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The Journal of Immunology, 2000, 164: 300-307.
Copyright © 2000 by The American Association of Immunologists

The Role of Sp Family Members, Basic Krupple-Like Factor, and E Box Factors in the Basal and IFN-{gamma} Regulated Expression of the Human Complement C4 Promoter1

Daniela Ulgiati2, Lily S. Subrata and Lawrence J. Abraham3

Department of Biochemistry, University of Western Australia, Nedlands, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The fourth component of human complement (C4) is a serum protein that is expressed in the liver and other organs. The promoter region of the C4 gene has been analyzed in reporter gene assays in two cell lines that represent hepatic (HepG2) and monocytic (U937) lineages. Analysis indicated that regions important for basal transcription in HepG2 cells included Sp1 and E box sites within the first 100 bp upstream of the transcription initiation site but not the nuclear factor-1 site important in the control of the mouse C4 gene. Also, a region encompassing -468 to -310 was able to repress activity 2-fold. However, when a CACCC or GT box sequence at -140 was mutated the repressive activity of the upstream region resulted in almost no activity. The -140 region consists of a series of four closely positioned GT boxes that were shown to bind Sp1, Sp3, and basic Krupple-like factor in EMSA. This novel two-part regulatory element may be involved in the regulated expression of C4. However, IFN-{gamma} a major activator of C4 expression did not signal through this two-part regulatory element. We were able to map the position of an IFN-{gamma} responsive element in U937. IFN-{gamma} was able to increase transcription by up to 20-fold with mutations in the E box sequence at -78 to -73, thus completely abolishing induction. We conclude that the E box binding factors, which appear to be distinct from upstream stimulatory factors 1 and 2, are totally responsible for IFN-{gamma} induction of C4.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The fourth component of human complement, C4, is a highly polymorphic serum protein consisting of 2 isoforms, C4A and C4B. These isotypes are encoded by two separate genes that are located 10 kb apart within the central portion of the MHC on chromosome 6p (1). The central MHC region also encodes genes for other complement components, C2 and factor B. Although two C4 genes are usually present, at least three genes have been found (2, 3, 4). The C4A and C4B genes are highly conserved both in coding and noncoding regions (5) but can differ in size depending on the presence or absence of a 6.5-kb retroviral insertion in intron 9 (6). Interest in the genomic arrangement of the C4 genes has arisen partly as a result of the association of the MHC with various autoimmune diseases, particularly systemic lupus erythematosus (SLE)4. An association with the MHC has been found in many studies. In particular, heterozygous and homozygous deficiencies of C4A have been strongly associated with SLE in many racial groups (7, 8, 9, 10, 11). As C4A and C4B differ in their ability to bind Ags (C4B has a greater haemolytic activity and is more reactive to hydroxyl groups than C4A), it would be expected that the number and type of functional C4 genes present may have consequences with respect to susceptibility to SLE. However, Alper’s group (11) has established that there were quantitative differences in C4 isotype present in serum depending on the MHC "extended" haplotypes present and not gene copy number alone. This implies that differences in expression are due to sequence polymorphism, and so it is possible that differences in the regulation of C4 expression may be relevant to SLE pathogenesis.

The promoters of both the human and murine C4 genes have been analyzed. In the mouse, three sequence motifs, an initiator element, an E box, and a nuclear factor-1 (NF-1) binding site, have been shown to be functionally relevant in directing constitutive C4 expression in cells of hepatic origin (12). In human, similar motifs are present, and it has been shown that the immediate upstream region from -178 to -39 is associated with maximal C4 expression in the hepatoma cell line HepG2 (13). This region contains an Sp1 site at -57 to -49 that is essential for activity and probably takes the place of the TATA motif in accurately initiating basal transcription. In addition, an E box motif at -78 to -73 is important, as mutation of this region results in an 8-fold reduction of activity in reporter gene assays.

