Departments of 1 Neuroscience and 2 Anesthesiology, College of Medicine, University of Florida, Gainesville, Florida 32610
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ABSTRACT |
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Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSOD promoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites. To address these findings in the context of the nucleoprotein environment, we first utilized chromatin immunoprecipitation (ChIP) to demonstrate the nuclear association of Sp1 with the MnSOD promoter region. To identify the precise location of Sp1 binding, we have modified the original protein position identification with nuclease tail (PIN*POINT) methodology, providing an approach to establish both the identity and binding occupancy of Sp1 in the context of the endogenous MnSOD promoter. These data, coupled with site-directed mutagenesis, demonstrate the functional importance of two of the Sp1 binding sites in the stimulus-specific regulation of MnSOD gene expression. We feel that the combination of ChIP and PIN*POINT analysis allows unequivocal identification and localization of protein/DNA interactions in vivo, specifically the demonstration of Sp1 with the MnSOD promoter.
protein-DNA interactions; chromatin immunoprecipitation; transcriptional regulation; manganese superoxide dismutase
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INTRODUCTION |
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OXYGEN FREE RADICALS produced as a byproduct of aerobic metabolism, as well as the inflammatory response, are detoxified through the action of the potent, cytoprotective antioxidant enzyme, manganese superoxide dismutase (MnSOD). The physiological significance of MnSOD was demonstrated when two groups independently ablated the MnSOD gene producing SOD null mice. These mutant mice all die within 10 days of birth with dilated cardiomyopathy and steatosis in liver and skeletal muscle, as well as metabolic acidosis (21, 26). In addition, it has become intuitively obvious that the endogenous cytoprotective potential of MnSOD (39, 38) is achieved through transcriptional induction by a variety of proinflammatory mediators as an adaptive response to inflammation. We have previously shown that stimulus-dependent transcriptional induction is mediated through the cooperative interaction between cis-elements and their cognate trans-acting factors within the MnSOD promoter and a complex intronic enhancer element (28).
Many of the approaches that attempt to address the underlying mechanisms controlling transcriptional regulation involve in vitro strategies to assess the interaction of trans-acting factors with their cognate binding sites. Such methodologies include electromobility gel shift assays (EMSA) (12) and in vitro footprinting studies (30). In vitro studies are unfortunately performed in the absence of the cellular milieu and, most importantly, without the influence of the nucleoprotein structure. Thus these methods only provide circumstantial evidence to support the physiological relevance and validity of in vivo protein-DNA interactions. Studies undertaken in vitro at ionic conditions, pH, and protein/DNA concentrations outside the physiological range dramatically affect the specificity of protein-DNA complexes, potentially allowing nonspecific interactions to predominate. Furthermore, in vitro studies are not performed in the context of a complex genome in which the size of the primary recognition sequence (4, 35, 36) and the local chromatin environment help to mediate the specificity of protein/DNA interactions (5).
The abundance of studies utilizing transient transfection assays has provided extensive information on promoter and transcription factor structure and function leading to the derivation of our present conceptual models of gene regulation. Transient expression studies presumably recreate the physiologically appropriate cellular environment to insure most levels of specificity necessary for valid intracellular protein/DNA complex formation. Recently, Lee et al. (23) developed a methodology that complements transient transfection assays, termed protein position identification with nuclease tail, or PIN*POINT. This methodology provides a mechanism to assess, in vivo, whether a specific trans-acting factor can indeed be placed directly at the scene of a putative binding sequence in cells. An expression vector encoding a chimera of a transcription factor and the nuclease cleavage domain of type IIS endonuclease FokI (18) is cotransfected into mammalian cells with a target plasmid containing the cognate DNA binding site for the chimeric trans-acting factor. This method has been employed transiently with a variety of transcription factor/FokI nuclease chimeras to demonstrate specific protein/DNA binding through adjacent nuclease cleavage (23, 22, 24, 25, 17). PIN*POINT can thus be an ideal approach to verify, in vivo, the identity of potential transcription factor candidates for functionally relevant cis-acting elements.
Unfortunately, the interpretation of transient transfection results is also limited by the inherent structure of plasmid DNA in transfected cells. Smith and Hager (32) have reviewed transcriptional regulation of mammalian genes in vivo and convincingly purported that although nonreplicating DNA may form structures, which include nucleosome deposition, the final structures are not organized in a manner similar to endogenous chromatin. This critical argument is best exemplified by the studies of Archer and colleagues (1, 2), which demonstrated that transfected, nonreplicating templates bind transcription factors constitutively, although these same factors will interact with endogenous sites only when the chromatin has been appropriately remodeled. Additionally, recent studies have underscored the central importance of chromatin structure (13, 40) and remodeling (14, 10, 11, 34) with respect to the specificity of trans-acting factor binding and the subsequent regulation of gene expression.
