Oct 18, 1993 - Garfinkel, S., Thompson, J. A,, Jacob, W. F., Cohen, R., and Safer, B. (1990) J. Bid. Chem. 265, 10309-10319. 23. Roy, A. L., Meistererust, M., ...
Val. 269, No. 13, Issue of April 1, pp. 9669-9674, 1994 Printed in U.S.A.
THEJOURNAL OF BIOLKICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the C1 Inhibitor Geneby yInterferon* (Received for publication, October 18, 1993, and in revised form, December 17, 1993)
Kamyar ZahediS,Anne E. Prada, and Alvin E. Davis I11 From the Division of Nephrology, Children's Hospital Research Foundation a n d the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-2899
Treatment ofthe hepatoma cell line, Hep3B, with y-in- unusually high number of Alu repeat elements (17 withinthe terferon(IFN)enhancedexpressionof C1 inhibitor gene) anda region of alternating purine, pyrimidine repeats in (ClINH)mRNA, primarily dueto an enhanced transcrip- its second intron, which may be capable of forming a Z-DNA tion rate. Hep3B cells transfected with reporter con- motif (7). It is possible that allof the above may play roles in structs containing various regions of the ClINH gene regulation of the C l I N H gene. In hepatoma cell lines, normal between positions -1182 and +587, and stimulated with human skin fibroblasts, blood monocytes, a n d monocytic cell y-IFN, expressed increased levels of chloramphenicol lines, ClINH mRNA and protein levels increase after stimulaacetyltransferase inthe presence of the first intron and tion witha number of cytokines (8-13). Tumor necrosisfactor-a! as few as 12 bases of the 5"flanking region. However, a and interleukin-6 enhanced ClINH synthesis (1.5- and5-fold 66%reduction in the inducibility of the constructs was increases in protein levels, respectively)(11). Treatment of huobserved when the upstream region between -582 and man peripheral blood monocytes with monocyte colony-stimu-252 was eliminated. Successive deletions mapped the lating factor led to a 15-fold increase in synthesis of C l I N H firstintronIFN-responsiveelements to aregion be(13).However, t h e effect of monocyte colony-stimulating factor tween +368 and +410. The data indicate that both the of class upstream and the first intron sequences can indepen- is thought to be mediated via the enhanced production dently enhance induction of ClINHgene expression. Ex- I interferons by monocyte colony-stimulating factor (13). The amination of the immediate upstream sequence of the most potent inducers of ClINH are types I a n d I1 interferons ClINH gene revealsthe absence of a TATA box. The pro- (8-10). Enhanced expression of ClINH mRNA in IFN-stimumoter ofthe ClINH gene was mapped to a region within lated cells was independent of de novo protein synthesis (14) 81 bases of the upstream sequence and the first exon. and, in allcell lines (withthe possible exception of monocytes), the increased mRNA levels were primarily due to an enhanced Further examination indicated two regions that were we examined the regulapotentially important for promoter activity as follows: 1) transcription rate (14). In this study, a G-C-rich region from -81 to -49, and 2) an initiator tory elements controlling the IFN-mediated enhancement of element at -3 to +5. The results indicate that the upthe minimal promoter ClINH gene expression and mapped stream sequences including -81 to -49 and the H-DNA activity within the immediate 5'-flanking regionof t h e gene. region between -48 and -17 are not necessary for promoter activity. The initiator element from -3 to +5 is MATERIALSANDMETHODS sufficient and necessary for promoter function. Cell Culture-The human hepatocellular carcinoma cell line, Hep3B 2.1-7 (ATCC)was cultured in Dulbecco's minimum essential medium, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, gentamicin (50 C1 inhibitor (ClINH)' isa member of the serine proteinase pg/ml) (Life Technologies,Inc.) and 10% fetal bovine serum (Hyclone). Construction of ReporterPlasmids-Using a full-length genomic inhibitor family. It is a M , 105000 glycoprotein (1)that reguclone of the ClINH gene containing approximately 10 kilobases of uplates the enzymatic activities of a variety of plasma proteases stream sequence, a 2.7-kilobase BumHI fragment was isolated, which including kallikrein, plasmin, factor XIa, and factor XIa and is contained 1182 base pairs of the 5"flanking region and 1109 bases pairs s of the clas- within the gene. This fragment was cloned into