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. 2010 May 28;285(22):16476-86.
doi: 10.1074/jbc.M109.058586. Epub 2010 Mar 29.

Sp1 Regulates Chromatin Looping Between an Intronic Enhancer and Distal Promoter of the Human Heme oxygenase-1 Gene in Renal Cells

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Free PMC article

Sp1 Regulates Chromatin Looping Between an Intronic Enhancer and Distal Promoter of the Human Heme oxygenase-1 Gene in Renal Cells

Jessy Deshane et al. J Biol Chem. .
Free PMC 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.

Figures

FIGURE 1.
FIGURE 1.
Characterization of the 220-bp minimal enhancer region in the human HO-1 gene by deletion analysis of the +12.5 kb enhancer in HK-2 cells. A, schematic showing the genomic structure of the human HO-1 gene and promoter in relation to the reporter gene constructs containing the −4.5 kb human HO-1 promoter with the intact +12.5 kb enhancer or with the indicated deletions (shown in open rectangles). The five exons are indicated (E1–E5). The restriction sites for BamHI (B), PstI (P), XbaI (X), and EcoRI (R) are indicated in the genomic map. The enhancer deletion constructs were derived from the parental plasmid, pHOGL3/4.5+12.5. Note that all enhancer fragments are cloned upstream of the −4.5 kb promoter in the SalI site of pGL3 as described under “Experimental Procedures.” B, HK-2 cells were transfected with the indicated constructs in a 10-cm dish using equimolar amounts of DNA and a batch transfection protocol as described under “Experimental Procedures.” 24 h after transfection, cells were split into two 12-well dishes and exposed to control (DMSO) or hemin (5 μm). Luciferase activity was measured 16 h later. Data are represented as mean ± S.E. (error bars) from four independent experiments with 8 replicates/group. *, p < 0.01 versus pHOGL3/4.5+12.5 hemin-stimulated samples. C, schematic shows the location of the 866- and 220-bp enhancer fragments for the pHOGL3/4.5+866 and pHOGL3/4.5+220 constructs, respectively. D, 866- and 220-bp intronic enhancer fragments show comparable reporter activity. Cells were transfected and treated with control (DMSO) or hemin (5 μm) as in B. Luciferase activity was measured 16 h later. Values are mean ± S.E. (error bars), n = 3 independent experiments with 8 replicates/group. *, p < 0.01 versus pHOGL3/4.5 kb control samples; #, p < 0.05 versus pHOGL3/4.5+12.5 hemin-treated samples. E, orientation-independent effects of the intronic enhancer. Cells were transfected in a 10-cm dish with the 5′–3′ and 3′–5′ orientations of the 866-bp enhancer. Cells were exposed to control (DMSO) or hemin (5 μm), and luciferase activity was measured 16 h later. Values are mean ± S.E., n = 2 independent experiments with 8 replicates/group. *, p < 0.001 versus control, vehicle-treated samples.
FIGURE 2.
FIGURE 2.
RNA interference and site-directed mutagenesis of Sp1 binding sites attenuate enhancer mediated reporter activity in HK-2 cells. A, schematic of the target sequences of Sp1 and CEBP-α for site-directed mutagenesis is shown for the wild-type as well as the mutated pHOGL3/4.5+220 construct. B, cells were transfected with the indicated pHOGL3/4.5+220 constructs containing mutations in the Sp1 and CEBP-α sequences. Data shown represent luciferase activity determined following stimulation with control (DMSO) or hemin (5 μm). Values are mean ± S.E. (error bars), n = 3 independent experiments, 12 replicates/group. *, p < 0.001 versus pHOGL3/4.5+220 CEBP-α deletion and wild-type constructs. C, HK-2 cells were transfected with siRNA targeted against Sp1 or mock siRNA as described under “Experimental Procedures.” Cells were allowed to recover for 16 h and then transfected with the pHOGL3/4.5+220 construct or DMSO controls. Five hours after transfection, cells were split into 12-well trays, and 24 h later, they were then induced with hemin (5 μm). Luciferase activity is presented as mean ± S.E., n = 3 independent experiments, 12 replicates/group. *, p < 0.001 versus other groups. D, decoy oligonucleotide targeting indicates that Sp1 consensus sequences are required for enhancer activity. HK-2 cells were transiently cotransfected with pHOGL3/4.5+220 and double-stranded WT or mutated decoy (Mut decoy) oligonucleotides. Transfected cells were analyzed for luciferase activity. Data are represented as mean ± S.E., n = 3 independent experiments. *, p < 0.001 versus other groups. E and F, Sp1 associates with the 220-bp enhancer region by a ChIP assay during hemin stimulation in HK-2 cells. E, sequence of the 220-bp enhancer region with the primers used for the ChIP assay. The Sp1 sequence is underlined. HK-2 cells were treated with 5 μm hemin for 2 h or with vehicle and processed for the ChIP assay with Sp1 antibody as described under “Experimental Procedures.” IgG was used instead of the primary antibody as a negative control. DNA isolated from the immunoprecipitation was amplified with primers (Table 1) for the regions containing the 220-bp intronic enhancer (F), the −4.0 kb HS-2 region (G), and the +2.2 kb region in intron 2 (H). Results shown are signal/noise ratio against input over background from three independent experiments (mean ± S.E.).
FIGURE 3.
FIGURE 3.
