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. 2010 May 5;24(7):1404–1412. doi: 10.1210/me.2010-0014

Farnesoid X Receptor Activation Mediates Head-to-Tail Chromatin Looping in the Nr0b2 Gene Encoding Small Heterodimer Partner

Guodong Li 1, Ann M Thomas 1, Steven N Hart 1, Xiaobo Zhong 1, Dequan Wu 1, Grace L Guo 1
PMCID: PMC2903909  PMID: 20444884

Abstract

As a unique nuclear receptor with only ligand-binding but no DNA-binding domain, small heterodimer partner (SHP) interacts with many transcription factors to inhibit their function. However, the regulation of SHP expression is not well understood. SHP is highly expressed in the liver, and previous studies have shown farnesoid X receptor (FXR) highly induces SHP by binding to a FXR response element (FXRRE) in the promoter of the Nr0b2 gene, which encodes SHP. The FXR-SHP pathway is critical in maintaining bile acid and fatty acid homeostasis. After genome-wide FXR binding by chromatin immunoprecipitation (ChIP) coupled to massively parallel sequencing (ChIP-seq), a novel FXRRE was found in the 3′-enhancer region of the Nr0b2 gene. This downstream inverted repeat separated by one nucleotide is highly conserved throughout mammalian species. We hypothesized that this downstream FXRRE is functional and may mediate a head-to-tail chromatin looping by interacting with the proximal promoter FRXRE to increase SHP transcription efficiency. In the current study, a ChIP-quantitative PCR assay revealed FXR strongly bound to this downstream FXRRE in mouse livers. The downstream FXRRE is important for FXR-mediated transcriptional activation revealed by luciferase gene transcription activation, as well as by deletion and site-directed mutagenesis. The chromatin conformation capture assay was used to detect chromatin looping, and the result confirmed the two FXRREs located in the Nr0b2 promoter and downstream enhancer interacted to form a head-to-tail chromatin loop. To date, the head-to-tail chromatin looping has not been reported in the liver. In conclusion, our results suggest a mechanism by which activation of FXR efficiently induces SHP transcription is through head-to-tail chromatin looping.

Farnesoid X receptor mediates a head-to-tail chromatin looping between promoter and downstream enhancer in the Nr0b2 gene encoding the small heterodimer partner in mouse liver.

Farnesoid X receptor (FXR) and small heterodimer partner (SHP) are both members of the nuclear receptor superfamily ( 1,2). Activation of FXR is essential in maintaining optimal bile acid levels by suppressing the transcriptional activation of genes in bile acid synthesis and uptake, as well as enhancing the transcriptional activation of genes in bile acid binding and efflux ( 3,4,5,6,7). The integrity of FXR-mediated regulation is also critical in regulating hepatic and systemic cholesterol, as well as fatty acid and triglyceride homeostasis ( 8,9).

Bile acid homeostasis is tightly regulated by feedback inhibition of bile acid synthetic enzymes and uptake transporters to protect the liver from overt bile acid toxicity, which is often observed during cholestasis. This inhibition is mediated, in part, by the FXR-SHP pathway ( 4,5). In this pathway, activation of FXR by bile acids, the endogenous ligands of FXR, or by synthetic FXR ligands, induces the expression of SHP. As a unique nuclear receptor with only a ligand-binding but no DNA-binding domain, SHP interacts with liver receptor homolog 1 (LRH-1) to inhibit transcriptional activation of genes encoding bile acid synthetic enzymes and uptake transporters. In addition, SHP has been shown to inhibit a number of other nuclear receptors that function as ligand-activated transcription factors, including the glucocorticoid receptor, estrogen receptor, thyroid hormone receptor, retinoic acid receptor, retinoid X receptor, constitutive androstane receptor, and pregnane X receptor ( 10).