pUC19 and sequenced. t h e sole regulator of t h e C l r a n d C lcomponents Various segments spanning the ClINH gene 5"flanking region to the sical complement pathway (2). Hereditary angioneurotic edema, which affects approximately1in 50-100,000 people, i s translation start site were amplified using the polymerase chain react h e clinical manifestation of C l I N H deficiency (3). C l I N H i s tion. Amplified segments were ligated into the linearized reporter gene vectors pCAT basic or pCAT enhancer (Promega).Sequences of all conencoded by a single 17-kilobase gene on chromosome 11 (4, 5); structs were confirmed by the dideoxy chain termination method. (15). i t consists of 8 exons and 7 introns (6). The ClINH gene does Dunsient Dansfection of Hep3B Cells and CAT Assays-Transient not possess a TATA box but has a region of potential H-DNA tranfections were performedon Hep3Bcultures using the calcium phosstructure extending from position -48 to -17. It contains an phate precipitation method of Wigler et al. (16). CAT constructs or CAT control plasmid(10 pg) were co-transfectedwith PSVP-galactosidase(3 pg) in duplicate plates. One plate of each set was induced with 1000 * This study was supported by United States Public Health Service unitdml of y-IFN. Cells were harvested and extracts prepared in 0.25 Grant HD22082 (to A. E. D.) and by a Children's Hospital Research mM Tris-HC1, pH 8.0. Cell extracts (30 pg/well) were assayed in dupliFoundation Trustee Grant (to K. Z.), The costs of publication of this article were defrayed in part by the payment of page charges. This cate for CAT protein levels using a CAT enzyme-linked immunosorbent article must therefore be hereby marked "advertisement" in accordance assay (Boehringer Mannheim). CAT concentrations were expressed as percent of pCAT control normalized against P-galactosidase activity of with 18 U.S.C. Section 1734 solely to indicate this fact. the extract. $ To whom correspondence should be addressed: Division of Nephrology, Children's Hospital Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-559-4531; Fax: 513-559-7407. RESULTS The abbreviations used are: ClINH, C1 inhibitor; IFN, interferon; Characterization of the ClINH Promoter Region-The immeCAT, chloramphenicol acetyltransferase; GAS, y-interferon activation sequence; ISRE, interferon-stimulated response element; ISGF, IFN- diate upstream region of the ClINH gene (Fig. 1)lacks a constimulated gene factor; PRDI, positive regulatory domain I. a polypurinelpolypyrimidine sensus TATA element but contains i=
9669
9670
y
Interferon Induction of C1 Inhibitor CTCETCT
GGAAAGGGAAGCG
TTCCTTTAA
FIG.1. Schematic diagram of the ClINH promoter region and y-IFN-responsiveelements. The CAAT boxes are at positions -103 to -100 and -61 to -58.0,H-DNAsequence (-48 to -17). The initiator element -3CTCAGTCT+5 is underlined with a broken line,the urrow denotes the transcription start site (+l), and the first and second exonsare enclosed by boxes. The GAS sequences involved in y-IFN response are at-346 to -338 and -332 to -324. The ISREs in the first intron are at positions +373 to +386 and +392 to +405.
region thought to be capable of assuming anH-DNA conforma- start site (+587) cloned adjacent to a promoterless CAT gene tion (-48 to -17) (7). It hasbeen hypothesized that theH-DNA were transfected into Hep3B cells. The transfected cells were sequence may play an important role in the function of the incubated for 18 h in mediumalone or in mediumsuppleClINH promoter, because similar sequences are involved in mented with y-IFN (1000 unitdml). All constructs were responregulation of the human C-myc gene (a TATA-driven gene) (7). sive to y-IFN (4.5-13.6-fold induction) (Fig. 4). The reporter The promoter region was characterized using reporter gene constructs were active in the presence of as few as 12 bases of constructs spanning positions -81, -48, and -3 to +210. The the 5’-flankingregion. However, deletion of the region between three constructs are transcriptionally active (Fig. 2 A ) but un- -582 and -252 led to 66% reduction in the y-IFN responsiveresponsive to y-IFN. Analysis of the immediate 5‘-flanking re- ness compared with the full-length clone. This indicates that gion and the first exon revealed a GC-rich region extending this region contains element(s) capable of mediating or modufrom position -81 to -60, a CAAT sequence (-61 to -581, and an lating a y-IFN-induced