Role of upstream DNase I hypersensitive sites in the regulation of human alpha globin gene expression

Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells.     

HS-48 alone has no enhancement role on the expression of human alpha-globin gene cluster

To investigate the in vivo function of the newly defined DNase I hypersensitive site HS-48 on the whole human alpha-globin gene cluster, the region containing all the other known 5 hypersensitive sites HS-4 to HS-40 was deleted from a 117 kb bacterial artificial chromosome clone bearing the whole human alpha-globin gene cluster. Transgenic mice were generated from this construct. The RNase protection assays showed that with HS-48 left and all the other 5 hypersensitive sites deleted, the expression of human alpha-like globin genes was completely silenced in embryonic, fetal and adult stages in all tissues. This finding indicates that HS-48 alone has no enhancer activity on the expression of human alpha-like globin genes, and that the region of HS-4 to HS-40 already contains all the upstream cis-elements needed for regulating human alpha-like globin genes.     

The expression of human α-like globin genes in transgenic mice mediated by bacterial artificial chromosome

After screening a bacterial artificial chromosome of human genomic DNA library with human HS-40, zeta-, alpha-, and theta-globin probes, a 110-kb clone bearing the whole human alpha-globin gene cluster was obtained and rare restriction endonuclease mapping was performed. The bacterial artificial chromosome DNA was isolated, and transgenic mice were generated. Three founders were detected from 35 newborn mice. The copy numbers were 1, 2, and 2, and the expression of human alpha-globin genes in various tissues at different developmental stages in the transgenic mice was assayed. The human alpha-globin mRNA can be detected in bone marrow, kidney, liver, brain, but not in muscle, testis, or thymus. The human zeta-globin genes were switched off, and the alpha-globin genes were switched at day 11.5 in mouse embryo, indicating that developmental stage-specific expression of the alpha-like globin genes was properly regulated. The human alpha-globin mRNA ranged between 17-68% of the endogenous mouse alpha-globin, suggesting that the expression of human alpha-globin genes is integration site-dependent in transgenic mice. The ratio of human alpha(2)- and alpha(1)-globin gene expression in adult transgenic mouse is about 2.5:1 similar to the expression in human.     

Analysis of the human alpha-globin gene cluster in transgenic mice.

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.     

The control of expression of the alpha-globin gene cluster

The alpha-globin gene cluster is located at the very tip of the short arm of chromosome 16. It produces the alpha-like globins, which is combined with the beta-like globins to form hemoglobin, and its mutants cause alpha-thalassemia, which is one of the most common genetic diseases. Its expression shows a tissue and developmental stage specificity that is balanced with that of the beta-globin gene cluster. In this article, we summarize the research on the control of expression of the alpha-globin gene cluster, mainly with respect to the alpha-major regulatory element (alpha-MRE): HS-40, the tissue-specific and developmental control of its expression, and its chromosomal environment. In summary, the alpha-globin gene cluster is expressed in an open chromosomal environment; HS-40, the 5'-flanking sequence, the transcribed region, and the 3'-flanking sequence interact to fully regulate its expression.     

Structure of the human 3-methyladenine DNA glycosylase gene and localization close to the 16p telomere.

We recently reported the presence of four genes lying between the human alpha-globin gene cluster and the telomere of the short arm of chromosome 16 (16p). We now report that one of these genes encodes 3-methyladenine DNA glycosylase, an enzyme important in the repair of DNA after damage by alkylating agents. The gene comprises five exons, representation of which differs in independently isolated cDNA clones. Although the gene is widely expressed, the abundance of its mRNA is considerably higher in a colon adenocarcinoma cell line (HT29) than in other cell lines that were tested. The major positive erythroid-specific regulatory element controlling alpha-globin gene expression lies equidistant between the promoters of the alpha-globin genes and the 3-methyladenine DNA glycosylase gene. Interestingly, in contrast to the alpha-globin genes, expression of the 3-methyladenine DNA glycosylase gene is not influenced by the regulatory element in the human erythroleukemia cell line K562.     

