Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5786-5790, June 1993 Genetics The androgen-dependent C4-Slp gene is driven by a constitutively competent promoter (liver /in vitro transcription/complement component C4/sexual dimorphism) YUKO MIYAGOE, ELENA GEORGATSOU*, NADINE VARIN-BLANK, AND TOMMASO MEOt Unit6 d'Immunog6n6tique et Institut National de la Sant6 et de la Recherche M6dicale, U. 276, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Communicated by Donald C. Shreffler, February 9, 1993

ABSTRACT The androgen-dependent liver Sip, be multiple intragenic recombinants comprising a 5' region together with its constitutively expressed dosely related iso- derived from C4 and a longer 3' side from C4-Slp (9, 17-19, form C4, provides a model to address the question of which 40). The unexpected observation that C4 and C4-Slp have minimal alteration in DNA can shut offthe expression ofa gene about 95% sequence identity fueled the evolutionary model of in a manner reversible by testosterone or by trans-acting independent duplications of the C4 gene in several mamma- mutations. Previous work indicated that sequences located at lian lineages (5, 14) and rekindled interest in the mouse -1.9, -0.45, and -0.25 kb from the transcription start site of system. the C4-Slp gene played a critical role in determining its unusual The sequences of the two nearly 15-kb-long genes have functional divergence from C4. Now, using quantitatively and been recently completed (9, 20) and functional assays have qualitatively controlied transfection assays in HepG2 human been carried out in two laboratories to identify regions of hepatoma cells and mouse L fibroblasts, we have observed that divergence upstream of the two genes. One group described the C4-Slp promoter is fufy effective and unhindered by a negative regulatory element 300-450 bp upstream of the upstream sequences and that the C4 promoter has a consistent transcription start site of both C4 and C4-Slp that depressed albeit modest superiority. The determinant ofthis nearly 2-fold the ability of the promoter to direct chloramphenicol acetyl- difference does not coincide with the sites highlighted in transferase (CAT) gene transcription in HepG2 liver cells (21) previous studies but lies within the most cap-site-proximal and could be overcome by further upstream sequences from , at positions -189 to +48. We have also established C4 but not from C4-Slp. A second group, driven by the conditions for cell-free transcription of C4 and C4-Slp from observation oftwo DNase I-hypersensitive sites (at -2.0 and and templates by using nuclear extracts from -2.3 kb) in liver associated with the hormonal rat and mouse liver of both sexes as well as from L cells. At regulation of C4-Slp (22), was led to the discovery next to variance with the rat a2.-globulin gene, C4-Slp transcription in C4-Slp of an ancient provirus, dubbed "imposon" (23) be- vitro does not require male factors, for it is expressed as cause it was to to the efficiently as C4 by all nuclear extracts. Further, the minimal purported impose androgen regulation promoter sequences required to direct accurate initiation ex- adjacent C4-Slp promoter (24). In stable transfectants in the tend not farther than the most proximal 19 nucleotides. Be- G221 subline of the S116 mouse mammary carcinoma, the cause L cells efficiently express transfected covering intact C4-Slp gene is apparently expressed constitutively and the whole C4 gene or C4/C4-Slp recombinants, as well as does not respond to androgens, whereas a fragment of its 5' carrying the C4-Slp promoter, but fail to express the flanking region (-2.9/+0.05 kb) encompassing the imposon full C4-Slp gene, we favor a model in which the expression of responds to testosterone with about 3-fold stimulation, but the gene is modulated intragenically. only when used in the reversed orientation to drive CAT expression. Subregions smaller than a 750-bp hormone- Sex-limited protein (Slp) is an isoform of the mouse comple- responsive fragment were inactive. This composite enhancer ment component C4 (1). Both are serum encoded in actually overrides the natural C4-Slp promoter in that tran- the S region of the H-2 complex by two tandemly organized scription