The mechanism by which FXR induces SHP expression has been reported through binding of FXR to a FXR response element (FXRRE), an inverted repeat separated by one nucleotide (IR1), in the proximal promoter region of the Nr0b2 gene, which encodes SHP ( 10). Recently, a vast database of nuclear receptor-binding sites has been established with the development of genome-wide discovery of transcription factor-binding sites by ChIP coupled to microarray technology and ChIP-seq (ChIP coupled to massively parallel sequencing) techniques ( 11,12). These data have shown that transcription factors tend to bind to multiple sites in the promoter and/or enhancer regions of target genes ( 13,14,15,16,17,18,19). A recent ChIP-seq study from our laboratory has identified a novel FXRRE in the downstream enhancer region of the Nr0b2 gene ( 20). It has been increasingly appreciated that the genomic DNA is not linear in vivo. Interactions occur between dispersed regulatory elements and/or genes. However, the mechanism by which these remote sites interact to coordinate gene transcription is not well established. One emerging theory is that the interaction of transcription factors with multiple response elements results in looping of interactive chromatin, which brings enhancer and promoter regions within close proximity to each other, thus providing a more efficient and effective chromatin environment to recruit RNA polymerase to initiate gene transcription ( 21,22). The most well-established method to determine chromatin looping is the chromatin conformation capture (3C) assay ( 23,24,25). In principle, protein/protein and protein/DNA cross-links are fixed, and the protein/DNA network is subject to restriction enzyme digestion, followed by ligation at very low DNA concentration. Ligation between intramolecular DNA fragments that are cross-linked is strongly favored over intermolecular DNAs that are not cross-linked. After ligation, the cross-links are reversed, and the ligation products are quantified by PCR. The amount of PCR product reflects the frequency with which two genomic sites interact. Thereby 3C provides information for native chromatin organization in vivo. In mammalian cells, most chromatin looping occurs between promoter and 5′-enhancers, and a head-to-tail chromatin looping between promoter and the 3′-enhancer is not common. However, this phenomenon has been reported in B cells ( 26).

In the current study, we have identified a novel FXRRE, IR1, located at the downstream 3′-enhancer region of the Nr0b2 gene. The function of this site was tested by FXR binding as well as reporter gene assay. Furthermore this novel IR1 site was found to interact with the IR1 identified at the promoter region to form a head-to-tail chromatin loop, which represents a novel mechanism that FXR uses to enhance Nr0b2 gene transcription.

Results

The novel IR1 in the downstream enhancer region of the Nr0b2 gene is conserved throughout mammalian species

It is well known that activation of FXR induces the expression of SHP via binding of FXR to an IR1 at the proximal promoter region of the Nr0b2 gene (−323 to −310 bp relative to transcription start site, TSS). By ChIP-seq analysis, our laboratory has identified multiple FXR-binding sites in the regulatory regions of the Nr0b2 gene ( 20). One strong binding site is located in the 3′-enhancer region (+4117 to +4130). In the current study, we have further analyzed this region and its contribution to SHP expression. This novel IR1 site was shown to be highly conserved in mammalian species, including mouse, rat, human, orangutan, dog, and horse (Fig. 1). The high degree of conservation indicates this novel FXRRE may be important in regulating SHP expression.

Figure 1.

Figure 1

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The novel FXRRE, IR1, at the downstream enhancer region of the Nr0b2 gene, is conserved throughout mammalian species. The IR1 identified at the downstream of the Nr0b2 gene is conserved through the mammalian species (mouse, rat, human, orangutan, dog, and horse). The novel IR1 is marked in the box.

Confirmation of FXR binding to the novel IR1 by regular ChIP-quantitative real-time PCR (qPCR)

Consistent with previous studies, activation of FXR by either 1% cholic acid containing diet or GW4064, a potent synthetic ligand of FXR, induced SHP expression at the mRNA level (Fig. 2A). To validate whether FXR binds to the novel IR1 identified downstream of the Nr0b2 gene, regular ChIP-qPCR was performed on livers of mice treated with vehicle or GW4064. Activation of FXR increases binding to the SHP promoter IR1, which is consistent with previous reports. Moreover, activation of FXR strongly increases binding to the 3′-enhancer region, flanking the novel IR1 located downstream of the Nr0b2 gene (Fig. 2B).

Figure 2.

