.=) 1992 Oxford University Press

Nucleic Acids Research, Vol. 20, No. 11 2795-2802

A negative transcriptional control region of a developmentally-regulated gene co-localizes with the origin of replication of an endogenous plasmid in Dictyostelium Jo Anne Powell, Jose Galindo and Richard A.Firtel* Department of Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634, USA Received February 13, 1992; Revised and Accepted April 22, 1992

ABSTRACT The endogenous nuclear plasmid Ddpl from the wildtype Dictyostelium discoideum strain NC4 has been cloned, its origin of replication has been localized (1,2), and plasmid-encoded genes have been mapped that are preferentially expressed during growth or development (3). Here we present an analysis of the regulation of the Ddpl -encoded gene d5, which, in wildtype cells, is expressed only during the multicellular stages of development. In this study, we show that sequences 3' to the d5 coding region are required to suppress constitutive expression of d5 from aberrant transcriptional start sites and that this regulatory region acts at a distance and in an orientation-independent manner. The cis-acting negative regulatory element(s) necessary for repression of aberrant d5 expression is either very tightly linked or identical to sequences required for extrachromosomal replication, such that all 3' deletions that cause constitutive d5 expression result in the integration of the plasmid into the D. discoideum genome. Placing d5 (without the 3' regions containing the Ddpl origin) on an extrachromosomal vector based on another endogenous plasmid (Ddp2) did not restore proper transcriptional regulation, suggesting that an extrachromosomal environment alone is not sufficient to confer proper transcriptional regulation to d5.

INTRODUCTION The cellular slime mold Dictyostelium discoideum grows vegetatively as individual ameobae. Upon starvation, the cells aggregate and undergo a defined pattern of multicellular development that culminates in the formation of a fruiting body containing spores and stalk cells. Dictyostelium is amenable to molecular manipulations and has proven to be a good system for studying basic eukaryotic processes such as chemotaxis, signal transduction, cellular differentiation, and developmental gene regulation (4,5).

*

To whom

correspondence

should be addressed

D. discoideum is one of the few eukaryotic organisms known to harbor endogenous extrachromosomal plasmids. Plasmids have been found in all wild-type strains that have been examined and range in size from 5.6 kb to 27 kb. The copy number of the plasmids varies, and molecular analysis has indicated that the different plasmids share no detectable sequence identity to each other or to chromosomal DNA as determined by standard hybridization techniques (6,7,8). However, substantial amino acid sequence identity has been found between a gene on the D. discoideum plasmid Ddp2 (9) and one on a plasmid from a related species (10). One of the best characterized of these plasmids is Ddpl, a 12.5 kb plasmid found at 50-100 copies per cell in the two wild-type strains NC4 and V12 (7). Ddpl is organized in chromatin and is localized in the nucleus (11). However, Ddpl was lost upon creation of the NC4-derived axenic laboratory strains KAx-3 and Ax2. Vectors carrying a cloned copy of Ddpl and an actin-neomycin resistance gene fusion (to confer G418 resistance) can be used to transform these axenic strains with high efficiency (12). These vectors replicate extrachromosomally and are stably maintained in transformants, even in the absence of G418 selection. The region containing the origin of replication has been identified and sequenced, and other elements that affect copy number and plasmid stability have been described (1,13,14). Possible developmental or regulatory roles of Ddpl and the genes it encodes are unknown. Ddpl is not essential for growth or development since axenic strains lacking the plasmid grow and develop normally. A transcription map of Ddpl has been constructed (3). Some of the Ddpl transcripts are preferentially expressed during growth, while others are induced during development. None of the transcripts is required for the stable replication of cloned Ddpl shuttle vectors in standard axenic laboratory strains of D. discoideum (1). This distinguishes Ddpl from Ddp2, which requires a plasmid-encoded trans factor for replication (9). In our studies of the maintenance and regulation of Ddpl, we have focused on one gene, dS, which has been shown to be preferentially expressed in later development. The dS gene is of interest because it maps very close to the origin of replication

2796 Nucleic Acids Research, Vol. 20, No. 11 (1,3) and is expressed at approximately the same time as many of the chromosomally-encoded late genes that are induced by cAMP (15-18). The gene has been sequenced, but homology searches revealed no clues to d5 function (13, 14). To gain insight into the possible role of dS and its developmental regulation, we have undertaken a molecular analysis of the transcriptional regulation of this gene. In this manuscript, we show that dS is inducible by cAMP and demonstrate that sequences 3' to the translational start are required for detectable induction of dS expression late in development. We have further localized the Ddpl origin of replication, which includes sequence directly 3' of the dS coding region. Our results suggest that the replication sequences co-localize to a negative regulatory element (or elements) that plays a role in promoter selection and temporal pattern of dS expression.

