Nucleic Acids Research, 1992, Vol. 20, No. 23 6235-6238

.!.) 1992 Oxford University Press

Transcriptional regulation by folate: inducible gene expression in Dictyostelium transformants during growth and early development Jurgen Blusch, Piero Morandini+ and Wolfgang Nellen* Max-Planck-Institut fOr Biochemie, Abt. Zellbiologie, 8033 Martinsried, Germany Received September 22, 1992; Revised and Accepted November 11, 1992

ABSTRACT The Dictyostelium discoidin genes are induced in bacteria-grown cells shortly before the onset of development but are also highly expressed during growth in axenic medium. We here show that axenically growing cells strongly respond to the extracellular signal folate by suppressing discoidin synthesis while cell growth and development is not substantially affected. Repression occurs via two previously identified promoter elements, the dIE and the dAXE. Removal of the signal molecules or setting cells up for development results in rapid reactivation of the promoter. Based on this observation, we constructed the transformation vector pVEII and describe a convenient method which allows for controlled expression of a gene of interest in growing cells and also for external modulation in early development. Deletion constructs of the discoidin promoter can be used in addition to vary transcriptional activity over about one order of magnitude. INTRODUCTION Transformation is being widely used in Dictyostelium to study the function of proteins either by overexpression (e.g. 1, 2) or by down-regulation via antisense RNA (e.g. 3, 4, 23). In addition, the application of Dictyostelium for the production of bioactive substances has recently been successfully initiated (5, 6). In many cases it is desirable to control the expression of the gene of interest by exogenous signals. This can be achieved by using promoters which are only activated in late development in specific cell types (e.g. 7) or promoters which can be activated by cAMP in early development (e.g. 8, 9). Except for an elegant, though complicated system based on induction of a suppressor tRNA (10), all inducible expression systems in Dictyostelium so far depended on the initiation of development and there was no possibility to modulate expression in growing cells. When cells grow by phagocytosis of bacteria as they usually do under natural conditions, the discoidin genes are induced some time before the onset of starvation by the extracellular factor PSF To whom correspondence should be addressed + Present address: MRC, Molecular Biology Laboratories,

*

Cambridge, UK

(11). At approx. 9 hrs of development they are down-regulated by cAMP. In axenically growing cells the genes are, however, highly active even in early log phase. This is, at least in part, due to a more sensitive response to PSF under these conditions. Based on observations by Alexander et al. (12) and by us (Blusch and Nellen, unpublished), we analyzed the response of axenically growing cells to the signal molecule folate. A transformation vector and appropriate conditions are described which allow for reversible down-regulation of the discoidin I gamma promoter in axenically growing cells. This provides a tool for controlled transcription of a gene of interest during growth, for transformation with potentially deleterious genes and for comparison of the 'on' and 'off' stage of antisense constructs under nearly identical culture conditions.

MATERIALS AND METHODS RNA preparation and Northern blots were as described by Maniak et al. (13) and CAT assays as specified in May et al. (14). Dictyostelium AX2 cell lines stably transformed with the PAV-CAT-Disc promoter deletion constructs described by Vauti et al. (15) were used. Cells were grown in AX medium containing 20 jig/ml G418 and developed in suspension culture (15). Gene repression was done by adding folate (Fluka) or cAMP to a final concentration of 1mM to cells growing in AX medium. Over a period of three days, cells were diluted daily with AX medium and fresh folate or cAMP was added. Cell density, as measured by counting in a Neubauer chamber, was always kept below 106 cells/ml. For monitoring morphogenesis during development, cells were plated at a density of 2.5 x 106 cells/cm2 on agar containing 17mM phosphate buffer and examined at different times after the onset of starvation. Western blots were done as described (16) using the monoclonal antibody 80-52-13 (17, Wetterauer et al. submitted) directed against discoidin I. The vector pVEII was constructed by replacing the actin6 promoter in pDNeo2 (18) by the discoidin I gamma promoter (19). Due to the multistep assembly, there are some differences in the polylinker region and in the framework of the vector

6236 Nucleic Acids Research, 1992, Vol. 20, No. 23 compared to pDNeo2. Important restriction sites and the arrangement of functional parts are shown in Fig. 1. The polylinker for inserting the gene of interest contains single restriction sites for XbaI, BamHI, KpnI, and Sacl downstream of the ATG. To confirm the reading frame of the insert, the oligonucleotide 5'-GAAAA ATTAA AATTT CATAC AAATT ATC-3' from -52 to -25 in the discoidin I gamma untranslated region was used for sequencing.