Figure 2

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FXR activation induces SHP by binding to the IR1s in both the promoter and the downstream enhancer region. A. Induction of SHP mRNA expression in liver after treatment with either cholic acid (CA)-containing diet or GW4064 in WT and FXR-KO mice, as described in Materials and Methods. *, P < 0.05 between vehicle and ligand treatment. B, Identification of FXR binding to 5′- and 3′-IR1s located in Nr0b2 gene by ChIP-qPCR. The relative intensity (fold) indicates fold increase over vehicle treatment. *, P < 0.05 between vehicle and ligand treatment.

Functional assessment of the novel FXRRE

Luciferase reporter gene assays were used to determine whether the downstream FXRRE in the Nr0b2 gene functions to enhance transcriptional activity (Fig. 3A). Consistent with previous studies, activation of FXR induced the luciferase activity driven by the proximal promoter. Compared with the proximal promoter region, the downstream enhancer region was more effective in inducing the luciferase activity (39-fold vs. 3-fold). We then combined these two regions in one luciferase vector by artificially placing the downstream enhancer region in front of the proximal promotion region to test whether the downstream region is indeed enhancing the promoter activity. The result showed an additive effect in inducing the luciferase gene expression (44-fold). For this experiment pGL4-23 luciferease vector was used, which has a lower gene activation background.

Figure 3.

Figure 3

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Binding of Activated FXR to the IR1 in the downstream enhancer functions to enhance gene transcription, revealed by the luciferase. A, Effect of downstream enhancer region of Nr0b2 on gene transcriptional activation and enhancing. On the left is a schematic representation of various constructs in the Nr0b2 gene that have been subcloned upstream of the pGL4-23 luciferase vector, which has a lower gene activation background. The sequence was numbered with respect to the TSS, which is designated as +1. The box represents FXR binding sites (IR1). The DNA constructs containing the proximal promoter only (−748 to +108), the downstream region only (+3639 to +4516), and the downstream region upstream of the proximal promoter (+3639 to +4516 plus −748 to +108) of the Nr0b2 gene were cloned into pGL4-23 firefly luciferase vector and transfected into HepG2 cells, which were then treated with vehicle or 1 μm GW4064 for 36 h, followed by evaluation of luciferase activity. The black bar is for cells treated with 0.1% dimethylsulfoxide, and the white bar is for cells treated with 1 μm GW4064. The relative luciferase activity (fold) indicates fold increase over empty vector by vehicle treatment. The fold induction of luciferase activity by these constructs with GW4064 treatment compared with dimethylsulfoxide is indicated next to the white bars. *, P < 0.05; **, P < 0.001 between vehicle and GW4064 treatment. B, Effect of various truncation of the downstream region of the Nr0b2 gene on luciferase activity. On the left is a schematic representation of various constructs of the downstream region of the Nr0b2 gene that have been subcloned upstream of the pGL4-TK luciferase vector. C, Site-directed mutagenesis of the novel IR1 comprised luciferase activity. On the left is a schematic representation of various constructs of the downstream region of the Nr0b2 gene that have been subcloned upstream of the pGL4-TK luciferase vector, with the IR1 site intact or mutated. *, P < 0.05 between wild-type construct and mutated construct after GW4064 treatment.

The downstream IR1 is important for FXR-mediated transcriptional activation revealed by deletion and site-directed mutagenesis

To further determine which region from +3639 to +4516 is mainly responsible for the strong induction of the luciferase activity, various subcloned fragments from region +3639 to +4516 were tested in the luciferase assay (Fig. 3B). Region +3639 to +4080, which does not have an IR1, appeared to be not responsible for luciferase induction. Regions +3639 to +4181, +4064 to +4181, and +4054 to +4255, which all cover the novel IR1, partially restored the luciferase activity. Furthermore, region +4064 to +4516, which covers the IR1 and further downstream sequence of the IR1, showed the highest activity in response to FXR activation. Surprisingly, a region from +4214 to +4516, which only covers sequences downstream of the IR1, but not IR1 itself, also showed moderate activity in inducing the luciferase gene expression upon FXR activation. To further validate this new IR1, the core sequence of this response element was mutated, and the results showed that the transcriptional activation was greatly diminished (Fig. 3C). For these assays, pGL4-TK vector was used.

