Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified 1, but others remain elusive. Ten of twenty-seven members from a Maltese family presented with HPFH. A genome-wide SNP scan followed by linkage analysis revealed a candidate region on chromosome 19p13.12–13. Sequencing identified a nonsense mutation in the KLF1 gene, p.K288X, ablating the DNA binding domain of this key erythroid transcriptional regulator 2. Only HPFH family members were heterozygote carriers of this mutation. Expression profiling on primary erythroid progenitors revealed down-regulation of KLF1 target genes in HPFH samples. Functional assays demonstrated that, in addition to its established role in adult globin expression, KLF1 is a critical activator of the BCL11A gene, encoding a suppressor of HbF expression 3. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

Three-dimensional organization of a gene locus is important for its regulation, as recently demonstrated for the ␤-globin locus. When actively expressed, the cis-regulatory elements of the ␤-globin locus are in proximity in the nuclear space, forming a compartment termed the Active Chromatin Hub (ACH). However, it is unknown which proteins are involved in ACH formation. Here, we show that EKLF, an erythroid transcription factor required for adult ␤-globin gene transcription, is also required for ACH formation. We conclude that transcription factors can play an essential role in the three-dimensional organization of gene loci. The mouse ␤-globin locus contains multiple ␤-like globin genes, arranged from 5Ј to 3Ј in order of their developmental expression (Fig. 1A). The adult-type ␤ maj -gene is transcribed at a very low level during primitive erythropoiesis in the embryonic yolk sac, but becomes expressed at high levels around day 11 of gestation (E11) when definitive erythropoiesis commences in the fetal liver (Trimborn et al. 1999). The ␤-globin locus control region (LCR) is essential for efficient globin transcription (Grosveld et al. 1987;Bender et al. 2000). It consists of a series of DNaseI hypersensitive sites (HS) located ∼50 kb upstream of the ␤ maj promoter (Fig. 1A). We have shown that the ␤-globin locus forms an Active Chromatin Hub (ACH) in erythroid cells (Tolhuis et al. 2002). The ACH is a nuclear compartment dedicated to RNA polymerase II transcription, formed by the cis-regulatory elements of the ␤-globin locus with the intervening DNA looping out. The ACH consists of the HS of the LCR, two HS located ∼60 kb upstream of the embryonic y-globin gene (5ЈHS-62/-60) and 3ЈHS1 downstream of the genes. In addition, the actively expressed globin genes are part of the ACH (Carter et al. 2002;Tolhuis et al. 2002). In erythroid precursors that do not express the globin genes yet, a substructure of the ACH, called a chromatin hub (CH) (Patrinos et al. 2004) is found, which excludes the genes and the HS at the 3Ј site of the LCR (Palstra et al. 2003).Expression of the ␤ maj -gene requires the presence of the erythroid Krüppel-like transcription factor EKLF, the erythroid-specific member of the Sp/XKLF-family (Miller and Bieker 1993). EKLF −/− mice die of anemia around E14, because of a deficit in ␤-globin expression (Nuez et al. 1995;Perkins et al. 1995). The ␤-globin locus contains a number of EKLF-binding sites, in particular in the LCR and the ␤ maj -globin promoter (Perkins 1999;Bieker 2001). Because ␤ maj -globin expression depends on the presence of EKLF, we were interested in determining whether EKLF is involved in the formation of the ACH. Results and DiscussionWe used chromatin conformation capture (3C) technology (Dekker et al. 2002) to investigate the three-dimensional conformation of the mouse ␤-globin locus in the absence of EKLF. Cells from E12.5 EKLF −/− and wild-type fetal livers were cross-linked with formaldehyde, followed by restriction enzyme digestion of the DNA. The samples were ligated ...

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The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of p-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein.

We describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified by both asymmetric and symmetric arginine methylation in vivo. Furthermore, Fop plays an important role in the ligand-dependent activation of estrogen receptor target genes, including TFF1 (pS2). Fop depletion results in an almost complete block of estradiol-induced promoter occupancy by the estrogen receptor. Our data indicate that Fop recruitment to the promoter is an early critical event in the activation of estradiol-dependent transcription.

Disruption of the gene for transcription factor EKLF (erythroid Krü ppel-like factor) results in fatal anaemiacaused by severely reduced expression of the adult β-globin gene, while other erythroid-specific genes, including the embryonic ε-and fetal γ-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the β-gene promoter, even though it is already present in embryonic red cells. Here, we show that a β-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF -/-embryos. Thus, embryonic β-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5ЈHS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5ЈHS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from γ-to β-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in β-globin gene activation.

The locus control region of the ␤-globin cluster contains five DNase I hypersensitive sites (5 HS1-5) required for locus activation. 5 HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5 HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5 HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5 HS3.

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing ␣-and ␤-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show that Fugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only ␣-globin genes (␣-locus), whereas the other also contains a ␤-globin gene (␣␤-locus). This is the first poikilothermic species analyzed in which the physical linkage of the ␣-and ␤-globin genes has been uncoupled, supporting a model in which the separation of the ␣-and ␤-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu ␣␤-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian ␤-globin clusters, may not be present in the Fugu ␣␤-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell