Adjacent Sequence Controls the Response Polarity of Nitric Oxide-sensitive Sp Factor Binding Sites*

Jianhua Zhang, Shuibang Wang, Robert A. Wesley and Robert L. Danner {ddagger}

From the Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, December 20, 2002 , and in revised form, May 6, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) and cAMP-dependent protein kinase (PKA) inhibitors up-regulate tumor necrosis factor {alpha} (TNF{alpha}) by decreasing Sp1 binding to a proximal GC box element. Here, elements flanking GC boxes were tested for their role in determining whether Sp sites act as activators or repressors. Promoter studies in receptive human cell lines demonstrated that NO down-regulated endothelial NO synthase (eNOS) but up-regulated TNF{alpha}. Like TNF{alpha}, Sp1 binding to the eNOS promoter was decreased by NO and a PKA inhibitor, H89, and increased by a PKA activator, dibutyryl cAMP (Bt2cAMP). For either promoter, mutation of Sp sites abolished NO responses. In contrast, mutation of an upstream AP1 site in the TNF{alpha} promoter (not present in eNOS) maintained NO responsiveness, but reversed the direction of NO and cAMP effects. Using artificial constructs, NO increased transcription when Sp and AP1 sites were both present (TNF{alpha}-like response), but decreased it when the adjacent AP1 site was disrupted (eNOS-like response). NO, H89, and Bt2cAMP were found to produce reciprocal protein binding changes at contiguous AP1 and Sp sites (p < 0.0001 for an interaction). Chromatin immunoprecipitation assays demonstrated that Sp1 and to a lesser extent Sp3 bound to the GC box regions of eNOS and TNF{alpha} in intact cells. Thus, this NO- and cAMP-responsive regulatory module has a Sp site sensor variably coupled to an adjacent element that determines response polarity. These results define a composite element that can utilize secondary inputs to convert off signals to on, thereby conferring complex functionalities to the same DNA binding motif.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO)1 is a free radical messenger that regulates vascular tone (13), inflammation (47), and gene expression (58). NO relaxes vascular smooth muscle by activating soluble guanylate cyclase (13), but NO signal transduction at the level of regulatory DNA sequence remains obscure. NO regulation of promoter activity, in some instances, has been linked to cGMP (810). However, many effects of NO on gene transcription have been ascribed to less well defined, cGMP-independent mechanisms (57, 11, 12), some of which involve the covalent inactivation of DNA-binding proteins (1317).

Recently, we described a GC box element that responds to NO independent of cGMP or NO-mediated transcription factor damage (18). Previous investigations had shown that NO donors, but not cGMP, up-regulated tumor necrosis factor {alpha} (TNF{alpha}) in human peripheral blood mononuclear cells (5) and neutrophil preparations (6). Furthermore, differentiated U937 cells, a human monoblastoid line, transfected with inducible NO synthase produced increased amounts of TNF{alpha} (19). Because U937 cells lack soluble guanylate cyclase, this cell line was subsequently used as a model system to explore cGMP-independent gene regulation by NO in phagocytes (19, 20). Initial studies linked NO up-regulation of TNF{alpha} mRNA and protein with decreases in intracellular concentrations of cAMP (20). Notably, NO effects were mimicked by H89, an inhibitor of cAMP-dependent protein kinase (PKA), and blocked by dibutryl cAMP (Bt2cAMP), a PKA activator (18, 20). PKA modulates gene transcription by phosphorylating nuclear proteins such as cAMP-response element (CRE) binding protein or Sp1 (21). The TNF{alpha} promoter has corresponding binding sites for both of these transcription factors (22). However, in reporter gene experiments, NO and cAMP responses were lost only when the Sp site (proximal GC box) was either deleted or mutated (18). NO and H89 were found to decrease, whereas Bt2cAMP increased Sp1 binding to the TNF{alpha} GC box (18). Thus Sp1 and its proximal GC box-binding site functioned as a genomic NO sensor and repressed TNF{alpha} transcription.

Proximal Sp factor binding sites are found in numerous genes that, unlike TNF{alpha}, lack TATA and CCAAT transcriptional start sites. In TATA- and CCAAT-less promoters, proximal Sp factor binding sites are necessary elements in the recruitment, positioning, and stabilization of the transcriptional complex (23, 24). For these genes, in contrast to TNF{alpha}, Sp factors often act as transactivators and NO-induced reductions in binding would be expected to turn-off rather than turn-on transcription.

The current investigation sought to generalize the NO responsive, GC box paradigm to other types of human promoters and to characterize how these NO sensors might function differentially in the context of larger composite regulatory elements. We tested whether NO-Sp factor-GC box signaling and promoter regulation might also apply to a Sp factor-dependent gene that lacks TATA and CCAAT motifs. Furthermore, we investigated the mechanisms by which NO-induced decreases in Sp factor binding might lead to either gene activation or repression. For the first question, NO regulation of endothelial NO synthase (eNOS) was studied in a human-hybrid endothelial cell line. The eNOS promoter lacks a TATA box, but has two overlapping Sp factor binding sites that are important for basal transcriptional activity (25, 26). Unlike TNF{alpha}, the proximal GC box of eNOS is not flanked by sequences that bind strong transactivating factors (26). For the second question, elements flanking the Sp binding site of TNF{alpha}, AP1, and AP2, were evaluated for their role in determining the directionality of NO-induced promoter responses.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Phorbol 12-myristate 13-acetate (PMA), S-nitroso-N-acetylpenicillamine (SNAP), Bt2cAMP, and H89 were purchased from Calbiochem (San Diego, CA). N-Acetylpenicillamine (NAP) was obtained from Sigma. The enhanced chemiluminescence immunoblot detection system was purchased from Amersham Biosciences. Polyvinylidene difluoride membranes, 4–20% Tris glycine gels, and 6% DNA retardation gels were purchased from Novex (San Diego, CA). Polyclonal anti-Sp1, anti-Sp3, anti-c-Jun/AP1, and anti-AP2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) or Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal anti-eNOS and polyclonal anti-mouse IgG conjugated to horseradish peroxidase were purchased from BD Biosciences.

