Characterization of trans-acting factor(s) regulating beta-globin gene expression by in vivo competition

A beta-globin/TK fusion gene was microinjected into non-erythroid cells (Ltk- cells) and erythroid cells (murine erythroleukemia (MEL) cells), and the interactions of the regulatory cellular factors with the beta-globin sequences were investigated by the in vivo competition experiment. The fusion gene was expressed efficiently in Ltk- cells. This expression was inhibited by a co-injection with a three-fold molar excess of the 5'-flanking sequence of the beta-globin gene or with a nine-fold molar excess of the mammary tumor virus LTR, but not with the alpha-globin gene. The fusion gene was expressed very poorly in the uninduced MEL cells and highly in the induced MEL cells. The co-injection of the beta-globin gene did not affect expression in the MEL cells in either uninduced or induced conditions.     

A novel regulatory element of the human alpha-globin gene responsible for its constitutive expression

The alpha-globin gene is expressed at a constitutively high level upon gene transfer into both erythroid and nonerythroid cells. The beta-globin gene, on the other hand, is dependent on the presence of a linked viral enhancer for its efficient expression upon transfer into heterologous cells. In this report, we describe a novel regulatory element within the structural alpha-globin gene which can activate its own promoter to result in a high level of expression in both erythroid and non-erythroid cells. This regulatory element does not appear to have the properties of a classical enhancer. While this element exerts a positive effect on its own promoter, we have demonstrated in a previous study that the same element exerts a negative effect on heterologous genes such as the beta- and gamma-globin genes. In this study, we localize this element to a 259 nucleotide fragment immediately downstream from the translation initiation codon which is partially overlapped by a DNase I hypersensitive domain only in erythroid cells. We propose that this element may activate the alpha-globin gene promoter in all cell types in vivo as it does in vitro. The specificity of erythroid expression of the alpha-globin gene in vivo is probably determined by a "permissive" chromatin configuration in erythroid cells and a "nonpermissive" configuration in non-erythroid cells.     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.     

CpG islands from the alpha-globin gene cluster increase gene expression in an integration-dependent manner.

In contrast to other globin genes, the human and rabbit alpha-globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer. This enhancer-independent expression of the alpha-globin gene requires extensive sequences not only from the 5' flanking sequence but also from the intragenic region. However, the features of these internal sequences that are responsible for their positive effect are unclear. We tested several possible determinants of this activity. One possibility is that a previously identified array of discrete binding sites for known and potential regulatory proteins within the alpha-globin gene comprise an intragenic enhancer specific for the alpha-globin promoter, but directed rearrangements of the sequences show that this is not the case. Alternatively, the promoter may extend into the gene, with the function of the discrete binding sites being dependent on maintenance of their proper positions and orientations relative to the 5' flanking sequence. However, the positive effects observed in gene fusions do not localize to a discrete region of the alpha-globin gene and the results of internal deletions and point mutations argue against a required role of the targeted discrete binding sites. A third possibility is that the CpG island, which includes both the 5' flanking and intragenic regions associated with the positive activity, may itself have a more general effect on expression in transfected cells. Indeed, we show that the size of the CpG island in constructs correlates with the level of gene expression. Furthermore, the alpha-globin promoter is more active in the context of a previously inactive CpG island than in an A+T-rich context, showing that the CpG island provides an environment more permissive for expression. These effects are seen only after integration, suggesting a possible mechanism at the level of chromatin structure.     

Structure and transcriptional regulation of the mouse ferrochelatase gene

Ferrochelatase (EC.4.99.1.1), the final step in the biosynthesis of heme, is widely expressed in various tissues and is induced in erythroid cells. We determined the structure of the mouse ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. The gene spans about 25 kb and consists of 11 exons. The exon/intron boundary sequences conform to consensus acceptor (GTn)/donor (nAG) sequences, and exons in the gene encode functional protein domains. The promoter region contains multiple Sp1 sites, a CACCC box and GATA-1 binding sites. Function analysis of the promoter by transient transfection assay demonstrated that one Sp1 binding site located at -37/-32 is essential for basic expression of the ferrochelatase gene in both mouse erythroleukemia (MEL) and non-erythroid EL4 cells. In addition, the region (-66/-51) containing a CACCC box and the neighboring GC box partly contributes to the inducible activity of the reporter in MEL cells upon induction with dimethylsulfoxide. It appears that at least two promoter regions of the mouse ferrochelatase gene function in basic and inducible expression.     

Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.