starts mostly from nonphysiological upstream lo- genes (2) that have played a major role in charting out the cations. genetic structure ofthe major histocompatibility complex (3). By a series of in vivo studies including endocrinological Although endowed with some biological activity (4-6), Slp is mutant and transgenic mice and surgically manipulated ani- defective as a complement component (7-9). Undetectable in mals, we have recently established that (i) the hormonal the serum of several inbred strains, in other strains Slp is induction of C4-Slp mRNA in the liver takes place in the normally found only in males and in varying levels correlated nucleus and (ii) the action of androgens on C4-Slp is indirect either with allelic changes (10) or with non-H-2 regulatory and mediated by growth hormone (25). Here we report genes (11). Furthermore, the rule of male-restricted expres- experiments showing that the promoters ofC4 and C4-Slp are sion is violated either by pseudoalleles (e.g., in the haplo- comparable in strength and that the C4-Slp regions implicated types H-2w7, H-2Wl6, and H-2W17; ref. 12) or by trans-acting in previous studies show no detectable effects. regulatory genes designated rsl, such as are found in strains FM, PL/J, and NZB (13). MATERIALS AND METHODS These "androgens-replacing" non-H-2 genes have not been identified (3), whereas the H-2-dependent constitutive Plasmids and Cosmids. The sequences of the 5' expression of C4-Slp was found to correlate with a locus flanking region of C4d (BamHI fragment of 2.7 kb) and amplification (14) brought about by extra gene copies akin to C4-Slp (15, 16). In fact, these C4-Slp-like genes turned out to Abbreviations: CAT, chloramphenicol acetyltransferase; TK, thy- midine kinase. *Present address: Institute of Molecular Biology and Biotechnology The publication costs of this article were defrayed in part by page charge Foundation ofResearch and Technology, P.O. Box 1527, Heraklion payment. This article must therefore be hereby marked "advertisement" 71110 Crete, Greece. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5786 Downloaded by guest on October 1, 2021 Genetics: Miyagoe et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5787 C4-Slpd (BamHI fragment of 2.9 kb) have been deposited in agarose gel. RNase protection was performed as described the GenBank data base (accession nos. X72494 and X72493). (25). For Si nuclease mapping, the hybridization mixtures The promoter-proximal two-thirds of these sequences can be digested with 0.2 unit of S1 nuclease in 200 ,ul of 30 mM compared with those reported for C4FM and C4-Slp FM, CH3COONa, pH 4.5/280 mM NaCl/45 mM ZnCl2. Protected respectively (21). Except for a small deletion in the proximal probes were resolved by 6% polyacrylamide/7 M urea gel (CA),, repeat of C4-Slpd, the sequences are essentially iden- electrophoresis. All hybridizations were performed under tical. verified excess probe conditions. For quantitative compari- Plasmid 17BG was derived from the promoterless pSBG sons, bands were excised from gels and radioactivity was (ref. 26; kindly provided by P. Herbomel, Institut Pasteur, measured by liquid scintillation. Paris), by inserting at the Sal I site an oligomer that introduces Nuclear Extracts and in Vitro Transcription. L-cell nuclear a Sma I and a Bgl II site and reconstitutes the Sal I site at both extract was prepared as described (32). Nuclei from mouse ends. Deletions of C4 and C4-Slp 5' flanking regions used in (BALB/c, 10-12 weeks old) and rat (Sprague-Dawley, 10-12 gene transfer experiments were obtained by cloning the weeks old) liver were prepared according to Gorski et al. (33), BamHI fragment of each gene in M13mp9 and submitting the except that in later preparations the following protease in- single-stranded DNA first to EcoRI digestion and then to hibitors were introduced: aprotinin (5 ,ug/ml), leupeptin (5 distributive "nibbling" by exonuclease (27). These nested ,ug/ml), pepstatin (5 p,g/ml), phenylmethanesulfonyl fluoride deletions terminating at the same BamHI site were cloned in (0.5 mM), benzamidine (2 ,uM), E-64 (2 ,uM), dichloroiso- the Sma I/Bgl II-treated vector 17BG and verified by sequenc- coumarin (20 ,uM). Nuclear proteins