Cytosine arabinoside plus hemin treatment of a human erythroid cell line, KMOE, strongly induces embryonic, fetal, and adult beta-like globin genes

A variety of regimens were utilized on KMOE cells to maximally raise globin mRNA levels for the purpose of improving the usefullness of this line for globin gene studies. Steady-state mRNA levels of embryonic (epsilon), fetal (gamma) and adult (beta) globin genes were assayed by the S1-nuclease protection method before and after exposure to inducing compounds. Exposure of KMOE cells to cytosine arabinoside and hemin leads to over 20-fold increases in beta- and gamma-globin mRNA steady-state levels, and an over 60-fold increase in epsilon-globin mRNA level. Exposure to cytosine arabinoside alone induced beta- and epsilon-globin but not gamma-globin gene expression. The alpha-like globin genes (zeta and alpha) were also monitored but found to be poorly expressed and not significantly inducible. The presence of epsilon-globin mRNA and the lack of alpha-globin mRNA distinguishes this line, KMOE-EL, from the KMOE sublines previously described.     

Theta, zeta, and epsilon globin messenger RNAs are expressed in adults

The theta globin gene is the most recently discovered member of the alpha globin gene family. Its pattern of expression during development is not fully defined, and its encoded protein has not yet been detected in vivo. The detection of theta globin messenger RNA (mRNA) in embryonic and fetal erythroid tissue but not in adults has suggested that theta is an embryonic globin gene. The present study further defines the pattern of theta globin gene expression. We use a modification of the highly sensitive polymerase chain reaction (PCR) technique to assess the levels of theta globin gene expression during development. We confirm the presence of the theta globin mRNA in embryonic and fetal erythroid tissue, and, in addition, we find theta mRNA in the peripheral reticulocytes of normal adults. Furthermore, using the same analytic approach, we detect low but significant levels of the embryonic zeta and epsilon mRNAs in reticulocytes of normal adults. Both zeta and theta gene expression appears erythroid specific in that neither mRNA species is detected in RNA isolated from brain tissue, peripheral blood mononuclear cells, or three nonerythroid cell lines (B-lymphocyte, T-lymphocyte, and hepatoma cell lines). The relative levels of zeta and theta gene expression were assayed during development by a coamplification technique. The results demonstrate the expected developmental regulation of zeta globin mRNA. In contrast, the level of theta globin mRNA fails to demonstrate the significant changes of the magnitude seen in other globin genes and remains low in embryonic, fetal, and adult life. The lack of zeta and epsilon globin proteins in normal adults using highly sensitive immunologic techniques, as reported by others, stands in contrast to these mRNA results and suggests a gap between mRNA and protein expression.     

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.     

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.     

Adult chicken alpha-globin genes alpha A and alpha D: no anemic shock alpha-globin exists in domestic chickens.

Three alpha-type globin genes have been identified in the alpha-globin linkage group of chickens. No other alpha-type genes hae been directly shown to be within 10 kilobase pairs of any of these three closely linked genes. These three genes have been conclusively identified by DNA sequence analysis. The gene at the 5' end of the linkage group is an embryonic alpha-type globin gene, pi or pi', and the central gene corresponds to the minor adult alpha- globin, alpha D. The 3'-terminal gene sequence corresponds to the sequence of cDNA clones previously described as "alpha S", presumed anemic shock-induced alpha-globin gene [Salser, W. A., Cummings, I., Liu, A., Strommer, J., Padayatty, J. & Clarke, P. (1979) in Cellular and Molecular Regulation of Hemoglobin Switching, eds. Stamatoyannopoulos, G. & Nienheis, A. (Grune and Stratton, New York), pp. 621-643; Richards, R. I. & Wells, J. R. E. (1980) J. Biol. Chem. 255, 9306-9311]. Several groups of workers have isolated alpha S-type cDNA clones but no one has identified a cDNA clone corresponding to the published amino acid sequence of the major chicken alpha-globin, alpha A. We have identified the alpha S-type sequence as the only abundant alpha-like globin sequence in cDNA clones made from reticulocyte mRNA isolated from nonanemic chickens. Therefore, we suggest that the alpha S-type sequence corresponds to the true alpha A-globin species.     

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.     

