The EMBO Journal vol.9 no.3 pp.929-937, 1990

Activity and interleukin 1 responsiveness of SV40 enhancer motifs in a rodent immature T cell line

E.Espel1, C.Fromental23, P.Reichenbachl and M.Nabholzl 'Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland and 2Laboratoire de Genetique Moleculaire des Eucaryotes du CNRS, Unite 184 de Biologie Moleculaire et de Genie Genetique de l'INSERM, Institut de Chimie Biologique, Facult6 de Medecine, 11 rue Humann, F-67085 Strasbourg, France 3Present address: Max Planck Institut fur Zuichtungsforschung, D-5000 Koln 30, FRG Communicated by B.Hirt We have analysed the enhancer activity and the interleukin 1 (ILl) responsiveness of individual motifs of the SV40 enhancer in an immature rodent T cell line, PC60. Transient transfection assays showed that tetramers of GT-I plus GT-HC motifs, the TC-ll or the P motif have significant enhancer activity in PC60, while neither Octamer nor SphI+II motifs have a detectable effect on promoter strength. Two motifs, TC-ll and P, strongly respond to stimulation by ILl. DNase I and methylation protection experiments with nuclear extracts show specific footprints in the TC-ll region of the SV40 enhancer. Exposure of PC60 cells to ILl increases their intensity. The TC-ll sequence forms several complexes detected in band shift assays. The molecules involved all have similar sequence specificity as NF-xB. Surprisingly, band shifts with extracts from control or ILl treated cells differ only slightly. However, if GTP is added to the binding reactions the intensity of bands formed by extracts from control cells is strongly reduced, whereas extracts from ILl treated cells fonn a single retarded complex that co-migrates with NF-xB from a pre-B cell line. The results suggest that in PC60 ILl induces NF-xB activity by activating molecules that are already in the nucleus. Key words: AP-1/interleukin I/NF-xB/SV40 enhancer/T lymphocyte

Introduction PC60.21.14.4 (hereafter referred to as PC60) is a hybrid derived from a cross between the rat thymoma C58 and the murine interleukin 2 (IL2) dependent cytolytic T cell (CTL) line B6. 1. Both C58 and PC60 grow independently of any exogeneously supplied growth factors (Conzelmann et al., 1982) and do not express detectable IL2 receptor light chains (Erard et al., 1984). Exposure of either cell line to a combination of interleukin 1 (ILl) and IL2 induces transcription of the IL2 receptor light chain gene (hereafter called the IL2 receptor gene) and accumulation of mRNA (G.Plaetinck, M.-C.Combe and M.Nabholz, manuscript in preparation) and protein (Erard et al., 1984; Plaetinck et al., 1987). PC60 cells express -25 ILl receptors/cell Oxford University Press

(R.Solari, personal communication). Recently we reported that in PC60 cells, stably transfected with genes under the control of the SV40 early promoter and enhancer, ILl induces a rapid, up to 30-fold increase in the transcription of these genes (Plaetinck et al., 1989). At least the early phase of induction is independent of protein synthesis. IL2 has no effect on the expression of the transfected genes. The SV40 enhancer The SV40 enhancer is a mosaic of cis-acting sequence motifs that serve as targets for transcription factors (for review, see Jones et al., 1988). Some motifs can increase transcription from a linked promoter in the absence of other enhancer elements (Jones et al., 1988). Others function only when present as tandem repeats or joined to a different motif (Fromental et al., 1988). The activity of several of the motifs is restricted to certain cell types (Nomiyama et al., 1987; Ondek et al., 1987; Pettersson and Schaffner, 1987; Schirm et al., 1987; Fromental et al., 1988) and correlates with the presence of tissue specific proteins binding to them (Davidson et al., 1986; Pettersson and Schaffner, 1987; Xiao et al., 1987). Sequences identical or closely related to two SV40 enhancer motifs, TC-ll and the Octamer, have been found also in immunoglobulin genes and have been implicated in B-cell specific expression of immunoglobulins (Lenardo et al., 1987). TC-ll is recognized by at least one ubiquitous DNA binding protein, and by the B cell specific factor NF-xB (Baldwin and Sharp, 1988). There is also one ubiquitous and several lymphocyte specific Octamer binding proteins [see Schreiber et al. (1988) for references]. At least one of these is expressed in certain T cells (Staudt et al., 1988), but its role in these cells is not yet clear. Several of the proteins binding to SV40 enhancer motifs can be induced by extracellular stimuli. NF-xB appears in pre-B cell lines upon stimulation with lipopolysaccharide (LPS) (Sen and Baltimore, 1986b; Atchison and Perry, 1987), where its appearance correlates with that of immunoglobulin light chain expression. Recently it has been shown that induction of NF-xB activity in the nucleus of LPS or phorbol myristic acetate (PMA) treated cells reflects transformation of an inactive, cytoplasmic form of the protein (Baeuerle et al., 1988a,b). This form is present also in non-lymphoid cells. NF-xB or a very similar protein has been shown to be induced in different cells by a number of different stimuli (Nabel and Baltimore, 1987; Leung and Nabel, 1988; Hiscott et al., 1989; Lenardo et al., 1989; Lowenthal et al., 1989; Osborn et al., 1989; Visvanathan and Goodbourn, 1989) including ILl (Osborn et al., 1989; Shirakawa et al., 1989). In addition to NF-xB, PMA can activate transcription factors AP-1, AP-2 and AP-3 that recognize different motifs in the SV40 enhancer (Angel et al., 1987; Imagawa et al., 1987; Lee et al., 1987). The AP-2 motif can also mediate a response to increases in cAMP levels (Imagawa et al., 1987). 929