To address some of the limitations in methods employed thus far to study gene expression, we present data that improve and extend the PIN*POINT methodology, allowing for detection of a specific trans-acting factor binding on an endogenous locus. Our laboratory has previously identified ten basal protein binding sites on the MnSOD promoter by dimethyl sulfate (DMS) in vivo footprinting (20). A number of these cis-acting elements show significant sequence similarity to the consensus Sp1-binding sequence (9). We have thus employed a chimeric Sp1-FokI nuclease expression vector to identify endogenous binding sites occupied by Sp1 in vivo. Furthermore, using site-directed mutagenesis, we evaluated the functional importance of Sp1 at these binding sites during inducible expression of MnSOD by proinflammatory stimuli.
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MATERIALS AND METHODS |
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Cell culture. L2 cells, a rat pulmonary epithelial-like cell line (ATCC CCL 149), were grown in Ham's modified F-12K medium (GIBCO) with 10% fetal bovine serum (Flow Laboratories), ABAM (penicillin G 100 U/ml, streptomycin 0.1 mg/ml, amphotericin B 0.25 mg/ml; Sigma), and 4 mM glutamine at 37°C in room air, 5% CO2.
Chromatin immunoprecipitation. L2 cells grown to 100% confluency were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with 125 mM glycine (3, 27). Cells were resuspended in cold swelling buffer (5 mM PIPES, pH 8.0, 85 mM KCl, and 0.5% NP-40) plus protease inhibitors, incubated on ice, and centrifuged, and the cell pellet was resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1, and protease inhibitors). The chromatin lysate was sonicated to ~200-500 bp, then diluted 1:10 in chromatin immunoprecipitation (ChIP) dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris, pH 8.1, and 167 mM NaCl). The diluted sonicated chromatin was subjected to immunoprecipitation using Sp1 specific antibodies (3; Upstate Biotech). Sp1 antibody or no antibody was added to precleared chromatin samples and rocked overnight at 4°C. Antibody-protein-DNA complexes were captured by incubation with protein A/G agarose beads (Santa Cruz) blocked with 20 µg of sheared herring testes sperm DNA. Captured complexes were washed successively [one wash of 0.1% SDS, 1% (vol/vol) Triton X-100, 20 mM Tris, pH 8.1, 2 mM EDTA, and 150 mM NaCl; one wash of 0.1% SDS, 1% (vol/vol) Triton X-100, 20 mM Tris, pH 8.1, 2 mM EDTA, and 500 mM NaCl; one wash of 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 10 mM Tris, pH 8.1, and 1 mM EDTA; and three washes of TE (10 mM Tris, pH 8.0, and 1 mM EDTA, pH 8.0)] and eluted with solution containing 1% SDS and 50 mM NaHCO3. The DNA-protein cross-links were reversed at 65°C for 4 h, followed by RNase A and proteinase K treatment. Purified DNA was then subjected to PCR with primers (top strand: 5'-CAAGGCGGCCCGAGAAGAGGCGGGG-3', bottom strand: 5'-CTTGGACACAGCTAGGCGCTGAC-3') specific to the MnSOD promoter, fractionated on an agarose gel, and transferred to a nylon membrane and hybridized with a random primed probe derived from the MnSOD promoter. ChIP fragments were visualized by autoradiography.
