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.     

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.     

A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse beta -globin locus

Developmental expression at the beta-globin locus is regulated in part by the locus control region, a region upstream of the genes containing at least five major DNase I hypersensitive sites (HSs) in mammalian erythrocytes. Sequences farther 5' of these HSs are conserved in mouse and human, and both loci are embedded within a cluster of functional odorant receptor genes. In humans, distant upstream sequences have been implicated in regulation of the beta-globin genes. In this study, the role of the 5'-most HSs and their adjacent sequence was investigated by deletion of an 11-kb region from the mouse locus, including 5'HS 4.2, 5'HS 5, 5'HS 6, and the 5'beta1 odorant receptor gene. Mice that were homozygous for this deletion were fully viable, and no significant effect on adult beta-globin gene expression was seen. 5'HSs 1-4, which are located downstream of the deletion, were still present in the mutant mice. In addition, two new upstream HSs, HS -60.7 and HS -62.5, were found in erythroid tissue of both wild-type and mutant mice. Therefore, although the possibility of a minor role still exists, neither the HSs nor the other regions deleted in this study are essential for beta-globin gene expression, and it is unlikely that chromatin structure is affected either upstream or downstream of the deletion. This is the largest deletion at the mouse locus control region to show no apparent phenotype, and focuses attention on the possible contribution of sequences even farther upstream.     

Proper developmental control of human globin genes reproduced by transgenic mice containing a 160-kb BAC carrying the human beta-globin locus

Four independent bacterial artificial chromosome (BAC) clones containing the human Beta-globin gene locus were obtained from a human genomic BAC library. A 160-kb clone (186D7) carrying the entire human Beta-globin locus including the Beta-globin gene family, locus control region (LCR), and 3' regulatory elements was used to transform mice. Four transgenic lines were generated by microinjecting the purified BAC DNA into the fertilized eggs. RNase protection analysis showed that the expression of human Beta-globin genes is tissue- and developmental stage-specific and the expression level is similar among the three independent transgenic lines which carry the entire human Beta-globin locus; however, no Beta-globin gene expression was detected in the transgenic mice lacking the LCR region. The results suggest that the transgenic mouse model system that we have produced and that uses BAC to study the complex human Beta-globin gene cluster is stable and reproducible. Our results also indicate that some newly characterized HSs upstream from the LCR appear not to play an important role in globin gene expression and switching, while the traditional LCR can ensure correct human Beta-globin gene expression in transgenic mice. The BAC-mediated transgenic system can be used for further studies to determine which kinds of cis-acting elements are included in regulating the developmental timing and the level of human Beta-globin gene expression.     

Proper Developmental Control of Human Globin Genes Reproduced by Transgenic Mice Containing a 160-kb BAC Carrying the Human β-Globin Locus

ABSTRACTFour independent bacterial artificial chromosome (BAC) clones containing the human B-globin gene locus were obtained from a human genomic BAC library. A 160-kb clone (186D7) carrying the entire human B-globin locus including the B-globin gene family, locus control region (LCR), and 3′ regulatory elements was used to transform mice. Four transgenic lines were generated by microinjecting the purified BAC DNA into the fertilized eggs. RNase protection analysis showed that the expression of human B-globin genes is tissue- and developmental stage-specific and the expression level is similar among the three independent transgenic lines which carry the entire human B-globin locus; however, no B-globin gene expression was detected in the transgenic mice lacking the LCR region. The results suggest that the transgenic mouse model system that we have produced and that uses BAC to study the complex human B-globin gene cluster is stable and reproducible. Our results also indicate that some newly characterized HSs upstream from the LCR appear not to play an important role in globin gene expression and switching, while the traditional LCR can ensure correct human B-globin gene expression in transgenic mice. The BAC-mediated transgenic system can be used for further studies to determine which kinds of cis-acting elements are included in regulating the developmental timing and the level of human B-globin gene expression.     

Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.

Several lines of evidence suggest that erythroid-specific DNase I hypersensitive sites (HS) located far upstream of the human beta-globin gene are important in regulating beta-globin gene expression. We used the polymerase chain reaction technique to amplify and clone an 882-base-pair DNA fragment spanning one of these HS, designated HSII, which is located 54 kilobases upstream of the beta-globin gene. The cloned HSII fragment was linked to a human beta-globin gene in either the genomic (HSII-beta) or antigenomic (HSII-beta) orientation. These two constructs and a beta-globin gene alone (beta) were injected into fertilized mouse eggs, and expression was analyzed in liver and brain from day-16 transgenic fetuses. Five of 7 beta-transgenic fetuses expressed human beta-globin mRNA, but the level of expression per gene copy was low, ranging from 0.93 to 22.4% of mouse alpha-globin mRNA (average 9.9%). In contrast, 11 of 12 HSII-beta transgenic fetuses expressed beta-globin mRNA at levels per gene copy ranging from 31.3 to 336.6% of mouse alpha-globin mRNA (average 139.5%). Only three fetuses containing intact copies of the HSII-beta construct were produced. Two of three expressed human beta-globin mRNA at levels per gene copy of 179.2 and 387.1%. Expression of human beta-globin mRNA was tissue-specific in all three types of transgenic fetuses. These studies demonstrate that a small DNA fragment containing a single erythroid-specific HS can stimulate high-level human beta-globin gene expression in transgenic mice.     

The mouse alpha-globin locus regulatory element

We have identified and cloned the major alpha globin locus regulatory element in the mouse (m alpha RE). This element shows a high level of sequence homology to its human counterpart (HS -40) and lies between the same two exons of an upstream, widely expressed gene in both species. Footprinting and band shift studies of the core element show conservation of many (but not all) of the protein binding sites identified as functionally important in HS -40. The functional equivalence of the mouse element was shown by attaching it to a human alpha globin gene and examining expression in transgenic mice. Readily detectable levels of human alpha mRNA were produced in these mice but they were lower than the endogenous gene expression and did not show copy number dependence. These results suggest that sequences additional to this major regulatory element may be necessary to obtain complete regulation of the alpha globin genes in both species.     

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.     

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.     

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.     

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.     

An erythroid-specific chromatin opening element reorganizes beta-globin promoter chromatin structure and augments gene expression.

In erythroid tissues the chromatin structure of the beta-globin gene locus is extensively remodeled. Changes include the formation of DNase I hypersensitive sites (HSs) over the promoters of actively expressed genes. To test the hypothesis that such "opening" of promoter chromatin structure is important for beta-globin gene expression, we placed a 101-bp erythroid-specific hypersensitive-site forming element (HSFE) from the core of LCR HS4 immediately upstream of a minimal beta-globin gene promoter. We then studied the effects of this element alone and in combination with other cis-acting elements on globin gene chromatin structure and gene expression in MEL cells and transgenic mice. Single or tandem HSFEs increased the size of the portion of the promoter accessible to DNase digestion, increased the proportion of promoters in an accessible conformation, and increased gene expression approximately 5-fold. These were equivalent to expression levels attained using a 2.8-kb microLCR construct. Inclusion of the LCR HS2 enhancer did not increase expression further. In transgenic mouse fetal liver cells the HSFE increased average expression 2.5-fold compared to the minimal promoter alone. These results indicate that a small cis-acting element is capable of remodeling local beta-globin promoter chromatin structure and producing expression similar to that seen with a microLCR construct.