RESULTS The promoter analysis vector PAV-CAT-Disc-411 contains the smallest (41 lbp) discoidin promoter fragment which still shows complete regulation in response to starvation and cAMP (15). In comparison to pVEII (Fig. 1) which harbours a 1.2 kb promoter fragment, PAV-CAT-Disc411 displays less than 25% of promoter activity. We have tested this and other promoter constructs on their response to cAMP and folate in axenic growth medium. Both substances down-regulate transcription from the complete promoter and folate also from all promoter deletions used. We have focussed on the effects of folate as the more potent, inexpensive and convenient additive to repress promoter activity. Figure 2a shows CAT expression of PAV-CAT-Disc-411 transformants in cells grown at a density below 106 for three days in standard axenic medium and in axenic medium supplemented with 1 mM folate. Cells were then washed and either resuspended in fresh medium without addition of folate or, to initiate development, in 17 mM phosphate buffer. The data demonstrate that CAT activity is strongly down-regulated when

folate is added. Over night culture in fresh axenic medium results in a complete reinduction of CAT expression to the levels seen in cells continuously grown in standard AX medium. When cells grown in the presence of folate are set up for development in suspension culture, the -411 promoter is induced after Shrs, a time similar to developmental promoter activation after growth on bacteria. Cells grown in axenic medium without folate, show high expression and no further induction of CAT activity in development. The difference between the induced and the noninduced state is even stronger when total cellular protein is assayed with an antibody against the endogenous discoidin I proteins: There is only a minor signal in the folate treated cells while in development or after axenic growth without folate, discoidin expression is high (Fig. 2b). Similar, but less pronounced effects are seen when cells are grown only over night instead of three days in the presence of folate (data not shown). The data show that both, the minimal -411 promoter and the complete endogenous promoter respond to the addition of folate to the growth medium by suppressing gene activity. Measurements on the level of protein expression may obscure the results due to differential protein stability. We therefore prepared RNA from cells treated as described above and hybridized with CAT and discoidin I specific probes. Fig. 3 demonstrates that both transcripts are down-regulated to very low or undetectable levels in the presence of folate. The effect of over night treatment (not shown) is more pronounced on the RNA level than on the protein level: after about 10 hrs of growth with

6161

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Figure 2. PAV-CAT-Disc-411 transformants were grown for 3 days in axenic ...

XbaI BamHI KpnI SacI ATTTATAAA ATG TCT AGA GGA TCC CCG GGT ACC GAG CTC G....

Figure 1. Circular map of the transformation vector pVEII. The sequence of the polylinker in respect to the AUG initiation codon is shown below.

medium without (- folate) and with (+ folate) the addition of 1mM folate. Cell density was always kept below 106 cells/ml. Aliquots were removed for CAT and Western analysis and cells were washed and either resuspended in fresh AX medium (AX -) or set up for development in suspension culture (dev). a) CAT assay showing activity of the -411 CAT construct. CAT activity is given as arbitrary units based on the linear range of the enzyme reaction. b) Western blot showing activity of the endogenous discoidin I genes.