Head-to-tail chromatin looping results from an interaction between the two FXRREs

To elucidate the significance of these two FXRREs in the Nr0b2 gene in response to FXR activation, 3C assay was used to determine whether chromatin regions containing these two IR1s interact. The genomic DNA was cut by MboI at multiple locations in the Nr0b2 gene. A forward primer, which is downstream to the proximal promoter IR1, was designed against the immediate upstream region of a MboI cutting site. A series of reverse primers were designed against regions immediately downstream of each MboI site, up to 140 kb from the TSS of the Nr0b2 gene (Fig. 4, A and B). Using two bacterial artificial chromosome (BAC) DNA clones that contain the Nr0b2 genomic region, we have shown that the efficiency of these primers was similar (Supplemental Fig. 1 published on The Endocrine Society’s Journals Online web site at http://mend.endojournals. org). The 3C assays were performed in livers of wild-type (WT) or FXR-knockout (KO) mice fed with control or cholic acid diet. The results gathered from the 3C assay showed that the overall trend of interaction between the proximal promoter region containing the IR1 and various downstream regions of chromatin decreased with distance (Fig. 5, A and B). At location +3840 to +4274 bp, where the novel downstream IR1 was located, an increased interaction with the proximal promoter IR1 region was observed after cholic acid feeding. This interaction was lost in FXR-KO mice (Fig. 5, A and B). One location, at +3640 bp kb where no IR1 was identified, also showed an increased interaction. However, this increased interaction was not dependent on activation of FXR, because neither treatment with cholic acid nor deficiency of FXR in mice affected the interaction frequency.

Figure 4.

Figure 4

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Chromatin looping experiment. A, Schematic representation of the 3C technology to test the head-to-tail chromatin looping in the Nr0b2 gene. Chromatin from formaldehyde-fixed cells is digested with a restriction enzyme, MboI, and diluted for intramolecular ligation with T4 DNA ligase. The resulting ligation products, reflecting interaction frequency, are evaluated by qPCR amplification. B, The locations of the two IR1s, MboI restriction enzyme cutting sites (vertical lines) and PCR primers (arrows) used in 3C analyses in the Nr0b2 gene.

Figure 5.

Figure 5

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The two IR1s in the Nr0b2 gene interact by chromatin looping mechanism. Interaction frequency of the two IR1s in WT mice (A) and in FXR-KO mice (B). The y-axis shows the relative interaction frequency of the different regions of the Nr0b2 gene. The value of interaction frequency between the IR1 in the proximal promoter region and the nearest downstream region, for which the first reverse primer was designed, in the Nr0b2 gene of the vehicle control group was set as 100%. The x-axis shows the distance of these regions relative to the TSS of the Nr0b2 gene. *, P < 0.05 between the control and cholic acid (CA)-treated group.

Discussion

The Nr0b2 gene consists of two exons separated by an intron. This overall genomic structure is conserved in all mammalian species. The protein SHP, encoded by the Nr0b2 gene, has important roles in regulating bile acid homeostasis, lipid metabolism, glucose balance, and liver carcinogenesis ( 9,10,27,28). FXR has been previously shown to induce the expression of SHP via binding to a FXRRE the proximal promoter region of the Nr0b2 gene. By genome-wide detection of FXR-binding sites via ChIP-seq technology, a novel FXRRE in the form of IR1was identified at the downstream enhancer region of the Nr0b2 gene ( 20). This response element is conserved throughout mammalian species, suggesting it may be critical in regulating SHP transcription. Our results showed that this downstream IR1 is bound by FXR with higher affinity compared with the one located in the proximal promoter region. Upon FXR activation, this response element strongly enhanced transcriptional activity either by itself or in combination with the proximal promoter, tested in a luciferase reporter gene assay. Deletion or mutation of this response element results in loss of activity. By 3C assay, we have shown that the two FXRREs located in the proximal promoter and downstream enhancer region facilitate the formation of a head-to-tail chromatin loop, which may be used to augment transcription efficiency.

