Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns

We have introduced the human fetal gamma- and adult beta-globin genes into the germ line of mice. Analysis of the resulting transgenic mice shows that the human gamma-globin gene is expressed like an embryonic mouse globin gene; the human beta-globin gene is expressed (as previously shown) like an adult mouse globin gene. These results imply that the regulatory signals for tissue- and developmental stage-specific expression of the globin genes have been conserved between man and mouse but that the timing of the signals has changed. Because the two genes are expressed differently, we introduced a hybrid gamma beta-globin gene construct. The combination of the regulatory sequences resulted in the expression of the hybrid gene at all stages in all the murine erythroid tissues.     

Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3'' enhancer in the stage-specific expression of globin genes.

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.     

CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells.

Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter.     

Developmentally regulated and erythroid-specific expression of the human embryonic beta-globin gene in transgenic mice.

Transgenic mice have proven to be an effective expression system for studying developmental control of the human fetal and adult beta-globin genes. In the current work we are interested in developing the transgenic mouse system for the study of the human embryonic beta-globin gene, epsilon. An epsilon-globin gene construction (HSII,I epsilon) containing the human epsilon-globin gene with 0.2 kb of 3' flanking sequence and 13.7 kb of extended 5' flanking region including the erythroid-specific DNase I super-hypersensitive sites HSI and HSII was made. This construction was injected into fertilized mouse ova, and its expression was analyzed in peripheral blood, brain, and liver samples of 13.5 day transgenic fetuses. Fetuses carrying intact copies of the transgene expressed human epsilon-globin mRNA in their peripheral blood. Levels of expression of human epsilon-globin mRNA in these transgenic mice ranged from 2% to 26% per gene copy of the endogenous mouse embryonic epsilon y-globin mRNA level. Furthermore, the human epsilon-globin transgene was expressed specifically in peripheral blood but not in brain or in liver which is an adult erythroid tissue at this stage. Thus, the HSII,I, epsilon transgene was expressed in an erythroid-specific and embryonic stage-specific manner in the transgenic mice. A human epsilon-globin gene construction that did not contain the distal upstream flanking region which includes the HSI and HSII sites, was not expressed in the embryos of transgenic mice. These data indicate that the human epsilon-globin gene with 5' flanking region extending to include DNase I super-hypersensitive sites HSI and HSII is sufficient for the developmentally specific activation of the human epsilon-globin gene in erythroid tissue of transgenic mice.     

BP1, a homeodomain-containing isoform of DLX4, represses the beta-globin gene

In earlier studies we identified a putative repressor of the human beta-globin gene, termed beta protein 1 (BP1), which binds to two silencer DNA sequences upstream of the adult human beta-globin gene and to a negative control region upstream of the adult delta-globin gene. Further studies demonstrated an inverse correlation between the binding affinity of the BP1 protein for the distal beta-globin silencer sequence and the severity of sickle cell anemia, suggesting a possible role for BP1 in determining the production of hemoglobin S. We have now cloned a cDNA expressing the BP1 protein. Sequencing revealed that BP1 is a member of the homeobox gene family and belongs to the subfamily called Distal-less (DLX), genes important in early development. Further analysis showed that BP1 is an isoform of DLX4. BP1 protein has repressor function towards the beta-globin promoter, acting through the two beta-globin DNA silencers, demonstrated in transient transfection assays. Strong BP1 expression is restricted to placenta and kidney tissue, with no expression in 48 other human tissues. BP1 exhibits regulated expression in the human erythroid cell line MB-02, where its expression decreases upon induction of the beta-globin gene. BP1 is thus the first member of the DLX family with known DNA binding sites and a function in globin gene regulation.     

A negative cis-element regulates the level of enhancement by hypersensitive site 2 of the beta-globin locus control region

The core of DNase hypersensitive site (HS) 2 from the beta-globin locus control region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5' and 3' ends of the core. Analysis of constructs with and without the 5' negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5' end of the HS2 core map the negative element within a 20-base pair region containing two conserved sequences.     

HS5 of the human beta-globin locus control region: a developmental stage-specific border in erythroid cells

Elements with insulator/border activity have been characterized most extensively in Drosophila melanogaster. In vertebrates, the first example of such an element was provided by a hypersensitive site of the chicken beta-globin locus, cHS4. It has been proposed that the homologous site in humans, HS5, functions as a border of the human beta-globin locus. Here, we have characterized HS5 of the human beta-globin locus control region. We have examined its tissue-specificity and assessed its insulating properties in transgenic mice using a lacZ reporter assay. Most importantly, we have tested its enhancer blocking activity in the context of the full beta-globin locus. Our results show that HS5 is erythroid-specific rather than ubiquitous in human tissues. Furthermore, HS5 does not fulfil the criteria of a general in vivo insulator in the transgene protection assay. Finally, a HS5 conditional deletion from the complete locus demonstrates that HS5 has no discernable activity in adult erythroid cells. Surprisingly, HS5 functions as an enhancer blocker in embryonic erythroid cells. We conclude that HS5 is a developmental stage-specific border in erythroid cells.