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The Journal of Immunology, 1999, 162: 7249-7255.
Copyright © 1999 by The American Association of Immunologists

The Promoter of the C1 Inhibitor Gene Contains a Polypurine·Polypyrimidine Segment that Enhances Transcriptional Activity1

Kamyar Zahedi2, John J. Bissler, Anne E. Prada, Jorge A. Prada and Alvin E. Davis, III

Division of Nephrology, Children’s Hospital Research Foundation and Department of Pediatrics, University of Cincinnati College of Medicine, Children’s Hospital Medical Center, Cincinnati, OH 45229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The C1 inhibitor (C1INH) promoter is unusual in two respects: 1) It contains no TATA sequence, but instead contains a TdT-like initiator element (Inr) at nucleotides -3 to +5; 2) it contains a polypurine·polypyrimidine tract between nucleotides -17 and -45. Disruption of the Inr by the introduction of point mutations reduced promoter activity by 40%. A TATA element inserted at nucleotide -30 in the wild-type promoter and in promoter constructs containing the mutated Inr led to a 2-fold increase in basal promoter activity. Previous studies suggested that the potential hinged DNA-forming polypurine·polypyrimidine tract might be important in the regulation of C1INH promoter activity. The present studies indicate that this region is capable of such intramolecular triple helix formation. Disruption of the polypurine·polypyrimidine sequence by substitution of 5 of the 23 cytosine residues with adenine prevented triple helix formation. Site-directed mutagenesis experiments demonstrate that the regulation of promoter activity is independent of hinged DNA-forming capacity but requires an intact AC box (ACCCTNNNNNACCCT) or the overlapping PuF binding site (GGGTGGG). The C1INH gene also contains a number of potential regulatory elements, including an Sp-1 and an hepatocyte nuclear factor-1 binding site and a CAAT box. The role of these elements in regulation of the C1INH promoter was examined. Elimination of the hepatocyte nuclear factor-1 site at nucleotides -94 to -81 by truncation reduced the activity of the promoter by ~50%. Similarly, site-directed mutations that disrupt this site reduce promoter activity by 70%.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The C1 inhibitor (C1INH),3 a member of the serpin family of proteinase inhibitors, regulates activation of the complement system, the contact system of kinin generation, and the intrinsic coagulation pathway. It is the sole inhibitor of the C1r and C1s components of the classical complement pathway and is the major regulator of factors XI and XII and of plasma kallikrein (1, 2, 3, 4, 5). The importance of the regulation of synthesis of C1INH is dramatically illustrated by hereditary angioedema, which develops in individuals who are heterozygous for a deficiency or dysfunction of C1INH (6). The reduction in the expression of C1INH leads to dysregulation of both the complement and contact systems. The resultant uncontrolled activation leads to further consumption of the remaining C1INH and to the generation of bradykinin (and perhaps other molecules), which mediates angioedema. Therefore, maintenance of C1INH levels above a critical level (~30% of normal) is required to maintain homeostasis.

C1INH is an acute phase reactant (7). Its transcription rate increases after stimulation with cytokines, including IFN-{alpha}, IFN-{gamma}, and IL-6 (8, 9, 10, 11). The IFN response elements map to the immediate 5' flanking region and the first intron of the C1INH gene (12, 13). The C1INH promoter is unusual in several respects. It lacks a TATA box, but depends upon an initiator element (Inr) (CTCAGTCT) at nucleotides -3 to +5 (Fig. 1Go). Inrs overlap the transcription start site and replace the TATA box; they are present in a number of housekeeping genes as well as in some complement genes (14, 15). The C1INH Inr belongs to the TdT family (consensus sequence CTCANTCT) (14). Previous data showed that the C1INH Inr, like the TdT Inr, directed transcription in the absence of any upstream sequence (12). The nature of the proteins that bind the Inr is not well understood. TATA binding protein, the TFII-D complex, and TFII-I bind to and protect the region containing the Inr against DNaseI digestion and may be components of the preinitiation complex that binds to the Inr (16, 17, 18, 19).