Western Blotting—EAhy 926 cells, a human-hybrid endothelial line generously contributed by Dr. John B. Graham, North Carolina University (27), were lysed after incubation for 24 h with one of the following reagents or combinations: NAP (control, 500 µM), SNAP (500 µM), Bt2cAMP (100 µM), and H89 (15 µM). Western blotting for eNOS was performed as described previously (19, 28). Briefly, soluble proteins (20 µg) from these different conditions were separated on 4–20% Tris glycine SDS gels (Novex, San Diego, CA) and transferred to polyvinylidene difluoride membranes. After overnight blocking with 5% nonfat dry milk containing 0.05% Tween 20 at 4 °C, membranes were treated with anti-eNOS 1:1,000 and developed with anti-mouse IgG conjugated to horseradish peroxidase.

Plasmid Construction—The two-plasmid reporter gene system used in this investigation has been described previously (18, 29). This reporter gene system is free of cryptic cAMP response elements and amplifies signals from low activity mutant promoters (29). Briefly, promoters of interest were inserted into the first plasmid, promoterless ptTA, to induce production of a transactivator (tTA) containing a tetracycline response element binding domain. This fusion protein binds to the second plasmid, pUHG10.3CAT (kindly provided by Dr. Rob Hooft van Huijsduijnen, Glaxo Institute for Molecular Biology, Geneva, Switzerland), thereby transactivating expression of a reporter gene, chloramphenicol acetyltransferase (CAT). The following promoter constructs were studied: human eNOS using wild type peNOS (from –1322 to +22 of the eNOS gene) and a Sp mutant peNOS(mSp), both graciously provided by Dr. Kenneth Wu, University of Texas, Houston Medical School (25); human TNF{alpha} using wild type pTNF (from –393 to +93 of the TNF{alpha} gene), a gift from Dr. James S. Economou, UCLA School of Medicine (18, 22), an AP1 mutant pTNF(mAP1), a Sp mutant pTNF(mSp), an AP2 mutant pTNF(mAP2), and finally an AP1 and Sp double mutant pTNF(mAP1)/(mSp); an artificial AP1/(Sp)3 promoter (18) using wild type p(AP1)/(Sp)3, an AP1 mutant p(mAP1)/(Sp)3, and a Sp triple mutant p(AP1)/(mSp)3; and two short artificial promoters spanning the AP1/Sp consensus sequence of the TNF{alpha} promoter (–71 to –45) in both the forward and reverse orientation, p(AP1)/(Sp) and p(Sp)/(AP1). Mutants of pTNF were generated using site-directed mutagenesis kits (Clontech, Palo Alto, CA). The wild type artificial promoter and its mutants were created by inserting synthetic double-stranded oligonucleotides, containing the corresponding sequences, into plasmid ptTA. The structures and site mutations for these promoters are outlined in Fig. 1. Mutated sequences were searched against the TRANSFACTM data base and did not match known binding sites for human transcription factors (30).



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FIG. 1.
Schematic of putative binding sites, promoter sequences, and site-directed mutations.

 

Cell Transfection and CAT Assay—Wild type and mutant constructs of the human eNOS promoter were transfected into the human-hybrid endothelial cell line EAhy 926 (27) using DOTAP transfection kits (Roche Diagnostics) according to the manufacturer's protocol. Wild type and mutant TNF{alpha} and artificial promoter constructs were transfected into the human monoblastoid U937 cell line using previously described methods (18). After transfection, U937 cells were incubated for 16 h with PMA (100 nM) to differentiate them into a TNF{alpha}-producing phenotype. CAT activity for all reporter gene experiments was measured after incubation for 24 h in the presence of various reagents by enzyme-linked immunosorbent assay kit (Roche Diagnostics). In all transfections, {beta}-galactosidase expressed by a cotransfected internal pSV{beta}-Gal control (Promega, Madison, WI) was used to normalize CAT. Promoter activity was calculated for each promoter construct as fold induction of CAT expression compared with the CAT expression obtained using the promoterless ptTA plasmid.

Electrophoretic Mobility Shift Assays (EMSA)—U937 cells were incubated with PMA (100 nM) for 16 h. Differentiated U937 or EAhy 926 cells were cultured for 24 h with one of the following reagents or combinations: NAP (control; 500 µM), SNAP (500 µM), Bt2cAMP (100 µM), and H89 (15 µM). EMSA were performed with 15 µg of nuclear extract and double-stranded DNA probes were labeled with 32P using previously described methods (31). The probes used included: Sp probe representing –108 to –83 section of the eNOS promoter (5'-GGGATAGGGGCGGGGCGAGGGCCAGC-3'); AP1-Sp probe representing the –71 to –44 section of the TNF{alpha} promoter (5'-GCTGGTTGAATGATTCTTTCCCCGCCCT-3'); and the Sp-AP2 probe representing the –52 to –25 section of the TNF{alpha} promoter (5'-CCCCGCCCTCCTCTCGCCCCAGGGACAT-3'). For supershift assays, 2 µg of the corresponding antibody was added to the binding reaction mixture and incubated for 20 min on ice prior to addition of the labeled probe. For competition assays, 100-fold molar excess of cold probe was added 10 min prior to the corresponding hot probe.

Chromatin Immunoprecipitation (ChIP) Assay—U937 cells were incubated with PMA (100 nM) for 16 h. Differentiated U937 or EAhy 926 cells were cultured for 24 h with one of the following reagents: NAP (control; 500 µM), SNAP (500 µM), or Bt2cAMP (100 µM). The ChIP assay was performed according to the manufacturer's instructions (Upstate Cell Signaling Solutions, Charlottesville, VA). Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Glycine was added to a final concentration of 0.125 M to stop cross-linking. After washing with ice-cold phosphate-buffered saline, cells were lysed for 10 min (1 x 106 cells/200 µl of SDS lysis buffer). The chromatin was sheared by sonication 4 times for 40 s at one-third of the maximum power with 1 min cooling on ice between each pulse. Cross-link released total chromatin was quantitated to determine the starting amount of DNA present in different samples (input chromatin). The remaining chromatin fractions were pre-cleared with salmon sperm DNA/protein A-agarose for 1 h followed by immunoprecipitation with either anti-Sp1 or anti-Sp3 (Upstate Biotechnology Inc.) overnight at 4 °C. Immune complexes were collected with salmon sperm DNA/protein A-agarose for 1 h, washed 5 times, and finally eluted in 1% SDS, 0.1 M NaHCO3. Sp1- or Sp3-DNA cross-linking was reversed at 65 °C overnight and digested with 100 µg of proteinase K at 45 °C for 1 h. DNA was then recovered in diethylpyrocarbonate-H2O for PCR using phenol/chloroform extraction and ethanol precipitation. PCRs were performed using the following promoter-specific primers: TNF-{alpha} (–127 to +29), 5'-CCACTACCGCTTCCTCCAGAT-3' and 5'-CTGTCCTTGCTGAGGGAGCGT-3'; eNOS (–170 to –7), 5'-CGGGCGTGGAGCTGAGGCTT-3' and 5'-CCAGCAGAGCCCTGGCCTT-3'. The PCR products were analyzed by electrophoresis on 2% agarose gels, stained with SYBR Green I (Molecular Probes), and quantified with Kodak Image Station 440 (Eastman Kodak Co.).