A novel 33.3 kb deletion (- -KOL) in the alpha-globin gene cluster: a brief report on deletional alpha-thalassaemia in the heterogeneous eastern Indian population

We have detected, in three unrelated eastern Indian individuals, a hitherto unreported alpha zero deletion, - -KOL, in the heterozygous state, encompassing the embryonic zeta2-globin and the duplicated alpha-globin genes extending from c. 1150 bp upstream of the zeta2 globin gene to c. 960 bp downstream of the theta1 gene. Other deletions present in 120 unrelated, eastern Indian, putative alpha-thalassaemia patients are -3.7 kb (16.25%), -4.2 kb (5%) and - -SEA (3.33%).     

The cellular basis for different fetal hemoglobin levels among sickle cell individuals with two, three, and four alpha-globin genes

Fetal hemoglobin (HbF) levels vary widely among individuals with sickle cell anemia (SS). Previous studies have suggested that HbF levels in SS individuals with alpha-thalassemia (two or three functional alpha- globin genes) are lower than HbF levels in SS individuals with four normal alpha-globin genes. Using immunocytochemical techniques, we studied F cell production as measured by % F reticulocytes, the amount of HbF per F cell, and the preferential survival of F cells versus non- F cells in 51 subjects with four alpha genes, 32 subjects with three alpha genes, and 18 subjects with two alpha genes. Comparison between alpha-globin gene groups was performed for the total sample as well as for a subset of 82 individuals who had replicate samples and a further subset of 39 age-matched individuals. %HbF levels were 6.8, 4.9, and 4.5 percent for the total four-, three-, and two-alpha-globin-gene groups, respectively. The percentage of F reticulocytes, percentage HbF per F cell, and the enrichment ratio (% F cell/% F reticulocytes) did not change significantly with alpha-globin gene number. Moreover, no correlation existed between alpha-globin gene number and the absolute number of F cells in any group studied. However, there was a strong inverse correlation (r = -0.407, P = .0001) between non-F cell levels (1.7 +/- 2, 2.2 +/- 5, 3.0 +/- 1.0 X 10(12)/L) and decreasing alpha- globin gene number. These data suggest that falling HbF levels among SS individuals with lessened numbers of alpha-globin genes reflect prolonged survival of non-F cells and are not due to intrinsic differences in F cell production or in the amount of HbF per F cell. The improved survival of non-F cells in SS alpha-thalassemia is presumed to be due to the lower MCHC observed in such individuals.     

DNA methylation in chicken alpha-globin gene expression.

We have investigated certain specific methylation sites of the chicken alpha-globin gene cluster in DNA from embryonic and adult erythroid cells as well as from brain and sperm cells. Eight contiguous DNA fragments of the alpha-globin gene cluster were subcloned from a recombinant lambda phage. The subclones were used as probes to map all the Msp I/Hpa II and Hha I sites in the unmethylated cloned DNA and specific sites of methylation in and around the alpha-globin gene cluster in chromosomal DNA. The data show that sperm DNA is totally methylated at these restriction sites in the globin gene region, as is brain DNA, with some exceptions. Interestingly, the methylation status of specific sites 5' to the coding sequences is correlated with expression of the embryonic or adult alpha-globin genes in different stages of erythroid development. Some sites showing partial methylation, however, do not conform to the model that transcribed genes are unmethylated or undermethylated. We also find a well-defined 3.5-kilobase region of DNA 5' to the alpha-globin gene cluster in which all C-C-G-G sites are resistant to Msp I digestion in all tissues. This "Msp block" is presumably caused by 5-MeCpC methylation.     

Molecular mechanisms of human hemoglobin switching: selective undermethylation and expression of globin genes in embryonic, fetal, and adult erythroblasts.

The globin chain synthetic pattern and the extent of DNA methylation within embryonic, fetal, and adult beta-like globin gene domains were evaluated in greater than or equal to 90% purified human erythroblasts from yolk sacs and fetal livers in the 6- to 12-wk gestational period as well as from adult marrows. The 6-wk erythroblasts produce essentially embryonic epsilon chains, whereas the 12-wk erythroblasts synthesize largely fetal gamma globin and the adult marrow erythroblasts synthesize almost exclusively adult beta chains. In all phases of ontogenic development, a strong correlation exists between DNA hypomethylation in the close flanking sequences of globin genes and their expression. These results suggest that modulation of the methylation pattern may represent a key mechanism for regulating expression of human globin genes during embryonic leads to fetal and fetal leads to adult Hb switches in humans. In ontogenic development this mechanism might in turn correlate with a gradual modification of chromatin structure in the non-alpha gene cluster, thus leading to a 5' leads to 3' activation of globin genes in a balanced fashion.     