E.Espel et al.

Interleukin 1 a and 3 Interleukin ILl (March et al., 1985) are mediators of inflammatory reactions. They affect expression of different genes in many different cell types. Both ILl species bind to a common surface receptor on T lymphocytes and fibroblasts (Sims et al., 1988), but recent evidence shows that ILl receptors on T lymphocytes and fibroblasts on the one hand, and B cells on the other, are different (Horuk et al., 1987; Bomsztyk et al., 1989; Chizzonite et al., 1989). Little is known about mechanisms by which ILl transmits signals to cells. Receptor bearing cells internalize ILl. T cells and fibroblasts accumulate ILl molecules in the nucleus (Mizel et al., 1987; Qwarnstrom et al., 1988). ILl does not provoke any rapid changes in soluble cytoplasmic calcium levels or phosphatidyl inositol turnover, nor does it activate protein kinase C (Abraham et al., 1987; Mukaida et al., 1987; Rosoff et al., 1988). In several cell lines of different tissue origin it has been shown to induce a rapid increase in cytoplasmic cyclic AMP (cAMP) levels (Shirakawa et al., 1988), whereas in other cells it appears to increase diacyl glycerol levels by accelerating the breakdown of phosphatidyl cholin (Rosoff et al., 1988) or phosphatidyl ethanolamine (Kester et al., 1989). IL 1 effects on T lymphocytes ILl has pleiotropic effects on lymphocytes. In a number of T cell lines it synergizes with agents that deliver signals through the T cell antigen receptor in inducing IL2 secretion or expression of IL2 receptor light chains (Kaye et al., 1984; Lowenthal et al., 1986; Hagiwara et al., 1987). However, its role in the stimulation of immune responses by normal mature T lymphocytes is not clear, and it has been difficult to demonstrate an effect of IL 1 on mature T lymphocytes under conditions that exclude involvement of other cell types present in the culture system. But recent results suggest that ILl may play a role in T cell differentiation: it has been shown, on the one hand, that thymic epithelial cells produce ILl (Le et al., 1986) and, on the other, that ILl can greatly

enhance IL2 responsiveness of purified CD4-CD8thymocytes stimulated with mitogenic lectins (Howe and MacDonald, 1988; MacDonald et al., 1988). C58 has phenotypic characteristics of a subclass of immature thymocytes (MacDonald et al., 1988). It expresses neither CD4 nor CD8 antigens (Silva et al., 1983) but carries a/$ T cell receptors on the cell surface (unpublished results). Thus, the ILl response of C58 and PC60 may reflect the one of normal immature thymocytes. In this paper we have compared the baseline enhancer activity and ILl responsiveness in PC60 of segments and individual motifs of the SV40 enhancer using plasmids whose expression has previously been analysed in other cell lines (Zenke et al., 1986; Fromental et al., 1988; Kanno et al., 1989). Two motifs, TC-II and P, mediate a strong response

ILl. ILl induces the appearance of a protein with the characteristics of NF-xB. But PC60 cells contain an additional constitutive DNA-binding protein with the same specificity as NF-xB, distinct from that of H2TFI. While the complex formation of NF-xB with its cognate motif is strongly increased by nucleoside triphosphates, the same treatment results in a drastic reduction of the binding by the constitutive factor. to

