REGULATED EXPRESSION OF HUMAN GLOBIN GENES AND NEIGHBORING SEQUENCES INTRODUCED INTO ERYTHROID AND NONERYTHROID CELLS (ACTIVATION, DNA-MEDIATED TRANSFER, NEGATIVE REGULATION, CHROMATIN DOMAINS, TAP)

Abstract

The regulation of human globin genes during hemopoietic differentiation was investigated by introducing these genes into mouse erythroleukemia (MEL) cells by chromosome transfer and by DNA-mediated gene transfer (DMGT). Human globin and other genes were also studied in mouse fibroblasts transfected by DMGT and in human erythroleukemia (HEL) cells.;MEL cells were fused to human lymphoblasts and fibroblasts. Hybrid cells retaining human chromosome 11 produced human (beta)(adult) and (gamma)(fetal) globin mRNAs indicating the activation of previously silent globin genes. Human globin mRNA levels were increased by dimethyl sulfoxide (DMSO), an inducer of MEL cell differentiation, to about 50-75% of mouse (beta)-globin mRNA. The (beta):(gamma)mRNA ratio was about 100:1, similar to that observed in adult erythroid cells and human (beta)-globin was readily detected. Thus, expression of the human (beta)-globin gene in cell hybrids is normal.;A plasmid, pTAG, having the human (alpha)- and (gamma)- globin and Herpes Simplex Virus thymidine kinase (TK) genes was introduced into mouse fibroblasts and MEL cells. Both globin genes were equally expressed in fibroblasts; the human (alpha)-globin gene was expressed at similar levels in MEL cells. However, human (gamma)-globin mRNA levels were very low in MEL transfectants. Fibroblast lines showed constitutive expression, while MEL lines displayed a marked decline (negative regulation) of human globin and TK mRNA levels after DMSO induction. Thus, in contrast to hybrid cells, normal regulation of human globin genes was not observed in MEL transfectants, although coordinate negative regulation was observed. A second form of coordinate regulation with pTAG was seen in fibroblast transfectants. The coordinate shutting off and turning on (modulation) of human (alpha)- and (gamma)- globin and TK genes was demonstrated by use of media that select against and for the expression of TK, respectively.;DNA sequences flanking the globin genes play a role in their regulation. To begin elucidating this role, transcription in the region upstream from the ('G)(gamma) and ('A)(gamma) genes was examined in embryonic-fetal HEL cells expressing these genes. Analysis of the upstream DNA sequence revealed a potential RNA polymerase II transcription unit. Transcription in this region was detected in HEL cells.