Nucleic Acids Research, 1992, Vol. 20, No. 23 6237 folate, CAT mRNA is barely detectable. Surprisingly, the developmental induction of the CAT gene is much stronger on the RNA level than expected from the enzyme activity measurements. Probably, this is due to reduced stability of the CAT protein during development. We also noted that CAT mRNA is more strongly expressed in axenic medium than discoidin mRNA. This is most obvious in Fig. 3, lane 3 (+ folate, AX -), but also in lane 6 (- folate, AX -). We then tested further promoter deletions on their ability to respond to folate. We have previously shown that promoter fragments shorter than -398 lack the developmental induction element (dIE) and are only active after growth in axenic medium but not after growth in bacterial suspension. Between -271 and -192 we had identified an element necessary for repression by cAMP (dNCE) and downstream of - 135 a further regulatory sequence (dAXE) sufficient for promoter activity in axenically growing cells (15). Fig. 4 shows that even the smallest active promoter fragment (-135) is still down-regulated, though less efficiently, than the -411 promoter. In agreement with these data, we find a low but significant (approx. 1.8fold) repression with the dIE in the context of the heterologous AlSdelBam promoter (15, data not shown). Using the appropriate constructs, expression levels in the induced state can thus be modulated between 100% and 5% (complete 1.2kb promoter and - 135 promoter respectively, see 15) without loosing the possibility to down-regulate by folate. To determine potential effects of folate on growth and development, the generation time in axenic medium with and without folate was monitored over three days and was found to be 8 hrs under both conditions. Cells grown in the presence and absence of folate for three days were plated on non-nutrient agar to examine morphogenesis during development. Treated cells were delayed by about 4hrs but completed the developmental cycle with no apparent abnormalities (not shown). Folate can therefore be used without any adverse consequences on cell growth and morphogenesis. The data demonstrate that the discoidin promoter can be efficiently employed to obtain inducible expression of genes in growing cells. We therefore constructed the expression vector +folate XSt

3.u4 X

-folate X

pVEII which contains the 1.2kb discoidin I gamma promoter fragment (15, 19), a multiple cloning site and the actin8 double terminator (21), the actinl5-Tn903 resistance cassette (20) serves as a selectable marker. The promoter carries the discoidin ATG start codon and single XbaI, BamHI, KpnI and SacI sites for cloning sequences of interest (Fig. 1). Our previous promoter deletion constructs (15) have shown that sequences upstream of -411 contribute only quantitatively to promoter activity under the conditions described here. If lower transcription activity is required, the CAT gene coding sequence can be removed from these vectors with an XbaI/NcoI digest and be replaced by the sequence of interest. Promoter deletions down to -411 can be down-regulated by cAMP in suspension development and, to a lower degree also during growth. This and our observation that cAMP and folate apparently function synergistically during growth (data not shown), provide further means to modulate expression.

DISCUSSION We have presented a tool for inducible expression of RNA and proteins during growth of Dictyostelium cells using the discoidin I gamma promoter and folate for repression. Being present in the normal environment of Dictyostelium, folate is expected to have a natural signal function rather than to non-specifically disturb cell behaviour and we have shown that growth and development proceed normally when cells are grown in the presence of folate. Folate may mimic the presence of bacteria and thus bring cells closer to the vegetative state than standard axenic medium. In axenically grown cells, several early developmental genes are already induced while they are, similar to the discoidin genes, silent in bacteria-grown cells. Thus, the observed delay in developmental gene expression after folate treatment could represent the difference between 'preinduced' axenically growing cells and cells grown on bacteria or folatesupplemented medium, which start the developmental cycle at time zero. Axenically growing cells respond to the PSF signal at much lower densities than cells grown on bacteria (11, 22). Folate is constitutively produced by bacteria and rather unstable. It therefore may be the substance which, in conjunction with PSF, enables Dictyostelium cells to compare their own population density with that of the bacterial food source.

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Figure 3. RNA was prepared from aliquots of the same samples used in Fig. 2 and separated on 1.2% agarose gels containing formaldehyde. After transfer to Biodyne A, the filter was first probed with a CAT specific in vitro transcript (CAT) and then with a discoidin specific transcript (disc.).

Figure 4. Promoter deletions -411, -271, -192 and -135 were tested on their response to folate. Repression factors were calculated by dividing CAT activity in cells grown without folate and CAT activity in cells grown in the presence of folate for three days.