HB Chiapas α2 114 PRO+ARG β2: Identification by High Pressure Liquid Chromatography

A variety of regimens were utilized on KMOE cells to maximally raise globin mRNA levels for the purpose of improving the usefullness of this line for globin gene studies. Steady-state mRNA levels of embryonic (?), fetal (γ) and adult (β) globin genes were assayed by the S1-nuclease protection method before and after exposure to inducing compounds. Exposure of KMOE cells to cytosine arabinoside and hemin leads to over 20-fold increases in β- and γ-globin mRNA steady-state levels, and an over 60-fold increase in ?-globin mRNA level. Exposure to cytosine arabinoside alone induced β- and ?-globin but not γ-globin gene expression. The α-like globin genes (ζ and α) were also monitored but found to be poorly expressed and not significantly inducible. The presence of ?-globin mRNA and the lack of α-globin mRNA distinguishes this line, KMOE-EL, from the KMOE sublines previously described.     

Use of yeast artificial chromosomes (YACs) for studying control of gene expression: correct regulation of the genes of a human beta-globin locus YAC following transfer to mouse erythroleukemia cell lines.

We demonstrate that transfer of a yeast artificial chromosome (YAC) containing 230 kb of the human beta-globin locus into mouse erythroleukemia cells by fusion results in correct developmental regulation of the human beta-like globin genes. Additionally, we show that early after hybrid formation, human embryonic epsilon- and fetal gamma-globin genes are coexpressed with the adult beta gene but that after 10-20 weeks in culture, globin gene expression switches to predominantly adult. Thus, in contrast to shorter gene constructs, the globin genes of the beta-globin locus YAC are regulated like the chromosomal globin genes. These results indicate that transfer of YACs into established cell lines can be used for the analysis of the developmental control of multigenic and developmentally regulated human loci.     

Characterization of a new α° thalassaemia defect in the South African population

A new alpha thalassemia defect has been detected in the South African population. Restriction mapping of the alpha globin gene cluster in affected individuals has established that the defect is associated with the removal of 22.8-23.7 kb of DNA, including the psi zeta 1, psi alpha 1, psi alpha 2, alpha 2 and alpha 1 globin genes. The 5' endpoint of the deletion has been localized between the zeta 2 and psi zeta 1 globin genes, and the 3' endpoint lies 4-5 kb 3' to the alpha 1 globin gene. We have called the deletion - -SA in order to distinguish it from alpha zero thalassaemia defects described in other populations.     

Functional effects of replacing human alpha- and beta-globins with their embryonic globin homologues in defined haemoglobin heterotetramers

Embryonic- and adult-stage globin subunits assemble into haemoglobin (Hb) heterotetramers that are expressed at low levels throughout human intrauterine development. These haemoglobins differ from adult Hb A (alpha2beta2) by the substitution of embryonic zeta for adult alpha globin (Hb zeta2beta2), or embryonic epsilon for adult beta globin (Hb alpha2epsilon2). Several key physiological properties of these 'semiembryonic' haemoglobins remain undefined, as ethical and methodological considerations have limited their availability from both human sources and conventional expression systems. The current study attempts to estimate how the physiological properties of semiembryonic and adult haemoglobins may differ, by determining whether the O2-binding characteristics of hybrid human/mouse haemoglobins change when human alpha- or beta-globin subunits are replaced by human embryonic zeta- or epsilon-globin subunits respectively. Each of the four human globins is expressed in transgenic mice that are nullizygous for either the endogenous mouse alpha- or beta-globin genes, resulting in the high-level expression of haemoglobins that can be studied either in situ in intact erythrocytes or in vitro. We showed that the exchange of human zeta-globin for human alpha-globin chains increased haemoglobin O2 affinity, both in the presence and in the absence of 2, 3-bisphosphoglycerate (2,3-BPG), and reduced the pH-dependent shift in its oxygen equilibrium curve (Bohr effect). By comparison, hybrid haemoglobins containing either human epsilon-globin or human beta-globin exhibited nearly identical O2-binding properties, both in situ and in vitro, regardless of 2,3-BPG levels or ambient pH. Neither the zeta-for-alpha nor the epsilon-for-beta substitutions substantially altered binding affinity for 2,3-BPG or cooperativity between globin subunits. These studies suggest that semiembryonic haemoglobins that assemble entirely from human subunits may exhibit properties that are similar to those of human Hb A.