNA-binding core of direct repeat erythroid definitive, a protein complex that binds to direct repeat elements in the embryonic and fetal beta-type globin gene promoters. Silencing of both the embryonic and fetal beta-type globin genes is delayed in definitive erythroid cells of Tr2 and Tr4 null mutant mice, whereas in transgenic mice that express dominant-negative TR4 (dnTR4), human embryonic epsilon-globin is activated in primitive and definitive erythroid cells. In contrast, human fetal gamma-globin is activated by dnTR4 only in definitive, but not in primitive, erythroid cells, implicating TR2/TR4 as a stage-selective repressor. Forced expression of wild-type TR2 and TR4 leads to precocious repression of epsilon-globin, but in contrast to induction of gamma-globin in definitive erythroid cells. These temporally specific, gene-selective alterations in epsilon- and gamma-globin gene expression by gain and loss of TR2/TR4 function provide the first genetic evidence for a role for these nuclear receptors in sequential, gene-autonomous silencing of the epsilon- and gamma-globin genes during development, and suggest that their differential utilization controls stage-specific repression of the human epsilon- and gamma-globin genes.

The five human beta-type-globin genes, epsilon, Ggamma, Agamma, delta and beta, are close together and are regulated by a locus control region (LCR) located at the 5' end of the locus. Here we investigate the functional consequences of this organization with respect to temporal regulation of the individual genes, by using recombination techniques to invert the order of either the genes or the LCR in vivo. Our analysis of transgenic mice bearing either normal or mutant transgenes leads to two new observations. First, the position of the epsilon-globin gene next to the LCR is mandatory for its expression during the yolk-sac stage of erythropoiesis. Second, LCR activity is orientation dependent, and so the LCR does not act as a simple enhancer to stimulate transcription of the globin genes. Thus, in the absence of any change in transgene integration position, transgene copy number, trans-acting factors or other resident genetic information, simple inversion of the human genes or the LCR fundamentally alters the transcription of beta-type globin genes.

We recently described an erythroid e-globin gene repressor activity, which we named DRED (direct repeat erythroid-de®nitive). We show that DRED binds with high af®nity to DR1 sites in the human embryonic (e-) and fetal (g-) globin gene promoters, but the adult b-globin promoter has no DR1 element. DRED is a 540 kDa complex; sequence determination showed that it contains the nuclear orphan receptors TR2 and TR4. TR2 and TR4 form a heterodimer that binds to the e and g promoter DR1 sites. One mutation in a DR1 site causes elevated g-globin transcription in human HPFH (hereditary persistence of fetal hemoglobin) syndrome, and we show that this mutation reduces TR2/TR4 binding in vitro. The two receptor mRNAs are expressed at all stages of murine and human erythropoiesis; their forced transgenic expression reduces endogenous embryonic ey-globin transcription. These data suggest that TR2/TR4 forms the core of a larger DRED complex that represses embryonic and fetal globin transcription in de®nitive erythroid cells, and therefore that inhibition of its activity might be an attractive intervention point for treating sickle cell anemia.