Statistical Analysis—Data are presented as mean ± S.E. of at least three independent experiments. All p values are two-sided unless noted otherwise, and considered significant if less than 0.05. Densitometry results from eNOS Western blots were analyzed by two-way analysis of variance (ANOVA), followed by Dunnett's post-hoc test to find which conditions significantly differed from the common control. Reporter gene experiments within each promoter were analyzed using two-way ANOVA (the two factors being group and experiment). If the overall ANOVA was significant, Fisher's least significant difference method was used to determine which groups differed. Stringently accounting for multiple comparisons was not considered critical because it was expected a priori (18) that the SNAP, H89, and SNAP/H89-treated groups would behave similarly as would the control, Bt2cAMP, and SNAP/Bt2cAMP-treated groups. Comparisons of control promoter activity across multiple wild type and mutant constructs were performed using two-way ANOVA, followed by the stringent post-hoc method of Games-Howell. ChIP assay results were analyzed using two-way ANOVA, followed by Bonferroni-adjusted pairwise comparisons. For pattern-response comparisons performed on the reporter gene element order and ChIP experiments, data was first rescaled to a common mean and then subjected to a 3-way ANOVA, computing the interaction term.

To evaluate reciprocal binding effects at adjacent AP1 and Sp or Sp and AP2 binding sites, Bonferroni-adjusted p values were computed for each condition separately by testing whether the change from control for Sp was in the opposite direction to the change from control for AP1 (or AP2). Evidence for a reciprocal effect was then examined over all conditions by summing these 5 p values and comparing this result to that expected under the null hypothesis of no reciprocal effect.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of NO on eNOS Protein Expression in EAhy 926 cells—We have previously shown that NO up-regulates TNF{alpha} in human phagocytic cells (6, 19) by decreasing intracellular cAMP levels and thereby decreasing Sp1 binding to a repressive GC box element in the TNF{alpha} proximal promoter (18, 20). However, other genes that have transactivating Sp sites would likely be down-rather than up-regulated by NO-induced decreases in Sp1 binding. To test this possibility, we examined eNOS, a TATA-less gene whose transcription is highly dependent on a proximal Sp transactivating element (25, 26). First, we assessed the effects of SNAP, a NO donor, H89, a PKA inhibitor, and Bt2cAMP, a PKA activator, on eNOS protein expression in EAhy 926 cells, a human-hybrid endothelial line, using Western blotting. Compared with control, eNOS protein expression was decreased by incubation with SNAP, H89, or SNAP plus H89 (Fig. 2; p < 0.01). In contrast, incubation with Bt2cAMP or SNAP plus Bt2cAMP compared with control nonsignificantly increased eNOS expression (p = 0.11 and 0.12, respectively).



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FIG. 2.
Effect of NO on eNOS protein expression in a human-hybrid endothelial cell line. A, densitometric measurements from Western blots; values represent the mean ± S.E. of four independent experiments. B, Western blot from a representative experiment. Cell lysates were prepared from EAhy 926 cells incubated in NAP (500 µM; control), the NO donor SNAP (500 µM), the PKA inhibitor H89 (15 µM), both SNAP (500 µM) and H89 (15 µM), the cell permeable cAMP analog Bt2cAMP (100 µM), or both SNAP (500 µM) and Bt2cAMP (100 µM).

 

NO Responsiveness of the Human eNOS Promoter; Dependence on the Proximal GC Box—Next we used a reporter gene system to determine whether NO and H89 decrease eNOS expression by suppressing transcription and, if so, whether these effects are transmitted through the proximal GC box element. To this end, we investigated the responses of both wild type and mutant constructs (Fig. 1). As shown in Fig. 3, SNAP, H89, or both decreased, whereas Bt2cAMP increased eNOS promoter activity (p < 0.0002 for all pairwise comparisons to control). Furthermore, Bt2cAMP blocked the down-regulatory effect of NO (p = 0.3 compared with Bt2cAMP alone). Mutation of the GC box, previously shown to bind Sp1 (25, 26), reduced overall promoter activity, and similar to TNF{alpha} (18), significantly reduced its ability to respond to either NO or cAMP pathway signals (p = 0.0012 compared with wild type promoter). These observations further confirm that the proximal GC box is an important transactivator of the human eNOS promoter. In addition, the results demonstrate that NO can negatively regulate eNOS transcription through this pivotal element.



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FIG. 3.
NO responsiveness of the eNOS promoter; dependence on the proximal Sp site. EAhy 926 cells were transfected with a two-plasmid reporter gene system containing one of the following eNOS promoter constructs: either wild type peNOS or the Sp mutant, peNOS(mSp). CAT activity was quantitated after incubation with NAP (500 µM; control), the NO donor SNAP (500 µM), the PKA inhibitor H89 (15 µM), both SNAP (500 µM) and H89 (15 µM), the cell permeable cAMP analog Bt2cAMP (100 µM), or both SNAP (500 µM) and Bt2cAMP (100 µM). Values represent the mean ± S.E. of three experiments each performed in duplicate.