930

Results The contribution of different motifs to baseline expression and IL 1 responsiveness of the SV40 enhancer in PC60 To determine the contribution of individual motifs of the SV40 enhancer to gene expression in PC60 we measured the amount of mRNA transcribed from plasmids containing different enhancer segments in cells grown in normal medium or with ILl. Figure 1 shows a description of the plasmids used, and the autoradiographs of one transfection experiment and a summary of the results. Figure lB gives an example of baseline and ILl induced expression of the rabbit ,B-globin gene containing tetramers of SV40 enhancer motifs 109 bp upstream from the transcription start site (Fromental et al., 1988). Quantification of the results (Figure IA) shows that transcription from the minimal f-globin promoter (pG1) is increased to detectable levels by the TC-II and the P wild-type motifs and by the enhancer segment containing the GT-IIC and the GT-I motifs. A mutation in the GT-IIC motif increases the enhancer effect of this segment even further. Neither the SphII+I nor the Octamer motif has any enhancing effect. The TC-ll and the P region confer strong ILl responsiveness. The response mediated by the TC-II motif is already close to maximal after 4 h ILl treatment and cannot be inhibited by cycloheximide (see legend to Figure IC). GU.B responds more slowly, whereas the ILl response due to the P motif is not yet detectable at 4 h. Mutations in the TC-II and P motifs abolish baseline expression as well as ILl inducibility. The plasmid containing intact Octamer motifs is expressed just above the limit of detection in cells cultured in ILl. Conversely ILl reduces to a slight but reproducible extent the expression of plasmids containing intact GT-I motifs. The results obtained with the pA plasmids (Zenke et al., 1986) (see Figure IC for a representative experiment and Figure lA for quantification) are somewhat more complex. As expected pAO responds to ILl, albeit much less than pG1 .B, whereas ILl has no effect on the expression of pA56. Deletion of the TC-II motif (pA59) reduces but does not abolish the ILl response. Removal of the P motif (pA62) has some effect on baseline expression (note that the concentration of the pAO plasmid is half that of pA62) but does not measurably reduce ILl inducibility. The plasmids lacking the segment between bp 256 and 346 show a much lower baseline expression than pAO and pA56. pA104 that contains both the TC-H and P motifs, responds very well to ILl, but the response is undetectable in pA223 and pA233 in which the TC-II but not the P element has been deleted.

DNA-binding studies In an attempt to identify transcription factors involved in the response of the SV40 enhancer to ILl we compared the proteins binding to the enhancer in nuclear extracts from ILl induced and non-induced PC60 cells. DNase I and dimethyl sulfate (DMS) protection analysis of the SV40 enhancer revealed reproducible patterns of protection in three different areas (see Figure 2). First, extracts from induced and non-induced cells protected a region upstream of the 72 bp repeat (hatched box) from DNase I digestion. This constitutive footprint was only seen when the early mRNA coding strand was labelled. It spans

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Fig. 1. Baseline and ILl induced transcription from promoters containing various segments of the SV40 enhancer. (A) The sequence of the wild-type SV40 enhancer containing only a single copy of the 72 bp repeat is shown at the top, with binding sites for some transcription factors (OBP: Octamer binding proteins). Nucleotides are numbered according to the BBB system (Tooze, 1982). Below are indicated the sequences present in the different plasmids used for transient transfection assays. pGl contains the rabbit (3-globin gene from position -109 to +1650 inserted into pBR322. In pG1.B a single copy of the indicated SV40 enhancer region was inserted upstream of the ,B-globin gene in the same orientation with regard to the transcription start site as in the SV40 early promoter. All other pGl plasmids contain head-to-tail tetramers of the framed sequence at position -109 (Fromental et al., 1988). Their orientation is opposite to the one in the SV40 enhancer. pAO contains the 346/5171 segment of the SV40 early promoter with a single copy of the 72 bp repeat inserted at position -9 of the rabbit ,B-globin gene. In the other pA plasmids the sequence in the black box has been deleted (Zenke et al., 1986). Transcripts from the transfected plasmids were detected by RNase protection assays and quantified by densitometry of suitably exposed films. The results are summarized on the right of the figure. Values used for normalization are boxed in. ND, not detectable; +, detectable signal but quantification not possible. 'Treatment for 4 h with ILl in the presence of cycloheximide results in 141-fold induction, whereas cycloheximide by itself induces a 10-fold increase in transcript levels. (B,C) Cells were transfected with 10 iLg pGl plasmids containing the indicated motif together with pA56 as internal control (B) or with different pA plasmids (C). ILl was present for the last 18 h of culture. Controls (Probe): ND, not digested; D, digested; pAO and pGl, protection with plasmid DNA. Fragments protected by transcripts from the 13-globin or the SV40 early early (SV40 EES) start sites are marked. The results obtained with cells transfected with pA104 are from a different experiment than the other lanes shown.

the binding motif for the factor GT-IIA found in all cell lines tested so far (Xiao et al., 1987). Second, two regions in the 72 bp repeat (white boxes) were protected against DNase and DMS by extracts from non-induced cells, but this protection was much more pronounced with extracts from ILI treated cells. These regions correspond to the binding sites for NF-xB and the Octamer binding factors. On the late strand the NF-xB site is protected against DNase I and DMS (G240, G244, G245) by extracts from induced as well as non-induced cells, but ILl strongly enhances the DNase I

and slightly the DMS footprint. Only extracts from induced cells clearly protect the early strand against DNase and DMS (G237, G238), but both types of extracts induce hypermethylation of G235. This effect is much stronger with extracts from induced cells. All these interactions can be specifically inhibited by addition of unlabelled TC-30 DNA, whereas the point mutation in TC-42 reduces the capacity to compete by at least one order of magnitude. This last result shows that the footprints observed are not due to AP-2, as the point mutation in TC-42 is outside the AP-2 binding site.