were extracted as de- ing. Further 5' deletion mutants of the C4 promoter tested in scribed (34). The standard transcription reaction mixture (20 cell-free transcription were obtained by PCR. The following ,ud) contained crude nuclear extract (60-100 ug), circular oligodeoxynucleotides 5'-GATATCGAATTCGICGiACCTC- DNA template (480-800 ng), and poly(dI-dC) (100-200 ng) in CCCAGCTCTGGCCTAGGGCCA-3' (-119/+48), 5'-GAT- 25 mM Hepes, pH 7.6/10% (vol/vol) glycerol/55.5 mM ATCGAATTCGTCGACGTTTCTTGGCCATCACGTGGT- KCI/6 mM MgCl2/0.125 mM EDTA/0.06 mM EGTA/0.5 TTC-3' (-88/+48), 5'-GATATCGAATTCGTfCACTCAT- mM dithiothreitol/0.6 mM NTPs. After incubation at 30°C for GGGGCAAAGGGAGGGGCAAG-3' (-60/+48), and 5'- 45 min, reactions were stopped by addition of 280 ul of 0.25 GATATCGAATTCGTCGjACGCCCCAACCTAGGTTGAT- M NaCl/1% SDS/20 mM Tris, pH 7.5/5 mM EDTA con- CAGAAG-3' (-19/+48) were used as 5' primers, together taining Escherichia coli tRNA and proteinase K to give final with the constant 3' primer 5'-GATATCAAGCTTG(iICfA-l concentrations of 60 and 100 ,ug/ml, respectively. After 30 CAGATCCAGGAA-3' (the Sal I site used for cloning site is min of incubation at 37°C, reaction mixtures were extracted underlined). Fragments amplified from cosmid 7.2 were di- with phenol/chloroform and ethanol-precipitated. Pellets gested with Sal I and cloned in front of the rabbit 831-globin were dissolved in 100 Al of 40 mM Tris HCl, pH 8.0/10 mM gene of the 17BG vector. For C4-17BG(-2645/+48), the C4 NaCl/6 mM MgCl2 and treated with 4 units ofDNase I at 37°C BamHI fragment was directly cloned in Bgl II-cut 17BG. for 30 min. After extraction, reprecipitated RNA was hybrid- Similarly, Rsa I-BamHI and Taq I-BamHI fragments were ized to 32P-labeled RNA probes (25). inserted in Sma I/Bgl II-cut 17BG to obtain C4-17BG(-382/ +48), C4-17BG(-189/+48), Slp-17BG(-284/+47), and Slp- RESULTS 17BG(-175/+47). Plasmid pCAJ6 (28) was used as internal control and was a generous gift of P. Herbomel. pAGO (29), The C4-Slp Promoter Resembles the C4 Promoter in Cel the plasmid carrying the herpes simplex virus thymidine Transfection Assays. In a first series of transient expression kinase (TK) gene used to derive stable transfectants, was a gift experiments in HepG2 and LTK- cells using C4dand C4-Slpd of A. Garapin (Institut Pasteur, Paris). 5' flanking regions joined to the CAT gene, we obtained Cosmids 7.2, 3.1 (C4), and 3.2 (C4-Slp) were isolated from results indicating that the poor constitutive expression of a BALB/c genomic library (30). Cosmid clones 744 and 703 C4-Slp in vivo could be indeed reproduced in vitro and traced contain natural C4/C4-Slp intragene recombinants (H-2w7R to the structure of its promoter. However, we could not haplotype), correspond to hybrids 1 and 3 reported by readily study the structure of CAT mRNA due to its very Pattanakitsakul et al. (19), respectively, and were derived rapid turnover and were compelled to use the rabbit 81-globin from a library prepared by M. Levy-Strauss in our laboratory gene as indicator (Fig. 1 A and C). To control for transfection from the B1O.W7R mouse strain. efficiency, the constructs were cotransfected into HepG2 Cell Culture and Transfections. HepG2 cells (human hep- cells together with the 61-globin plasmid pCAJ6, containing atoma cells) and LTK- cells (TK-negative mouse L fibro- the simian virus 40 enhancer and the rabbit f31-globin pro- blasts) were grown in Dulbecco's modified Eagle's medium moter region. C4-Slpd and C4d have major transcriptional supplemented with 10% fetal bovine serum and . start sites that are indistinguishable (data not shown) from Transfections were performed by the calcium phosphate those reported for the FM haplotype (21). Accurately initi- technique (31). For assays of transient transfection, cells ated transcripts from the C4d promoter template and pCAJ6 were harvested 48 hr after transfection, and cytoplasmic yielded the expected 202-nt and 135-nt protected fragments, RNA was isolated by the citric acid method as described (25). respectively (Fig. 1B). The transcriptional efficiencies of C4d Stable L-cell transformants were selected, cloned, and main- and C4-Slpdpromoters normalized to those ofpCAJ6 plasmid tained in medium with