 

Effect of NO on Sp Proteins; Binding to the Proximal GC Box of the eNOS Promoter—Our previous studies demonstrated that NO decreased intracellular cAMP levels leading to reduced Sp1 binding to the human TNF{alpha} promoter (18, 20). Notably, this effect of NO on Sp1 binding was simulated by PKA inhibition. To investigate whether the effect of NO on eNOS promoter activity occurred through a similar mechanism, EMSA were performed using 32P-labeled probe containing the Sp consensus sequence of the proximal eNOS promoter (Fig. 4). Nuclear extracts from endothelial EAhy 926 cells formed two main complexes (a major Sp complex and a minor band) with the probe (Fig. 4B). The presence of Sp1 in the major complex was demonstrated by anti-Sp1 polyclonal antibody, which partially supershifted the band (Fig. 4B). Furthermore, unlabeled Sp oligonucleotide was shown to compete off protein from the major complex. Incubation with SNAP, H89, or SNAP plus H89 (Fig. 4A) similarly decreased the formation of the Sp major complex compared with nuclear extract from control cells (p < 0.01 for all). Conversely, Bt2cAMP increased Sp factor binding (Fig. 4A; p < 0.01) and interfered with the ability of SNAP to reduce Sp complex formation (Fig. 4A; p = 0.12 compared with Bt2cAMP alone by paired t test).



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FIG. 4.
Effect of NO on Sp protein binding to the GC box element in the proximal eNOS promoter. A, densitometric measurements from EMSA gels; values represent the mean ± S.E. of four independent experiments. B, EMSA gel from a representative experiment. C, relative densitometric results from ChIP assay gels; values were normalized to DNA inputs and represent the mean ± S.E. of four independent experiments. D, ChIP assay gel from a representative experiment. EAhy 926 cells were treated with NAP (500 µM; control), the NO donor SNAP (500 µM), the PKA inhibitor H89 (15 µM), and the cell-permeable cAMP analog Bt2cAMP (100 µM) alone, or in combination as indicated.

 

To obtain direct in vivo evidence of a NO effect on Sp protein binding to the GC box in the proximal eNOS promoter, a ChIP assay was performed using EAhy 926 endothelial cells. PCR primers for the ChIP assay were designed to amplify a 163-bp sequence encompassing the proximal GC box in the eNOS promoter. As seen in Fig. 4, C and D, the GC box-containing region of eNOS binds Sp1 and to a lesser degree Sp3 protein in vivo. Importantly, effects of NO and Bt2cAMP on protein binding were significantly different from each other for both Sp1 (p = 0.026) and Sp3 (p = 0.03). Furthermore, Sp1 and Sp3 had highly similar patterns of response across the three conditions (p = 0.89 for a difference). These results by in vitro EMSA and in vivo ChIP assay demonstrate, as previously shown for TNF{alpha} (18), that NO decreases Sp1 binding to the proximal GC box element of the human eNOS promoter.

NO Responsiveness of the TNF{alpha} Promoter; Role of AP1 and AP2 Sites Flanking the GC Box—Next, we re-examined the TNF{alpha} promoter to determine whether GC box flanking sequences somehow cause Sp1 or other Sp proteins to behave functionally as transcriptional repressors. As shown in Fig. 5, the NO donor, SNAP, and the PKA inhibitor, H89, both increased while the PKA activator, Bt2cAMP, decreased the wild type TNF{alpha} promoter activity (p < 0.0001 for all). Note that all responses are opposite to those seen for eNOS. Consistent with its function as a repressor element, mutation of the Sp binding site enhanced TNF{alpha} promoter activity (p < 0.01 comparing pTNF(mSp) to control pTNF). Furthermore, the up-regulatory effect of NO on the TNF{alpha} promoter was blocked by either the addition of Bt2cAMP (p > 0.20 compared with Bt2cAMP alone) or Sp mutation (p = ns for overall test of group differences by ANOVA). In contrast, AP1 mutation, as seen in Fig. 5, left, the TNF{alpha} promoter NO-responsive, but completely reversed the direction of responses to both NO and cAMP pathway signals (eNOS-like pattern) compared with the wild type promoter; SNAP and H89 alone and in combination decreased, but Bt2cAMP increased the activity of pTNF(mAP1) (p < 0.0001 for each compared with control). Although the AP2 mutation (pTNF(mAP2)) decreased overall promoter activity, the pattern of responses to NO, H89, and Bt2cAMP were unchanged compared with that of the wild type promoter (Fig. 5). Double mutations of AP1 and Sp (pTNF(mAP1/mSp), like the Sp mutation alone, rendered the TNF{alpha} promoter unresponsive to either NO or Bt2cAMP (Fig. 5). These data support the conclusion that the GC box (Sp factor binding site) in the proximal TNF{alpha} promoter functions as a NO sensor, whereas the upstream AP1 site controls response polarity.



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FIG. 5.
NO responsiveness of the TNF{alpha} promoter; dependence on the proximal Sp and upstream AP1 sites. U937 cells were transfected with a two-plasmid reporter gene system containing one of the following TNF{alpha} promoter constructs: wild type pTNF, AP1 mutant pTNF(mAP1), Sp mutant pTNF(mSp), AP2 mutant pTNF(mAP2), or AP1 and Sp double mutant pTNF(mAP1)/(mSp). Cells were then exposed to PMA (100 nM) to differentiate them into a TNF{alpha}-producing phenotype. CAT activity was quantitated after incubation with NAP (500 µM; control), the NO donor SNAP (500 µM), the PKA inhibitor H89 (15 µM), both SNAP (500 µM) and H89 (15 µM), the cell permeable cAMP analog Bt2cAMP (100 µM), or both SNAP (500 µM) and Bt2cAMP (100 µM). Values represent the mean ± S.E. of five experiments each performed in duplicate.

