Developmental programs of human erythroleukemia cells: globin gene expression and methylation (original) (raw)
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Blood, 1985
In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or de...
DNA methylation patterns of the γδβ-globin genes in human fetal and adult erythroid tissues
American Journal of Hematology, 1992
An investigation of the correlation between the y->p-globin switch and DNA methylation was carried out. The restriction patterns obtained with methylation-sensitive and-insensitive enzymes indicated hypomethylation in the promoter region of the r-globin genes in fetal liver DNA but high methylation of the same region in all other samples (except in the presence of an elevated erythroblast count or leukemia). All samples appeared to be partially hypomethylated at the 5' end of the &globin gene and hypomethylated at the 3' region of the p-globin gene. Although consistent with a role for DNA methylation in globin gene regulation, the results also suggest that other factors besides methylation may be required for regulation of the level of expression, and switching of the globin genes.
American Journal of Hematology, 1990
Human theta (01)-globin gene represents a member of the d i k e globin gene family residing on chromosome 16. el-Specific transcripts have been detected so far only in erythroid tlssues and in erythroleukemia K562 cells. To investigate systematically its inducible expression and developmental specificity, we analyzed at the RNA level five additional human erythroleukemia cell lines with diverse developmental globin programs, two somatic cell hybrids between K562 and mouse erythroleukemia (MEL) cells, a human fetal liver x MEL somatic cell hybrid, and reticulocytes and bone marrow cells from normal adults. el-Globin gene was expressed in all cell types. Inducible expression (twoto sixfold) was documented both in HEL and K562 erythroleukemia cells after 5azacytldine treatment. Like K562 cells, HEL cells also displayed hemin-Inducible el-globin gene expression. Following transfer of human chromosome 16 from embryonicifeta1 K562 to the adult MEL cells, el-globin gene remained active but lost its potential for Inducibility, suggesting probably a trans regulation mechanism. Higher levels of 81 mRNA were found in fetal liver cells compared with trace amounts in reticulocytes and normal adult bone marrow cells. These data clearly show that in contrast to the embryonic and adult patterns of expression of 5 and a-globin genes, respectively, el-globln gene displays a different profile, being active predominantly during the early stages of ontogeny, switching to lower levels of expression in adulthood.
Experimental Cell Research, 1978
Stimulation of the T3C12 clone of Friend erythroleukemia cells with 1.2% dimethyl sulfoxide (DMSO) results in progressive increase in the concentration of globin mRNA sequences in the total cellular RNA of treated cells, as measured by nucleic acid hybridization employing a globin cDNA probe. The greatest increment in the content of globin RNA occurs between 30 and 40 h after addition of DMSO. Globin cDNA was also used to measure the concentration of globin-specific sequences in the DNA and RNA of transcriptionally active and inactive chromatin fractions prepared from these cells by the DNase II -MgCl, procedure of Gottesfeld et al. [ 161. Essentially equal concentrations of globin sequences are present in the DNA isolated from active and inactive chromatin fractions of cells grown in the presence of 1.2 % DMSO for 50 h (the time of initiation of hemoglobin synthesis). Furthermore, there are no significant differences in the globin gene concentrations between the active chromatin fractions from DMSO-treated and control cultures at either 50 or 120 h after initiation of DMSO treatment. However, chromatin-associated RNA isolated from the active chromatin of cells synthesizing maximum amounts of hemoglobin (120 h) contains a higher concentration of globin sequences than RNA from the active chromatin of control cells. Chromatin fractions from untreated cells also contain a significant amount of RNA which hybridizes to the globin cDNA probe. These observations suggest that both transcriptional and post-transcriptional control mechanisms are involved in hemoglobin gene expression in T3C12 erythroleukemia cells.
Proceedings of the National Academy of Sciences, 1983
The globin chain synthetic pattern and the extent of DNA methylation within embryonic, fetal, and adult f-like globin gene domains were evaluated in 290% 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 E chains, whereas the 12-wk erythroblasts synthesize largely fetal gamma globin and the adult marrow erythroblasts synthesize almost exclusively adult /3 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-fetal and fetal-adult Hb switches in humans. In ontogenic development this mechanism might in turn correlate with a gradual modification of chromatin structure in the non-a gene cluster, thus leading to a 5'-* 3' activation of globin genes in a balanced fashion.