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  		FIGURE 1. Schematic diagram of the C1INH promoter region and the reporter constructs used in this study. The potential HNF-1 (dashed box), Sp-1 (dashed oval), and CAAT (solid oval) sites are at positions -94 to -81, -80 to -72, and -62 to -59, respectively. The H-DNA sequence (solid box) spans the -48 to -17 region. The Inr is underlined at position -3 to +5, and the transcription start site at +1 is designated by the arrow. The reporter constructs are listed below the diagram of the promoter region. Mutated bases are underlined.

 
The C1INH promoter also contains a potential hinged DNA (H-DNA)-forming region (nucleotides -45 through -17) (Fig. 1Go). A similar sequence is present in the c-myc promoter at nucleotides -141 through -115 (20). Such polypurine·polypyrimidine tracts make up ~1% of the human genome (21, 22). They have the capacity to form intramolecular triple helices as indicated by their nuclease hypersensitivity (23, 24, 25, 26, 27, 28). Sequences such as these appear to play a role in replication (29, 30) and transcriptional regulation (31, 32, 33). In some genes, such as c-myc, TGF-ß3, and Drosophila hsp26, their effect appears to be stimulatory (20, 34, 35), whereas in others, such as {gamma}-globin and the rat neural cell adhesion molecule and androgen receptor genes, their effect appears to be suppressive (25, 36, 37, 38, 39). The mechanisms by which these sequences mediate their diverse effects have not, as yet, been determined.

In the current studies, we have shown that the C1INH gene requires an intact Inr to maintain its normal transcription level. In addition, other upstream elements, in particular an hepatocyte nuclear factor-1 (HNF-1) element at -94 through -82, are required for normal basal transcription. The results indicate that the triplex-forming region enhances the transcription of the C1INH gene only in the presence of other upstream elements. Furthermore, the activity of this region depends upon the presence of a potential PuF element (23) and/or an AC box (20) and is independent of triplex formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

PCR reagents, restriction enzymes, bacterial culture and tissue culture media, antibiotics, and reagents were obtained from Life Technologies (Gaithersburg, MD). FBS was purchased from BioWhittaker (Walkersville, MD). The human hepatocellular carcinoma cell line Hep3B2.1–7 as well as the T lymphoblastoid CEM cells were obtained from the American Type Culture Collection (Manassas, VA). Oligonucleotide primers were synthesized at the University of Cincinnati DNA Core Facility (Cincinnati, OH). The pCAT enhancer and other vectors were purchased from Promega (Madison, WI). The ELISA kit for chloramphenicol acetyltransferase (CAT) protein levels was purchased from Boehringer Mannheim (Indianapolis, IN).

Cell culture

Hep3B2.1–7 cells were grown in DMEM supplemented with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 µg/ml gentamicin, and 10% FBS.

Construction of reporter plasmids

A 2.7-kb BamHI fragment of the C1INH gene containing 1182 bp of the 5' flanking region was used as a PCR template to amplify various segments of the upstream region. Amplified fragments were digested with appropriate restriction endonucleases and ligated into a linearized pCAT enhancer vector containing the CAT reporter gene and an SV40 enhancer. Sequences of all constructs were confirmed by dideoxy chain termination sequencing.

Transient transfection of Hep3B cells and CAT assays

Transient transfection of Hep3B cells was performed using the calcium phosphate precipitation method (12). Duplicate plates of Hep3B cells were cotransfected with 3 µg of pSVß-gal and 10 µg of CAT constructs, pCAT enhancer, or pCAT control plasmid (contains the SV40 promoter and enhancer regions upstream of the CAT reporter gene). Cells were harvested at 48 h posttransfection, and extracts were prepared by lysis of the cells using repeated freeze-thawing (three times). Cell extracts were cleared by centrifugation (10,000 x g) and assayed for ß-galactosidase (ß-gal) activity (40). CAT protein levels in 30 µg of cell extract were determined by ELISA. The ratio of pg CAT/µg protein to pg of ß-gal/µg protein was determined for all samples. The final results were normalized against the ratio of pg CAT/µg protein to pg of ß-gal/µg protein for pCAT control samples.

