Functional roles of in vivo footprinted DNA motifs within an alpha-globin enhancer. Erythroid lineage and developmental stage specificities.
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.
Zhang
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Q
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Rombel
I
I
Reddy
G N
GN
Gang
J B
JB
Shen
C K
CK
D000328:Adult; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000502:physiology; D004742:Enhancer Elements, Genetic; D004912:Erythrocytes / Q000502:physiology; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D005914:Globins; D004247:DNA