1992
HE HUMAN (Y and p globin gene clusters are well- T characterized gene families that have been extensively used in studies of tissue-specific and developmental gene expression.' In both complexes the genes are arranged in the order in which they are expressed during development (5'-52-(~2-a1-3', 5'-~-~y-*y-6-P-3') and both are under the control of sequences lying several kilobases upstream of the
Blood, 1985
Analysis of methylation at the beta-globin gene cluster was carried out on DNA derived from nucleated RBCs (orthochromatic normoblasts) isolated from peripheral blood of patients with beta-thalassemia major or other congenital hemolytic anemia after splenectomy. A procedure to separate these normoblasts from the other nucleated cells of the peripheral blood was developed, providing us with a convenient source of DNA for investigating parameters related to human erythroid differentiation. Blood samples were obtained from six adult patients who express their gamma-globin genes at different levels. Inverse correlation between methylation and gene activity was consistently observed for five of the eight sites analyzed. A site 3' to the beta gene was always unmethylated, two sites flanking the epsilon gene were always found to be methylated, and two sites 5' to the two gamma genes, G gamma and A gamma, were hypomethylated in correlation with gamma gene activity of the individual ...
Quantitative analysis of globin gene induction in single human erythroleukemic cells
Nucleic Acids Research, 2000
The mechanisms involved in the normal developmental regulation of globin gene expression, and the response to pharmacological agents that elevate fetal hemoglobin, may be expected to involve either changes in each cell or a selection process affecting subsets of differentiating erythroid cells. To study these mechanisms we have developed assays to measure mRNA levels in single erythroid cells. The assay involved the use of globin-specific probes, with no detectable cross-reactivity, in real-time, fluorescence-based quantitative PCR (Q-PCR). We had previously used this Q-PCR method to measure globin mRNA levels in cultures of primary erythroid cells demonstrating that drugs like hydroxyurea, 5-azacytidine and butyric acid each yielded increases in gamma/( gamma + ss) mRNA ratios, with differential effects on ss-globin levels. We have now extended this approach to measure globin mRNA levels in single K562 cells, a human erythroleukemic cell line, with and without 30 microM hemin treatment. Hemin exposure increases total hemoglobin levels by approximately 9-fold and total alpha-, epsilon- and gamma-globin mRNA levels by 1.5-2.3-fold. Single cell analyses showed initial wide distributions of each of the three individual globin mRNA levels with most cells having detectable but very low levels of each globin transcript. Hemin induction shifted the distributions to higher levels, with a tendency to residual left skewing as some cells remained with very low expression levels despite the effect of hemin in increasing expression in most of these low expressing cells. Thus transcriptional heterogeneity remains a crucial variable, even in this extensively used model of human erythroid biology, and clearly influences strongly the response to inducing agents. These methods may enable us to define better possible molecular and/or cellular models of globin gene modulation.
DNA sequences required for regulated expression of β-globin genes in murine erythroleukemia cells
Cell, 1984
We introduced into MEL cells rabbit /3-globin gene deletion mutants and two sets of hybrid genes constructed from the inducible human @-globin gene and noninducible human y-globin gene or the murine H-2K"'"' class I MHC gene. Sl nuclease analysis of gene transcripts before and after MEL differentiation showed that induction of the rabbit @-globin gene did not require more than 58 bp of DNA 5' to the transcription initiation site. Hybrid genes were constructed with human B-globin DNA sequences from either 5' or 3' of the translation initiation site linked to the complementary parts of they or H2Kbm' genes. Both types of constructs were inducible during MEL differentiation. The relative rates of transcription of the 5'7-3'8 and 5'H2-3'8 hybrid genes show that induction of the hybrid gene transcripts results at least in part from transcriptional activation of the genes. We suggest that DNA sequences that regulate 8-globin gene transcription during MEL differentiation are located both 5' and 3' to the translation initiation site.