The liver is the main source of C4 in the blood and constitutive expression is enhanced during the acute phase response. Unlike most other acute phase reactants, IFN-{gamma} is the only known inducer of C4 expression in liver and serves to increase mRNA and protein expression by ~50%. In HepG2 cells, the induction by IFN-{gamma} is predominantly through stabilization of C4 mRNA (14). Many other cell types express C4, including glomerular and proximal tubule cells of the kidney (15, 16), synoviocytes, fibroblasts (17), and monocytes, including the myelo-monocytic U937 cell line (18).

To determine the requirements for basal and regulated expression of C4, we have analyzed 1137 bp of C4 promoter sequence by testing the effects of introduced deletions and mutations on basal and IFN-{gamma}-induced expression of a reporter gene. EMSA were also conducted to identify factors that interact with transcriptionally relevant regions of the promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions

Human hepatoma derived HepG2 (HB-8065) and myelo-monocytic U937 (CRL-1593) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture medium at 37°C with 5% CO2 consisting of either Eagles MEM with Earle’s salts and L-glutamine supplemented with 10% FBS, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (HepG2) or RPMI 1640 with L-glutamine supplemented with 10% FBS (U937). Both media contained 100 µg/ml streptomycin and 100 IU/ml penicillin.

Construction of C4 promoter deletion and mutant luciferase fusion constructs

A SmaI/BamHI fragment of the C4 promoter containing -1137 to +44 was cloned into the luciferase reporter pGL3-basic vector (Clontech, Palo Alto, CA). Site-directed mutagenesis was performed using the Quickchange Mutagenesis kit (Stratagene, La Jolla, CA), which enabled the incorporation of NheI restriction sites extending 3' from the positions -316, -141 (CACCC), -101 (NF-1), -78 (E box), and -36. Deletion constructs were then prepared utilizing the newly incorporated NheI site together with the NheI site situated in the pGL3-basic vector. Restriction enzyme digestion of the mutant plasmids with NheI resulted in varying lengths of upstream C4 promoter sequence being deleted from the full-length construct.

Transfections and detection of promoter activity

Before each transfection, HepG2 cells were grown to ~70–85% confluency and U937 were grown to 7 x 105 cells/ml and then electroporated with plasmid DNA prepared using Qiagen Maxiprep-500 columns (Qiagen, Clifton Hill, Australia). To each cuvette (Bio-Rad, Hercules, CA) was added 15 µg full-length promoter construct or equimolar amounts of deletion constructs, 300 ng pRL-TK (Promega, Madison, WI) transfection control vector and 400 µl of cells (2.5 x 106–2.5 x 107 cells/ml). The cells were electroporated using a Bio-Rad gene pulser (240 V and 960 µF) and distributed into a 6-well culture tray containing 5 ml of medium and allowed to recover for 18 h. Cell lysates were then prepared following a further 24 h. Cell lysates from the transfected cells were prepared and assayed for both firefly and Renilla luciferase according to the manufacturer’s instructions (Promega) All transfection data are representative of three independent transfections using at least two independent preparations of both DNA and plasmid clones. Promoter activity is expressed as relative firefly luciferase activity normalized against Renilla luciferase activity.

EMSA

Approximately 8 x 107 cells were used to make nuclear extracts according to a standard method (19). Extracts were frozen in liquid N2 and stored at -80°C. Determination of protein concentration was performed using the Bio-Rad protein assay kit. For EMSA, nuclear extracts (10–20 µg protein) were preincubated on ice for 10 min together with 1 µg poly(dI-dC) in a binding buffer consisting of 4% Ficoll, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, and 50 mM KCl. When required, competitor oligonucleotides or supershift basic Kruppel-like factor (BKLF) (20), or commercial Sp1, Sp3 or upstream stimulatory factor-1/2 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were then incubated with the nuclear extract for 30 min on ice. The nuclear extract was then incubated with 80 fmol of 32P-labeled oligonucleotide for 30 min on ice before loading onto a 6% polyacrylamide gel. The gel was then electrophoresed at 150 V using 0.25x Tris-taurine-EDTA as the running buffer. EMSA gels were then dried under vacuum and exposed to x-ray film. Coordinates of the sequences of the double-stranded wild-type CAC/GT box and E box oligonucleotides utilized in these experiments are given in Results and correspond to the sequences shown in Fig. 1Go. USF supershift control experiments were done using a consensus USF binding site from Santa Cruz Biotechnology and was of the sequence CACCCGGTCACGTGGCCTACACC. All double-stranded oligonucleotides had an additional single 5' G overhang on the lower strand to allow end labeling with [32P]dCTP and Klenow DNA polymerase.