hypoxanthine, aminopterin, and thy- were determined in two independent experiments (Fig. 1C). midine. The -189/+48 fragment of C4 is sufficient to direct consid- Probes, RNase Protection, and Si Mapping. C4 and C4-Slp erable levels of accurately initiated transcription, and no RNA probes described in the text were transcribed from additional upstream regulatory elements appear to be re- inserts cloned downstream of the T3 or T7 RNA polymerase quired. From the intensity of the signals corrected for the promoter in pBS(+/-) (Stratagene). Linearized structure ofthe probes and the number ofthe template copies templates were transcribed in vitro by T3 or T7 RNA poly- transfected we calculate that the strength of the C4 promoter merase in the presence of [a-32P]CTP (3000 Ci/mmol; 1 Ci = is about 1/10th that ofthe simian virus 40-enhanced ,B3globin 37 GBq) under recommended conditions (Stratagene). Ho- promoter. Surprisingly, like C4d, C4-Slpd has an efficient mogeneously labeled probe for 51 nuclease analysis was promoter, has a similarly located transcription initiation site, synthesized with the Klenow fragment of DNA polymerase is devoid of upstream regulatory elements, and is able to I from single-stranded M13 template carrying C4 HindIII- direct constitutive levels of transcription that are marginally Sma I fragment (-1882/+ 126) and purified from a denaturing but consistently lower (=-2-fold) than those of C4. Like Downloaded by guest on October 1, 2021 5788 Genetics: Miyagoe et al. Proc. Natl. Acad. Sci. USA 90 (1993)

A C Relative promoter efficiency C4 -17BG 7* C4-17BG constructs C4-7B B-globin genel EXP. I EXP. 2 +48 RNA probe - spurious -2645 [ i5 -globin 0.13 0.11 -79 (202nt) start sites -1748 +1 0.32 0.21 protected fragment -843 0.49 0.12 Sip -17BG -479 0.13 0.36 ,' |1- globm gene I. - C4 -382 0.21 0.36 +47 RNA probe 202nt -189 +48 0.44 0.22 -93 ( 202 nt ) protected fragment I ,,,, C4-Slp-17BG constructs pCAJ6 (co-transfected control) -2465 i -globin i 0.13 0.07 -1959 +1 SV40 0.09 0.12 Enhancer L; _ -1084 0.08 0.07 PI- globin gene p-i - I globin -916 0.18 ( 135 nt) 135nt 0.21 -284 0.17 0.13 protected fragment -175 +47 P1 234 0.14 0.17 FIG. 1. Deletion analysis of C4 and C4-Slp promoter activity in HepG2 cells. (A) Outlines of the chimeric test plasmids (C4-17BG and Slp-17BG), the f-globin control plasmid (pCAJ6), and the antisense RNA probes used to assess the structures and yields of mRNA expressed in transfectants. Arrows indicate the positions ofthe cap sites. SV40, simian virus 40. (B) Transfection assays with C4 deletion mutants. Correctly initiated transcripts generate 202-nt and 135-nt protected from test and control plasmids, respectively. The doublet at '202 nt was seen with natural mRNA as well as with in vitro transcripts. Lane 4, transfectants receiving only pCAJ6; lane P, the labeled RNA probe. (C) Relative strength of 5' deletion mutants of C4 and C4-Slp promoters. Relative promoter efficiency was calculated by normalizing the radioactivity in the bands of the correctly initiated transcripts from the test templates relative to the transcription of the intemal reference plasmid pCAJ6 [(cpm test - cpm background)/(cpm control - cpm background)]. C4-Slpd, the C4d gene has similarly located but longer (CA),, As expected (18), the expression of the recombinant genes repeats at about -200 bp and -1600 bp relative to the cap site was easily detected in L cells. Although L fibroblasts and (21). Neither the proviral 5' long terminal repeat at -1.9 kb HepG2 hepatoma cells cannot be regarded as a surrogate of from C4-Slp nor the (CA)n repeat at -200 bp exerts a detect- normal liver cells, it is significant that they both mirror the able influence on the promoter activity (compare the con- pattern of female livers when transfected with the full genes structs -189/+48 and -382/+48 and the constructs -1748/ or recombinants thereof. +48 and -843/+48 in Fig. 1C). In addition, the results Equivalence in Strength and Minimal Structure Require- obtained with the same constructs in transfection experiments ments of C4 and C4-Slp Promoters in Cell-Free Transcription of L fibroblasts were wholly akin to those shown here for Systems. To assess the efficiency of the C4-Slp promoter HepG2 