 

Functional Analysis of Sp and AP1 Binding Sites in Artificial Promoters—Next, we used an artificial promoter p(AP1)/(Sp)3-tTA (Fig. 1) to further explore the behavior of NO- and cAMP-responsive GC box motifs. Three potential Sp protein binding sites were used in the artificial construct to create a promoter with sufficient length and activity (18). As shown in Fig. 6A, SNAP, H89, or SNAP plus H89 increased (p < 0.0001 for each), whereas Bt2cAMP or SNAP plus Bt2cAMP decreased (p < 0.001 for both) the activity of the wild type artificial promoter construct (TNF{alpha} promoter-like responses). Likewise, mutation of the AP1 site reversed the direction of all observed responses; SNAP, H89, or SNAP plus H89 decreased (p < 0.0001 for all), while Bt2cAMP or SNAP plus Bt2cAMP increased (p < 0.0001 for both) the activity of the AP1 mutant promoter (Fig. 6A). Thus, the AP1 mutant demonstrated eNOS-like responses to NO and cAMP pathway signals. Again, simultaneous mutation of all Sp sites abolished the effects of SNAP, H89, and Bt2cAMP (Fig. 6A).



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FIG. 6.
Functional analysis of Sp and AP1 sites in artificial promoter constructs. U937 cells were transfected with a two-plasmid reporter gene system containing one of the following artificial promoter constructs: A, wild type p(AP1)/(Sp)3, AP1 mutant p(mAP1)/(Sp)3, or Sp triple mutant p(AP1)/(mSp)3; B, p(AP1)/(Sp) or p(Sp)/(AP1). Cells were then exposed to PMA (100 nM) to differentiate them into a TNF{alpha}-producing phenotype. CAT activity was quantitated after incubation with NAP (500 µM; control), the NO donor SNAP (500 µM), the PKA inhibitor H89 (15 µM), both SNAP (500 µM) and H89 (15 µM), the cell permeable cAMP analog Bt2cAMP (100 µM), or both SNAP (500 µM) and Bt2cAMP (100 µM). Values represent the mean ± S.E. of five (A) or three (B) experiments, respectively, each performed in duplicate.

 

Unlike the TNF{alpha} promoter, disruption of the AP1 site in the artificial construct did not decrease, but rather slightly increased base-line artificial promoter activity. This suggests that the AP1 mutation may have increased cooperation among the tandem Sp sites. Consistent with this concept, simultaneous mutation of all Sp sites did not increase base-line transcription, indicating that the transcriptional activity of this construct reflects the net effect of Sp repression and transactivation across the three sites. Nonetheless, the responses of this artificial construct to NO and cAMP analogs were similar to those of the TNF{alpha} promoter and the same functional relationship between AP1 and the GC box elements was demonstrated.

To investigate whether the AP1 site and GC box order affects NO responsiveness, two additional artificial promoter constructs were studied. The first, p(AP1)/(Sp)-tTA, contains the 26-bp sequence from the TNF{alpha} promoter (–71 to –45) that spans the AP1 site and GC box. For p(Sp)/(AP1)-tTA, an identical sequence was used but inverted, placing the GC box upstream to the AP1 site (Fig. 1). As shown in Fig. 6B, SNAP increased (p < 0.001), while Bt2cAMP or SNAP plus Bt2cAMP decreased the activity of both promoters (p < 0.001), a pattern of responses identical to those of the TNF{alpha} promoter. Interestingly, element order was not important for module function. The overall pattern of response to NO and cAMP analogs was preserved independent of the element order (p = 0.71 for a difference).

A Binding Interaction Analysis of AP1, Sp, and AP2 Sites in the Proximal TNF{alpha} Promoter—To further investigate how AP1, Sp, and AP2 sites may interact to convert NO signals (through decreased Sp protein binding) from off to on, we studied transcription factor binding behavior in the presence and absence of NO and cAMP pathway signals. EMSA were performed using combined AP1-Sp or Sp-AP2 32P-labeled probes spanning corresponding sequences in the TNF{alpha} promoter. Nuclear extracts for these experiments were prepared from PMA-differentiated U937 cells after incubation under various conditions (Fig. 7).



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FIG. 7.
A binding interaction analysis of AP1, Sp, and AP2 sites in the proximal TNF{alpha} promoter. A, densitometric measurements from EMSA gels, using the AP1-Sp probe. Values represent the mean ± S.E. of four independent experiments. B, AP1-Sp probe EMSA gel from a representative experiment. C, densitometric measurements from EMSA gels, using the Sp-AP2 probe. Values represent the mean ± S.E. of four independent experiments. D, Sp-AP2 probe EMSA gel from a representative experiment. Nuclear extracts were prepared from PMA-differentiated U937 cells incubated with NAP (500 µM; control), the NO donor SNAP (500 µM), the PKA inhibitor H89 (15 µM), both SNAP (500 µM) and H89 (15 µM), the cell permeable cAMP analog Bt2cAMP (100 µM), or both SNAP (500 µM) and Bt2cAMP (100 µM).

 

Two major DNA-protein complexes were detected with both probes. The AP1-Sp probe formed Sp and AP1 binding complexes (Fig. 7B) and the Sp-AP2 probe formed Sp and AP2 binding complexes (Fig. 7D). The presence of Sp1, AP1, and AP2 in their corresponding complexes was confirmed by super-shift assays with specific antibodies. Sequence specificity was demonstrated using appropriate competition assays with cold probes (Fig. 7, B and D). Interestingly, blocking Sp protein binding to the 32P-labeled probe with excess cold Sp oligonucleotide increased AP1 binding in vitro (Fig. 7B). Conversely, blocking AP1 binding with excess cold AP1 oligonucleotide increased Sp protein binding (Fig. 7B). Thus, even simple competition testing supported the notion that strong binding interactions exist between these two sites. Likewise, incubation with SNAP, H89, or SNAP with H89 similarly decreased Sp binding, but reciprocally increased AP1 binding (Fig. 7, A and B; one-sided p < 0.0001 for an overall reciprocal Sp/AP1 effect). Although NO has been reported to enhance DNA binding of AP1 through a cGMP-dependent signaling pathway (8), this effect cannot be invoked here because U937 cells have been shown to lack NO-sensitive guanylate cyclase (19, 20). However, in cells with an intact NO-cGMP signaling pathway, combined NO effects through cGMP-dependent increases in AP1 binding and cGMP-independent decreases in Sp protein binding might be expected to synergistically activate TNF{alpha}-like promoters.