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  		FIGURE 1. Sequence of the -1137 to +41 region of the C4 promoter. The region was cloned into the pGL3-basic reporter vector via the terminal CCCGGG SmaI and GGA (TCC not shown) BamHI restriction enzyme sites. Boxed sequences represent cis-acting promoter elements as indicated. Overlined sequences denote putative upstream binding sites for GATA and Nkx transcription factors. The double-underlined 6-bp sequences indicate regions that were mutated by replacement with a NheI restriction site, GCTAGC. The shaded regions represent sequences reminiscent of IFN-{gamma} response elements.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Requirements for high level basal activity lie within the first 138 bp of the human C4 promoter

To determine promoter regions that contribute to expression of C4, a series of 5' deletion constructs were generated by introducing mutations into a reporter construct that consisted of the -1137 to +44 region of the C4 gene (Fig. 1Go) linked to the luciferase coding region of pGL3-basic. Deletion of the regions between the mutations and the NheI restriction site in the pGL3 vector created the 5' deletion series. These constructs were used in transient transfection assays to determine the minimum promoter region required for basal expression of human C4 in the hepatic (HepG2) environment (Fig. 2Go). The full-length -1137 construct was able to mediate high level expression in HepG2. Truncation of the C4 promoter from -1137 to -310 (Fig. 2GoA) caused a 1.6-fold increase in promoter activity over the full-length construct. Further truncation to position -136 resulted in the maintenance of elevated basal transcription with a 2-fold increase observed when compared with the full-length -1137 construct. Recent studies by Vaishnaw et al. (13) have established the presence of negative regulatory elements in the -1043 to -178 region in HepG2 cells. We have now defined the region to between -1043 and --310. Further truncation to -95 had a minor effect. However, truncations to position -72 resulted in a marked (2-fold) decrease in promoter activity (Fig. 2GoA) compared with the full-length construct. Further truncation of the full-length construct to position -30, leaving only the Inr and downstream elements, resulted in a 40-fold decrease in basal transcriptional activity. These later results are consistent with the requirements seen in human (13) and mouse hepatic cells (12), as the Inr and downstream elements are sufficient for start site selection and low level activity, with the immediate upstream region containing the Sp1 site at -57 to -49, conferring elevated basal transcription.



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  		FIGURE 2. Transcriptional activity of the full-length (-1137 WT) C4 promoter/luciferase reporter compared with deletion and mutation derivatives in transiently transfected HepG2 cells. Indicated on the constructs are the locations of the polyadenylation site of the upstream RP1 gene, the CACCC box, NF-1, E box, and downstream Sp1 sites relative to the INR at the transcriptional initiation site (bent arrow). A, Normalized transcriptional activity of the C4 promoter deletion series is shown. The results represent mean luciferase activity data ± SE. B, Transcriptional activity of linker-scan mutants compared with the wild-type full-length construct -1137 WT. The crosses and associated coordinates indicate the positions at which the NheI mutations were introduced. The results represent mean data ± SE.