Site-directed mutagenesis

C1INH promoter elements were mutated using PCR-mediated site-directed mutagenesis (41). Primers containing specific base alterations were designed to mutate the Inr, H-DNA, CAAT, Sp-1, and HNF-1 sequences within the C1INH promoter (Fig. 1Go). The PCR-amplified DNA fragments were digested with PstI and XbaI, gel-purified, and cloned into pCAT enhancer. The presence of the mutations was confirmed by DNA sequence analysis.

Generation of topoisomers

Functionally active topoisomerase was isolated from CEM cells as described previously (42, 43). Topoisomers were generated by incubation of 30 µg supercoiled plasmid DNA for 18 h at 37°C with a functionally active topoisomerase preparation in 600 µl of buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 50 mM NaCl, either in the absence or presence of 12.5 µg/ml ethidium bromide. After removal of the ethidium bromide and topoisomerases by phenol-chloroform extraction, the DNA was precipitated with ethanol and dissolved in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA.

Chemical probing

Before chemical modification, 2.5 µg of either relaxed or supercoiled plasmid was incubated at 42°C for 60 min in 40 µl of 13.5 mM Tris acetate (pH 4.0 or 8.0) and 10 mM magnesium acetate. Samples were treated with either 0.1% dimethyl sulfate (DMS), 4% chloroacetaldehyde (CAA), or 0.9 mM KMnO4. All reactions were performed at 25°C. After 10 min, DMS and KMnO4 reactions were halted by the addition of 2.5 M 2-ME. CAA reactions were stopped after 2.5 h by extraction with diethyl ether. After ethanol precipitation, modified DNAs were digested with PvuII and extracted with phenol. Samples were dissolved in 20 µl of 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA; A total of 5 µl was used in a primer extension reaction with a Stoffel fragment of Taq DNA polymerase. Products were resolved on a 7% denaturing polyacrylamide gel in 90 mM Tris borate (pH 8.3) and 2.5 mM EDTA with sequencing markers generated by dideoxy termination sequencing using Taq DNA polymerase. Gels were dried and exposed to a PhosphorImager plate for analysis with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Statistical analysis

Statistical analyses included univariate statistics and tests of hypotheses. Descriptive univariate statistics for each outcome included means, SDs, and normality tests. A statistical analysis was performed using Student’s t test. A one-way ANOVA was performed to analyze differences between the 5'-truncated promoter constructs. In ANOVA instances, Levene’s test was used to compare the equality of the variance. The independent pairwise multiple factor comparisons between means were based on Tukey’s test. Analysis was conducted with a personal computer using SigmaStat statistical software (version 2.03, 1997, SPSS, Chicago, IL). Data are reported as the mean ± SD of the mean (SDM). A p value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of C1INH promoter truncation mutants

Examination of the C1INH 5' flanking region (Fig. 1Go) suggests the presence of a number of elements that can potentially contribute to the activity of the promoter. In addition to the Inr and the potential triplex-forming region, three additional potentially important regulatory elements (HNF-1, Sp-1, and CAAT) were identified. Truncated reporter constructs were developed that progressively deleted these elements at nucleotides -98, -81, and -48. The ability of these constructs to direct the activity of a CAT reporter gene was examined. All three constructs were able to direct the expression of CAT (Fig. 2Go). Comparison of their activities indicated that the -98 -> +9 construct was 1.8-fold more active than the -81 -> +9 construct and 2.3-fold more active than the -48 -> +9 construct. However, the difference in activity between the 81 -> +9 and the -48 -> +9 constructs was not statistically significant, whereas the differences between the -98 -> +9 construct and each of the others were significant. These results suggest that the Sp-1 and CAAT elements do not play a major role in regulation of the C1INH promoter. To determine the role of different regulatory elements identified in the -98 to -48 region, constructs containing mutations that disrupt the potential HNF-1, Sp-1, and CAAT elements were prepared. As expected from the experiments with the truncation constructs, clones with mutations in the Sp-1 or CAAT consensus sequences did not reveal any significant reduction in CAT expression compared with the wild-type (wt) C1INH promoter constructs (data not shown). The construct containing a mutated HNF-1 (HNF-1Mu) sequence, however, was one-third as active as the wt C1INH promoter construct (Fig. 3Go). The data from truncation constructs and site-directed mutagenesis studies indicate that HNF-1 enhances the activity of the C1INH promoter.