6238 Nucleic Acids Research, 1992, Vol. 20, No. 23 We have previously described two independent promoter elements, the dIE and the dAXE which stimulate discoidin expression (15). Only the dIE but not the dAXE is activated by PSF (Nellen and Saur, unpublished, Nellen and Clarke, in preparation). Under axenic growth conditions, expression is induced by both elements, arguing for a PSF dependent and a PSF independent process. Folate represses CAT activity from the -411 promoter, which contains the dIE, about 6 fold but only about 3.5 fold from shorter promoter fragments lacking this element (Fig. 4). This indicates that the signal functions on both, the dIE and the dAXE which is confirmed by the observation that the dIE alone confers a negative response to folate in the context of a heterologous promoter (not shown). The data in Fig. 4 show that the negative cAMP element dNCE, located between -192 and -271 (15), has no effect on repression by folate. Deletion of the dNCE does, however, abolish downregulation by cAMP during growth (data not shown). It should be emphasized that the repression factors in Fig. 4 are calculated from CAT activity. Comparison of Fig. 2a and b demonstrates that different proteins can have different half lifes under different developmental conditions. CAT and discoidin serve as examples, other proteins may display other variations in stability. The essential prerequisite for controlled gene expression is, however, the strong down-regulation of mRNA. The developmental induction of both, CAT and discoidin mRNA is seen in treated and untreated cells. While this is clearly reflected in the expression of discoidin protein, CAT activity is significantly less affected: after growth with folate, there is only a moderate increase and no additional activation is observed after growth in AX medium without folate. As we have previously suggested (9), this is probably due to decreasing stability of the CAT protein during development compared to discoidin. The inducibility of the discoidin promoter by PSF (11, 22, Nellen and Clarke in preparation) implies that this signal can also be used to control expression from pVEII and similar vectors. In fact, Clarke and coworkers have used PSF to induce expression of a calmodulin antisense fragment in pVEII to study the effects of calmodulin depletion in Dictyostelium cells. (23). The possibility to down-regulate activity of the discoidin promoter during growth is an excellent tool for the expression of sequences which are potentially harmful for the organism. This could be genes encoding toxic products or antisense constructs interfering with the expression of vital proteins. Since growth is normal in the presence of folate, cells can be prepared, transformed and selected under conditions where the discoidin promoter is repressed. One has to take care, however, to keep the cells below 106/ml since higher densities override the downregulation by folate (data not shown). Reinduction of genes by transfer of cells to folate-free medium is unexpectedly higher for the -411 promoter-CAT construct than for the endogenous discoidin genes (Fig. 3, lane 3 and 6). We do not know the reason for this but it could be due to a yet undetected negative regulatory element upstream of -411 which reduces expression in axenic medium. In addition, it has to be taken into account that the discoidin probe recognizes transcripts from all members of the discoidin I gene family which are slightly different in their regulation (24, 15). For the purposes discussed here, the stronger induction of the -411 promoter is advantageous for an inducible system.

Inducible expression will prove useful to examine the effects of protein overexpression or antisense-mediated down-regulation since it allows for a direct comparison of the 'on' and 'off' state

of the promoter within the same strain and under very similar growth conditions. The discoidin promoter system could also be advantageous to employ Dictyostelium for the production of biologically active substances. A recently isolated discoidin overproducer mutant (Wetterauer and MacWilliams, submitted) may allow for the translation of introduced genes to up to 10% of total cellular protein. Such high amounts of a foreign protein are often deleterious for the cell and down-regulation of the promoter by folate may circumvent this problem. An additional advantage is that promoter repression does not interfere with developmental induction. It is thus possible to grow large amounts of cells under repression conditions and then induce production by removal of the growth medium and development in phosphate buffer.

ACKNOWLEDGEMENTS We thank G.Gerisch and B.Wetterauer for providing the mAB 80-52-13. We also thank M.Clarke, B.Wetterauer, H.MacWilliams and S.Alexander for stimulating discussions and for communicating results prior to publication. J.B. is a recipient of a Boehringer Ingelheim Fonds grant. This work was supported by grants of the European Community and the DFG (SFB 190) to W.N.

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