 
Mutation analysis demonstrates important roles for the -141 CACCC box and -78 E box motifs in maintaining basal expression of C4

The 6-bp NheI mutations used to create the 5' deletion series were designed to introduce changes into putative transcription factor binding sites in the C4 promoter. These mutant constructs were used to identify critical elements involved in basal C4 transcription. Both the full-length promoter construct and the site-directed mutants were transiently transfected into HepG2 cells and assayed for transcriptional activity. The full-length C4 promoter and the construct containing a 6-bp linker mutation at location -316 (Fig. 2GoB) produced high levels of basal transcriptional activity. In contrast, a promoter construct carrying a 6-bp mutation at position -141 in a CACCC box (or GT box) regulatory element produced a 94% decrease in transcriptional activity compared with the full-length construct in HepG2 (Fig. 2GoB), suggesting an important role for this site in maintaining efficient levels of basal activity although the deletion results gave no indication of this. Mutation of the putative NF-1 site at position -101 did not significantly effect transcription, although this site has been shown to be protected by HepG2 nuclear extracts in DNase I footprinting (13). In contrast to our results, the equivalent NF-1 site in the mouse C4 promoter is required for full activity C4 promoter activity. Mutation of the E box motif (position -78) resulted in a 86% decrease in transcriptional activity when compared with the full-length construct. These results indicate a major role also for the E box motif in basal C4 activity. Mutation in a region starting at position -36 had the effect of decreasing transcriptional activity by 64%. This region may be protected by HepG2 extracts in DNase I footprinting (13) and serves a modulatory role in C4 promoter activity in both cell types.

Characterization of factors binding to the E box motif

The transfection results from both the deletion and mutation constructs identified the E box as a major transcriptional element. The E box sequences in human and mouse are identical and in mouse it has been shown that this site is a major element driving basal expression of the C4 gene (12, 13). The murine results also suggest that members of the USF family distinct from USF-1 or USF-2 bind to the rodent sequence (21). To determine the nature of the factors interacting with the human E box sequence, EMSA were conducted using extracts derived from HepG2 cells and the -89 to -64 sequence containing the E box motif as a probe (Fig. 3Go). Two major complexes (A and B) were seen (Fig. 3GoA), both of which could be substantially competed away using 10-fold molar excess of unlabeled E box probe. When a consensus USF binding site was used as a competitor, the lower complex B could be competed away using 10-fold excess, indicating that it contained factors that recognized the USF binding site. However the major complex, A, was not substantially removed by USF site competition at 250-fold molar excess, indicating that this complex was not recognizing the USF binding site but may recognize a site that overlaps with the E box site in the C4 promoter. To determine whether complex B was USF-1 or USF-2, supershift assays were done with a USF-1/2 Ab. Neither pre- or postincubation of the extract with Ab caused any discernible change in the gel retardation profile seen in the absence of Ab (Fig. 3GoB), whereas control reactions using a consensus USF binding site showed substantial reactivity with bound complexes (data not shown), suggesting that complex B did not contain proteins antigenically related to USF-1 or USF-2.



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  		FIGURE 3. EMSA analysis of the -89 to -64 E box region with the 26-bp probe, Ebox, using HepG2 nuclear extracts. The two major complexes interacting with the E box recognition site, A and B are indicated by the arrows. A, Competition EMSA using either the Ebox probe or a consensus USF binding site. Fold molar excess (x) of unlabeled competitor is as indicated. B, Supershift EMSA using USF-specific Ab. Ab was added either before or after incubation of the probe with the nuclear extract as indicated.