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  		FIGURE 2. Analysis of the activity of 5'-truncated C1INH promoter constructs. Hep3B cells were transfected with C1INH reporter constructs, cells were harvested, and extracts were prepared. The level of CAT expression was determined by ELISA and normalized against ß-gal expression for each sample. The CAT levels are presented as a percentage of the CAT expressed by the pCAT control plasmid. Values are the average of four independent experiments (mean ± SDM; *, p <= 0.05 by ANOVA).

 


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  		FIGURE 3. Examination of the effect of HNF-1 disruption on the activity of the promoter. Hep3B cells were transfected with C1INH reporter constructs -98 to +9 and HNF-1Mu that contains a mutated HNF-1 element; next, cells were harvested, and extracts were prepared. The level of CAT expression was determined by ELISA and normalized against ß-gal expression for each sample. The CAT levels are presented as a percentage of the CAT expressed by the pCAT control plasmid. Values are the average of four independent experiments (mean ± SDM; *, p <= 0.05 by Student’s t test; wt vs mutant).

 
Role of the Inr in regulation of the C1INH promoter

The C1INH promoter lacks a TATA box, which in many genes anchors the assembly of the general transcription factors and directs positioning of the polymerase. Previous studies using truncation constructs indicated that an Inr at -3 to +5 is sufficient to maintain basal promoter activity (12). To determine the role of the Inr, reporter clones were constructed that contained the following: 1) a mutated Inr, 2) a TATA box at -36 to -30, or 3) a TATA box at -36 to -30 together with the mutated Inr. These were examined, in comparison with the wt C1INH promoter construct, for their ability to regulate CAT expression (Fig. 4Go). Disruption of the Inr led to a 40% drop in the basal activity of the promoter. Addition of a TATA box led to an ~2-fold enhancement of transcription of the reporter gene in the presence of either the wt or the mutated Inrs. These results indicate that the Inr is important in maintaining the normal transcriptional level of the C1INH gene, and that it may be replaced by an appropriately located TATA element.



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  		FIGURE 4. Examination of the role of the Inr in the regulation of promoter activity. Hep3B cells were transfected with C1INH reporter constructs, cells were harvested, and extracts were prepared. The level of CAT expression was determined by ELISA and normalized against ß-gal expression for each sample. The CAT levels are presented as a percentage of the CAT expressed by the pCAT control plasmid. The activity of the reporter constructs containing a mutated Inr and a TATA box (INITMu + TATA), a mutated Inr (INITMu), a normal Inr, and a TATA box (+TATA) was compared with the wt. Values are the average of at least four independent experiments (mean ± SDM; *, p <= 0.05 by Student’s t test; wt vs mutants).

 
The -48 to -17 region is capable of intramolecular triple helix formation

To determine whether the polypurine·polypyrimidine region is able to form a triplex DNA structure, supercoiled pUC18 constructs containing nucleotides -81 to +9 of the C1INH promoter region with either the wt or mutated -48 to -17 region (H-1Mu, Fig. 1Go) were examined using chemical modification followed by primer extension. The chemical modification patterns of supercoiled plasmid identified a structural transition at a pH of 4.0 that was not found at a pH of 8.0 (Fig. 5Go). Treatment with CAA, which modifies unpaired adenines, cytosines, and, to a lesser extent, guanines (44), identified 11 bases that were modified only at a low pH in the wt construct (Fig. 5GoA). DMS, which recognizes the unprotected N7 of guanine and, to a lesser extent, the N3 of adenine, showed a pattern of protection on the purine-rich complementary strand of the wt plasmid that was pH- and superhelical density-dependent (Fig. 5GoB). KMnO4 modification of supercoiled plasmid at a pH of 4.0 (Fig. 5GoB) also supports the results obtained by chemical modification with CAA and DMS. The results of the chemical modification experiments and the modified bases involved in the potential triplex are shown in Fig. 5GoC. Examination of the chemical modification results of the mutant construct with a disrupted H-DNA sequence did not reveal any alternative secondary structures (data not shown). These data indicate that the region spanning nucleotides -48 to -17 can form a triple helical structure.