SHP physically interacts with numerous transcription factors and nuclear receptors to repress their transcriptional activation. For example, SHP interacts with LRH-1 and suppresses LRH-1 mediated transcriptional activation of cyp7a1 gene, which encodes cholesterol 7-α hydroxylase, the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver ( 4,5). The expression of SHP is regulated by several transcription factors and members of the nuclear receptor superfamily, including FXR ( 4), steroidogenic factor-1 (SF-1) (29), LRH-1 ( 5), hepatocyte nuclear factor-4α ( 30), c-Jun (31), and sterol-regulatory element-binding protein 1c ( 32). Furthermore, each of these factors has been shown to regulate SHP expression by binding to a single site in the proximal promoter region of the Nr0b2 gene, ranging from −570 to −48 relative to the TSS, except for SF-1, which has five binding sites scattered from −570 to −48. The significance of five SF-1 binding sites and potential interaction among these sites is not clear. Currently, the only reported FXRRE has been located at −323 to −311 (GGGTTAaTGACCT), relative to the TSS ( 4). No regulatory region has been previously identified in the downstream enhancer region. We have identified a novel FXRRE, IR1, in the downstream region of the Nr0b2 gene by a genome-wide ChIP-seq approach ( 20). When compared with the previous identified IR1 in the proximal promoter region, this downstream IR1 appears to be bound by FXR with a higher intensity and was quite strong in activating transcriptional activation. Furthermore, this downstream region is functional as a strong enhancer because when combined with the proximal promoter, an additive effect in activating luciferase gene expression upon FXR activation resulted. Interestingly, its activity was strongly enhanced by the presence of further downstream sequences (+4214 to + 4516), indicating FXR, or other factors associated with FXR activation, may have interacted with these downstream sequences to further enhance Nr0b2 gene transcription.

It is well known that interactions among multiple transcription factor-binding sites could increase transcriptional activation. Recently, several studies have identified genome-wide nuclear receptor binding sites by ChIP coupled to microarray technology or ChIP-seq analysis ( 13,14,15,16,17,18,19). The results from these studies indicate that, in addition to interacting with the proximal promoter region of target genes, nuclear receptors also bind at sites located at distal promoter regions, introns, and downstream gene-regulatory regions on genes, regions that are collectively referred to as “enhancers.” Furthermore, many of the target genes have multiple nuclear receptor-binding sites, including regions that are large distances away from genes. Chromatin looping is one of the proposed models to explain the significance of enhancers to facilitate transcription activation across chromatin. This model suggests that enhancers can physically interact with the promoter region, allowing distant chromatin regions to be brought within close proximity to the transcriptional machinery by a looping mechanism to facilitate transcriptional activation ( 21,22). Most of the reported chromatin loops form between 5′-enhancers and promoter, over long distances. The head-to-tail loop formed between promoter and 3′-enhancers is common in the yeast but has been only reported in B cells in the mammalian species ( 26,33). We speculate that head-to-tail looping may not only activate transcription initiation but also increase transcription efficiency by moving the transcriptional machinery in a rolling circular manner. In the current study, a head-to-tail chromatin loop has been observed between the promoter and the downstream region of the Nr0b2 gene, where the two IR1s are identified. The interaction was increased upon FXR activation and was lost in the FXR-KO mice, thus providing direct in vivo evidence that looping between these two sites is a direct result of FXR activation. This loop is small because the Nr0b2 gene has only two exons and one intron. In future studies, it will be interesting to test the significance of this looping in regulating tissue-specific expression of SHP by interaction with epigenetic machinery as well as transcription cofactors.