Genetic polymorphism of the major regulatory element HS-40 upstream of the human alpha-globin gene cluster

The highly conserved 350-bp major regulatory element HS-40 (or alphaMRE) upstream of the human alpha-globin gene cluster is involved in the regulation of alpha-globin gene expression. The study of alphaMRE differences between human populations and the evolution of alphaMRE sequences in mammals may lead to a better understanding of the function and importance of this element in the regulation of expression of the downstream alpha-cluster. Denaturing gradient gel electrophoresis was used to determine the sequence heterogeneity of the alphaMRE region in 276 unrelated individuals, representing seven different populations. Furthermore, we analysed the alpha major regulatory elements of chimpanzee, orang-utan and rhesus monkeys and compared them with the equivalent human and murine sequences. Six different alphaMRE haplotypes (labelled A to F) were found in humans. Haplotype frequencies between the seven populations showed a gradual shift to a higher haplotype A distribution from west to east, being the highest in Indonesians. The African sample shows the largest divergence in haplotypes. Five out of six different haplotypes were present, three of which were exclusively found in Africans. The high prevalence of the haplotype A in humans, together with the conservation of this haplotype in apes, suggests that it is the ancestral one. The alphaMRE fragment appears to be a highly polymorphic marker, which could be used in combination with the regular markers in the alpha-cluster to extend the haplotype and to follow segregation of alpha-thalassaemia genes in population studies more accurately.     

Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: optimization of a gamma-globin gene expression cassette

Progress toward gene therapy of beta-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the Agamma- and beta-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that the Agamma-globin promoter could be deleted to -159 without affecting expression, while deleting the beta-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal beta-globin gene promoter was consistently higher than that from the optimal Agamma-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the beta-globin locus control region (termed a muLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the alpha-globin locus. All three enhancers increased expression from the beta-globin gene to roughly the same extent, while the HS-40 element was notably less effective with the Agamma-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Agamma-globin gene linked to the beta-globin promoter. Inclusion of extended 3' sequences from either the beta-globin or the Agamma-globin genes had no significant effect on expression. A 714-bp internal deletion of Agamma-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 beta-globin promoter, an Agamma-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, gamma-globin expression averaged 166% of murine alpha-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine alpha-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for gamma-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.     

alpha-thalassemia resulting from a negative chromosomal position effect

To date, all of the chromosomal deletions that cause alpha-thalassemia remove the structural alpha genes and/or their regulatory element (HS -40). A unique deletion occurs in a single family that juxtaposes a region that normally lies approximately 18-kilobase downstream of the human alpha cluster, next to a structurally normal alpha-globin gene, and silences its expression. During development, the CpG island associated with the alpha-globin promoter in the rearranged chromosome becomes densely methylated and insensitive to endonucleases, demonstrating that the normal chromatin structure around the alpha-globin gene is perturbed by this mutation and that the gene is inactivated by a negative chromosomal position effect. These findings highlight the importance of the chromosomal environment in regulating globin gene expression.     

De novo deletion within the telomeric region flanking the human alpha globin locus as a cause of alpha thalassaemia

We have identified and characterized a Scottish individual with alpha thalassaemia, resulting from a de novo 48 kilobase (kb) deletion from the telomeric flanking region of the alpha globin cluster which occurred as a result of recombination between two misaligned repetitive elements that normally lie approximately 83 kb and 131 kb from the 16p telomere. The deletion removes two previously described putative regulatory elements (HS-40 and HS-33) but leaves two other elements (HS-10 and HS-8) intact. Analysis of this deletion, together with eight other published deletions of the telomeric region, showed that they all severely downregulated alpha globin expression. Together they defined a 20.4-kb region of the human alpha cluster, which contains all of the positive cis-acting elements required to regulate alpha globin expression. Comparative analysis of this region with the corresponding segment of the mouse alpha globin cluster demonstrated conserved non-coding sequences corresponding to the putative regulatory elements HS-40 and HS-33. Although the role of HS-40 as an enhancer of alpha globin expression is fully established, these observations suggest that the role of HS-33 and other sequences in this region should be more fully investigated in the context of the natural human and mouse alpha globin loci.     

Developmental switch in the relative expression of the alpha 1- and alpha 2-globin genes in humans and in transgenic mice.

Human alpha-globin is encoded by two adjacent genes, alpha 2 and alpha 1. Despite their remarkable level of structural identity, the more 5' (alpha 2) gene is the major alpha-globin locus in the normal adult, expressed at 2.6-fold higher levels than the adjacent and more 3' (alpha 1) globin gene. In light of the well-characterized pattern of gene activation in the human alpha- and beta-globin gene clusters during development, we considered the possibility that the relative expression of these two alpha-globin loci might be developmentally controlled. Analysis of human embryonic and early fetal erythroid RNA samples confirmed this possibility; levels of mRNA encoded by the two alpha-globin loci are equal in the embryo and subsequently shift to dominant expression of the alpha 2-globin locus at week 8 in utero. In transgenic mice carrying the entire human alpha-globin cluster (except for the theta gene) we show the same shift from equal expression of the alpha 1- and alpha 2-globin loci at the embryonic stage to predominance of the alpha 2-globin locus in the adult. These data demonstrate a switch in the expression of the two adjacent alpha-globin genes during the embryonic-to-fetal switch in erythroid development and provide an experimental system for its further characterization.     