response. initiator element (CTCAGTCT)at -3 to +5. This sequence exy-IFN Responsiveness of 3‘-Duncation Constructs-In order actly matches the element (CTCANTCT) described by Smale to determine the role of the first intron in y-IFN induction, and co-workers (17, 18) that is important in theregulation of constructs containing progressively shorter intronic segments transcription by many TATA-less genes, such as terminal de- were used. Computer analysis of the first intron revealed two oxynucleotidyl transferase. In orderto boost the activity of the potential ISREs at positions +373 to +386 and +392 to +405. reporter gene, comparable constructs containing the SV40 en- Comparison of the activity of constructs spanning-582 to +438, hancer were used. The consequence of the deletion of the H- -582 to +410, and -582 t o +368 revealedthat elimination of the DNA sequence wasdetermined by examining the -81, -48, and region between +368 and +438 reduced the responsiveness of the reporterconstruct by approximately 52% (Fig. 5 A ) . In order -3 to +210 enhancer-containing constructs. These constructs directed transcription of the CAT gene (Fig.2B ). An important to eliminate the contribution of the sequences between -582 element in the regulation of the ClINH promoter is located and -252 to y-IFN responsiveness (Figs. 3 and 4) and to conbetween -81 and -48; elimination of this region leads to a 75% centrate on the role of the intronic sequences,Hep3B cells were reduction in promoter activity. The region spanning -48 to -3, transfected with constructs spanning -80 to +438, -80 to +410, which includes the H-DNA region, may exert a negative effect and -80 to +368. Examination of y-IFN responsiveness of these on the initiator-drivenactivity of the ClINHpromoter. The -81 constructs confirmed the data in Fig. 5A. Elimination of the to -48 region may function to override the negative regulation +368 to +410 region diminished the inducibility of the reporter exerted by the H-DNA sequence. However, the above regions constructs to nearbackground levels (Fig. 5 A ) . The role of the are not requiredfor basal transcription, because the construct first intron in y-IFN induction was examined further using spanning -3 to +210 was sufficient to direct transcription of the constructs containing nucleotides +207 to +564 cloned downstream from the CAT gene in reporter constructs with nuclereporter gene (Fig. 2B 1. otides -582 to +38 or -252 to +38, 5’ to the CAT gene. Comy-IFN Enhances Danscription of the ClINH GeneStimulation of the human hepatoma cell line, HepSB, with parison of the split constructs(-582 ”-f +38/+207 + +564 and y-IFN (1000 unitdml) led to a 15-fold increase in the ClINH -252 + +38/+207 + +564) to the -582 to +38 and -252 to +38 mRNA level (Fig. 3A). The increase was primarily due to an constructs revealed that allexcept the -252 to +38 respondedto enhanced transcription rate, as shown by nuclear runoff assays y-IFN stimulation (Fig. 5B). Furthermore, comparison of the +38/+207 to previously reported three inducible constructs revealed that the -252 (Fig. 3B ). These results are similar those in HepG2, PLC/PRF/5, U937, and normal human fibroblasts + +564 and -582 + +38 were not as responsive as the-582 + hypothesis (11, 14, 19-21). Analysis of the -1182 t o +587 region of the +38/+207 + +564 construct. These data support the ClINH gene revealed the presence of two y-interferon activa- that the intronic elements (+368 to +410) and the upstream elements (-582 to -252) both are involved in y-IFN induction of tion sequences (GAS) in the 5”upstream region (-346 to -338 the ClINH gene. and -332 to -3241, while two potential IFN-stimulatedresponse elements (ISREs)were revealed in the first intron (+373 DISCUSSION to +386 and +392 to +405)(Fig. 1). Inordertodetermine The studies presented here have addressed two important whether the sequences present in the clone extending from -1182 to +587 were sufficient to mediate a response to y-IFN, aspects of the regulation of ClINH synthesis as follows: proHep3B cells were transfected with reporter constructs contain- moter characterization and y-IFN responsiveness. As pointed gene lacks a TATA element. ing this segment of the gene cloned upstream from a promot- out by Carter et al. (7), the ClINH erless CAT gene and stimulated with y-IFN (1000 units/ml) The immediate upstreamregion (-81 to +l), however, contains a CAAT element (-61 to +58), a