931

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Fig. 2. DNase I and DMS footprints on the SV40 enhancer. End-labelled DNA samples were incubated with nuclear extracts from PC60 cells and treated with DNase I or dimethyl sulfate (DMS) and piperidine. G: presence of 3 mM GTP during binding reaction. Nuclear extracts from non-induced (N) cells and cells treated for 4 h with ILI (I). CH, cells were treated concomitantly with cycloheximide. COMPETITOR, cold DNA containing the TC-II motif (w) or its point mutation (m) shown in Figure IA. Numbers indicate the molar excess of cold over labelled DNA. White boxes indicate regions protected by extracts from ILl-induced cells. The hatched box indicates a region protected by extracts from control as well as induced cells. For each box the limits of the protected areas are given. The position of the single 72 bp and the 21 bp repeats of the SV40 enhancer are shown. 0, Gs protected against methylation by DMS; 0, hypermethylated Gs or As.

Sen and Baltimore (1986b) have shown that in pre-B cells cycloheximide by itself induces the appearance of NF-xB and has a synergistic effect on NF-xB induction by LPS. Similarly, it increases the levels of TC-II binding proteins in PC60 revealed either by footprint (Figure 2) or gel retardation assays (see below). Another recently described characteristic of NF-xB is that nucleoside triphosphates enhance its binding affinity (Lenardo et al., 1988). Figure 2 shows that presence of GTP in the binding reaction enhances the methylation protection of G240, G244 and G245 in the TC-ll region on the late strand by extracts from induced cells, while the protection of the same bases by extracts from non-induced cells is diminished. Neither in the presence or absence of GTP did extracts from non-induced cells give a clear DNase I footprint on the early strand. But GTP does enhance the footprint by extracts from induced cells, and extends it to include nucleotide 233. The DMS protection of G237 and G238 observed with extracts from induced but not from non-induced cells is increased by GTP. Conversely, GTP enhances hypermethylation of G235 by non-induced extracts. Gel retardation assays confirm the footprint analysis. Figure 3A shows that extracts from non-induced as well as from ILl treated cells produce several retarded complexes with an oligonucleotide containing an NF-xB site. All the bands disappear when an excess of unlabelled TC-30 DNA is added. The mutation in TC-42 abolishes the capacity to compete. We observed no specific competition for the formation of any of the complexes by a DNA fragment 932

containing the complete AP-3 binding site of the SV40 enhancer (not shown). Surprisingly, ILl induces only a slight increase in all of these complexes. However, addition of GTP to the binding reaction virtually abolishes complex-formation by extracts from non-induced cells, whereas extracts from ILl treated cells now form a single sharp band co-migrating with the fastest complex (band 1) seen in the absence of GTP. This band co-migrates precisely with NF-xB complexes from LPS treated 70Z/3 cells (Figure 3C). Maximal levels of this complex are found in extracts from cells exposed for 20 min or less to ILl. Upon longer treatment the intensity of this band declines somewhat to reach a stable level maintained for at least 6 h (Figure 3C and results not shown). As reported (Lenardo et al., 1988) GTP has no effect on the migration or intensity of complexes formed by the same cell extracts with a fragment containing the Octamer motif (not shown). Figure 3D shows that different ribo- and deoxyribonucleoside triphosphates induce the NF-xB-like complex in PC60 extracts with similar efficiency, with the exception of UTP which is less effective. Nucleoside monophosphates are inactive but, in contrast to Lenardo et al. (1988), we found that diphosphates are as active as triphosphates. We think that this discrepancy is due to minor technical differences as we obtained exactly the same result with extracts from 70Z/3 cells (Figure 3D). There is a very striking correlation between the increase in the intensity of the NF-xB-like complex and the reduction of the group of complexes formed in the absence of nucleotides.

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Fig. 3. Gel retardation assays with TC-II fragment. (A) A fragment containing a single TC-ll motif (see Materials and methods) was end-labelled and incubated with nuclear extracts from control (N), ILI treated (I) or cycloheximide treated (CH) PC60 cells, in the presence or absence of 3 mM GTP. F, free DNA. Competitor DNA (w: pGl.TC-30, m: pGl.TC-42) was added in 8-fold molar excess over the probe. The retarded complexes are referred to in the text by the numbers shown. (B) Competition of PC60 proteins binding to labelled TC-II probe by fragments with different NF-xB binding sites. Unlabelled DNA containing either the TC-II motif or the H2TF1 motif of the H-2Kb gene (XbaI-SphI fragment) was added to the binding reactions at the indicated molar ratio between competitor DNA and probe. (C) Comparison of TC-II binding proteins in extracts from 70Z/3 cells (Z) treated for 1 h with ILI and extracts from PC60 cells incubated in ILI for different periods. A, 3 mM ATP added to binding reaction. (D) Effect of addition of varying concentrations of different nucleotides to binding reactions of extracts from ILl treated PC60 or 70Z/3 cells.