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  		FIGURE 5. Examination of the H-DNA region by chemical modification. The sequence ladder was generated by the dideoxy chain termination method. Modified bases on the complementary strand were identified by primer extension. Lanes containing relaxed closed circular plasmid are designated with an (o). Lanes containing supercoiled plasmid are designated with a (s). The numbers indicate the pH. Above the pH is the agent used for chemical modification of the DNA. A, Chemical modification of the pyrimidine-rich strand; • identify CAA modification. B, Chemical modification of the purine-rich strand; {circ} identify bands caused by KMnO4 modification; {blacktriangleup} denote DMS protection by reduced band intensity. C, Schematic representation of chemical modification results. The pyrimidine-rich strand is depicted in black, and the purine rich strand is shown in gray. • denote CAA-modified bases, {circ} represent KMnO4 modification, and the arrowheads indicate bases protected by DMS. The vertical lines attached to the symbols for CAA and KMnO4 modification represent the magnitude of modification.

 
Role of the H-DNA region in regulation of the C1INH promoter

The polypurine·polypyrimidine region spanning nucleotides -48 to -17 is similar to a sequence found at nucleotides -141 to -115 in the c-myc promoter (45). In addition to its ability to form a DNA triple helix, this segment contains three overlapping potential transcription factor binding elements (Fig. 6Go). The first element is a PuF binding site (GGGTGGG) (23) on the noncoding strand. The second element, an AC box, consists of two repeats of ACCCT separated by a run of five bases (CCCTG), which may bind a ribonucleoprotein (20). The third element is a palindromic sequence (GGGAGGG) on the noncoding strand that is identical with the Myc-associated zinc finger protein (MAZ) binding site of the c-myc gene (46). To examine the role of this region in the regulation of the C1INH promoter, the following constructs were prepared (Fig. 1Go): 1) wt; 2) disrupted H-DNA with mutated PuF, AC box, and MAZ elements (H-1Mu); 3) disrupted H-DNA with an intact PuF element and AC box and a mutated MAZ element (H-2Mu); and 4) an enhanced H-DNA (C- and T-rich) sequence with a mutated PuF element and AC box and an intact MAZ binding sequence (H-3Mu). The effects of these mutations were examined in constructs spanning nucleotides -98 to +9 (Fig. 7Go). Disruption of the H-DNA together with mutations of the MAZ binding element had very little, if any, effect on promoter activity. Alternatively, disruption of the PuF element and the overlapping AC box reduced the activity of the promoter by >85% in comparison with the wt construct. This was the case in constructs containing either a disrupted or an enhanced H-DNA sequence. These results indicate that the PuF element and/or the AC box are important for the optimal activity of the promoter.



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  		FIGURE 6. Schematic diagram of the polypurine·polypyrimidine segment within the C1INH promoter (enclosed within the rectangle). The putative PuF binding sequence is indicated by boldface underlining, the AC box is indicated by the broken overline, and the potential MAZ binding sequence is indicated by the dotted underline.

 