PIN*POINT analysis. L2 cells, grown to 70-90% confluency in 100-mm tissue culture plates, were transiently transfected with an expressing vector for the fusion protein composed of the Sp1 DNA binding domain and the nuclease cleavage domain of FokI endonuclease (kindly provided by Dr. J. H. Chung) using FuGENE 6 (Roche Diagnostics) as a transfection reagent following the manufacturer's instructions. The control samples were treated with FuGENE 6 but not the Sp1-FokI expression vector. The genomic DNA was isolated and purified by phenol-chloroform extractions. The protein-free, untreated genomic DNA from L2 cells was used for the in vitro guanine-specific reaction performed as described (8). All samples were then subjected to BamHI digestion, followed by ligation-mediated PCR (LMPCR) (15) using Vent DNA polymerase (New England Biolabs). The primers used for LMPCR are, for the top strand, primer 1, 5'-GGCCGTTCGCTAGCAGCCGCGCGTC-3', primer 2, 5'-CGTCTGCTCTGC-GGCGTCCGCCCGGCGTCC-3'; for the bottom strand, primer 1, 5'-GGCCCCTGATTA-CGCCATGGCTCT-3', primer 2, 5'-CTCTGACCAGCAGCAGGGCCCTGGCTTCCC-3'. The PCR products were size fractionated on a 6% denaturing polyacrylamide gel, electrotransferred to a nylon membrane, and covalently cross-linked to the membrane by ultraviolet (UV) irradiation (8). Two oligos, 5'-CGTCCGCTTGGACACAGCTAGGCGCTGACC-3' and 5'-GGCTTCCCGGAGG-AAAGTCTCTGGGGCTTTCC-3', were used to make 32P radioisotope probes as described (29) for visualization of the top and bottom strands, respectively.
Site-directed mutagenesis and construction of expression vectors.
The MnSOD promoter from 420 to +74 relative to the
transcriptional start site was used to construct wild type and each of the mutant clones in a human growth hormone expression plasmid also
containing the rat MnSOD intronic enhancer element
(28). PCR-based, site-directed mutagenesis was used to
create each mutant clone, as previously described (16).
PCR primers used to produce mutated binding sites (mutated base pairs
underlined) were as follows: MI:
5'-CGGCCCGAGAAGAGGCTTTTCCTAGTCTGAGG-3',
5'-CCTCAGACTAGG-AAAAGCCTCTTCTCGGGCCG-3'; MV:
5'-GTGGCCACACTATTTTCGTTTCCGTGG-CAAGCCC-3', 5'-GGGCTTGCCACGGAAACGAAAATAGTGTGGCCAC-3'; MIX: 5'-GTGTCGCGGTCCTAAAATCCGTTGATGGG-3',
5'-CCCATCAACGGATTTTAGGA-CCGCGACAC-3'. Two outside primers
containing either HindIII or XbaI restriction sites are as follows: HindIII primer:
5'-AAGCTTGAAGGCCCCTGATTACGCCATGGC-3' XbaI primer:
5'-TCTAGATGCTGAGGCGCCCACGA-3'. Vent DNA polymerase (New
England Biolabs) was used to produce a blunt-ended PCR product, which
was cloned into the pPCRscript sk(+) plasmid using PCR-Script Amp
Cloning Kit (Stratagene). The plasmid was then digested with HindIII and XbaI, and the insert was cloned into
a HindIII and XbaI predigested promoterless,
pUC 12-based human growth hormone expression vector pØGH
(Nichols Institute Diagnostics), which also contained the rat
MnSOD intronic enhancer element (from +1,268 to +2,187)
defined by our laboratory (28). The final mutagenesis plasmids were verified by sequencing.
Transient transfection with vectors containing Sp1 mutations in
the MnSOD promoter.
To control for transfection efficiency, transfections were carried out
using a batch transfection method, as described previously (28), as well as cotransfection with pcDNA 3.1/HisB/LacZ
(Invitrogen). Cells were grown as monolayers in 150-mm tissue culture
plates until 70-90% confluent. The cells were transfected with 10 µg of each expression vector using the DEAE-dextran method
(19). After 24 h, cells from each 150 mm were
trypsinized and plated onto four separate 100-mm tissue culture plates.
After 24 h, inflammatory mediators were added to each plate with
final concentrations of 0.5 µg/ml Escherichia coli
lipopolysaccharide (LPS, E. coli serotype 055:B5, Sigma), 10 ng/ml TNF- (a gift from Genetech), or 2 ng/ml IL-1
(a gift from
the National Cancer Institute). Total RNA for Northern analysis was
isolated 24 h after treatment by the acid guanidinium
thiocyanate-phenol-chloroform extraction method described by
Chomczynski and Sacchi (7) with modifications
(28). RNA was size-fractionated on a 1% agarose
formaldehyde gel and electrotransferred to a charged nylon membrane
(Zetabind) and UV cross-linked. Membranes were hybridized with
32P-labeled human growth hormone (hGH) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes made by
primer extension and subjected to autoradiography. GAPDH was used
as the RNA loading control. NIH image was used to analyze the signal
density of messages.
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RESULTS |
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Localization of Sp1 to the MnSOD promoter by ChIP.