 
Factors binding to the -141 CACCC box region include Sp1, Sp3, and BKLF

The reporter gene analysis of mutation within the -141 CACCC box indicated that it was very important in regulating expression from the C4 promoter. To determine the nature of the transcription factors that interacted with this region, EMSA were conducted using double-stranded oligonucleotides that corresponded to the -152 to -113 region of the promoter. Inspection of this 40-bp region indicated that a total of four GT boxes were present, two in the reverse (CACCC box) orientation (Fig. 4GoA). The region has the potential to form a variety of palindromic structures involving adjacent GT box and CACCC box sequences. Complexes binding to the region were identified in EMSA using oligonucleotides representing either the entire 40-bp region (4XGT), a truncated region including the upstream 33 bp but missing the proximal 3' GT box (3XGT), and two mutant sequences that had either -141 to -137 (Umut) or -128 to -123 (Dmut) changed to a NheI restriction site GCTAGC (Fig. 4GoA). Using HepG2 nuclear extracts, 4XGT bound four major complexes A, B, D, and E (Fig. 4GoB). Self competition indicated that complex B could be removed with a 10-fold excess and complex A with a 50-fold excess of unlabeled 4XGT. However removal of complexes D and E required a 250-fold excess and may indicate that very high concentrations of these factors are present in the extract and/or represents nonspecific interactions. Use of the shortened 3XGT probe in EMSA (Fig. 4GoC), indicated that only complex B was able to bind to the upstream region. Introduction of the mutation at -128 to -123 (probe Dmut) to the 4XGT sequence had the effect of abolishing binding of complex E and substantially reducing formation of complex B (Fig. 4GoC). The upstream GT box mutation (Umut) abolished formation of complexes B, D and E. Taken together, the results indicated that complex A was interacting with the -118 to -112 region and complex B was interacting with the -141 to -127 region but was influenced by downstream sequences. Surprisingly, complex D was interacting with the -141 to -127 region but also required the downstream region -118 to -112 to bind. Complex E appeared to require the entire 40-bp region for binding, interacting with the -141 to -127 region, the -127 to -122 region and the -118 to -112 region deleted in the 3XGT oligonucleotide.



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  		FIGURE 4. EMSA analysis of the -141 CACCC box/GT box region using HepG2 nuclear extracts. A, Probes used in EMSA showing the positions of the GT and GT/CACCC boxes in the 4XGT sequence. Also, the positions of introduced mutations Umut and Dmut are underbracketed. B, Self-competition EMSA using the 4XGT probe as competitor at the indicated fold excess (x). The positions of the major complexes A-E are indicated by the arrowheads. C, EMSA using the wild-type probes 3XGT and 4XGT and the mutant Dmut and Umut probes. D, Cross-competition EMSA. The probes are as indicated above the bars and the unlabeled competitors are shown below the bars. The unlabeled competitors were used at 250-fold molar excess over probe. E, Supershift analysis using the 4XGT or 3XGT probe as indicated above the bars, and either an Sp1, Sp3, or BFLF Ab as indicated below the bars. The position of an Sp1 supershifted band is indicated by the small arrowhead.

 
To establish whether Kruppel-like Sp (22) or EKLF (20) family members were involved in complex formation within the -140 region, competition EMSA were conducted using cognate GC and GT box sequences as well as the GC-rich AP2 consensus sequence (Fig. 4GoD). Both the GC and GT box Sp1 consensus competitors were able to specifically remove complex A only; complex B, D and E were unaffected. The consensus AP2 competitor had the effect of removing complex A but also reduced the binding of complexes B and E. Considering the fact that 250-fold molar excess of the AP2 consensus could not completely prevent formation of these complexes indicated that complexes B and E may be interacting nonspecifically with the AP2 oligonucleotide (Fig. 4GoD). The identity of complex A as containing Sp family members was confirmed using Abs specific for either Sp1 or Sp3 (Fig. 4GoE). Both Abs were able to interfere specifically with complex A formation indicating that complex A contained both Sp1 and Sp3; furthermore, both proteins were required for complex formation as either Ab almost completely abolished complex A binding. As complex B was interacting specifically with the CACCC box sequence at -141 to -127, supershift analyses were conducted using an anti-BKLF Ab. BKLF has been shown to be a major CACCC box binding activity in liver (20). The BKLF-specific Ab was able to interfere with the formation of complex B (Fig. 4GoE), suggesting that complex B was BKLF.

