Activation of Developmentally Mutated Human Globin Genes by Cell Fusion

Papayannopoulou, Thalia; Enver, Tariq; Takegawa, Susumu; Anagnou, Nicholas P.; Stamatoyannopoulos, George

doi:10.1126/science.2461587

Cited by 28 publications

(16 citation statements)

References 10 publications

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“…Mutated (HPFH) human fetal globin genes are activated in stable somatic cell hybrids (29). In this report, we show that in transient heterokaryons formed by fusion of MEL cells with cells from several different HPFH cell types, the mutated (HPFH) fetal genes are rapidly activated.…”

mentioning

confidence: 74%

Regulated expression of human alpha- and beta-globin genes in transient heterokaryons.

Baron

Maniatis

1991

Mol. Cell. Biol.

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We have examined the expression of human a-and P-like globin genes in transient heterokaryons formed by fusion of human nonerythroid cells with terminally differentiating mouse erythroleukemia (MEL) cells or with a MEL cell variant (GM979) in which the endogenous mouse embryonic 1-globin genes are activated. In both the parental MEL cells and the heterokaryons, the a-globin genes were activated at least 12 h earlier than the embryonic, fetal, and adult P-globin genes. These results suggest that kinetic differences in the activation of aand 1-like globin genes are not simply the result of different rates of accumulation of erythroid-specific regulatory factors but may reflect differences in the mechanisms governing the transcriptional activation of these genes during erythroid cell differentiation. In mouse GM979 x human nonerythroid heterokaryons, the human embryonic 1-globin gene was activated, consistent with our previous demonstration that erythroid cells contain stage-specific trans-acting regulators of globin gene expression. Moreover, a dramatic increase in the ratio of human fetal to adult 13-globin transcription was observed compared with that seen in MEL-human nonerythroid hybrids. This ratio change may reflect competition between the fetal and adult 1-globin genes for productive interactions with erythroid cell-specific regulatory elements. Finally, we demonstrate that the behavior of naturally occurring mutations that lead to aberrant hemoglobin switching in humans also leads to aberrant expression in transient heterokaryons. Therefore, erythroid cells must contain trans-acting factors that interact with mutated regulatory elements to induce high-level expression of the human fetal globin genes.

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mentioning

confidence: 74%

Regulated expression of human alpha- and beta-globin genes in transient heterokaryons.

Baron

Maniatis

1991

Mol. Cell. Biol.

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“…Lymphoid cells from a Hispanic thalassemia individual, immortalized with Epstein-Barr virus, were fused with MEL cells, selected, and propagated as described (Takegawa et al 1986;Papayannopoulou et al 1988). Briefly, MEL cells were mixed with human lymphoid cells at 1 : 10 ratio, and 50% polyethylene glycol (PEG, vol/vol) was added in RPMI-1640 media at 37~ Cells were then washed, resuspended in RPMI plus 10% fetal calf serum, and cultured for 48 hr prior to selection for hybrid phenotype.…”

Section: Somatic Cell Hybridsmentioning

confidence: 99%

“…Previous work has shown that these hybrids faithfully reproduce normal and mutant globin gene expression and thereby constitute a model system to study globin gene regulation (Papayannopoulou et al 1988).…”

mentioning

confidence: 99%

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Forrester¹,

Epner²,

Driscoll³

et al. 1990

Genes Dev.

461

281

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Naturally occurring deletions that remove sequences located -60 kb upstream of the human adult {3-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of c/s-linked globin genes. This regulatory region located at the 5' end of the {3-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human I~-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire 13-globin locus and sequences -100 kb 5' and 3' of the adult 13-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.

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“…Hybrid aliquots which had been cryopreserved before the onset of the ␥-to-␤ switch were used in this study. Because addition of sodium butyrate to the culture medium induces terminal differentiation of MEL cells (15,16,21), we used ␣-ABA instead of sodium butyrate; ␣-ABA is a compound which does not, in our experience, trigger terminal differentiation. Cells were grown in the presence of 20% FCS or 20% charcoal-absorbed FCS.…”

Section: Resultsmentioning

confidence: 99%

“…(29,34). When lymphoid cells from persons with hereditary persistence of fetal hemoglobin due either to ␥ gene promoter mutations or to ␦-and ␤ gene deletions are fused with MEL cells, human fetal hemoglobin is produced in the hybrids (21). When erythroid cells from fetuses are fused with MEL cells, the HFE ϫ MEL hybrids produce predominantly human fetal hemoglobin; however, after several weeks in culture, they switch to adult human globin production (20).…”

Section: Discussionmentioning

confidence: 99%

Effects of Butyrate and Glucocorticoids on γ- to β-Globin Gene Switching in Somatic Cell Hybrids

Zitnik

Peterson

Stamatoyannopoulos

et al. 1995

Molecular and Cellular Biology

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Butyrate and its analogs have been shown to induce fetal hemoglobin in humans and primates and in erythroid cell cultures. To obtain insights concerning the cellular mechanisms of butyrate action, we analyzed the effects of butyrate on human globin gene expression in hybrids produced by fusing mouse erythroleukemia cells (MEL) with human fetal erythroid cells (HFE). These hybrids initially express human fetal hemoglobin but subsequently switch to adult globin expression after several weeks in culture. We found that ␣-aminobutyric acid, a butyrate analog which does not induce terminal maturation, strikingly delays the rate of the ␥-to ␤-globin gene (␥-to-␤) switch in the HFE ؋ MEL hybrids. The effect of butyrate on globin expression is transient, with the result that the delay of globin gene switching requires the continuous presence of this compound in culture. Furthermore, butyrate fails to induce fetal hemoglobin expression in hybrids which have switched, suggesting that the effect of this compound on ␥-globin expression is due to inhibition of ␥ gene silencing rather than to induction of ␥ gene transcription. Since in other cellular systems, glucocorticoids antagonize the action of butyrate, the effect of dexamethasone on the ␥-to-␤ switch in HFE ؋ MEL hybrids was examined. Dexamethasone strikingly accelerated the ␥-to-␤ switch, and its effect was irreversible. The effects of dexamethasone and butyrate on the ␥-to-␤ switch of the HFE ؋ MEL hybrids appear to be codominant. These results indicate that steroids can have a direct effect on globin gene switching in erythroid cells.

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Activation of Developmentally Mutated Human Globin Genes by Cell Fusion

Cited by 28 publications

References 10 publications

Regulated expression of human alpha- and beta-globin genes in transient heterokaryons.

Regulated expression of human alpha- and beta-globin genes in transient heterokaryons.

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Effects of Butyrate and Glucocorticoids on γ- to β-Globin Gene Switching in Somatic Cell Hybrids

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