The data presented so far show that ILI induces in PC60 an increase in a DNA binding protein with the characteristics of NF-xB. To determine whether any of the constitutive complexes seen in the gel retardation assays are related to H2TF1, a protein with very similar specificity, found in HeLa cells and fibroblasts, we compared the relative capacity of the H2TF1 motif in the H-2 gene and the TC-II motif to compete for the TC-IH binding proteins in PC60 extracts. In such assays the H-2 motif competes much better than the TC-H motif for H2TF1 (Baldwin and Sharp, 1987). Figure 3B shows that all the complexes formed by induced and non-induced cell extracts are equally well competed by either sequence. Similarly, oligonucleotides containing the NF-xB motif of the Igx chain gene competed with the same efficiency as the H2TF1 oligonucleotide for all complexes formed with the TC-II motif (not shown). NF-xB and H2TF1 also differ in their contacts with guanines in the binding site (Baldwin et al., 1988). Figure 4 shows that binding of the protein forming band 1 in the presence of GTP is inhibited strongly by methylation of G236, 237 or 238, and much less by methylation of G235. Thus this protein behaves like NF-xB. Formation of the complexes seen in non-induced extracts is less sensitive to methylation of any of the bases, but quantitative evaluation of such gels shows that the relative sensitivity to methylation of the different guanines remains the same.

Within the region containing the SphI and II and the Octamer motif, guanines G209 and G204 on the early strand are protected against methylation by extracts from induced as well as control cells, suggesting the presence of a constitutive factor binding to this region. However, the DNase footprint of this strand shows a clear protection of this region only with induced extracts. Furthermore, adenine A212 on the late strand is hypermethylated only in extracts from induced cells (Figure 2). Surprisingly, addition of excess unlabelled TC-30 DNA abolishes most of the changes in this region, suggesting that they are due to binding of TC-II specific proteins to the probe. In gel-retardation assays we have found the same Octamer specific complexes in both induced and non-induced extracts. The most parsimonious explanation of these results is that proteins binding to the TC-II motif can induce a conformational change in the Octamer motif that changes its reactivity with DNase I and DMS.

Discussion Activity of different SV40 enhancer motifs in

an

immature T cell line Analysis of transient expression in PC60 of rabbit f-globin gene recombinants containing tetramers of different SV40 enhancer motifs (pG1) shows that in these cells the TC-H

933

E.Espel et at.

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Fig. 4. Methylation interference analysis of the contact points of TC-II binding proteins. DNA containing the TC-II motif was end-labelled in the early strand and partially methylated. It was incubated with nuclear extracts from non-induced (N) or ILI treated (I) cells. In the latter case GTP was added. Free (F) and bound (B) DNA molecules were cleaved with piperidine and run on a sequencing gel. On the right the quantified and normalized signals from two experiments are shown. By densitometry signals from bound DNA were compared to corresponding signals from free DNA. A guanine outside the TC-II motif (4) served to correct for differences in the amount of DNA loaded.

and the P motifs augment baseline expression and, in addition, confer strong ILl responsiveness. Induction via the TC-fl motif is faster than the pGl.B response and not inhibited by cycloheximide. Kovacs et al. (1986) have reported that ILl increases c-fos transcription in normal human T cells. Since the present manuscript was submitted, Muegge et al. (1989) have published that ILl induces transcription of c-jun but not c-fos in the murine T lymphoma line LBRM-331A5. Both effects can lead to an increased activity of AP-1 (Chiu et al., 1988; Rauscher et al., 1988; Sassone-Corsi et al., 1988), and it seems likely that the ILl response of the P motif in PC60 occurs via one of these pathways. Muegge et al. (1989) found that in LBRM cells a promoter containing three AP-1 sites shows a 3.4-fold response to ILl. Note that the Octamer motif which accounts for most of the SV40 enhancer activity in myeloma cells (Fromental et al., 1988) is inactive in non-induced PC60 cells, but we could detect expression of the plasmid carrying this motif after ILl treatment. In band shift experiments we have observed an increase in an Octamer binding protein in cells exposed for 18 h to ILl (results not shown). Conversely, the GT-HC+GT-I region increases baseline transcription in PC60. This enhancement is still observed when the GT-IIC motif is mutated (GT-HICm+GT-I) and thus is probably due to GT-I. Notably, the GT-IICm+GT-I sequence has no enhancer activity in HeLa, MPC 11 myeloma or F9 embryonal carcinoma cells, although the GT-I motif cooperates with GT-IICwt to form a potent enhancer in HeLa and F9 cells (Fromental et al., 1988). Neither GT-IIC nor GT-I confer ILl responsiveness. In fact, ILl reproducibly decreases expression of the plasmids containing an intact GT-I motif. 934

