Kollia P, Noguchi C T, Fibach E, Loukopoulos D, Schechter A N
First Department of Medicine, University of Athens, Laikon General Hospital, Greece.
Proc Assoc Am Physicians. 1997 Jul;109(4):420-8.
We are interested in the genetic mechanisms whereby several classes of drugs increase fetal hemoglobin (HbF) in patients with sickle-cell anemia or beta-thalassemia. Recently, we have shown (Kollia et al., Proc. Natl. Acad. Sci. U.S.A. 93: 5693, 1996) that cultured primary human adult erythroid cells (hAEC) offer a useful model for the study of transcriptional and posttranscriptional regulation of globin gene expression. We have found also that hemin markedly increases HbF levels in these cells. We report here the effect of hemin on globin gene transcription and RNA processing in hAEC. Quantitative reverse transcriptase-polymerase chain reaction analysis showed that the gamma-globin message levels in the cytoplasm and nucleus were increased two-fold by hemin. In the untreated cells, only spliced gamma-transcripts were detected in the cytoplasm, indicating that only completely processed gamma-RNA is transported to the cytoplasm, whereas approximately half of the nuclear gamma-globin RNA transcripts were unspliced. After treatment with hemin, correctly spliced gamma-transcripts increased in the cytoplasm and nucleus, while the unprocessed gamma-transcripts decreased in number in the nucleus. We also studied epsilon-globin RNA transcripts; in the cytoplasm of untreated cells, only correctly processed transcripts were present, whereas the nuclear epsilon-globin RNA transcripts were unspliced. In hemin-induced cells, unspliced nuclear epsilon-transcripts decreased in number. In contrast to the gamma- and epsilon-globin genes, the levels of full-length, correctly spliced beta-globin message are not affected by hemin. Nuclear run-on transcription assays confirmed the increase in the rate of transcription of gamma- and epsilon-globin genes in hemin-treated versus untreated hAEC. These results indicate that hemin affects the expression of embryonic and fetal globin genes by acting both at the transcriptional and posttranscriptional levels. These results may be relevant to the action of other agents that affect the hemoglobin phenotype of human erythroid cells.
我们对几类药物增加镰状细胞贫血或β地中海贫血患者胎儿血红蛋白（HbF）的遗传机制感兴趣。最近，我们已经表明（科利亚等人，《美国国家科学院院刊》93: 5693, 1996），培养的原代成人人类红系细胞（hAEC）为研究珠蛋白基因表达的转录和转录后调控提供了一个有用的模型。我们还发现血红素能显著提高这些细胞中的HbF水平。我们在此报告血红素对hAEC中珠蛋白基因转录和RNA加工的影响。定量逆转录聚合酶链反应分析表明，血红素使细胞质和细胞核中的γ珠蛋白信使水平增加了两倍。在未处理的细胞中，细胞质中仅检测到剪接后的γ转录本，这表明只有完全加工后的γRNA才被转运到细胞质中，而细胞核中约一半的γ珠蛋白RNA转录本未被剪接。用血红素处理后，正确剪接的γ转录本在细胞质和细胞核中增加，而未加工的γ转录本在细胞核中的数量减少。我们还研究了ε珠蛋白RNA转录本；在未处理细胞的细胞质中，仅存在正确加工的转录本，而细胞核中的ε珠蛋白RNA转录本未被剪接。在血红素诱导的细胞中，未剪接的细胞核ε转录本数量减少。与γ和ε珠蛋白基因不同，全长、正确剪接的β珠蛋白信使水平不受血红素影响。细胞核连续转录分析证实，与未处理的hAEC相比，血红素处理的hAEC中γ和ε珠蛋白基因的转录速率增加。这些结果表明，血红素通过在转录和转录后水平发挥作用来影响胚胎和胎儿珠蛋白基因的表达。这些结果可能与其他影响人类红系细胞血红蛋白表型的药物的作用有关。
