Mechanism of silencing of the human epsilon globin gene

Ili B. Peters, E. Fibach, A. N. Schechter, C. T. Noguchi

Research output: Contribution to journalArticlepeer-review

Abstract

To study the mechanism of silencing of the human embryonic e-globin gene, a component of hemoglobin "switching" apparently under autonomous gene control, we have used transient transfection assays in human primary cell cultures and cell lines. We find that the e-globin gene is completely silenced in primary human adult erythroid cell cultures, but not in embryonic cell lines, and that mutation of the canonical silencer leads to strong expression in these adult cells. The silencer reduces expression of an SV40luciferase (SV40-luc) construct by 50% in this system. These cells appear to be the optimal assay for the silencer. The silencer only slightly inhibited SV40-luc expression in the K562 embryonic cell line but did so strongly in HeLa cells. Mutation of the GATA-1 site of the silencer decreased E-globin transcription in K562 cells while mutation of the YY1 site increased trancription. Co-transfection assays indicate that GATA-1 overexpression increases E-globin transcription in K562 cells, while GATA-2 over-expression has little effect, suggesting that GATA-2 does not act as a transactivator of the silencer. Over-expression of YY1 did not appear to affect the action of the silencer. These results suggest that developmentally specific proteins interact with YY1 and/or GATA-1 to modulate the negative effect of the silencer. Deletion mutation analyses have identified further negative regulatory regions at -4 kb, -3 kb, -2.2 kb and -1.6 kb relative to the canonical cap site. The region near -3 kb could be separated into several subrogions with positive or negative effects. These regions showed strong evolutionary conservation (as high as 55%) and GATA motifs, similar to the properties of the canonical silencer. Downregulation of E-globin gene expression during erythroid development appears to involve interactions of these several negative transcription elements.

Original languageEnglish
Pages (from-to)215a
JournalJournal of Investigative Medicine
Volume44
Issue number3
StatePublished - 1996
Externally publishedYes

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