Functional interaction between the -141 CACCC box region and upstream sequences

Results from the 5' deletion experiments indicated that the GT box sequences at -141 were not required for basal expression of C4, because deletion of the region from -1137 to -141 including the reverse strand GT box sequence CACCC, had no significant effect on promoter activity. However, mutation of the CACCC site to a NheI site in construct mut-141 almost completely abolished activity. These results taken together indicated that sequences upstream of the GT box were important and had a strongly negative effect on promoter activity but only in the absence of the GT element. A possible explanation is that deletion of both these elements nullifies the balancing effect of the factors binding at the two sites and results in activated transcription generated by sequences downstream including the Sp1 and E box motifs. To further delineate the upstream region responsible for the negative effect, a series of deletions were introduced into the full-length construct containing the mutation of the GT box CACCC site at -141 (Fig. 2Go, construct mut-141). When the constructs were tested by transfecting HepG2 cells, it was determined that the region responsible for the repressive effect was located between -468 and -310, as deletion to -468 had no effect on the very low activity of the parent construct, mut-141, but deletion to -310 restored high level expression to above wild-type levels (Fig. 5Go). Analysis of the region between -472 and -310 using TFSEARCH version 1.3 (40) revealed the presence of potential transcription factor binding sites for GATA and Nkx2 (see Fig. 1Go); cognate binding activities for both GATA-6 (23) and Nkx2.8 (24) are expressed in liver. Members of the Nkx and GATA families of transcription factors have been shown to interact in other tissue types (25) and may potentially interact in hepatocytes.



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  		FIGURE 5. Localization of the upstream repressor region that interacts with the -140 CACCC box region. The activity of the full-length mut-140 parent construct was compared with the derivative deletion series which carried the -140 CACCC mutation and 5' truncations to the nucleotide shown. Also shown is the activity of the wild-type -1137 WT construct which was the parent of mut-140. All constructs were used to transfect HepG2 cells and subsequently assayed for luciferase activity. Normalized transcriptional activity of the C4 promoter deletion series is shown. The results represent mean luciferase activity data ± SE.

 
IFN-{gamma} responsiveness of the C4 promoter

Previous data have indicated that expression of C4 is increased in response to IFN-{gamma} in cells of both hepatic and myeloid origin. In the hepatic cell line HepG2, C4 expression is expressed constitutively at high levels. Mitchell et al. (14) found that C4 expression was increased in response to IFN-{gamma} primarily via posttranscriptional events. In myeloid cells such as peripheral blood monocytes and the U937 cell line, however, expression is undetectable but is strongly increased in response to IFN-{gamma}. Part of this increase presumably involves transcriptional activation of the C4 promoter (18). To determine whether the C4 promoter played a role in the increased C4 synthesis seen upon induction with IFN-{gamma}, the responsiveness of the various C4 promoter constructs were tested in U937 cells.

Treatment of cells transfected with the full-length, -1137 WT construct with varying concentrations of IFN-{gamma} produced a dose- and time-dependent increase in luciferase production when compared with the transcriptional activity obtained for uninduced samples with maximal induction (20- to 25-fold) occurring at a concentration of 100 U/ml of IFN-{gamma} (data not shown). Induction using 100 U/ml IFN-{gamma} was half-maximal at 5 h (Fig. 6GoA).



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  		FIGURE 6. Effect of IFN-{gamma} on the expression of C4 in U937 cells. A, U937 cells were transfected with construct -1137 WT and then treated with 100 U/ml of recombinant human IFN-{gamma} for 3, 6, 12, or 24 h before assaying for luciferase activity. Shown are mean induction ± SE from three experiments. B and C, Activities of deletion and mutant constructs were compared with the parent -1137 WT construct with or without IFN-{gamma} treatment for 24 h. The results represent the mean luciferase activity data ± SE for three experiments.

 
To localize the region responsible for the IFN-{gamma} up-regulation observed, both the deletion and mutant constructs (see Fig. 2Go) were tested in U937 cells. Of significant interest were deletion constructs and site-directed mutants that eliminated putative IFN-{gamma} responsive elements of the type identified in the TRANSFAC search. These elements were located at -327 to -320, -316 to -309, and -39 to -32 (see Fig. 1Go) and were of the type CTG/TG/TANNY found in HLA class II (26, 27, 28) and FcR (29) genes to mediate IFN-{gamma} responsiveness. When the deletion constructs were transiently transfected into U937 cells, the data revealed that only 95 bp of upstream promoter sequence was required for full inducibility by IFN-{gamma} (Fig. 6GoB). Deletion of two of the putative {gamma} response elements at positions -327 to -320 and -316 to -309 (Fig. 6GoB, construct -136) did not affect induction significantly. Truncation of the promoter to position -72 (Fig. 6GoB) resulted in the elimination of IFN-{gamma} induction, indicating that an element required for IFN-{gamma} inducibility was positioned between -95 and -72.

