Sp1 regulates chromatin looping between an intronic enhancer and distal promoter of the human heme oxygenase-1 gene in renal cells.

Academic Article

Abstract

  • 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.
  • Published In

    Keywords

  • Apoptosis, Cell Line, Chromatin, Chromatin Immunoprecipitation, Enhancer Elements, Genetic, Gene Expression Regulation, Enzymologic, Heme Oxygenase-1, Hemin, Humans, Kidney, Mutagenesis, Plasmids, Promoter Regions, Genetic, RNA, Small Interfering, Sp1 Transcription Factor, Transfection
  • Digital Object Identifier (doi)

    Author List

  • Deshane J; Kim J; Bolisetty S; Hock TD; Hill-Kapturczak N; Agarwal A
  • Start Page

  • 16476
  • End Page

  • 16486
  • Volume

  • 285
  • Issue

  • 22