As expected for the combined Sp-AP2 probe, SNAP or H89, or both consistently decreased whereas Bt2cAMP increased Sp protein binding (Fig. 7, C and D). However, none of these reagents significantly altered AP2 binding, and therefore changes in Sp protein binding were not reciprocally mirrored by AP2 (one-sided p = 0.5). Collectively, these results suggest that AP1 and Sp factor sites in the proximal TNF{alpha} promoter antagonize each other by competing for binding space and demonstrate that NO inhibition of Sp protein binding reciprocally enhances AP1 binding, thus increasing promoter activity.

Effect of NO on the in Vivo Binding of Sp1 and Sp3 to the Proximal TNF{alpha} Promoter—To further investigate NO effects on Sp protein binding to the proximal GC box of the TNF{alpha} promoter in vivo, ChIP assays were performed on PMA-differentiated U937 cells using PCR primers designed to amplify a 156-bp sequence encompassing the proximal GC box in the TNF{alpha} promoter. Consistent with the in vitro EMSA results, NO and Bt2cAMP had directionally opposite effects that differed significantly from each other (Fig. 8) for both Sp1 (p = 0.006) and Sp3 (p = 0.0004). Again, the pattern of effects on Sp1 and Sp3 across the three conditions were very similar (p = 0.85 for a difference), suggesting that both Sp1 and Sp3 may be involved in GC box-mediated TNF{alpha} gene regulation by NO.



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FIG. 8.
Effect of NO on the in vivo binding of Sp1 and Sp3 to the proximal TNF{alpha} promoter. A, relative densitometric results from ChIP assay gels; values were normalized to DNA inputs and represent the mean ± S.E. of four independent experiments. B, ChIP assay gel from a representative experiment. PMA-differentiated U937 cells were treated with NAP (500 µM; control), SNAP (500 µM), and Bt2cAMP (100 µM) as indicated.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
NO is reported to regulate a growing list of seemingly unrelated genes in a wide variety of cell types and species (520). However, the specificity and underlying mechanisms of these events at the level of regulatory DNA sequence are controversial. Some NO-regulated genes are arguably affected through specific pathways that involve cGMP-dependent protein kinase (810). In contrast, NO appears to regulate many genes through less well characterized, cGMP-independent mechanisms that include stress-related mitogen-activated protein kinase signaling (3234), peroxisome-activated receptor {gamma} activation (11), and direct effects on cysteine-rich, but otherwise unrelated transcription factors (12, 13, 1517, 35, 36). Notably, S-nitrosylation has been proposed to alter gene expression by modifying the DNA binding affinity of many nuclear proteins including heat shock factor 1 (12), AP1 (13), NF-{kappa}B (1417), Oct 1 (35), and Sp1 (36). However, the conditions under which covalent interactions become biologically relevant remain uncertain. Collectively, these studies suggest a somewhat nonspecific model of cGMP-independent NO signaling. The regulatory consequences of these diverse mechanisms are likely to be highly context dependent and difficult to predict based on DNA sequence.

Here, we describe a modular NO-responsive logic gate-like mechanism containing a GC box-actuator variably coupled to an adjacent AP1 inverter element that determines response polarity. This putative regulatory module was shown to decrease eNOS but increase TNF{alpha} expression through similar effects on Sp factor binding. For both promoters, GC box disruption abolished NO and cAMP responsiveness. In contrast, mutating the AP1 site in the TNF{alpha} promoter maintained promoter sensitivity to NO and cAMP signaling, but completely reversed the direction of their effects. These structure-function relationships were further confirmed using artificial promoter constructs and supported by both in vitro EMSA and in vivo ChIP experiments. Sp factor binding sites were found to be necessary for NO sensitivity while the presence or absence of an AP1 site determined whether NO produced on (TNF{alpha}-like) or off (eNOS-like) responses, respectively. NO- and cAMP-induced changes in promoter activity correlated with strong reciprocal binding interactions between AP1 and Sp proteins. Thus, nearby regulatory sequence and corresponding nuclear protein binding events can switch the behavior of GC box motifs from that of activator to repressor without fundamentally altering its binding relationship with Sp factor family members. Together, these results suggest a general logic whereby secondary inputs not only modulate output, but also can convert off signals to on.

The finding that NO and cAMP can regulate eNOS through its proximal, canonical Sp1 binding site has several important implications for vascular physiology. First, it suggests a negative feedback loop mechanism whereby eNOS can turn off its own transcription. Furthermore, unfettered NO production by inducible NOS might down-regulate eNOS through this pathway in conditions associated with vascular inflammation such as sepsis and atherosclerosis (37, 38). Finally, vasoactive mediators that affect intracellular cAMP such as angiotensin II and catecholamines (39) may thereby alter eNOS expression in the vasculature.

The relationship between cAMP-induced PKA activation and increases in Sp factor binding that were repeatedly demonstrated in this investigation are not invariably supported by the existing literature. Sp1 dephosphorylation in some reports has been associated with increased Sp1 binding to GC box motifs (40, 41). Importantly, eNOS, itself, has been shown to be induced by protein phosphatase 2A-mediated dephosphorylation of Sp1 (25). In contrast, C-terminal Sp1 phosphorylation has been associated with its activation during the cell cycle (42). This suggests that Sp1 phosphorylation or dephosphorylation might have different effects depending on the site specificity of the enzyme involved. Consistent with our observations, Rohlff et al. (18, 21, 43) and others have demonstrated that Sp1 is a cAMP- and PKA-responsive transcription factor, which can be activated by phosphorylation (21, 42, 44).

Notably, the regulation of Sp factor-dependent genes through PKA could potentially explain some effects of cAMP signaling on cell growth and inflammation. For example, previous reports have shown that cAMP signaling suppresses TNF{alpha} transcription (29, 45, 46). The current investigation identifies the proximal GC box element as a final target for this anti-inflammatory effect. However, it is important to note that GC box effects on TNF{alpha} promoter activity, as reported here, were generally modest relative to control values. The TNF{alpha} promoter has numerous cis-elements including NF-{kappa}B, Ets, CRE, NF-AT, and C/EBP{beta} that contribute to and control its transcriptional activation (47, 48).