cells. The failure to reproduce in promoter assays the under experimental conditions approximating those of the contrasting expression levels of C4 and C4-Slp could be normal liver parenchymal cells, we established an in vitro ascribed inter alia to the lack of relevant control sequences in transcription assay using crude nuclear extract from rat liver; the constructions or to unsuitable host cell types. We set out this system has been employed for the transcription of to test both hypotheses by studying (i) cosmids spanning the liver-specific genes from other mammals (33, 36). More entire length of both genes and (ii) in vitro transcription with notably, rat liver nuclear extracts have proven useful to nuclear extracts from liver and other cell types. reproduce in vitro the sexually differentiated and develop- Lack of C4-Slp mRNA Expression in L Cells Transfected mentally regulated expression of the a2u-globulin gene (37). with Gene-Spanning Cosmids Carrying 5' king Regions of Initially, we tested whether the C4 template C4- Variable Length. We cotransfected C4 or C4-Slp cosmids 17BG(-2645/+48), shown schematically in Fig. 1C, could be (Fig. 2A) into mouse LTK- cells together with the selectable transcribed correctly and efficiently (Fig. 3A). Transcription TK gene (29). After isolation of stable transformants, mRNA vivo levels were assessed by S1 nuclease protection assays (Fig. started at the same location previously determined in 2B) using a probe that detected both classes oftranscripts. As and was inhibited by low levels of a-amanitin (2 pg/ml), expected from previous work (5, 18, 35), the C4d cosmids (7.2 indicating an RNA polymerase II product; as expected, the and 3.1) were transcribed in L cells (Fig. 2B, lanes 1 and 2) upstream-initiated transcripts were only partially sensitive. and produced high quantities of functional protein (data not Further, the 5' extragenic region of C4 could be largely shown). By contrast, C4-Slp mRNA could not be detected in eliminated without affecting the accuracy or the yield of L cells stably transfected with the C4-Slp cosmid 3.2 (Fig. 2B, specific transcripts (Fig. 3B). By using a ,B-globin template as lane 3). Moreover, unlike the C4 message, C4-Slp mRNA was an internal standard in the transcription assay of the trun- undetectable by immunofluorescence in numerous transient- cated C4 plasmids, we could reliably conclude that the basic transfection assays (data not shown). This expression defect, promoter of C4 was unusually short. While the minimal observed also in monkey COS cells, cannot be ascribed to the promoter structure able to confer full expression had its 5' insufficient length of upstream regulatory regions, because end not further than 189 nt upstream from the transcription the C4-Slp cosmid 3.2 carries a 6-kb-long 5' flanking se- start site, additional deletions in the 3' direction did not quence, far longer than that in the expressed C4 cosmids. In abolish initiation. A promoter structure reduced to the 19 the hope of removing putative inhibitory domains, we pro- most proximal nucleotides was still able to direct the syn- duced an EcoRI-truncated version of cosmid 3.2 carrying thesis of correctly initiated transcripts, albeit with 10-fold only 0.9 kb of the 5' flanking region, a size adequate for lower efficiency (Fig. 3C). As no major difference could be plentiful expression ofC4, but failed to restore the expression revealed between C4 and C4-Slp templates featuring -180-bp of C4-Slp in transfected cells (data not shown). We also promoter regions, the deletion experiments were carried only transfected into L cells cosmids 703 and 744 (C4/C4-Slp on C4. However, we also tested templates covering very long recombinant genes) isolated from B1O.W7R mice (Fig. 2C). 5' flanking regions ofboth genes (2645 bp for the C4 template Downloaded by guest on October 1, 2021 Genetics: Miyagoe et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5789

cosmids and A a-alflanitin N r s 0%7 A probes B o0co 00 r-r--to.- C,0% cl _n t vc o 0 M C4 2 50 M .I 00R 00~2 p SRT -18kb - 7.2 C4d *"imp.- - C4 * -4kb 3.1 C4d 202 nt ili;- C4 -6kb 3.2 C4-Sipd 202 H S SI probe (C4 and C4-Slp) S -C4 protected fragment (126nt) 202 n S H-2 W7R 703 C4/C4-Slp rec. H-2 W7R 744 C4/C4-Slp rec. 44OSI, -p-globin 191nt 1 35nt RNA Probe (Sip)