GC-rich sequence (-81 to -611, (Fig. 3C). y-IFN stimulated CAT expressionapproximately 9-fold. Fidelity of the cap site was determined by rapid ampli- and a polypurine/polypyrimidine or H-DNAregion (-48 to -17). gene possesses the sequence CTfication of 5”cDNAends followed by sequence analysis. Thecap Furthermore,theClINH site was identical to that demonstratedby Carter et al. (7) and CAGTCT from position -3 to +5, which is similar to the initiapositions corresponded to position +1 of the construct -1182 to +587 tor element sequence (CTCATTCT) inthesame shown by Smale andco-workers (17,18) tobe important in the (data not shown). regulation of the terminal deoxynucleotidyl transferase gene. y-IFN Responsiveness of 5’-Duncation ConstructsSegments of the 5’-flankingregion extending to the translation The initiator element in the absence of any other sequences +
y
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Interferon Induction of C l Inhibitor
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FIG.2. ClINJ3 promoter analysis. Hep3B cells were co-transfected with ClINH reporter constructs (10 pg) and the PSVP-galactosidase reference plasmid (3 pg). Sixteen hours after transfection, cells were treated with y-IFN (1000 units/ml). Cells were harvested 24 h after stimulation, and CAT levels in cell extracts (30 pg) were determined and normalized against P-galactosidase activity. The CAT levels are expressed as the percentage of CAT expressed by pCATcontrol plasmid. The values are the average of three independent experiments. The solid bar indicates y-IFN-induced and the cross-hatched indicates unstimulated samples. A, ClINH reporter constructs containing positions -81 to +210, -48 to +210, and -3 to +210 were tested in transient transfection assays. B , constructs containing nucleotides -81 to +210. -48 to +210, and-3 to +210 of the ClINH gene upstream from an SV40 enhancer were examined for their ability to direct CAT expression.
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capable of binding general transcriptionfactors, such as TATA, CAAT, or SP1 elements, was sufficient for directing baseline transcription of the terminaldeoxynucleotidyl transferase gene (18).It was later demonstrated that the consensus sequencefor the terminal deoxynucleotidyl transferase family of initiator elements is CTCANTCT (171, a perfect match with the above ClINH sequence. The terminal deoxynucleotidyl transferase family of initiator elements was also shown to bind factors such as TFII-I and Cap binding factor (22, 23), both of which can interact with general transcription factors (24, 25). A number of TATA-lessgenes aredriven by different families of initiator elements (26). These genes share two characteristics as follows: 1) virtually all are considered housekeeping genes, and 2) most possess a single transcription start site. A number of studies demonstrate that disruptionof initiator elements leadsto the use of multiple transcription initiation sites (17, 18, 27). ClINH is an initiator-drivengene, because as few as three upstream bases can direct minimal transcription, and constructs with 3"termination at the transcription start site are inactive (data not shown). However, further studies are required to fully characterize thecore promoter activityand the role of other elements (i.e. H-DNA and CAAT box) in its regulation. Upstream regions may play significant, but not essential, roles in ClINHpromoter activity. These includethe -81 to -48 region, which contains a CAAT box and a GC-rich sequence. Bothof these may augment theactivity of the initiator. In addition, the -48 to -4 region, which includes the H-DNA region (-48 to -17) may, in the absence of the -81 t o -49 segment, actas a negative element reducing the activity of the initiator element. This is supported by data that suggest that the regulatoryeffect of H-DNA is a function of its length (28). as negative H-DNA segments 31-35 bases in length tend to act regulatory elements,while shorter H-DNA segments may augment transcription. Although most described initiator-driven genes have been housekeepinggenes, it has recently been shown that the T1-8d gene, a class I MHC gene in mouse that is TATA-less and y-IFN-responsive, and the human androgen receptor gene are also initiator-driven (29, 30). These data, together with the data reported here, may indicate a more prevalent role for initiator elements in gene regulation.
