Beta-globin mRNA Levels Research Articles

Spontaneous δ- to β-Globin Switching in K562 Human Leukemia Cells

Abstract Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta- globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching.

Spontaneous delta- to beta-globin switching in K562 human leukemia cells

Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta-globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching.

Novel metabolism of several beta zero-thalassemic beta-globin mRNAs in the erythroid tissues of transgenic mice.

Mice that are transgenic for human beta zero-thalassemic beta-globin alleles were generated in order to study how beta zero-thalassemic mutations affect beta-globin RNA metabolism in erythroid tissues. Three thalassemic alleles were studied, each of which harbors either a frameshift or a nonsense mutation. These mutations result in the premature termination of beta-globin mRNA translation and an abnormally low level of beta-globin mRNA in the peripheral blood of thalassemic patients. Comparative studies of mice that express any of the beta zero-thalassemic transgenes with mice that express a normal human beta-globin transgene demonstrated that all three thalassemic mRNAs are metabolized in erythroid tissues abnormally. RNA blotting and S1 nuclease transcript mapping revealed for each thalassemic transgene that (i) the full-length mRNA is abnormally short-lived and (ii) in addition to full-length mRNA, three more stable yet smaller RNAs are present. These smaller RNAs are polyadenylated and lack the mRNA 5' end.

Molecular analysis of the human beta-globin locus activation region.

Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human epsilon-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human beta-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase beta-globin mRNA levels 8- to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed.

Nonsense mutations in the human beta-globin gene affect mRNA metabolism.

A number of premature translation termination mutations (nonsense mutations) have been described in the human alpha- and beta-globin genes. Studies on mRNA isolated from patients with beta zero-thalassemia have shown that for both the beta-17 and the beta-39 mutations less than normal levels of beta-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human beta-globin mRNA.) In vitro studies using the cloned beta-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. We have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, we have studied the effect of five nonsense mutations and two missense mutations on the expression of human beta-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.

Identification of a stable nuclear RNA complementary to the 3'-end flanking sequences of the mouse beta-major globin gene.

A stable nuclear RNA of approximately 1600 nucleotides (nt) isolated from dimethyl sulfoxide-induced Friend erythroleukemia cells has been characterized. This RNA has been shown to be homologous to a region of unique sequences situated 3' to the mouse beta-major globin gene between the poly(A) addition site and a BglII site located 1400 nt farther downstream. It is transcribed from the same DNA strand as the beta-major globin mRNA, and the amount of this RNA present in the cell is directly proportional to the level of beta-globin mRNA. Therefore, the 1600-nt RNA appears to be related to the large primary transcript of the beta-major globin gene. We feel that this RNA species is an unusually stable intermediate product of the early processing event which cleaves the primary transcript at the poly(A) addition site. The observed stability and discrete length which are contrary to the expected properties of such a processing intermediate may reflect peculiarities of the transformed state of the Friend erythroleukemia cells.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression.

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression.

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

DNase I hypersensitive sites of globin genes of uninduced Friend erythroleukemia cells and changes during induction with dimethyl sulfoxide.

We compared the DNase I sensitivity of beta-globin genes in dimethyl sulfoxide-treated and untreated Friend erythroleukemia cells to determine whether the induced globin synthesis in these cells is associated with any change in the chromatin conformation of the globin genes. The beta-globin genes were preferentially sensitive to DNase I digestion in both uninduced and induced cells as compared to the alpha-fetoprotein gene and bulk DNA. Additionally, we detected specific cleavage sites in the region of the beta-major globin gene when nuclei from uninduced cells were digested with DNase I. Following induction, a 2- to 4-fold increase in DNase I digestion at one of these hypersensitive sites was observed while digestion at the other sites was greatly reduced. We have localized this primary hypersensitive site to the 5' end of the beta-major gene. To determine when during induction these observed changes in chromatin structure occur, nuclei were digested with DNase I after 6 to 120 h of induction with dimethyl sulfoxide. Some change in chromatin conformation was detectable by 12 h of induction and the configuration characteristic of the fully induced state was established by 24 h of induction. Since it has been reported that increases in beta-globin mRNA levels are not detected until 18-20 h of induction, it appears that alterations in the chromatin conformation of the globin genes may precede the changes in other biochemical parameters associated with differentiation and may be an early reflection of commitment to the erythroid pathway.

Beta zero thalassemia in Sardinia is caused by a nonsense mutation.

We report the characterization of a molecular lesion of beta thalassemia in Sardinia. Beta thalassemia in this area is predominantly the beta zero type with low levels of beta-globin mRNA. Translation assay of this messenger RNA in a cell-free system showed beta-globin chain synthesis only with the addition of an amber (UAG) suppressor transfer RNA. Double-stranded complementary DNA prepared from reticulocyte mRNA from a Sardinian patient was cloned in a bacterial plasmid and a beta-globin complementary DNA containing clone was isolated and sequenced. At the position corresponding to amino acid number 39, a single nucleotide mutation converted a glutamine codon (CAG) to an amber termination codon (UAG). We previously reported an amber nonsense mutation at amino acid 17 as a cause of Chinese beta zero thalassemia. Thus, beta zero thalassemia in Sardinia represents the second example of a nonsense mutation, and we predict that other beta zero thalassemias with mutations at various points along the beta-globin chain will be found to form a discrete subgroup of beta zero thalassemia. These experiments further illustrate the heterogeneity of lesions that lead to defective globin chain synthesis in beta thalassemia.

Structure and expression of a cloned beta o thalassaemic globin gene.

We have cloned the single beta-globin gene from an Italian patient who is a double heterozygote for beta o/delta beta o thalassaemia. RNA isolated from nucleated red cells from this patient can be translated in vitro to give detectable levels of A gamma- G gamma and alpha-globin, but no beta-globin. S1-mapping transcription studies show that beta-globin mRNA is present at about 1-3% of the level of alpha- and gamma-globin mRNA. In addition, the expression of this gene has been studied by reversed genetics. SV40-plasmid-beta o-globin gene recombinants have been transfected into Hela cells and analysed for beta-globin mRNA. In contrast to the results obtained with mRNA isolated directly from the blood of this patient, in the transfected Hela cells the same level of beta-globin mRNA is seen for the beta o thalassaemic globin gene and for a normal beta-globin gene. To elucidate the nature of the lesion, the entire DNA sequence of the beta-globin gene of this patient has been determined. The sequence shows that this gene contains a termination codon at position 39 (CAG - greater than UAG). Otherwise, there is a remarkable conservation of the entire DNA sequence.