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.     

A hormone response element in the human apolipoprotein CIII (ApoCIII) enhancer is essential for intestinal expression of the ApoA-I and ApoCIII genes and contributes to the hepatic expression of the two linked genes in transgenic mice

We have generated transgenic mice carrying wild-type promoters of the human apolipoprotein A-I (apoA-I)-apoCIII gene cluster or promoters mutated in their hormone response elements. The wild-type cluster directed high levels of apoA-I gene expression in liver and intestine, moderate expression in kidney, and low to minimal expression in other tissues. It also directed high levels of chloramphenicol acetyltransferase (CAT) expression (used as a reporter for the apoCIII gene) in liver, low levels in intestine and kidney, and no expression in other tissues. Mutations in the apoCIII promoter and enhancer abolished the intestinal and renal expression of the apoA-I gene, reduced hepatic apoA-I expression by 80%, and abolished CAT expression in all tissues. A similar pattern of expression was obtained by mutations in the apoCIII enhancer alone. Mutations in the proximal apoA-I promoter reduced by 85% hepatic and intestinal apoA-I expression and did not affect CAT expression. The findings suggest that a hormone response element within the apoCIII enhancer is essential for intestinal and renal expression of apoA-I and apoCIII genes and also enhances hepatic expression. The hormone response elements of the proximal apoA-I promoter or the apoCIII enhancer can promote independently low levels of hepatic and intestinal expression of the apoA-I gene in vivo.     

Regulation of human microsomal prostaglandin E synthase-1 by IL-1β requires a distal enhancer element with a unique role for C/EBPβ

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1β (interleukin 1β) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPβ (CCAAT/enhancer-binding protein β) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPβ binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPβ in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPβ highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.     

The SP1 sites of the human apoCIII enhancer are essential for the expression of the apoCIII gene and contribute to the hepatic and intestinal expression of the apoA-I gene in transgenic mice

We have generated transgenic mice carrying wild-type and mutant forms of the apolipoprotein (apo)A-I/apoCIII gene cluster. Mutations were introduced either in one or in three SP1 binding sites of the apoCIII enhancer. In mice carrying the wild-type transgene, major sites of apoA-I mRNA synthesis were liver and intestine and minor sites were kidney and, to a lesser extent, other tissues. The major site of chloramphenicol acetyl transferase (CAT) activity (used as a reporter for the apoCIII gene) was liver and minor sites intestine and kidney. A mutation in one SP1 binding site reduced the expression of the apoA-I gene to ~23 and 19% in the liver and intestine, respectively, as compared to the control wild-type. The hepatic expression of the CAT gene was not affected whereas the intestinal expression was nearly abolished. Mutations in three SP1 binding sites reduced the hepatic and intestinal expression of the apoA-I and CAT genes to 14 and 4%, respectively, as compared to the wild-type control, and abolished CAT expression in all tissues. The findings suggest that the SP1 sites of the apoCIII enhancer are required for the expression of the apoCIII gene and also contribute significantly to the hepatic and intestinal expression of the apoA-I gene in vivo.     

Activity of the rat liver-specific aldolase B promoter is restrained by HNF3.

Although it contains binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the aldolase B gene is surprisingly weak when tested by transient transfection in differentiated hepatoma cells. This low activity could be due to overlapping between HNF1 and HNF3 binding sites in element PAB, from -127 to -103 bp with respect to the cap site. Replacement of the PAB region by a consensus HNF1 binding site unable to bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained by HNF3 binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter by excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 binding site is weakly transactivated by HNF1. The inhibitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition between these two factors for binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 binding site. A construct in which PAB has been replaced by an exclusive HNF3 binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.