B C4 C4-Slp C--S< m 703 7.2 3.1 3.2 - ,,ScS,,,.744 ,*# -P-globin -~~~ a 135iit I p 10 27 50 94100 Q _ _~~ dw Efliciency d- I C4-Slp (v/) io OM= -191nt 126nt- 99 FIG. 3. Cell-free transcription ofC4 gene constructs with rat liver Go nuclear extracts. (A) Plasmid C4-17BG(-2645/+48) (480 ng, lane C4) was transcribed in vitro with an optimal concentration (3 Ag/pl) of 1 2 3 4 5 6 7 8M liver nuclear extract from male rats, and the RNA was detected with RNase protection the C4-specific RNA probe shown in Fig. 1A. The accurately initiated but not the spuqous transcripts were sensitive to a low dose 1 2 3 4 ofa-amanitin (2 jig/ml). Hpa II-digested pBR322 DNA provided size SI protection markers (lane M). SRT, spurious and read-through transcripts. (B and C) Deletion mutant templates contained promoter sequences FIG. 2. Detection ofC4 and C4-Slp mRNA in cosmid-transfected from nt +48 relative to the cap site to the 5' deletion endpoint L cells. (A) Outlines ofcosmids and probes for S1 nuclease or RNase indicated above each lane. All reaction mixtures contained 380 ng of protection studies. rec., Recombinant. (B) Cytoplasmic RNA iso- C4 template and 67 ng of the internal control template (pCAJ6). lated from stable uncloned transfectants (lanes 1-3) and untrans- Relative transcription yields under each lane were obtained by fected cells (lane 4) was hybridized (25 sg) to the 32P-labeled S1 densitometric determinations of the C4 and (-globin signals and probe, which covered the transcriptional initiation site but did not represent the average of two experiments. discriminate between C4 and C4-Slp transcripts. The length of 126 nt ofthe major protected fragment seen in the ladder oftranscripts from 7.2 and 3.1 is as expected from the location ofthe cap sites of C4 and deletion analysis, using transient-transfection assays con- C4-Slp. (C) Expression in L cells of cosmids carrying the natural trolled for transfection efficiency and accuracy of transcrip- recombinant C4/C4-Slp genes detected with an RNA probe com- tion initiation, revealed only a <2-fold superiority of the C4 plementary to the region from +4171 to +4363 of the Slp mRNA. Control protections were done with RNA from BALB/c adult female C4 C4-Slp and male livers (lanes 1 and 2, respectively). The longer protected fragment contains an unnatural oligo(dG) stretch only partially EXTRACT ( Ag ) 'S 0 x °58 removed by RNase A. RNA from 7.2 transfectants (lane 3) provided ' ' 00 < TEMPLATE (ng) L00 n Vm 00 0x a control for the specificity of the C4-Slp RNA probe. In contrast to cosmid 3.2 (lane 4), cosmids 744 (three independent transfections, lanes 5-7) and 703 (lane 8) were efficiently transcribed. Lane M, Hpa II-cut pBR322 used as size markers. *""" - * ~ C4/C4-Slp and 2465 bp for the C4-Slp template) and compared extracts from male and female rats. Further, to eliminate possible pitfalls inherent in the use ofheterologous assay systems, we prepared transcriptionally active nuclear extracts from ~ ~~ ~ ~ ~ ~ ~ ~ ~ ~ .. mouse livers of both sexes. Finally, the observation that, in -' I I" s ! a female liver-like fashion, L cells expressed C4 but failed to M123 45678 91011121314 Rat Mouse L Rat Mouse express the entire C4-Slp gene prompted us to establish a WLJ transcription assay also with nuclear extracts from L cells. The results of these experiments, exemplified in part by Fig. 4, uniformally point to the same conclusion: C4-Slp features C4-17BG + a constitutively competent promoter essentially indistin- Sip-i 7BG +48 guishable in strength and quality from that of C4. We also +47 tested cosmids carrying the entire C4 and C4-Slp genes as templates in in vitro transcription reactions. Although the FIG. 4. C4-17BG(-2645/+48) and C4-Slp-17BG(-2465/+47) yield of correctly initiated RNA relative to the spurious templates were transcribed in vitro by liver nuclear extracts from transcripts was somewhat lower for cosmid DNA template male rat (lanes 1, 3, 4, 9, and 10) or BALB/c female mouse (lanes 5, (data not shown), the results were qualitatively identical. 