Egr-1 mediates hypoxia-inducible transcription of the NDRG1 gene through an overlapping Egr-1/Sp1 binding site in the promoter.
N-myc down-regulated gene 1 (NDRG1/Cap43) is inducible by a variety of environmental stressors, including hypoxia. The present study identified a cis-acting element mediating the transactivation of the NDRG1 gene in murine RAW264.7 macrophage cells treated with hypoxia or deferoxamine, an iron chelator mimicking hypoxia. Through a series of deletions of the promoter of NDRG1 luciferase constructs, a minimal cis-acting element conferring inducibility by hypoxia and deferoxamine was localized to an early growth response 1 (Egr-1) and Sp1 overlapping binding site. Electrophoretic mobility shift assay, antibody supershift assay, and mutations of the Egr-1 binding site confirmed the specific binding of Egr-1 protein to this Egr-1/Sp1 motif. In addition, hypoxia increased the level of Egr-1 protein that correlated with induction of NDRG1 expression at both RNA and protein levels. Transient transfection of the Egr-1 gene into HeLa cells also resulted in up-regulation of the NDRG1 mRNA. The role of Egr-1 was further verified by mutations in the Egr-1 binding site, which reduced promoter inducibility by hypoxia and deferoxamine. Furthermore, the induction of NDRG1 expression by hypoxia and deferoxamine was diminished by RNA interference knockdown of Egr-1 gene expression and in Egr-1-/- mouse embryonic fibroblasts (MEF) compared with Egr-1+/- MEFs. These results showed for the first time that Egr-1 regulates NDRG1 transcription through an overlapping Egr-1/Sp1 binding site that acts as a major site of positive regulation of the NDRG1 promoter by hypoxia signaling.
Zhang
Ping
P
Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987, USA.
Tchou-Wong
Kam-Meng
KM
Costa
Max
M
D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics*; D015687:Cell Hypoxia / Q000235:genetics; D045744:Cell Line, Tumor; D003676:Deferoxamine / Q000494:pharmacology; D051766:Early Growth Response Protein 1 / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008264:Macrophages / Q000378:metabolism / Q000502:physiology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019319:Okadaic Acid / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation; D000099259:N-myc Downstream-Regulated Gene 1 Protein
D018797:Cell Cycle Proteins; D003676:Deferoxamine; D051766:Early Growth Response Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D019319:Okadaic Acid; D016329:Sp1 Transcription Factor; D000099259:N-myc Downstream-Regulated Gene 1 Protein