Furthermore, the 3′-enhancer IR1 in the Nr0b2 gene is found to be conserved throughout the mammalian species including humans, mice, rats, orangutans, dogs, and horses. Although it will be more convincing if the 3C experiment is confirmed in other species, such as in humans, the currently available human hepatoma cell lines are not suitable for this experiment. This is because, in contrast to the in vivo situation in which normally a high degree of SHP induction is observed after FXR activation in mice (4- to 6-fold), induction of SHP mRNA in several commonly used human hepatoma cell lines, including HepG2, Hep3B, and Huh 7, can only reach 1.3- to 2-fold, even after addition of other boosting reagents, including 9-cis retinoid acid or estrogen ( 34,35). Using primary human hepatocytes may be a better alternative approach due to high induction of SHP mRNA by FXR activation ( 4,36), but because large amount of cells are required for the 3C assay, and also because huge individual variations are associated with primary human hepatocytes, performing the 3C assay in human cells will be considered when better human models are available.

In summary, the current study has shown that a novel FXRRE in the downstream regulatory region of the Nr0b2 gene is functional and facilitates the formation of a head-to-tail chromatin loop.

Materials and Methods

Animals and treatments

Wild-type (WT) and FXR knockout (KO) male mice in the C57BL/6 genetic background were used in this study (8–10 wk of age, n = 5–6 per group). C57BL/6 mouse breeders were ordered from The Jackson Laboratory (Bar Harbor, ME) and were bred and maintained in the University of Kansas Medical Center. All mice were housed in pathogen-free animal facilities under a standard 12-h light, 12-h dark cycle with access to regular rodent chow and autoclaved tap water ad libitum. All protocols and procedures were approved by the University of Kansas Medical Center Animal Care and Use Committee. Mice were fed with the cholic acid-containing diet or regular diet for 1 d for 3C experiment to determine chromatin interaction or for 3 d to determine SHP mRNA expression by qPCR. The 1% (wt/wt) cholic acid-containing diet was made in house by mixing cholic acid (Sigma Chemical Co., St. Louis, MO) with regular rodent chow diet, which was completely dried. Mice were also orally gavaged with 75 mg/kg GW4064 dissolved in vehicle (PBS containing 1% methocellulose and 1% Triton-100) or vehicle, twice at 1800 h and 0600 h, to determine SHP mRNA expression in liver. GW4064 is a potent and specific FXR synthetic ligand ( 37), which was synthesized by the Chemical Discovery laboratory at the University of Kansas (Lawrence, KS). To determine FXR binding to IR1s, by ChIP-qPCR, in the promoter or downstream of the Nr0b2 gene, mice were orally gavaged with 75 mg/kg GW4064 or vehicle for 4 h. Mice were killed after these treatments, and livers were quickly removed, snap frozen in liquid nitrogen, and stored in −80 C freezer.

RNA isolation and qPCR

Frozen livers collected from mice treated with 1% cholic acid or GW4064 were used to isolate total RNA using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The concentration of the total RNA was determined by spectrophotometry, with the integrity confirmed by 3[N-morpholino]propanesulfonic acid gel electrophoresis. Expression of SHP mRNA was quantified by real-time qPCR using SYBR green chemistry and normalized to 18s RNA levels. The primer sequences for detecting SHP and 18s are presented in the Supplemental Table 1.

ChIP assay

ChIP assays were performed on livers of mice treated with GW4064 or vehicle once for 4 h using the EZ CHIP KIT (Millipore Corp., Temecula, CA). Briefly, flash-frozen livers were minced and fixed in 1% formaldehyde for 15 min and then quenched with 0.125 m glycine. The cells were lysed and centrifuged. The nuclei pellet was resuspended in nuclear lysis buffer with protease inhibitors. Nuclear extracts were sonicated to yield 500- to 1000-bp DNA fragments. Sonicated chromatin was aliquoted and chromatin (30 mg tissue equivalents) was used for each immunoprecipitation assay. Samples were precleared with Protein agarose G-salmon sperm DNA beads (Millipore) before incubation with an IgG antibody or the anti-FXR antibody (H-130x; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Samples were incubated with prepared Protein agarose G-salmon sperm DNA beads to extract antibody-chromatin complexes. Complexes were washed and eluted with immunoprecipitation elution buffer. DNA fragments associated with the FXR antibody were released by boiling samples in a 450 mm NaCl solution. RNA and protein were degraded by treating chromatin with ribonuclease A and proteinase K. DNA fragments were purified by standard DNA column purification. The purified DNA fragments that were bound by FXR were analyzed by qPCR with primers amplifying FXRREs (IR1s) located in the proximal promoter and downstream region of the Nr0b2 gene. The sequences for the primers for ChIP assay are presented in Supplemental Table 1.