6, 11, and 12) livers. Transcripts were hybridized with the C4 or C4-Slp RNA probe outlined in Fig. 1A. The amounts of templates and protein extracts are indicated above each lane. The same DISCUSSION templates were also incubated with a nuclear extract from mouse L cells (lanes 7, 8, 13, and 14). Control reactions in the absence of We have provided several lines of evidence supporting the template or of extract (lanes 1 and 2) prove the nature of the conclusion that C4 and C4-Slp are driven by almost indistin- protecting transcripts. Lane M, Hpa TI-cut pBR322 used as size guishable transcriptional control elements. A systematic 5' markers. Downloaded by guest on October 1, 2021 5790 Genetics: Miyagoe et al. Proc. Natl. Acad. Sci. USA 90 (1993) promoter, too modest to account for the >1000-fold differ- Colten, H. R. & Seidman, J. G. (1983) Proc. Natl. Acad. Sci. USA 80, 6947-6951. ence in the steady-state levels of nuclear precursors of C4 and 3. Shreffler, D. C. (1988) J. Immunol. 141, 1791-1798. C4-Slp observed in female hepatocytes (25). It is interesting 4. Meo, T. & Tosi, M. (1985) Ann. Inst. Pasteur/Immunol. 136C, that the element responsible for the slight in vitro difference 225-243. must reside within the -189/+48 region and that its activity 5. Tosi, M., Levi-Strauss, M., Georgatsou, E., Amor, M. & Meo, T. is largely insensitive to the presence of farther upstream (1985) Immunol. Rev. 87, 151-183. 6. van den Berg, C. W., Demant, P., Aerts, P. C. & van Dijk, H. (1991) DNA segments. Our findings are not in agreement with those Complement Inflammation 8, 236 (abstr.). of Nonaka et al. (21), who traced the difference in transcrip- 7. Ferreira, A., Nussenzweig, V. & Gigli, I. (1978) J. Exp. Med. 8, tional regulation between C4 and C4-Slp genes to a region 1186-1197. 1.0-kb upstream that was able in C4 but not in C4-Slp to 8. Karp, D. R., Capra, J. D., Atkinson, J. P. & Shreffler, D. C. (1982) a sequence present on both J. Immunol. 128, 2336-2441. overcome the effects of negative 9. Ogata, R. T. & Zepf, N. A. (1991) J. Immunol. 147, 2756-2763. genes between nt -437 and -333. Nor do our data agree with 10. Atkinson, J. P., Karp, D. R., Seeskin, E. P., Killion, C. C., Rosa, their later conclusions (38) that the region between nt -472 P. A., Newell, S. L. & Shreffler, D. C. (1982) Immunogenetics 16, and +44 is transcriptionally permissive in C4 and inhibitory 617-620. in C4-Slp. The causes of the discrepancies may be traced to 11. Bruisten, S. M., Demant, P. & Robins, D. M. (1989) Immunoge- variables deemed by Yu et al. (38) to account for the netics 29, 340-345. the 12. Shreffler, D. C., Atkinson, J. P., Chan, A. C., Karp, D. R., Killion, irreproducibility of the results-i.e., fluctuations in cell cul- C. C., Ogata, R. T. & Rosa, P. A. (1984) Philos. Trans. R. Soc. ture conditions and construct artifacts. However, in our London Ser. B 306, 395-403. opinion a major share can be attributed to the irregular but 13. Brown, L. J. & Shreffler, D. C. (1980) Immunogenetics 10, 19-29. substantial transcription activity initiating at nonphysiologi- 14. Levy-Strauss, M., Tosi, M., Steinmetz, M., Klein, J. & Meo, T. cal sites, which give rise to uncontrolled amounts of active (1985) Proc. Natl. Acad. Sci. USA 82, 1746-1750. 15. Levy-Strauss, M., Georgatsou, E., Tosi, M. & Meo, T. (1985) CAT protein. Immunogenetics 21, 397-401. Our data, both from transfection and transcription assays, 16. Rosa, P. A., Sepich, D. S., Shreffler, D. C. & Ogata, R. T. (1985) also fail to support the role attributed to the NF-KB motif J. Immunol. 135, 627-631. present at about -200 only on C4 and shown to bind a HepG2 17. Nakayama, K., Nonaka, M., Yokoyama, S., Yuel, Y. D., Pattana- nuclear protein (39). Moreover, the abit-ity of C4-Slp to direct kitsakul, S. & Takahashi, M. (1987) J. Immunol. 138, 620-627. transcription initiation as accurately and efficiently as C4 18. Stavenhagen, J., Loreni, F., Hemenway, C., Kalff, M. & Robins, source nuclear extracts D. M. (1987) Mol. Cell. Biol. 7, 1716-1724. regardless of the of the cell-free 19. Pattanakitsakul, S., Nakayama, K., Takahashi, M. & Nonaka, M. demonstrates that the sexual dimorphism of C4-Slp must (1990) Immunogenetics 32, 431-439. involve processes other than those implicated in the male- 20. Ogata, R. T., Rosa, P. A. & Zepf, N. E. (1989) J. Biol. Chem. 264, restricted expression of the rat a2.