Analysis of the expression of the pA plasmids in which the ,B-globin gene is transcribed from the SV40 early promoter confirms the conclusions drawn above, but also indicates that other parts of the SV40 enhancer affect expression of these plasmids in PC60. Comparison of the baseline expression of pAO with that of pA56 on the one hand, and of pA 104 and 223 on the other indicates that constitutive enhancer elements between bp 346 and 271 make a much greater contribution to the activity of the whole enhancer than in HeLa cells (Zenke et al., 1986). One motif in this region that may enhance baseline transcription is the purine-rich box identified by Pettersson and Schaffner (1987). Another contribution may be due to the constitutively expressed protein binding to the GT-IIA motif (Figure 2). A role for this protein is suggested by the fact that the deletion in pA233 which extends exactly to the border of this footprint reduces expression to the level observed with pA223. The high constitutive expression of the plasmids containing the segment between bp 346 and 271 (pAO, pA59, pA62) may, at least in part, be responsible for their relatively small response to ILl, compared to that of pA104. The ILl responsive sequences are restricted to the segment from bp 272 to 101 of the wild-type enhancer. Deletion of the TC-II element in pA59 very strongly reduces the ILl response. A predominant role for the TC-II element in the response of the intact SV40 enhancer is also suggested by the comparison of the expression of pA104 and pA223, and by the negligible effect of deleting the P motif (in pA62). The data obtained with the pGl plasmids suggest that the small but reproducible response of pA59 is due to the P element. We could not detect any ILI response of pA223 and pA233, both containing intact P motifs, but their baseline expression is too low to permit detection of a small increase. Analysis of cell surface markers and T cell receptor (TCR) gene expression suggests that the rat thymoma C58 and its derivative PC60 are representative of CD4-CD8- ct/3 TCR+ thymocytes (MacDonald et al., 1988). In adult mice these cells represent 0.3 % of thymic lymphocytes. When triggered with concanavalin A they proliferate in response to IL2, but only if ILI is added as well. This effect of ILl may be due to its stimulation of IL2 receptor light chain expression as suggested by the ILl response of T cell lines like PC60. But it remains to be tested whether ILl activates gene expression in normal CD4-CD8- cxIl TCR+ thymocytes via the TC-H or the P motif. Previous work from other groups has shown that the promoter of the human IL2 receptor light chain gene contains a binding site for NF-xB that mediates stimulation of the expression of this gene by the HTLV-I protein tax, (Leung and Nabel, 1988; Ruben et al., 1988). However, the role of this motif in the response of the IL2 receptor light chain gene to other stimuli that induce NF-xB is controversial (Bohnlein et al., 1988; Leung and Nabel, 1988; Cross et al., 1989). -

Analysis of DNA binding proteins The transcriptional response of a promoter containing TC-II motifs to ILl correlates with the appearance of a nuclear protein that shares with NF-xB specificity, response to cycloheximide, sensitivity to GTP and mobility on nondenaturing gels. For the remainder of the discussion we will refer to this protein as NF-xB. In addition to its interaction with NF-xB the TC-II motif forms complexes with other proteins present in similar

ILl responsiveness of SV40 enhancer motifs

amounts in extracts of untreated and ILl treated PC60 cells. The same complexes are formed by nuclear mini-extracts of C58 and B6. 1, the parental cells of PC60, but not by extracts from 70Z/3, prepared by the same method. Although the TC-II motif in the SV40 enhancer overlaps with binding sites for transcription factors AP-2 and AP-3 our experiments render any role of these factors in the formation of the TC-ll complexes very unlikely. In fact, these complexes are due to proteins with a specificity indistinguishable from that of NF-xB. The competition and methylation interference experiments make it improbable that the band shifts obtained with extracts from non-induced cells are due to H2TF1, a protein constitutively expressed in a number of cell lines (Nomiyama et al., 1987) and probably identical to KBF 1 (Yano et al., 1987). Additional arguments against this possibility are that H2TF1 is not sensitive to nucleotides (Lenardo et al., 1988), and gives rise to a sharp bana that migrates faster than NF-xB complexes (Macchi et al., 1989). HeLa cells contain a factor, named EBP1, that recognizes a sequence in the TC-II motif (Clark et al., 1988). The relationship of this factor to NF-xB and H2TF1 is, at present, not clear, but it may be a different protein with similar specificity. We cannot exclude that the bands formed by PC60 extracts in the absence of nucleotides are due to EBP1. However, extracts from HeLa cells, prepared and tested under identical conditions, do not give rise to similar complexes (not shown). Thus, PC60 cells contain, besides NF-xB, other DNAbinding proteins with very similar specificity. These proteins may be T cell specific as we have found them, so far, in two T cell lines (C58 and B6. 1) but not in the pre-B cell line 70Z/3 or in HeLa cells. The striking correlation between the nucleotide-induced appearance of the band due to NF-xB and the reduction of the constitutive TC-H complexes in extracts from IL1 treated cells suggests that the latter are related to NF-xB and that nucleotides transform the proteins giving rising to these complexes into NF-xB. We are presently attempting to test this hypothesis. Mechanism of action of IL 1 The appearance of NF-xB or a similar protein in the nucleus is induced in different cell types by a number of extracellular stimuli (see Introduction). In those cases where this has been tested this response is independent of the synthesis of new proteins. Recent work has shown that in cells which do not contain NF-xB in their nucleus the protein is present, in a form which has no detectable DNA-binding activity, in the cytoplasm, coupled to another molecule, called IxB (Baeuerle et al., 1988a,b). Active NF-xB can be recovered from the complex by mild denaturation. Treatment of cells with PMA or cycloheximide induces translocation of NF-xB into the nucleus, but it is not clear whether the only effect of these reagents is the dissociation of NF-xB from its cytoplasmic inhibitor, or whether other changes are required to reveal its DNA binding activity. In any case, evidence that natural stimuli induce appearance of NF-xB by the same mechanism has so far only been published for the case of viral infection of L929 cells (Lenardo et al., 1989). Many natural stimuli may only induce translocation of a small fraction of cytoplasmic NF-xB into the nucleus and comparison of NF-xB activity in nuclei treated with ILI or with cycloheximide (Figure 3A) indicates that ILl does not induce a quantitative translocation of NF-xB into the nucleus