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  		FIGURE 7. Analysis of the role of potential structural and transcription factor binding elements in the H-DNA region on the activity of the C1INH promoter. Hep3B cells were transfected with C1INH reporter constructs, cells were harvested, and extracts were prepared. The level of CAT expression was determined by ELISA and normalized against ß-gal expression for each sample. The CAT levels are presented as a percentage of the CAT expressed by the pCAT control plasmid. The activity of the wt construct was compared with constructs containing: 1) disrupted H-DNA with mutated PuF, AC box, and MAZ elements (H-1Mu); 2) disrupted H-DNA with an intact PuF element and AC box and a mutated MAZ element (H-2Mu); and 3) an enhanced H-DNA (C- and T-rich) sequence with mutated PuF element and AC box and an intact MAZ binding sequence (H-3Mu). The effects of these mutations were examined in constructs spanning nucleotides -98 to +9. Values are the average of four independent experiments (mean ± SDM; *, p <= 0.05 by Student’s t test; wt vs mutants).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies presented here have characterized the roles of several components that are important in regulation of the activity of the C1INH promoter: the Inr, the HNF-1 element, and sequences within the polypurine·polypyrimidine segment. The Inr is highly homologous to the TdT Inr (consensus sequence, CTCANTCT) (47). Inrs function in the absence of a TATA sequence and do not require specific upstream sequences to direct basal levels of transcription (14). Mutations in the C1INH Inr led to a 40% decrease in CAT expression by the reporter construct. Although most Inr mutations drastically reduce transcription, a substantial number only partially reduce promoter activity (48), as observed with the C1INH Inr mutants. In addition to an absolute decrease in promoter activity, in other model systems, mutations similar to the ones introduced into the C1INH Inr decrease the use of the normal transcription start site by 38–100% (48). Therefore, it is likely that in these experiments, some of the transcripts that initiate from other start sites are normally translated into CAT and contribute to the measured enzyme levels. Our results also support previous observations that insertion of a TATA sequence at the appropriate position upstream from the transcription start site can enhance Inr activity or can substitute for a mutated Inr (47).

Data from deletion constructs indicate that the region from nucleotide -98 to -81 is important with regard to the activity of the basal promoter. Examination of the 5' flanking region of the C1INH gene indicates the presence of a potential HNF-1 element between -98 and -81 and the presence of potential Sp-1 and CAAT sites at -81 to -72 and -62 to -58, respectively. Deletion or disruption of the potential HNF-1 binding site reduced promoter activity by 50–70%, whereas mutation or deletion of the Sp-1 or CAAT sites had no effect. HNF-1 is a POU (Pit/Oct/Unc) and homeodomain protein that is highly expressed in the liver and kidney, whereas low levels of its transcript are found in the intestine, spleen, and thymus (49, 50). HNF-1 is important in the regulation of a variety of liver-specific genes (e.g. fibrinogen, {alpha}1-antitrypsin, and albumin) (51, 52). Its level of expression correlates with the differentiation state of the hepatocyte (53, 54). HNF-1 is also involved in gene regulation in other organs (e.g., the guanylin gene in the intestine and phosphoenolpyruvate carboxykinase in the kidney) (49, 55). These data indicate that HNF-1 plays an important role in the regulation of C1INH transcription in hepatocytes; however, its role in extrahepatic expression of C1INH requires further investigation. The results rule out the Sp-1 and CAAT sites as potential regulatory elements in the C1INH promoter.

In their description of the structure of the C1INH gene, Carter et al. identified a polypurine·polypyrimidine region that may assume an H-DNA structure (-48 to -17), and postulated that it might be important in the regulation of C1INH transcription (45). Polypurine·polypyrimidine sequences make up ~1% of the human genome (21, 22). The function of these triplex DNA-forming regions in replication and transcription is not yet clear, and the mechanism of their action is not known. Studies that examine the role of triplexes in gene regulation indicate that they may be important regulatory elements that enhance the transcription of some genes (c-myc and human decorin) and act as repressors of transcription of others (neural cell adhesion molecule, human {gamma}-globin, and the mouse androgen receptor) (20, 25, 34, 35, 36, 37, 38, 39). The differences in the effect of the H-DNA region on promoter activity may be due to differences in the cell lineages in which these elements were examined (i.e., a hepatoma cell line vs lymphoblastoid cells) or due to the position of the element in relation to other regulatory factors such as the TATA box and other transcription factor binding sites. The -48 to -17 region of the C1INH gene is highly homologous to the H-DNA region of the c-myc gene (45). It is capable of forming a DNA triple helix only at a low pH in a highly supercoiled plasmid (Fig. 5Go). A model of the triplex, based on the chemical modification data, is presented in Fig. 8Go. The extremely high degree of supercoiling required for extrusion of the triplex does not negate the potential for the formation of such a structure in normal physiological conditions, because significant local stress may be exerted on DNA by the binding of transcription factors (56).