An important aspect of the current experiments is that the consequences of NO or cAMP signaling-induced changes in Sp protein binding to GC box elements was placed in a larger context. A nearby binding site and the presence or absence of sequence-specific protein binding to this adjacent element was shown to ultimately determine whether Sp factor-dependent events would activate or repress transcription. Although the Sp protein family has been reported to interact with many transcription factors including NF-{kappa}B (49), AP1 (50), AP2 (51), NF-Y (43), and Egr-1 (52), our data provide details of a flexible mechanism that can be configured to produce either up- or down-regulation in response to a single environmental signal. Furthermore, we identify two forms of this molecular circuit in naturally occurring, but dissimilar promoters. The version of this regulatory module with Sp-dependent repressor function may provide a useful model for understanding similar observations in other genes with putative GC box repressor elements (5357). This paradigm may also have relevance for cAMP signaling events that have been shown to exert opposite effects depending on the activation state of secondary transcription factors (46). Collectively, our results imply a promoter logic that can generate off, on, or indifferent responses depending on the physical and functional presence of secondary inputs.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Critical Care Medicine Dept., Bldg. 10, Rm. 7D43, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, Tel.: 301-496-9320; Fax: 301-402-1213; E-mail: rdanner{at}nih.gov.

1 The abbreviations used are: NO, nitric oxide; TNF{alpha}, tumor necrosis factor {alpha}; PKA, cAMP-dependent protein kinase; Bt2cAMP, dibutyryl cAMP; CRE, cAMP-response element; eNOS, endothelial nitric-oxide synthase; PMA, phorbol 12-myristate 13-acetate; SNAP, S-nitroso-N-acetylpenicillamine; NAP, N-acetyl-D-penicillamine; tTA, tetracycline-controlled transactivator; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; ANOVA, analysis of variance. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Palmer, R. M., Ashton, D. S., and Moncada, S. (1988) Nature 333, 664–666[CrossRef][Medline] [Order article via Infotrieve]
  2. Huang, P. L., Huang, Z., Mashimo, H., Bloch, K. D., Moskowitz, M. A., Bevan, J. A., and Fishman, M. C. (1995) Nature 377, 239–242[CrossRef][Medline] [Order article via Infotrieve]
  3. Ma, P., and Danner, R. L. (2002) Crit. Care Med. 30, 947–949[CrossRef][Medline] [Order article via Infotrieve]
  4. Kubes, P., Suzuki, M., and Granger, D. N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4651–4655[Abstract]
  5. Lander, H. M., Sehajpal, P., Levine, D. M., and Novogrodsky, A. (1993) J. Immunol. 150, 1509–1516[Abstract/Free Full Text]
  6. Van Dervort, A. L., Yan, L., Madara, P. J., Cobb, J. P., Wesley, R. A., Corriveau, C. C., Tropea, M. M., and Danner, R. L. (1994) J. Immunol. 152, 4102–4109[Abstract/Free Full Text]
  7. Corriveau, C. C., Madara, P. J., Van Dervort, A. L., Tropea, M. M., Wesley, R. A., and Danner, R. L. (1998) J. Infect. Dis. 177, 116–126[Medline] [Order article via Infotrieve]
  8. Pilz, R. B., Suhasini, M., Idriss, S., Meinkoth, J. L., and Boss, G. R. (1995) FASEB J. 9, 552–558[Abstract/Free Full Text]
  9. Zaragoza, C., Soria, E., Lopez, E., Browning, D., Balbin, M., Lopez-Otin, C., and Lamas, S. (2002) Mol. Pharmacol. 62, 927–935[Abstract/Free Full Text]
  10. Gudi, T., Lohmann, S. M., and Pilz, R. B. (1997) Mol. Cell. Biol. 17, 5244–5254[Abstract]
  11. Von Knethen, A., and Brune, B. (2002) J. Immunol. 169, 2619–2626[Abstract/Free Full Text]
  12. Xu, Q., Hu, Y., Kleindienst, R., and Wick, G. (1997) J. Clin. Invest. 100, 1089–1097[Abstract/Free Full Text]
  13. Melino, G., Bernassola, F., Catani, M. V., Rossi, A., Corazzari, M., Sabatini, S., Vilbois, F., and Green, D. R. (2000) Cancer Res. 60, 2377–2383[Abstract/Free Full Text]
  14. Peng, H. B., Rajavashisth, T. B., Libby, P., and Liao, J. K. (1995) J. Biol. Chem. 270, 17050–17055[Abstract/Free Full Text]
  15. delaTorre, A., Schroeder, R. A., Bartlett, S. T., and Kuo, P. C. (1998) Surgery 124, 137–142[CrossRef][Medline] [Order article via Infotrieve]
  16. Marshall, H. E., Merchant, K., and Stamler, J. S. (2000) FASEB J. 14, 1889–1900[Abstract/Free Full Text]
  17. Marshall, H. E., and Stamler, J. S. (2001) Biochemistry 40, 1688–1693[CrossRef][Medline] [Order article via Infotrieve]
  18. Wang, S., Wang, W., Wesley, R. A., and Danner, R. L. (1999) J. Biol. Chem. 274, 33190–33193[Abstract/Free Full Text]
  19. Yan, L., Wang, S., Rafferty, S. P., Wesley, R. A., and Danner, R. L. (1997) Blood 90, 1160–1167[Abstract/Free Full Text]
  20. Wang, S., Yan, L., Wesley, R. A., and Danner, R. L. (1997) J. Biol. Chem. 272, 5959–5965[Abstract/Free Full Text]
  21. Rohlff, C., Ahmad, S., Borellini, F., Lei, J., and Glazer, R. I. (1997) J. Biol. Chem. 272, 21137–21141[Abstract/Free Full Text]