Construction of plasmids for reporter gene luciferase assay

Fragments containing an IR1 in the promoter region of the Nr0b2 gene, located −748 to 108 relative to the TSS, and in the downstream regulatory region of the Nr0b2 gene, from +3639 to +4516 relative to the TSS, were amplified from mouse genomic DNA by PCR using pairs of primers containing XhoI, BglII, KpnI, and NheI restriction enzyme sites, respectively (the sequences of the primers are listed in Supplemental Table 1). The PCR products, named pGL748 or pGL3639, respectively, were subcloned upstream of the luciferase gene into firefly luciferase vectors pGL4-23 and pGL4-TK. To evaluate enhancer activity of the downstream genomic region, constructs cloned into the pGL4-23 vector were used. To determine which region in the downstream enhancer is responsible for transcriptional activation of the luciferase gene, constructs cloned into the pGL4-TK vector were used. In detail, various subcloned fragments upstream of the luciferase gene in the pGL4-TK vector from region +3639 to +4516 were tested in the luciferase assay. A total of six fragments were cloned from +3639 to +4516, downstream of the Nr0b2 gene: +3639 to +4080, +3639 to +4181, +4064 to +4181, +4054 to +4255, +4064 to +4516, and +4214 to +4516. The sequences of these constructs were confirmed by DNA sequencing.

Site-directed mutagenesis of FXRRE in the downstream enhancer

QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to generate mutations of the IR1 site in pGL3639 and pGL4064-4516, from gggtgactgacct to gttcgtctgagat, according to the manufacturer’s protocol. Primers for site-directed mutagenesis are given in Supplemental Table 1. The desired mutation was verified by DNA sequencing.

Cell culture, transient transfection, and luciferase reporter gene assays

HepG2 cells were cultured in high-glucose DMEM supplemented with 1% penicillin/streptomycin, 1% l-glutamine, and 10% fetal bovine serum (Omega Scientific, Tarzana, CA). Cells were grown to 90% confluency in 96-well plates and were transiently transfected with various reporter gene constructs as well as pCMV-ICIS human FXR (Open Biosystems, Huntsville, AL), pSG5 human retinoid X receptor-α, and pCMV-Renilla luciferase vector (Promega Corp., Madison, WI). Transient transfection was done using ExGen 500 (Fermentas, Glen Burnie, MD) according to the manufacturer’s instructions. Cells were treated with 1 μm GW4064 or 0.1% dimethylsulfoxide (control) 4 h after transfection. Firefly luciferase and Renilla luciferase activities were quantified 36 h later using a Dual-Glo Luciferase Kit from Promega with a Synergy-HT plate reader (Bio-Tek Instruments, Inc., Winooski, VT). The firefly luciferase activity value was normalized as a ratio to that of Renilla luciferase and expressed as fold over the empty vector control. The data were presented as the average of six wells, and the experiments were repeated at least twice.