of PC60 cells. This was confirmed by experiments in which NF-xB activity was measured in deoxycholate plus formamide treated cytoplasmic extracts of these cells (not shown). In addition, the same stimulus may well activate NF-xB via different pathways in different cell types. This possibility is suggested by a comparison of the ILl response of PC60 and of the pre-B cell line 70Z/3. Recent reports suggest that in the latter ILl activates NF-xB by raising cAMP levels (Shirakawa et al., 1989). In PC60, however, ILl does not trigger a detectable increase in cAMP concentration (R.Solari, personal communication). Interestingly the same appears to be true for Jurkat, another ILl responsive T cell line (Shirakawa et al., 1988). Our interpretation ofthe gel retardation assays wih PC60 extracts suggests that in these cells the principal response to ILl may not be translocation of cytoplasmic NF-xB into the nucleus, but activation of NF-xB molecules that are already in the nucleus. This activation would result in a different sensitivity to nucleoside triphosphates. Whereas both inactive and activated molecules can bind their cognate DNA motif in the absence of nucleotides, inactive molecules lose their binding activity after a conformational change induced by nucleotides.

Materials and methods Cell cultures and transfections The PC60.21.14.4 cells have been described in previous publications (Conzelmann et al., 1982; Silva et al., 1983). They were transfected according to Queen and Baltimore (1983) with the indicated amounts of plasmid DNA in 20 1Lg/ml DEAE-dextran (Pharmacia). After (330 h culture an excess (final concentration 1 ng/ml) of human recombinant ILl (a gift of Glaxo) was added to half the cells. Four or 18 h later cells were harvested. In some experiments cycloheximide (200 mg/ml) was added 30 min prior to ILl. In the initial experiments we extracted total RNA with the guanidinium isothyocyanate-acid phenol method, according to Chomczynski and Sacchi (1987). As we found it was difficult to remove all plasmid DNA from such preparations we later switched to extraction of cytoplasmic RNA using NP-40 (Ausubel et al., 1976). Plasmids Plasmids were extracted either by standard methods and purified on CsCl gradients. Alternatively they were prepared by a method developed by A.N.Shakov. Briefly, bacteria from a 200 ml culture are treated with lysozyme, followed by SDS in NaOH. After precipitation with 5 M potassium acetate 1/2 volume (- 13 ml) 40% polyethylene glycol (PEG) (mol. wt 6000) in H20 is added to the supematant. Following a 30 min incubation on ice the precipitate is spun down for 30 min at 3000 g and dissolved in 4 ml H20. Two volumes of 7.5 M NH4-acetate are added and the solution incubated for 30 min at -20°C. After a 30 min spin at 10 000 r.p.m. the supernatant is extracted with 1 vol chloroform-phenol. Nucleic acids are precipitated from the aqueous phase with isopropanol, redissolved in 600 i1 TE and transferred to an Eppendorf tube. PEG (40%; 200 ILI) in 2.5 M LiCl is added. The tube is incubated for 30 min at room temperature and spun for 10 min. The supematant is carefully removed and the pellet washed twice with ethanol. In transfection experiments plasmids prepared in this manner gave the same results as CsCl gradient purified preparations. The pA and most of the pG1 plasmids have been described previously (Zenke et al., 1986; Fromental et al., 1988; Kanno et al., 1989) (see also Figure 1). pGl.P(SV40) and pGl.P(SV40)m are described in Figure 1. The sequences of the DNA preparations were checked before transfection. Plasmid pSPTA was constructed by insertion of the 500 bp Asp7l8-TaqI fragment of pA56 into Asp7l8 and AccI digested SPT19. This fragment contains the SV40 promoter joined to the -9/ + 307 fragment of the rabbit start site (EES) in (3-globin gene. Spliced transcripts from the early earlywhereas processed pA plasmids will protect -220 bp of this probe, transcripts starting at position + 1 of the (-globin gene in pGl plasmids will give a protected fragment of 144 bp. A plasmid containing a single copy of the TC-ll motif (pSPT-TCII) was prepared by cloning the oligonucleotide gatccTGTGGAAAGTCCCCAGg into the BamHI site of SPT18.