-globulin genes, elegantly 16565-16572. shown by Sarkar and Feigelson (37) to be transcribed in vitro 21. Nonaka, M., Kimura, H., Yeul, Y. D., Yokoyama, S., Nakayama, only by adult male liver nuclear extract. The apparent K. & Takahashi, M. (1986) Proc. Natl. Acad. Sci. USA 83, 7883-7887. inconsistency of L cells in being able to transcribe C4-Slp 22. Hemenway, C. & Robins, D. M. (1987) Proc. Natl. Acad. Sci. USA cosmids in nuclear extracts but not in intact cells may 84, 4816-4820. indicate that in vivo an inhibitory control sets in after RNA 23. Stevenhagen, J. B. & Robins, D. M. (1988) Cell 55, 247-254. initiation. Alternatively, the cell-free system is inadequate to 24. Loreni, F., Stevenhagen, J. B., Kalff, M. & Robins, D. M. (1988) reproduce the compositional or structural requirements crit- Mol. Cell. Biol. 8, 2350-2360. ical for inhibition. At any rate, a significant upshot of the 25. Georgatsou, E., Bourgarel, P. & Meo, T. (1993) Proc. Natl. Acad. Sci. USA 90, 3626-3630. cell-free transcription experiments has been the discovery 26. Heard, J. M., Herbomel, P., Ott, M.-O., Mottura-Rollier, A., that the promoter sequence minimally required for accurate Weiss, M. & Yaniv, M. (1987) Mol. Cell. Biol. 7, 2425-2434. transcription extends not further than 19 nt from the cap site. 27. Dale, R. M. K., McClure, B. A. & Houchins, J. P. (1985) Plasmid Indeed, as will be described elsewhere (Y.M., E.G., M. D. 13, 31-40. Galibert, G. Fourel, and T.M., unpublished work) this min- 28. Bohmann, D., Keller, W., Dale, T., Scholer, H. S., Tebb, G. & that set it apart from other Mattai, I. W. (1987) Nature (London) 325, 268-272. imal promoter has features 29. Colbere-Garapin, F., Horodniceanu, F., Kourilsky, P. & Garapin, TATA-less genes. In sum, the data reported here converge A.-C. (1981) J. Mol. Biol. 150, 1-14. toward the conclusion that the failure of C4-Slp to be ex- 30. Steinmetz, M., Malissen, M., Hood, L., Om, A., Maki, R. A., pressed in vivo and in transfected cells cannot be attributed Dastoomikko, G. R., Stephan, D., Gibb, E. & Romaniuk, R. (1984) to a debilitated promoter. EMBO J. 3, 2995-3003. If the lack of expression of C4-Slp in L cells and in female 31. Graham, F. L., Bacchetti, S., McKinnon, R., Stanners, C., Cordell, B. & Goodman, M. (1980) Introduction of Macromolecules into hepatocytes is due to one and the same mechanism, it is Viable Mammalian Cells, The Wistar Symposium Series, eds. tempting to propose that the expression of C4-Slp is inhibited Baserga, R., Croce, C. & Rovera, G. (Liss, New York), pp. 3-25. at a postinitiation step by a factor present in various cell types 32. Shapiro, D. J., Sharp, P. A., Wahli, W. W. & Keller, M. (1988) which can be counteracted by a mechanism set in action DNA 7, 47-55. either by hormones or by the rsl mutations. 33. Gorski, K., Carneoro, M. & Schibler, U. (1986) Cell 47, 767-776. 34. Dignam, J. D., Levovitz, R. M. & Roeder, R. G. (1983) Nucleic and C. Bazzali-Hernandez Acids Res. 11, 1475-1489. We are grateful to C. Chereau-Pauliac 35. Chaplin, D. D., Sackstein, R., Perlmutter, D. H., Weis, J. H., for invaluable assistance with the cell culture work, to C. Duponchel Kruse, T. A., Coligan, J., Colten, H. R. & Seidman, J. G. (1984) for synthesis of the , and to P. Bourgarel for helping Cell 37, 569-576. with the preparation of nuclear extracts. We acknowledge the 36. Monaci, P., Nicosia, A. & Cortese, R. (1988) EMBO J. 7, 2075- support of the Association pour la Recherche sur le Cancer and the 2087. Fondation pour la Recherche Medicale. Y.M. is the recipient of a 37. Sarkar, P. & Feigelson, P. (1989) Mol. Endocrinol. 3, 342-348. Poste Vert fellowship awarded by the Institut National de la Sante et 38. Yu, D. Y., Nonaka, M. & Takahashi, M. (1988) J. Immunol. 141, de la Recherche Medicale. 4381-4387. 39. Yu, D. Y., Huang, Z. M., Murakami, S., Takahashi, M. & Nonaka, 1. Meo, T., Krasteff, T. & Shreffler, D. C. (1975) Proc. Natl. Acad. M. (1989) J. Immunol. 143, 2315-2400. Sci. USA 72, 4536-4540. 40. Rosa, P. A., Sepich, D. S., Robins, D. M. & Ogata, R. T. (1987) J. 2. Chaplin, D. D., Woods, D. E., Whitehead, A. S., Goldberger, G., Immunol. 139, 1568-1577. Downloaded by guest on October 1, 2021