ClINH production is increased in several different cell types following treatment with anumber of cytokines (8-14). In uitro, y-IFN is the most potent inducer of ClINH synthesis (8-10, 14). This increased production results primarily from an enhanced transcription rate(14). Two regions contribute to y-IFN inducibility as follows: 1)a segment spanningpositions -582 to -252, and 2) an area in the first between intron +368 and +410. Both of the above sequences were independently active but also appeared to be additive. Examination of the first intron indicated the presence of two ISREs (+373 to +386 and +392 to +405) potentially involved in the binding of IFN-inducible DNA-binding proteins. Both ISREs contain a perfect half-site (GAAANN) and a half-site with a one-base mismatch (Fig. 1). Removal of the region from +368 to +410 decreasedthe responsiveness ofCAT constructs in the presence or absence of the 5"flanking region between -80 and -582 (Fig. 5A). GAAANN multimers were shown to be important in theactivity of transcriptional enhancers of interferons and interferon-inducible genes (31,321. Furthermore, the ISREs important in theregulation of types I and 11IFN-responsive genes were composed of two direct repeats of the GAAANN hexamer (32). These elements bind factors such as ISGF-2 and ISGF-3, which are involved in theinduction of interferon-responsive genes (33-36). The 5"flanking region of the ClINH gene between -582 and -252 also contains interferon-responsive elements. Elimination of this region leads to a 50-75% loss of inducibility (Fig. 4., A X ) . Two potential GAS elements (TTC/ACNNNAA)(37) are present at -346 to -338 and -329 to -321. These bind to y-IFN activation factor or ISGF-3 (37, 38). Activation of y-IFN activation factornSGF3 after y-IFN stimulation and itsbinding to the GAS is important in the induction of the guanylate-binding protein gene (39-411, which is regulated by both types I and I1 interferons (39) and the F,yRI (42,43) and the mouse Mig gene (441, which are responsive to type I interferonsonly. This region also contains a single PRDI sequence (-461 to -456) (45). Multimers of the PRDI hexamer areinvolved in the viral induction of p-interferon (46). Theactive sequence in this induction was later shown to be a dimeric repeat ofGAAANN (32). Palindromic repeats of GAAANN are involved in y-IFN responsiveness of the F,yRI and interferon regulatory factor-1 genes (47,
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FIG.3. y I F N enhances transcriptionof the ClINH gene. A, Hep3B cells were stimulated for24 h with a-IFN(1000 unitdml), y-IFN(1000 unitdml), and interleukin-6 (100 units/ml). Total RNAwas size-fractionated and analyzed for the presence of ClINH mRNA. Autoradiographs were analyzed by laser densitometry. Results were normalized against a-actin mRNA levels. The data are expressed as the ratio of normalized ClINH mRNA signals of induced to uninducedcells. B , nuclear runoff assays were performed to determine the transcription of rate the ClINHgene. The results were normalized against the transcription rate of the a-actin gene.C, Hep3B cells were co-transfected witha ClINH reporter construct (10 pg) containingthe -1182 to +587 segment of the gene and the pSVP-galactosidase reference plasmid(3 pg). Transfected cells were stimulated with
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FIG.4. Analysis of the ClINH y I F N regulatory elements. Serial 5’ deletion fragments between -912 and +587 of the ClINH gene were cloned into pCAT basic. Hep3B cells were co-transfected withClINH CAT constructs and the PSVp-galactosidase reference plasmid. Transfected cells were treated with y-IFN (1000 unitdml). Cell extracts (30 pg) were assayed for CAT expression. The resultsof CAT assays were normalized against p-galactosidase levels of the same sample. The CAT levels are expressed as the percentageof CAT expressed by pCAT control plasmid. The values are the average of three independent experiments. The solid bar indicates y-IFN-induced and thecross-hatched burindicates unstimulated samples.
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FIG.5. y-IFN responsiveness of 3‘4runcation constructs. 3’ deletion fragments of the ClINH gene were cloned upstream of a promoterless CAT gene. Hep3B cells were co-transfected with reporter constructs and the pSVp-galactosidase reference plasmid. Transfected cells were stimulated with y-IFN (1000 units/ml). Each cell extract (30 pg) was assayed for CAT expression. CAT levels were normalized against the P-galactosidase activityof the sample. TheCAT levels are expressed as thepercentage of CAT expressed by pCAT control plasmid. The valuesare the averageof three independent experiments. The solid bur indicates y-IFN-induced and the cross-hatched bur indicates unstimulated samples. A , reporter constructs containing 3’ deletion fragments from -582 to +438 and-81 t o +438 were used to examine the role of intronic sequences in y-IFN induction of the ClINH gene. B , ClINH constructs containing upstream regions (-582 to +38 and -252 to +38) and split constructs to +564) cloned downstream of the reporter gene were examined t o further determine the containing the aboveregions with intronic regions (+207 role of the ISREs in the y-IFN induction of the ClINH gene.