3C assay

Preparation of the 3C samples

The 3C templates were generated following a standard protocol with minor modifications ( 24,25,38). Briefly, liver tissues were dissected from WT and FXR-KO mice, rinsed with PBS, minced, and sieved through a 70-μm nylon cell strainer (BD Biosciences, Palo Alto, CA) into PBS. Approximately 1 × 107 liver cells were centrifuged and resuspended in 10 ml of PBS. The suspended cells were cross-linked by 1% formaldehyde for 10 min at room temperature with tumbling, followed by the addition of 0.125 m glycine for 15 min at room temperature to quench the formaldehyde. The cells were lysed in ice-cold lysis buffer [10 mm Tris (pH 7.5), 10 mm NaCl, 0.2% (vol/vol) Nonidet P 40] containing protease inhibitors cocktail tablets (Roche, Indianapolis, IN) for 15 min. Nuclei were harvested and resuspended in 0.5 ml of restriction buffer 4 (New England Biolabs, Inc., Ipswich, MA) containing 0.3% (wt/vol) sodium dodecyl sulfate (SDS) by incubation at 37 C for 1 h with gentle shaking. Triton X-100 was then added to 2.0% (vol/vol), and the nuclei were further incubated for 1 h at 37 C with gentle shaking to sequester the SDS. The cross-linked DNA was digested overnight at 37 C with 600 U MboI (New England Biolabs) to achieve >80% digestion. Then, SDS was added to a final concentration of 1.6%, and the restriction enzyme was inactivated at 65 C for 45 min. The reaction was further incubated at 37 C for 1 h after dilution with 6.5 ml of T4 DNA ligation buffer (Fermentas), and Triton X-100 was added to 1%. DNA fragments were ligated with 400 U T4 ligase (Fermentas) for 5 h at 16 C and 1 h at room temperature. A volume of 50 μl of 10 mg/ml proteinase K (Invitrogen) was added to the ligation complex followed by incubation at 65 C overnight to reverse the cross-links. The next day, the samples were incubated for 1 h at 37 C with 30 μl of 10 mg/ml deoxyribonuclease-free ribonuclease A (Fermentas), and the DNA was purified by phenol-chloroform extraction and ethanol precipitation. Purified DNA was dissolved in nuclease-free water.

Preparation of random template control

Generation of the control 3C template using BAC DNA clones were essentially the same as described above. BAC clones RP24-375L21 and RP23-315M19 (BACPAC Resources Center at Children’s Hospital Oakland Research Institute, Oakland, CA), were used, which contain the Nr0b2 gene and downstream 160-kb region. A total of 20 μg of equal molar amounts of two BAC clones were used to generate 3C control templates. PCR primer efficiency was measured by amplifying 0.1–50 ng of mixed BACs with a fixed amount (50 ng) of digested genomic DNA. All primer combinations showed a linear correlation between BAC templates and PCR products (Supplemental Fig. 1, a–c). To control for differences in the 3C efficiency in different samples, an internal control was used within two MboI sites in the β-actin locus.

qPCR analysis of the ligation products

qPCRs were performed by the SYBR green chemistry with appropriate primers using 3C templates as well as the control template generated. The linear range of amplification was determined for 3C samples by serial dilution. An appropriate amount (50 ng) of 3C DNA within the linear range was added to each PCR. PCR cycling parameters were as follows: 40 cycles at 95 C for 30 sec, 64 C for 30 sec, and 72 C for 30 sec. All primers were designed to have an annealing temperature of 65–70 C, and they all yielded a product of 150–300 bp when used with the control templates. Sequences of the 3C PCR primers are shown in Supplemental Table 2. All quantitative 3C assay results are presented as the average and standard deviation from at least five independent preparations of 3C DNA, followed by triplicate qPCR analysis.

Statistical analysis

All data were presented as mean ± sd. Statistical differences between two groups were analyzed by Student’s t test. P < 0.05 is considered significant.

Supplementary Material

[Supplemental Data]
me.2010-0014_index.html (781B, html)

Footnotes

This work was supported by National Institutes of Health Grants DK031343-01 (to G.L.G.), 5P20-RR021940 (to G.L.G. and X.B.Z.), T32ES007079 (to A.M.T.), and a Madison and Lila Self graduate fellowship from the University of Kansas (to S.N.H.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online May 5, 2010

Abbreviations: BAC, Bacterial artificial chromosome; 3C, chromatin conformation capture; ChIP, chromatin immunoprecipitation; ChIP-seq, ChIP coupled to massively parallel sequencing; FXR, farnesoid X receptor; FXRRE, FXR response element; IR1, inverted repeat separated by one nucleotide; KO, knockout; LRH-1, liver receptor homolog 1; qPCR, quantitative real-time PCR; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; SHP, small heterodimer partner; TSS, transcription start site; WT, wild type.

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