  22. Rhoades, K. L., Golub, S. H., and Economou, J. S. (1992) J. Biol. Chem. 267, 22102–22107[Abstract/Free Full Text]
  23. Pugh, B. F., and Tjian, R. (1990) Cell 61, 1187–1197[Medline] [Order article via Infotrieve]
  24. DiMario, J. X. (2002) Int. J. Mol. Med. 10, 65–71[Medline] [Order article via Infotrieve]
  25. Cieslik, K., Zembowicz, A., Tang, J. L., and Wu, K. K. (1998) J. Biol. Chem. 273, 14885–14890[Abstract/Free Full Text]
  26. Karantzoulis-Fegaras, F., Antoniou, H., Lai, S. L., Kulkarni, G., D'Abreo, C., Wong, G. K., Miller, T. L., Chan, Y., Atkins, J., Wang, Y., and Marsden, P. A. (1999) J. Biol. Chem. 274, 3076–3093[Abstract/Free Full Text]
  27. Edgell, C. J., McDonald, C. C., and Graham, J. B. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3734–3737[Abstract]
  28. Wang, W., Wang, S., Yan, L., Madara, P., Del Pilar Cintron, A., Wesley, R. A., and Danner, R. L. (2000) J. Biol. Chem. 275, 16899–16903[Abstract/Free Full Text]
  29. Ollivier, V., Parry, G. C., Cobb, R. R., de Prost, D., and Mackman, N. (1996) J. Biol. Chem. 271, 20828–20835[Abstract/Free Full Text]
  30. Wingender, E., Chen, X., Fricke, E., Geffers, R., Hehl, R., Liebich, I., Krull, M., Matys, V., Michael, H., Ohnhäuser, R., Prüß, M., Schacherer, F., Thiele, S., and Urbach, S. (2001) Nucleic Acids Res. 29, 281–283[Abstract/Free Full Text]
  31. Zhang, J., and Ding, X. (1998) J. Biol. Chem. 273, 23454–23462[Abstract/Free Full Text]
  32. Wang, W., Wang, S., Nishanian, E. V., Del Pilar Cintron, A., Wesley, R. A., and Danner, R. L. (2001) Am. J. Physiol. 281, C544–C554
  33. Bauer, P. M., Buga, G. M., and Ignarro, L. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12802–12807[Abstract/Free Full Text]
  34. Kibbe, M. R., Li, J., Nie, S., Watkins, S. C., Lizonova, A., Kovesdi, I., Simmons, R. L., Billiar, T. R., and Tzeng, E. (2000) J. Vasc. Surg. 31, 1214–1228[CrossRef][Medline] [Order article via Infotrieve]
  35. Liu, X. K., Abernethy, D. R., and Andrawis, N. S. (1998) Life Sci. 62, 739–749[CrossRef][Medline] [Order article via Infotrieve]
  36. Berendji, D., Kolb-Bachofen, V., Zipfel, P. F., Skerka, C., Carlberg, C., and Kroncke, K. D. (1999) Mol. Med. 5, 721–730[Medline] [Order article via Infotrieve]
  37. MacNaul, K. L., and Hutchinson, N. I. (1993) Biochem. Biophys. Res. Commun. 196, 1330–1334[CrossRef][Medline] [Order article via Infotrieve]
  38. Jeremy, J. Y., Rowe, D., Emsley, A. M., and Newby, A. C. (1999) Cardiovasc. Res. 43, 580–594[CrossRef][Medline] [Order article via Infotrieve]
  39. Volicer, L., and Hynie, S. (1971) Eur. J. Pharmacol. 15, 214–220[CrossRef][Medline] [Order article via Infotrieve]
  40. Borellini, F., Aquino, A., Josephs, S. F., and Glazer, R. I. (1990) Mol. Cell. Biol. 10, 5541–5547[Medline] [Order article via Infotrieve]
  41. Daniel, S., Zhang, S., DePaoli-Roach, A. A., and Kim, K. H. (1996) J. Biol. Chem. 271, 14692–14697[Abstract/Free Full Text]
  42. Black, A. R., Jensen, D., Lin, S. Y., and Azizkhan, J. C. (1999) J. Biol. Chem. 274, 1207–1215[Abstract/Free Full Text]
  43. Cote, F., Schussler, N., Boularand, S., Peirotes, A., Thevenot, E., Mallet, J., and Vodjdani, G. (2002) J. Neurochem. 81, 673–685[CrossRef][Medline] [Order article via Infotrieve]
  44. Alroy, I., Soussan, L., Seger, R., and Yarden, Y. (1999) Mol. Cell. Biol. 19, 1961–1972[Abstract/Free Full Text]
  45. Eigler, A., Siegmund, B., Emmerich, U., Baumann, K. H., Hartmann, G., and Endres, S. (1998) J. Leukocyte Biol. 63, 101–107[Abstract]
  46. Galea, E., and Feinstein, D. L. (1999) FASEB J. 13, 2125–2137[Abstract/Free Full Text]
  47. Zagariya, A., Mungre, S., Lovis, R., Birrer, M., Ness, S., Bayar, T., and Pope, R. (1998) Mol. Cell. Biol. 18, 2815–2824[Abstract/Free Full Text]
  48. Krämer, B., Wiegmann, K., and Krönke, M. (1995) J. Biol. Chem. 270, 6577–6583[Abstract/Free Full Text]
  49. Lim, S. P., and Garzino-Demo, A. (2000) J. Virol. 74, 1632–1640[Abstract/Free Full Text]
  50. Noti, J. D., Reinemann, B. C., and Petrus, M. N. (1996) Mol. Cell. Biol. 16, 2940–2950[Abstract]
  51. Xu, Y., Porntadavity, S., and St. Clair, D. K. (2002) Biochem. J. 362, 401–412[CrossRef][Medline] [Order article via Infotrieve]
  52. Du, B., Fu, C., Kent, K. C., Bush, H., Jr., Schulick, A. H., Kreiger, K., Collins, T., and McCaffrey, T. A. (2000) J. Biol. Chem. 275, 39039–39047[Abstract/Free Full Text]
  53. Roman, D. G., Toledano, M. B., and Leonard, W. J. (1990) New Biol. 2, 642–647[Medline] [Order article via Infotrieve]
  54. Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332–6340[Abstract/Free Full Text]
  55. Tang, Q. Q., Jiang, M. S., and Lane, M. D. (1999) Mol. Cell. Biol. 19, 4855–4865[Abstract/Free Full Text]
  56. Ogra, Y., Suzuki, K., Gong, P., Otsuka, F., and Koizumi, S. (2001) J. Biol. Chem. 276, 16534–16539[Abstract/Free Full Text]
  57. Li, R., Hodny, Z., Luciakova, K., Barath, P., and Nelson, B. D. (1996) J. Biol. Chem. 271, 18925–18930[Abstract/Free Full Text]