As summarized in Fig. 1A, we
have previously used DMS in vivo footprinting coupled with
ligation-mediated PCR (LMPCR) to identify ten basal protein binding
sites on the MnSOD promoter (20). A computer
analysis of these binding sites shows that a subset of these basal
binding sites (sites I-V) has a high degree of similarity to the
consensus DNA binding sequence of Sp1 (9), GKLF
(31), as well as other GC-box binding proteins (6,
37). To demonstrate that Sp1 does interact with sequences in
this region, we utilized ChIP (27) with Sp1-specific
antibodies (3). Figure 1B shows the results of
ChIP analysis, illustrating that Sp1 can specifically bind to a region
of the MnSOD promoter. These results implicate Sp1 as one of
the constituents of the endogenous MnSOD promoter.
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Identification of specific Sp1 binding sites on the endogenous MnSOD promoter by in vivo PIN*POINT analysis. ChIP analysis allows for the determination of endogenous interactions between specific trans-acting factors and chromatin. The limitation of this methodology is the inability to define the precise location of the respective binding site due to the dependence on the extent of sonication, which usually results in fragments ranging from 100 to 500 bp. To determine the identity of a transcription factor bound to a specific DNA sequence at single nucleotide resolution, Lee et al. (23) recently developed protein position identification with nuclease tail or PIN*POINT methodology, which directly localizes transcription factor binding in an intact cell. A chimeric protein containing the transcription factor Sp1 fused to the FokI nuclease provides a cleavage marker along the DNA, which demonstrates sequence-specific binding. These investigators have employed this methodology to demonstrate the interaction of a variety of transcription factors with their cognate binding sites in a cellular milieu (23, 22, 24, 25, 17). This methodology has so far only been implemented in conjunction with a transiently transfected binding site on a target plasmid. As previously discussed, transiently transfected plasmid DNA does not always assume a chromatin structure that facilitates transcription factor binding on the endogenous nuclear DNA binding site (1, 2). Smith and Hager (32) have recently developed a cogent argument that transiently transfected plasmid DNA does not always serve as an appropriate "stand in" for the endogenous regulatory sequences.
We therefore enhanced the original PIN*POINT protocol to allow detection of chimeric Sp1/FokI nuclease binding on the endogenous MnSOD promoter. Pulmonary epithelial cells (L2) were transiently transfected with a mammalian expression vector to drive the expression of the Sp1/FokI nuclease in these cells. The specific binding of this transcription factor/nuclease chimeric protein to the endogenous MnSOD promoter was then visualized at single nucleotide resolution by amplification of the purified genomic DNA using LMPCR (15), followed by strand-specific hybridization. Figure 2, A-C, illustrates the results of PIN*POINT analysis demonstrating specific Sp1 binding to protein sites III, V, and IX (Fig. 1A) as defined previously by in vivo footprinting (20). Kim and Chandrasegaran (18) have previously shown with in vitro studies that transcription factor chimeras with the nuclease cleavage domain of FokI cause strand cleavage between 3 and 13 nucleotides 5' from the potential DNA binding site. In addition, our logic is based on the observation by Smith et al. (33) that the existence of two adjacent transcription factor binding sites exclusively leads to double-strand cleavage. We systematically searched for double-strand cleavage for all of our cleavage sites but only observed single-strand cleavage in all cases. Therefore, we concluded, based on previous results in the literature, that the observed cleavages were a consequence of the binding site 3' to the cleavage, i.e., sites III and V. As shown in Fig. 2, A and B, strand cleavage occurs in vivo 2-4 nucleotides 5' to protein binding sites III and V. Figure 2A also illustrates the specificity of the chimeric cleavage in that this ~140 bp is extremely GC-rich, providing numerous opportunities for nonspecific Sp1 interaction, yet only one cleavage is observed adjacent to the previously localized binding site. Interestingly, we observed two cleavage sites near site IX, the first occurring 4 bases 5' to the potential binding site, whereas the second observed cleavage was 29 bases 5' to the site IX. On closer analysis of our previous in vivo footprinting data, we identified additional putative protein contacts 3' to this latter cleavage site, which may define an additional in vivo Sp1 binding site.
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Sp1 sites important to stimulus-dependent MnSOD gene expression.
To evaluate the functional importance of Sp1 binding sites III, V, and
IX, defined in vivo by PIN*POINT analysis, we employed PCR-based
site-directed mutagenesis to construct mutant clones of these sites.