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  		FIGURE 8. Model of the potential DNA triplex formed by the -48 to -17 region based on chemical modification data.

 
Like c-myc, the C1INH H-DNA region also contains potential protein binding elements that include an AC box (20) and binding sites for the transcription factors PuF (GGGTGGG) (23) and MAZ (GGGAGGG) (Fig. 6Go) (46). AC boxes are composed of two ACCCT sequences separated by a spacer region (20). The AC box in the C1INH promoter overlaps the PuF binding site. The c-myc H-DNA region is important in the mediation of the activity of its promoter (20, 57). This region may form a triplex structure, and it binds a number of proteins, including PuF and MAZ, in addition to a ribonucleoprotein that binds to the AC box (20, 23, 46). The role of triplex formation in the regulation of c-myc promoter activity and in the binding of the above factors is not clear. However, increased transcriptional activity of c-myc promoter mutants correlated with an enhanced ability to form triple-stranded DNA, which suggested that H-DNA formation might play a role in the regulation of c-myc (57). In these studies, we have demonstrated that C1INH promoter activity appears to be dependent upon the presence of the potential PuF element and/or the AC box, and does not appear to be affected by the potential MAZ binding element or by triplex formation (Fig. 7Go).

In previous studies, we observed a significant inhibitory effect on the activity of the C1INH promoter when the nucleotides immediately upstream of the H-DNA region (-81 to -48) were removed (12). This inhibitory activity was overcome after removal of the -47 to -3 region. Based on those observations, we proposed that the triplex-forming region might negatively affect the activity of the promoter (12). However, the current results using mutations within the -98 -> +9 construct indicate that the triplex-forming region contains positive regulatory elements and that its activity is independent of its ability to form H-DNA. This difference between the two studies may be due to a requirement for elements upstream of nucleotide -81 by the factors that bind to the H-DNA region to enhance the activity of the basal promoter. The transcription factor PuF may bind to this region of the C1INH gene. Recombinant PuF, although capable of binding to the c-myc promoter, was unable to enhance its transcription in the absence of other cofactors (23). Therefore, regulation of the C1INH promoter via a similar mechanism may not be surprising. In the c-myc gene, progressive deletion starting at the 3' end of the triplex-forming region indicates that removal of the first 11 bp, which leaves the triplex-forming region intact, reduces the activity of the construct to 26% of the wt. Further deletions, which remove the triplex region completely, slightly enhance the activity of the promoter to 35% of the wt (20). These observations indicate that this region may act as a weak negative regulatory element (20). This finding is similar to the response observed in our previous studies and suggests the possibility that this region functions to fine-tune the activity of the promoter. Deletion of the triplex-forming region may therefore allow the Inr to act as an independent promoter element without relying on its interaction with other upstream regulatory elements. A role for triplex formation as a dynamic structural element regulating the binding of specific transcription factors still cannot be ruled out and requires further examination. Identification of the factors and characterization of their potential interactions is important if the mechanisms by which polypurine·polypyrimidine sequences influence gene expression are to be defined. In addition, such knowledge may lead to techniques to enhance the expression of C1INH in vivo.


   Footnotes
 
1 This study was supported by U.S. Public Health Service Grants HD33727 and HD22082. Back

2 Address correspondence and reprint requests to Dr. Kamyar Zahedi, Division of Nephrology, Children’s Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: Back

3 Abbreviations used in this paper: C1INH, C1 inhibitor; Inr, initiator element; wt, wild type; SDM, SD of the mean; CAT, chloramphenicol acetyltransferase; DMS, dimethyl sulfate; CAA, chloroacetaldehyde; ß-gal, ß-galactosidase; HNF, hepatocyte nuclear factor; MAZ, Myc-associated zinc finger protein; H-DNA, hinged DNA. Back

Received for publication January 11, 1999. Accepted for publication March 24, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sim, R. B., A. Reboul, G. J. Arlaud, C. L. Villiers, M. G. Colomb. 1979. Interaction of 125I-labeled complement components C1r and C1s with protease inhibitors in plasma. FEBS Lett. 97:111.[Medline]
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