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

Abstract

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.

Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells.

“…1. Human or mouse erythroid cells were fused with nonerythroid cells of the other species to form intertypic heterokaryons (4,5). After 1 to 2 days in culture, cells were harvested for isolation of total cellular RNA or for fluorescence microscopy to confirm formation of heterokaryons.…”

“…After 1 to 2 days in culture, cells were harvested for isolation of total cellular RNA or for fluorescence microscopy to confirm formation of heterokaryons. RNA was examined for de novo activation of GATA-1 in the nonerythroid nuclei by quantitative RNase mapping (4,5) or by a semiquantitative RT-PCR analysis. The RT-PCR assay was developed to increase the sensitivity of detection of gene expression in transient heterokaryons.…”

“…MEL, K562, HeLa, and Raji (human T-cell line) cells, Epstein Barr virusimmortalized normal human B cells (line DTC), C127 mouse fibroblasts, and mouse embryo fibroblasts were maintained in culture and heterokaryons were prepared as described previously (4,5). The mouse ES cell line CCE (40) was maintained as previously described (43).…”

“…The first direct evidence that erythroid cell-and developmental stage-specific trans-acting regulatory factors are involved in the regulation of globin gene expression was provided by analyses of transient heterokaryons (4). Following fusion of erythroid with nonerythroid cells, globin genes of different developmental stages can be turned on in a variety of nonerythroid cell types of multiple embryonic lineages (4,5).…”

Structural and Functional Cross-Talk between a Distant Enhancer and the ɛ-Globin Gene Promoter Shows Interdependence of the Two Elements in Chromatin

“…Digestion was carried out at a 1/30 dilution of RNase A/T 1 for 60 min at 37°C. The ε-globin and ␥-actin (loading control) probes used have been described elsewhere (7,28). Transcription of the ε-globin gene was normalized to the ␥-actin message level and corrected for copy number of the minichromosome.…”