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E.Espel et al. RNase protection

Transcripts derived from the transfected plasmids were detected by RNase protection essentially as described by Zinn et al. (1983), using as probe 32P-labelled T7 transcripts from EcoRI digested pSPTA. The protected probe fragments were separated in 6% acrylamide/urea gels and quantified by densitometric scanning of suitably exposed autoradiographs. The signal of transcripts from pG 1 plasmids was standardized by comparing it to the signal from 15 Ag of co-transfected pA56. Expression of pA plasmids was standardized by comparison with the signal from co-transfected pG1.TC-30. We compared baseline expression (in the absence of ILl) of different pGl plasmids to that of pG 1 .B, and of pA plasmids to that of pA56 (see Figure IA). As expression of pA56 is not affected by ILl, inducibility of pGl plasmids is equal to the ratio between the standardized expression in ILl treated and in control cells. Inducibility of pA plasmids was calculated by multiplying the ratio between the standardized expression in ILl treated and control cells with the inducibility of pGl.TC-30 as determined in the same

experiment by co-transfection with pA56.

Nuclear extracts and gel-retardation assays Exponentially growing PC60 cells were induced by addition of an excess of recombinant human IL1I3 during 4 h or for the times indicated. Where mentioned cycloheximide was added to a final concentration of 20 sg/ml, 10 min before addition of ILl. Nuclear extracts were prepared either according to Shapiro et al. (1988) or according to Schreiber et al. (1989) (mini-extracts). Binding reactions were carried out essentially as described by Sen and Baltimore (1986a). Plasmid SPT-TCII was linearized with EcoRl, end-labelled and cut with HindIII. The TC-ll fragment was purified on a polyacrylamide gel. In each binding reaction 0.7 Mg nuclear extract was incubated in 5 mM Tris (pH 8.0), 5 mM HEPES (pH 7.9), 25 mM KCI, 25 mM NaCl, 0.5 mM dithiothreitol (DTT), 0.4 mM EDTA, 5% glycerol, 1.0 Mg poly(dI.dC).poly(dI.dC) (Pharmacia) and 5.0 Mg BSA in 5 td. After 15 min on ice GTP and unlabelled competitor DNA were added. Five min later 0.2 -2.0 ng of probe was added. The reaction mixture (final volume 8 Ml) was incubated for 30 min at 4°C and fractionated by electrophoresis on a non-denaturing 4% gel in 0.5 x TBE at 9 V/cm during 2 h. DNase I and DMS footprints The SV40 enhancer was cut out from pAO with NcoI, the isolated fragment was labelled either at its 3' ends with Klenow DNA polymerase, or its 5' ends with T4 polynucleotide kinase. To obtain probes labelled only at one end, the insert was digested with HaeIII and the HaeHII(331)-NcoI(40) fragment (BBB nucleotide numbering system; Tooze, 1982; Zenke et al., 1986) isolated. For each reaction 25 Mg nuclear extract and 3 ng of probe were incubated in a total volume of 10 Ml containing 3 mM Tris (pH 8.0), 14 mM HEPES (pH 7.9), 68 mM KCI, 7 mM NaCl, 0.4 mM EDTA, 1.4 mM DTT, 11% glycerol and 1.5 Mug poly(dI.dC).poly(dI.dC). The incubation procedure was the same as for gel retardation assays. Prior to addition of DNase I or DMS, reactions were preincubated for 1 mmn at 20°C. For DNase I footprints 6 U (in 1.5 ul containing 20 mM MgCl2) of enzyme (Worthington) was added and the sample incubated for 1 min at The reaction was stopped and samples were phenol extracted. For 20°C. DMS footprints, 1.5 M1 of 5% DMS in water were added and the mixture incubated for 1 min at 20°C. The reaction was terminated, and the nucleic acids extracted and incubated in 1.0 M piperidine for 30 min at 90°C. DNA was analysed by fractionation on 6% sequencing gels. DNA methylation interference was performed as described previously (Sen and Baltimore, 1986a).

Acknowledgements We would like to thank P.Chambon for suggesting to us the use of the pG I plasmids. J.-A.Garcia-Sanz helped us to set up the RNase protection assay. The protocol for plasmid preparation with PEG was developed by A.N.Shakov. Claudine Ravussin provided secretarial assistance, and P.Dubied and M.Allegrini prepared the figures. R.Solari permitted us to quote his unpublished results. Martine Collart provided us with a sample of 70Z/3 nuclear extract and 70Z/3 cells. Glaxo Institute for Molecular Biology SA, Geneva, supplied us with generous amounts of recombinant ILl. This work was supported in part by grants from the Swiss National

Science Foundation to M.N.

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