Sp1 regulates chromatin looping between an intronic enhancer and distal promoter of the human heme oxygenase-1 gene in renal cells.
HO-1 (heme oxygenase-1) is an inducible microsomal enzyme that catalyzes the degradation of pro-oxidant heme. The goal of this study was to characterize a minimal enhancer region within the human HO-1 gene and delineate its role in modulating HO-1 expression by participation with its promoter elements in renal epithelial cells. Deletion analysis and site-directed mutagenesis identified a 220-bp minimal enhancer in intron 1 of the HO-1 gene, which regulates hemin-mediated HO-1 gene expression. Small interfering RNA, decoy oligonucleotides, site-directed mutagenesis, and chromatin immunoprecipitation assays confirmed the functional interaction of Sp1 with a consensus binding sequence within the 220-bp region. Mutations of regulatory elements within the -4.5 kb promoter region (a cyclic AMP response and a downstream NF-E2/AP-1 element, both located at -4.0 kb, and/or an E-box sequence located at -44 bp) resulted in the loss of enhancer activity. A chromosome conformation capture assay performed in human renal epithelial (HK-2) cells demonstrated hemin-inducible chromatin looping between the intronic enhancer and the -4.0 kb promoter region in a time-dependent manner. Restriction digestion with ApaLI (which cleaves the 220-bp enhancer) led to a loss of stimulus-dependent chromatin looping. Sp1 small interfering RNA and mithramycin A, a Sp1 binding site inhibitor, resulted in loss of the loop formation between the intronic enhancer and the distal HO-1 promoter by the chromosome conformation capture assay. These results provide novel insight into the complex molecular interactions that underlie human HO-1 regulation in renal epithelial cells.
Deshane
Jessy
J
Department of Medicine, Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Kim
Junghyun
J
Bolisetty
Subhashini
S
Hock
Thomas D
TD
Hill-Kapturczak
Nathalie
N
Agarwal
Anupam
A
D017209:Apoptosis; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry*; D047369:Chromatin Immunoprecipitation; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D051547:Heme Oxygenase-1 / Q000096:biosynthesis*; D006427:Hemin / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D016296:Mutagenesis; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014162:Transfection
D002843:Chromatin; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D006427:Hemin; D051547:Heme Oxygenase-1