The developmental switch in hemoglobin synthesis, from embryonic (including epsilon globin) to fetal (gamma) to adult (beta) hemoglobins, that occurs during ontogeny is of great interest because of its fundamental biology as well as its relevance to the treatment of hemoglobinopathies. We have been studying transcriptional regulation of globin genes to clarify the molecular mechanisms of this switch. Transcriptional activity of globin genes is determined by a variety of cis-acting regulatory DNA sequences and trans-acting proteins. One of the cis-acting elements is the silencer located in the region between - 177 and -392bp 5' to the epsilon globin gene, which was characterized in this laboratory. To delineate the molecular mechanism of the silencer and its possible role in the developmental regulation of epsilon gene expression, silencing of the epsilon gene in human adult erythroid cells (hAEC) was studied. The K562 erythroleukemia cell line constitutively expresses low levels of embryonic and fetal, but not adult hemoglobin and can serve as a control. We have now shown that in hAEC the mRNA level of each exon of the epsilon gene is present in low and varied amounts but the full length epsilon message is not present. In contrast, in K562 cells, where epsilon-globin gene is expressed, the full length epsilon- message is detectable and the mRNA level among the three exons is high and similar. Detection of the other globin messages (gamma and beta) showed that in hAEC beta-mRNA exon levels are high and gamma-mRNA levels are low. In K562 cells beta-mRNA levels are very low. As expected, in these cells which express high levels of gamma-globin, gamma-mRNA levels are high. Moreover, all or the majority of the globin transcripts for the highly expressed globin genes in both cell types are correctly spliced in the cytoplasm and the nucleus. The globin-transcripts, detected in cells where are not expressed, are unspliced (all or part of the transcripts). The above results may indicate that during erythropoiesis when erythroid cells switch from expressing epsilon-to- gamma -globin and then to beta-globin gene, changes in the transcription rate of epsilon-transcripts lead to the suppression of epsilon-gene transcription. These changes may activate inappropriate RNA splicing mechanism, thus unspliced epsilon-transcripts are detected when epsilon- gene is silent. 5' flanking sequences of the epsilon-globin gene, like the epsilon-globin gene silencer may contribute to the above changes.
