Abstract
Transcriptional regulation of the human alpha-like globin genes, embryonic zeta 2 and adult alpha, during erythroid development is mediated by a distal enhancer, HS-40. Previous protein-DNA binding studies have shown that HS-40 consists of multiple nuclear factor binding motifs that are occupied in vivo in an erythroid lineage- and developmental stage-specific manner. We have systematically analyzed the functional roles of these factor binding motifs of HS-40 by site-directed mutagenesis and transient expression assay in erythroid cell cultures. Three of these HS-40 enhancer motifs, 5'NF-E2/AP1, GT II, and GATA-1(c), positively regulate the zeta 2-globin promoter activity in embryonic/fetal erythroid K562 cells and the adult alpha-globin promoter activity in adult erythroid MEL cells. On the other hand, the 3'NF-E2/AP1 motif is able to exert both positive and negative regulatory effects on the zeta 2-globin promoter activity in K562 cells, and this dual function appears to be modulated through differential binding of the ubiquitous AP1 factors and the erythroid-enriched NF-E2 factor. Mutation in the GATA-1(d) motif, which exhibits an adult erythroid-specific genomic footprint, decreases the HS-40 enhancer function in dimethyl sulfoxide-induced MEL cells but not in K562 cells. These studies have defined the regulatory roles of the different HS-40 motifs. The remarkable correlation between genomic footprinting data and the mutagenesis results also suggests that the erythroid lineage- and developmental stage-specific regulation of human alpha-like globin promoters is indeed modulated by stable binding of specific nuclear factors in vivo.
Highlights
We have systematically analyzed the functional roles of these factor binding motifs of HS-40 by site-directed mutagenesis and transient expression assay in erythroid cell cultures
The enhancer effects of these HS-40 mutants on the b'2-globin promoter activity in K562 cells and on the a-globin promoter activity in Me2SO-induced MEL cells were measured by transient expression assay
Since only transient expression assay has been used, our data most likely reflect the structure-function relationship during the final stage of transcriptional activation when the globin promoters are presumably physically associated with the HS-40 enhancer
Summary
(Received for publication, December 23, 1994, and in revised form, January 31, 1995). Similar to the regulation of the (3-like globin genes by (3-LCR, the a-LCR or HS-40 confers high levels of erythroid-specific expression of linked human ~2- or a-globin genes in transgenic mice or stably integrated cell lines (Jarman et al, 1991; Sharpe et al, 1992; Vyas et al, 1992; Sharpe et al, 1993; Gourdon et al, 1994) It behaves as a classical enhancer for the promoter activity of ~- or a-globin genes in transiently transfected erythroid cell culture (Pondel et al, 1992; Ren et al, 1993; Zhang et al, 1993). It did not allow us to assess the functional significance of the developmental stage-specific difference of nuclear factor binding patterns within HS-40, as seen in previous genomic footprinting analysis of HS-40 in adult versus embryonic/fetal erythroid cells (Strauss et al, 1992; Zhang et al, 1993). The functionality of these motifs correlates remarkably well with their erythroid lineage- and developmental stage-specific occupancies by nuclear factors in vivo
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