Results obtained from the mutant constructs confirmed that the -95 to -72 region and specifically the E box was the major element controlling IFN-{gamma} responsiveness (Fig. 6GoC). All of the other mutations retained significant responsiveness to IFN-{gamma}, although expression levels differed. Interestingly, the promoter construct containing a mutation in the CACCC box at position -141, which was shown to cause a marked decrease in basal transcriptional activity, was still inducible by IFN-{gamma} (Fig. 6GoC, construct mut-141). These results indicate that, as well as being an important element contributing to basal C4 transcription, the E box was the major element mediating IFN-{gamma} responsiveness.

To determine whether IFN-{gamma} induction was correlated with changes in the nature of the nuclear proteins interacting with the E box region, EMSA analysis was undertaken. Using the E box probe and nuclear extracts derived from U937 cells, EMSA results indicated that similar complexes to those seen in HepG2 extracts were formed but complex A was less abundant than that seen in the HepG2 extract (Fig. 7Go). U937 cells also were incubated with 100 units IFN-{gamma} for 0, 0.5, 3, or 6 h before preparation of extracts. The EMSA results showed that both complexes A and B increased following induction with IFN-{gamma} (Fig. 7Go), and an additional complex B' appeared at 3 h. Following 6 h of IFN-{gamma} treatment, all three complexes decreased when compared with the 3 h treatment.



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  		FIGURE 7. EMSA analysis of the E box region following IFN-{gamma} induction. E box probe binding activity from nuclear extracts of untreated or 0.5, 3, or 6 h IFN-{gamma} treated U937 cells were analyzed as indicated. The major complexes A and B (indicated by arrowheads) that were seen in HepG2 extracts (Hep) were also present in the U937 extracts. An additional IFN-{gamma} inducible complex B' is indicated by the arrowhead.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Investigation of the requirements of the human C4 promoter for expression revealed that high levels of basal transcription were attained with a region comprising 141 bp of upstream promoter sequence. Promoter constructs containing only the initiator and downstream elements resulted in a decrease in activity to a very low but measurable level. These results are comparable to data from mouse studies which found that initiator and downstream elements were sufficient for minimal transcription and start site selection; however, promoter activity was enhanced with the presence of both the NF1 and E box motifs (12). Within the immediate upstream promoter region of the human gene, two elements have been identified recently as being important in driving expression, an Sp1 site (-57 to -49) and an E box motif (-78 to -73) (13). Our data confirm these results and, in addition, identify an additional regulatory site downstream of the Sp1 site in the -36 to -31 region. In contrast to the mouse studies, we have found evidence for only a minor involvement of the NF-1 site in driving transcription of the human gene. This is despite the fact that DNase I footprint analysis indicates that proteins are interacting with the putative NF-1 site of the human C4 gene (13). It is possible that, if NF-1 is involved in transcriptional control, then it may have a role other than in basal transcription in hepatic cells and may be involved in cell-type or activation-specific activities.

Our results indicate a very important function for the E box element located at -78 to -72. The E box motif forms part of a growing number of proteins, termed basic helix-loop-helix (bHLH) transcription factors which bind the consensus CANNTG. The E box element located within the C4 promoter is characterized as class B due to the cognate binding sequence, CACGTG. Included within this family are c-MYC (30) and USF (31). Galibert et al. (21) have characterized the binding of a transcription factor as similar, albeit distinct, from USF interacting with the mouse C4 E box motif. Our EMSA data show that two nuclear protein complexes interact with the region; however, only one of these (complex B, Fig. 3Go) recognizes the consensus USF binding site, suggesting that it is a member of the USF family. However, supershift analysis indicated that it was not antigenically related to USF-1 or USF-2 and may be another member of the class B bHLH family.