48). Studies are inprogress to further characterize thisregion. The ClINH gene is unusual in that itcontains y-IFN-responsive elements both in the 5”flanking region and in the first intron and that the two can act either independently or together. However, the presence of transcriptional enhancers in introns or the 3‘ region of genes is not unusual and, by definition, enhancer elements areactive regardless of their position or direction. In summary, regulation of the ClINHgene by y-IFN, its most
potent inducer, reveals that ClINHis an initiator-driven gene and that interferon induction is regulated by two sets of elements present in the upstream region and in the first intron. The regulatory elements of the ClINH gene supplement the observation of Yan and Tamm (49) regarding the structure of IFN-inducible genes based on their ability to respond to different IFNs. Type I IFN-responsive genes contain a TATA-driven promoter, and the IFN-responsive element is located 5‘ to the TATA element. The ( 2 ’ 3 ’ )oligoadenylate synthetase ME-12
y-IFN (1000unitdm11 for 24 h. Cell extracts (30 pg) of stimulated and unstimulated cells were examined for CAT expression. CAT expression was normalized against p-galactosidase activity of the same sample. The CAT levels are expressed as the percentage of CAT expressed by pCAT control plasmid. The values are the average of three independent experiments. The solid bar indicates y-IFN-induced andthe cross-hatched barindicates unstimulated samples.
9674
y
Interferon Induction
gene respondsto both types I and I1 IFNs, containsno TATA, is initiator-driven, and contains anIFN-responsive element at a position corresponding to theTATA site. An additional element appears to be required for its IFN responsiveness. ClINH, an initiator-driven gene that is responsive to both types I and I1 IFNs, partially fits this pattern. However, it differs in that it contains two interferon-responsive regions that are independently active and are separated by the core promoter of the gene. Experiments in progress are further characterizing the elements involved in promoter activityand inresponsiveness to types I and I1 interferons as well as mapping those elements involved in androgen and interleukin-6 induction. REFERENCES 1. Haupt, H., Heimburger, N., Kranz, T., and Schwick, H. G. (1970) Eur. J . Biochem. 17,254-261 2. Davis, A. E., 111 (1988) Annu. Reu. Immunol. 6, 595-628 3. Donaldson, V. H., and Evans, R. R. (1963)Am. J . Med. 3 5 , 3 7 4 4 4. Bock, S. C., Skriver, K., Nielsen, E., Thogersen, H. C., Wiman, B., Donaldson, V. H., Eddy, R. L., Marrinan, J., Radziejewska, E., Huber, R., Shows, T. B., and Magnusson, S. (1986)Biochemistry 25,4292-4301 5. Davis, A. E., 111, Whitehead, A. S., Harrison, R. A,, Dauphinais, A., Bruns, G. A., Cicardi, M., and Rosen, F. S. (1986) Proc. Natl. Acad. Sci. U. S. A . 83, 3161-3165 6. Carter, P. E., Dunbar, B., and Fothergill, J. E. (1988) Eur. J . Biochem. 173, 163-169 7. Carter, P. E., Duponchel, C., %si, M., and Fothergill, J. E. (1991) Eur. J . Biochem. 197,301-308 8. Lotz, M., and Zuraw, B. L. (1987) J . Zmmunol. 139,3382-3387 9. Hamilton, A. O., Jones, L., Morrison, L., and Whaley, K. (1987) Biochem. J . 242,809-815 10. Heda, G. D., Mardente, S., Weiner, L., and Schmaier, A. H. (1990) Blood 75, 2401-2407 11. Katz, Y., and Strunk, R. C. (1989) J. Zmmunol. 142,2041-2045 12. Zuraw, B. L., and Lotz, M. (1990) J . B i d . Chem. 265, 12664-12670 13. Schmidt, B., Gyapay, G., Valay, M., and Fust, G. (1991)Immunology 74,677679 14. Lappin, D. F., Guc, D., Hill, A,, McShane, T., and Whaley, K. (1992) Biochem. J . 281,437-442 15. Sanger, F., Nicklen, S., and Coulson,R. A. (1977)Proc. Natl. Acad.Sci. U. S. A. 74, 5463-5467 16. Wigler, M., Silverstein, S., Lee, L. S., Pellicer, A., Cheng, Y. C., and Axel, R. (1977) Cell 11,223-232 17. Smale, S. T.,Schmidt, M. C., Berk, A. J., and Baltimore, D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 45094513 18. Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113 19. Moms, K. M., Aden, D. P., Knowles, B. B., and Colten, H. R. (1982) J . Clin.
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