Mutations in the human HO-1 promoter attenuate enhancer activity in HK-2 cells. A, schematic showing the −4.5 kb human HO-1 promoter with exons 1 (E1) and 2 (E2), including the 220-bp enhancer in intron 1. The regulatory elements in the −4.0 kb HS-2 region (containing the CREB and AP-1 sites) and the −44 bp HS-1 region (containing the E box region, which binds to USF-1/2) with the wild-type sequences and mutated sequences are shown. P, PstI; X, XbaI. ΔCRE, deletion of the CRE; the E-box M3 and the ARE-5 point mutations are indicated by the lowercase t. B, cells were transfected with the indicated plasmid constructs containing mutations in the HO-1 promoter (ΔE box, ΔCRE, and ARE-5 mutation) or combination of ARE-5 + E box deletion and deletion of CRE + ARE-5 mutation. Mutations or deletion of the regulatory regions in the promoter are indicated by X. Luciferase activity was determined following stimulation with vehicle (DMSO) or hemin (5 μm). Values are mean ± S.E. (error bars), n = 2 independent experiments with 12 replicates/group. *, p < 0.001 versus all other groups by analysis of variance.
FIGURE 4.
FIGURE 4.
Time-dependent chromatin looping between the −4.0 kb promoter region and the 220-bp intronic enhancer region by the 3C assay. A, HK-2 cells were treated with 5 μm hemin or vehicle (DMSO) for 2 h, and the 3C assay was performed as described under “Experimental Procedures.” 300 ng of the sample was amplified by PCR. A human bacterial artificial chromosome (HO-1 BAC; GenBankTM accession number Z82244) clone was used as a positive control, and non-cross-linked genomic DNA from HK-2 cells (HK-2 DNA) was used as a negative control. Reverse-cross-linked DNA was then amplified with primers (Table 1 and schematic in D). PCR products were electrophoresed in a 1% agarose gel. B, extracted DNA was amplified with primers F3 and R1 by real-time PCR to quantitate the cross-linking efficiency between the fragment containing the −4.0 kb promoter and 220-bp enhancer region. Relative Ct values from each sample were normalized with products obtained from primers amplifying the internal control region, which contains no BglII restriction site. Results are shown as -fold increase over vehicle from three independent experiments performed in triplicate each time (mean ± S.E. (error bars)). *, p < 0.01. C, 3C assay was performed on HK-2 cells stimulated with a single exposure to 5 μm hemin for the indicated times. Quantitative real-time PCR using F3 and R1 primers was performed to measure the cross-linking efficiency. Results are shown as -fold increase over time 0 from three independent experiments performed in triplicate each time (mean ± S.E.). *, p < 0.05 versus 0, 10, and 30 min and 16 h time points. D, schematic of HO-1 promoter and gene showing location of HS-2 in the −4.0 kb region (closed oval) and the enhancer in intron 1 (trapezoid). The arrows indicate the primers used to detect the interactions between various regions. Interaction between the fragment containing the −4.0 kb promoter region and that containing the 220-bp intronic enhancer was identified from the PCR product of F3 and R1 primer pairs. B, BglII restriction sites. The gray rectangles represent the exons (E1–E3) in the HO-1 gene. DNA from the PCR product of hemin-treated sample with the F3/R1 primer pairs was extracted, TOPO TA-cloned, and then sequenced. The sequence of the PCR product containing one end of the −4.5 kb promoter-containing fragment, BglII restriction site in the middle (boldface type), and the other end of the fragment that contains the 220-bp enhancer is shown.
FIGURE 5.
FIGURE 5.
Confirmation of chromatin looping by 3C assay with BglII and/or ApaLI restriction enzyme. A, schematic showing potential interaction between the fragments containing HS-2 region and the internal enhancer (square) and transcription factor(s) (TF). B, BglII restriction site. A, ApaLI restriction site in the enhancer. B, HK-2 cells were treated with 5 μm hemin or vehicle (DMSO) for 2 h, and a 3C assay was performed with BglII and/or ApaLI. 300 ng of the sample was amplified by standard PCR with primer F3 and R1 (Table 1). M, 1-kb DNA ladder; V, vehicle (DMSO); H, hemin. C, DNA extracted after 3C assay was amplified with real-time primers F3 and R1 (Table 1) by real-time PCR to quantitate the cross-linking efficiency between the fragment containing the −4.5 kb promoter and the 220-bp enhancer region. Relative Ct values from each sample were normalized with the product obtained from primers amplifying the internal control region, which contains no BglII restriction site. Results are shown as -fold increase over vehicle from two independent experiments performed in triplicate each time (mean ± S.E. (error bars); *, p < 0.01).
FIGURE 6.
FIGURE 6.
Sp1 is required for chromatin looping between HS-2 region of the distal HO-1 promoter and the intronic enhancer. Shown are Sp1 protein (A) and mRNA (B) analyses in HEK293 cells following transfection with oligofectamine (control) alone, mock siRNA, or Sp1 siRNA. C, schematic of real-time PCR primers F3 and R1 relative to the HS-2 region and the 220-bp enhancer used for 3C assay (top). B, BglII 3C assay performed on HEK293 cells stimulated with 5 μm hemin following transfection with oligofectamine alone, mock siRNA, or Sp1 siRNA. Quantitative real-time PCR was performed using F3 and R1 primers showing reduction in cross-linking efficiency with Sp1 siRNA. Results are shown as -fold increase from three independent experiments performed in triplicate each time (mean ± S.E. (error bars)). *, p < 0.05. D, Western blot showing reduction in hemin-mediated increase in HO-1 protein expression in HEK293 cells following treatment with 10 and 100 nm mithramycin A, a GC binding inhibitor of Sp1. Actin levels were used as loading controls. E, 3C assay performed on HEK293 cells in the presence or absence of 100 nm mithramycin A and stimulated with control (vehicle) or 10 μm hemin. Quantitative real-time PCR using F3 and R1 primers was performed to measure the cross-linking efficiency after the 3C assay Results are shown as -fold increase from three independent experiments performed in triplicate each time (mean ± S.E.). *, p < 0.05.

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