High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors.

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40-containing vector encoding a hybrid human beta/gamma-globin gene led to 43% to 113% human gamma-globin expression/copy of the mouse alpha-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.     

Isolation of hybrid cell clones that contain deletion and non-deletion defects of alpha-thalassemia in man

We have succeeded in isolating hybrid mouse erythroleukemia cell clones from a patient with hemoglobin H disease, which exhibit either deletion or nondeletion mutations of the human alpha-globin genes. Analysis of one of these hybrid clones that had retained a human chromosome 16 from the patient's cells showed that both human alpha-globin had been deleted. Several clones of another hybrid cell had retained a human chromsome 16 from the patient's cells, which contained both human alpha- globin genes on an EcoRI fragment of 23 kilobases (kb). These latter hybrid clones showed the presence of human alpha-globin chains at detectable but low levels. These studies show that there are two different types of human chromosome 16 in this patient and that the nondeletion mutation of human alpha-globin genes leading to hemoglobin H diseases in this patient acts in cis to the two alpha-globin genes remaining in his cells. The close correlation between the pattern of human alpha-globin gene expression in the patient and in the hybrid cells suggests that this method of transfer of human globin genes to rodent cells will be a useful one for study of mutations affecting the expression of differentiated genes that lead to disease in man.     

Inactivation of human alpha-globin gene expression by a de novo deletion located upstream of the alpha-globin gene cluster.

Synthesis of normal human hemoglobin A, alpha 2 beta 2, is based upon balanced expression of genes in the alpha-globin gene cluster on chromosome 16 and the beta-globin gene cluster on chromosome 11. Full levels of erythroid-specific activation of the beta-globin cluster depend on sequences located at a considerable distance 5' to the beta-globin gene, referred to as the locus-activating or dominant control region. The existence of an analogous element(s) upstream of the alpha-globin cluster has been suggested from observations on naturally occurring deletions and experimental studies. We have identified an individual with alpha-thalassemia in whom structurally normal alpha-globin genes have been inactivated in cis by a discrete de novo 35-kilobase deletion located approximately 30 kilobases 5' from the alpha-globin gene cluster. We conclude that this deletion inactivates expression of the alpha-globin genes by removing one or more of the previously identified upstream regulatory sequences that are critical to expression of the alpha-globin genes.     

Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer.

We describe a cell-lineage marking system applicable to the vertebrate nervous system. The basis of the technique is gene transfer using the retroviral vector system. We used Escherichia coli beta-galactosidase as a marker gene and demonstrate a high level of expression of this marker from the viral long terminal repeat promoter, with simultaneous expression of the Tn5 neo gene from the simian virus 40 early promoter. This expression has allowed us to detect individual infected cells histochemically. We applied this marking technique to the study of lineage relationships in the developing vertebrate nervous system, both in vivo and in culture. In the rat retina, we injected virus in vivo and histochemically identified clones of marked neural cells. In addition, we used this virus to infect cultures of rat cerebral cortex and have analyzed the clonal relationships of morphologically different neural cell types. The host range of the marking system extends to avian as well as mammalian species. Thus, this system should have broad applicability as a means of gene transfer and expression in the nervous system.     

High-level erythroid expression of human alpha-globin genes in transgenic mice.

The human alpha 1-globin gene was fused downstream of two erythroid-specific DNase I super-hypersensitive sites that are normally located upstream of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and expression was analyzed in 16-day fetal livers and brains. All 11 fetuses that contained intact copies of the transgene expressed correctly initiated human alpha-globin mRNA in the erythroid fetal liver but not in brain. Levels of expression ranged from 4% to 337% of endogenous mouse beta-globin mRNA. A human alpha-globin construct that did not contain super-hypersensitive sites was not expressed. These results demonstrate that human beta-globin locus activation sequences can stimulate high levels of human alpha-globin gene expression in erythroid tissue of transgenic mice. The results also provide a foundation for experiments designed to coexpress human alpha- and beta-globin genes in transgenic mice and suggest a feasible approach for production of a mouse model for human sickle cell disease.