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.     

Binding of USF to a non-canonical E-box following stress results in a cell-specific derepression of the lama3 gene

Expression of the lama3 gene, encoding the laminin α3A chain, is restricted to specialized epithelia. We previously showed that lama3 gene expression is controlled by an epithelial enhancer through the cooperative effect of AP-1 binding sites. In fibroblasts, there is no lama3 expression because of the recruitment of a repressor complex absent or inactive in epithelial cells. In this paper, we show evidence that this repression of the lama3 gene is relieved by exogenous and UV-induced USF-1 through its interaction with a non-canonical E-box site. Using a chromatin immunoprecipitation assay, we find that UV stress induces USF to bind to the lama3 promoter in vivo. We further demonstrate that this loss of cell specificity is directly related to the accessibility of the E-box, resulting in a strong induction in fibroblasts, while expression remains constitutively high in keratinocytes. This accessibility appears to be dependent upon the recruitment of a fibroblastic repressor complex. Therefore, we speculate that anchorage of this repressor complex in fibroblasts modifies the enhancer geometry, allowing USF to interact under stress-inducing conditions with its heptameric binding site.     

Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites

Deletion analysis of the human PRL promoter in endometrial stromal cells decidualized in vitro revealed a 536-bp enhancer located between nucleotide (nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragment ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cells. DNase I footprint analysis of decidualized endometrial stromal cells revealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind activator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 complex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 536-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer activity by approximately 70%. Conversely, coexpression of Fra-2 in combination with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. Introduction of JunD and Fra-2 into nondecidual cells is sufficient to confer enhancer activity. JunD and Fra-2 protein expression was markedly increased in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These investigations indicate that the 5'-flanking region of the human PRL gene contains a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding sites within this enhancer region are critical for activity.     

Frequency and Distribution of AP-1 Sites in the Human Genome

The AP-1–binding sequences are promoter/enhancer elementsthat play an essential role in the induction of many genes inmammalian cells; however, the number of genes containing AP-1sites remains unknown. In order to better address the overalleffect of AP-1 on expression of genes encoded by the entiregenome, a genome-wide analysis of the frequency and distributionof AP-1 sites would be useful; yet to date, no such analysisof AP-1 sites or any other promoter/enhancer elements has beenperformed. We present here our study of the consensus AP-1 siteand two single-bp variants showing that the frequency of AP-1sites in promoter regions is significantly lower than theiraverage rate of occurrence in the whole genomic sequence, aswell as the frequency of a random heptanucleotide suggestingthat nature has selected for a decrease in the frequency ofAP-1 sites in the regulatory regions of genes. In addition,genes containing multiple AP-1 sites are more prevalent thanthose containing only one copy of an AP-1 site, which againmay have evolved to allow for greater signal amplification orintegration in the regulation of AP-1 target genes. However,the number of AP-1–regulated genes identified in variousstudies is far smaller than the number of genes containing potentialAP-1 sites, indicating that not all AP-1 sites are activatedin a given cell under a given condition, and is consistent withthe prediction by others that cellular context determines whichAP-1 sites are targeted by AP-1.     

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.     

Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.