REGULATIN OF GLOBIN GENE EXPRESSION BY 5' DNA SEQUENCES

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In order to better understand the switching of human globin synthesis from embryonic to fetal to adult globin, we are studying a human cell line, K562, blocked in the transition from fetal to adult globin synthesis. K562 cells are unable to synthesize beta-globin mRNA. Previous studies from this laboratory have shown the molecular defect in K562 cells appears to be in a trans-acting factor. The most likely possibilities are that a repressor is continuously synthesized which binds to DNA near the beta-globin promoter, preventing transcription, or that an activator which is necessary for beta-globin transcription is not synthesized, or that K562 cells both synthesize a repressor and lack an activator. To test these hypotheses we are using a plasmid, p-beta-GLCAT, which contains the human beta-globin promoter and additional 5' DNA fused to an easily assayable gene, CAT (chloramphenicol acetyl transferase). The fusion gene, beta-CAT, is inactive in K562 cells so we are attempting to activate it using several approaches. Our experiments to activate beta-CAT either in cis, with the SV40 enhancer, or in trans, with the E1A protein, have been unsuccessful in K562 cells but successful in Chinese hamster cells, consistent with the presence of a repressor in K562 cells. Further support for this model comes from 5' deletion mutants of beta-CAT, which indicate that deletion of certain 5' sequences does allow beta-CAT expression in K562 cells. Thus, there seems to be a negative regulatory region near the beta-globin promoter. Experiments are underway to try to identify the DNA binding site of the putative repressor and to identify the repressor molecule itself.