The residues chosen for mutagenesis were based on the protein contacts
originally defined by our DMS in vivo footprinting (20).
The MnSOD promoter from 420 to +74 was subcloned into a
hGH reporter vector, also containing the MnSOD enhancer from
positions +1,268 to +2,187 (28). Reporter vectors
containing wild-type and site-specific promoter mutants were
transiently transfected into L2 cells and stimulated with
proinflammatory mediators. Reporter gene expression levels were
evaluated by Northern analysis. Transfection efficiency was evaluated
by cotransfection pcDNA 3.1/HisB/LacZ and found to be equal between
independent experiments. For comparison, the mutagenesis results for
site I are shown as a negative control (Fig.
3A). Representative results for sites V and IX are shown in Fig. 3B. The data
demonstrate that Sp1 sites V and IX play relevant functional roles in
the induction of MnSOD gene expression by proinflammatory
stimuli. In addition, we also generated reporter plasmids, which
contained mutations at both sites V and IX as shown in Fig.
3C, illustrating an additional loss of stimulus-dependent
induction. Mutagenesis of site III had no effect on either basal or
stimulated reporter gene expression (data not shown).
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DISCUSSION |
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Our previous in vivo footprinting studies have defined the existence of ten basal protein binding sites (20) within the proximal MnSOD promoter (Fig. 1A). Our original results (20) also included in vitro footprinting analysis using commercially available Sp1 protein, which implied that Sp1 could interact with sites I-V. Unfortunately, at the time, no methodologies existed to unequivocally demonstrate the association of Sp1 with the potential binding sites in the promoter within the context of the nucleus.
The development of PIN*POINT analysis by Chung and coworkers (23) provided the first avenue to address the critical question of trans-acting factor occupancy within the nucleoprotein environment. The PIN*POINT strategy, however, as originally described (23), involves detection of protein/DNA interactions only on a cotransfected target plasmid. Unfortunately, mounting evidence would indicate that regulatory sequences on transiently transfected plasmid DNA may not be a suitable model for studies on the regulation of the endogenous gene given the lack of appropriate chromatin structure on transfected DNA (32). The importance of chromatin structure is of special significance to MnSOD regulation in that we have previously demonstrated that both functionally relevant promoter sequences, as well as the intronic enhancer element, reside in independent DNase I-hypersensitive sites (20). We therefore modified the original PIN*POINT methodology to directly demonstrate the occupancy of a cis-element by a specific trans-acting factor within the context of the endogenous chromatin structure.
The goals of these studies were to establish whether Sp1 could truly interact with MnSOD promoter sequences as predicted by both consensus sequence comparisons and in vitro footprinting results. We first utilized ChIP analysis to establish the association of Sp1 with the analogous region of the endogenous MnSOD promoter (Fig. 1B). Our enhancements of the PIN*POINT strategy allow detection in vivo (Fig. 2), demonstrating that Sp1 occupies three of the ten protein binding sites (sites III, V, and IX; Fig. 1A) originally defined by in vivo footprinting. Interestingly, these results are only in part consistent with predictions of Sp1 binding through either computer-predicted consensus sequences or our in vitro footprinting studies (20), which showed binding to each of sites I-V. The differences in the results are a testimony to the longstanding arguments that purport the importance of the cellular and nuclear milieu in establishing the specificity of protein/DNA interactions (4, 36, 35).
Our mutagenesis results also provide convincing evidence that cis-elements and their cognate transcription factors within the promoter are necessary for the stimulus-dependent function of the MnSOD intronic enhancer element. Mutation of two of the Sp1 sites (V and IX) causes a significant reduction in stimulus-dependent expression, thus providing evidence linking the MnSOD promoter with the distant, intronic enhancer element. We therefore suggest that, by coupling ChIP with our in vivo PIN*POINT strategy, one can address and support the inadequacies of existing in vitro and transient transfection approaches. In summary, we have developed a strategy that can unequivocally reveal the identity of a transcription factor and establish its occupancy on a cis-element within the context of the nucleoprotein environment, thus allowing for a more physiologically relevant assessment of MnSOD expression.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-39593 to H. Nick.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. S. Nick, Dept. of Neuroscience, College of Medicine, Univ. of Florida, Gainesville, Florida 32610 (E-mail: hnick{at}ufl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 16, 2002;10.1152/ajpcell.00356.2002
Received 10 August 2002; accepted in final form 11 October 2002.
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