The experiments performed to investigate IFN-{gamma} inducibility of the human C4 promoter indicated a dose-dependent increase in transcriptional activity of the full-length construct when treated with IFN-{gamma}. The kinetics of induction showed enhanced transcriptional activity reminiscent of that previously seen in MHC class II genes (32). An increased response was observed at 3 h, with maximal activity shown between 6 and 12 h. After an induction time of 24 h, the transcriptional activity was decreasing but still elevated over basal levels. Even taking into account the 1-h lag time in the synthesis of luciferase protein (33, 34), these data suggest that the IFN-{gamma} response element functional in the C4 promoter would be similar to a IFN-{gamma} regulatory element ({gamma}-IRE) (32) rather than an immediate response IFN-{gamma} activation site (GAS) element, where induction with IFN-{gamma} is abolished after 3 h (35, 36). However, investigation of the putative {gamma}-IREs within the C4 promoter demonstrated that none of the classical IFN-{gamma} elements were functional. Instead the E box at -78 was totally responsible for induction.

The identity of the transcription factors binding to the E box remain unknown but, as outlined above, EMSA indicates that one of them (complex B) recognizes consensus USF binding sites and the other (complex A) does not. However complex A does recognize at least part of the E box sequence as mutations at this site abolish binding. Both of these complexes increase dramatically upon IFN-{gamma} stimulation and may compete for binding at overlapping cognate sites. In addition, a new complex (B') appears following IFN-{gamma} treatment. Further work is required to determine whether increased transcriptional activity is due to an increase in the concentration of the proteins or to phosphorylation or other modifications of the proteins involved. However, given that the kinetics of induction of the binding activities precedes transcriptional activity, de novo synthesis of components of the EMSA complexes is likely, as in the case of IFN-{gamma} activation of the MHC class II genes (32).

A novel two-part regulatory element was discovered within the human C4 promoter which appears critical for basal activity in hepatic cells. The reporter gene analysis results indicated the presence of repressor sequences between -468 and -310 (which contain putative binding sites for GATA and Nkx2) that had the effect of decreasing promoter activity by almost 50%. In addition, these distal elements appeared to be acting in concert with a complex of Sp1/3 and BKLF-binding GT box elements around -140. This interaction has the effect of masking the very strong negative effects due to the distal region. The mechanism for this masking effect is currently unknown, but our hypothesis is that interaction with the -140 region prevents interaction of the upstream element with the proximal basal elements. Ablation of the -140 sequences allows interaction of the repressor region with the proximal elements that has the effect of preventing transcriptional activity. It is of interest to determine whether extracellular inflammatory or other signals are able to impinge upon the factors interacting at the -140 and/or upstream repressor sites and regulate expression of C4 in a similar way. Also, given the role of BKLF and other EKLF family members in the cell type-specific regulation of a number of genes (20, 37, 38, 39), it is likely that this complex regulatory element may play a role in the tissue-specific expression of the C4 gene.


   Acknowledgments
 
We thank Joanna Guy for expert technical assistance and Dr. Merlin Crossley for provision of the BKLF Ab and helpful advice.


   Footnotes
 
1 This work was funded by grants from the Australian Arthritis Foundation and the Australian Kidney Foundation to L.J.A. D.U. was the recipient of an Australian Postgraduate Research (Industry) Award. Back

2 Current address: Division of Rheumatology, University of Colorado Health Sciences Center, 4200 E 9th Avenue, Denver, CO 80262. Back

3 Address correspondence and reprint requests to Dr. L. J. Abraham, Department of Biochemistry, University of Western Australia, Nedlands, WA 6907, Australia. E-mail address: Back

4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; BKLF, basic Kruppel-like factor; EKLF, erythroid Kruppel-like factor; NF-1, nuclear factor-1; USF, upstream stimulatory factor; WT, wild type. Back

Received for publication June 11, 1999. Accepted for publication October 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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