MATERIALS AND METHODS Growth and development of D. discoideum Wild-type NC4 and the derived axenic strains KAx-3 and Ax-2 were used in these studies. NC4 cells were grown shaking in E. coli B/r that had been grown to saturation in L broth and then washed and concentrated two fold in 12 mM Na+/K+ phosphate buffer, pH 6.1. Axenic cells were grown in standard HL5 culture medium. Logrithmically growing cells at a density of 1-3 x 106 cells/ml were harvested for development and, in the case of NC4, freed from bacteria by repeated low-speed centrifugations. Cells were washed 3 times and resuspended in cold 2mM KCI, 1.2mM MgSO4, 12mM K2HPO4, 13mM H2PO4, pH6.1 (PDF) and then plated for development on Whatman 50 filters (5 x 106 cells/filter) suspended over buffer as described previously (19) Axenic cells were transformed by standard calcium phosphate or electroporation procedures, and transformants were selected for G418 resistance (20,21,22).

Construction of plasmids The construction of the Ddpl-based extrachromosomal vectors Ddpl-20 and C1KSBB was described previously (1). The dS-containing KpnI-EcoRl Ddpl fragment (see Figure 2) was cloned into pSP73 (Promega). It was excised as a BgflI-Sall fragment and cloned into the BamHI and SalI sites of BIOSX, a previously described vector carrying the actin-neomycin resistance fusion gene (12), to make pC2KRR. The dS KpnI-PstI fragment was cloned into pSP73 to form pC2KP73. This vector was then digested with Clal, which cut once in the Ddpl sequence and once in the polylinker. The vector was then religated to exclude the KpnI-ClaI fragment 5' of dS, making pC2CP73. pX2KP was made by digesting pC2KP73 with BgIII and SphI and cloning the d5-containing fragment into the BamHI and SphI sites of pnDeA I (9,23), a shuttle vector based upon the endogenous plasmid Ddp2. Similarly, the Ddpl sequences were cut out of pC2CP73 with BgEl and SphI and cloned into pnDeAl to make pX2CP. The d5-containing KpnI-BgllI fragment of C1KSBB was cloned into the KpnI and BamnHI sites of pSP73. This vector was digested with XbaI and SphI. Unidirectional ExoIII deletions were made into the region 3' of d5, and SalI linkers were added to create the C2KBA vectors. Deletion endpoints were determined by sequencing. The Ddpl sequence was excised with BgflI and Sall or, for A8,A 12, and A27 which did not receive Satl linkers, with BglII and XhoI. The fragments were then cloned into the BamHI and Salt sites of BIOSX to make the C2KBRA deletion series.

To make the C2KBRA4APstI-EcoRI and C2KBRA12APstIEcoRI constructs, the region between the PstI and &oRI sites downstream of dS was removed from C2KBRA4 and

C2KBRA12, respectively. The ends were filled in with the Klenow fragment of DNA polymerase I aid were ligated. The 1.4 kb AccI-HindIl genomic fragment containing d5 upstream and 172 bp coding sequence was cloned into pGEM3 (Promega). The d5 sequences were liberated from this vector with BamHI and HindIH and cloned into the BamHI and HindI]m sites of the luciferase expression vector CathLucl3 (21). The resulting vector was digested with Hindu, and the ends were filled in. When the plasmid was religated to form pHAl.4L, the dS-luciferase fusion gene was in the form of a continous open reading frame. pHA2.5L was made by cloning the pC2KP73 BglI-AccI fragment which contains the d5 upstream sequence into the BglI and AccI sites of pHA1.4L. To create the constructs C2KRR+ 1.2 and C2KRR+2.2r, The Ddpl origin was excised from C2KBA12 as a 1.2 kb HindM fragment and was cloned in both orientations into the Bluescript II vector. The origin-containing sequences were then cut out with

A

NC4

Figur 1. d5 expression in NC4 cells and in Ddpl-20 trainsformants. A. RNA blot illustrating d5 expression during a developmiental time course. RNA was isolated from the cells at the time points shown and analyzed as described in the Materials and Methods. For NC4 cells, the aggregation stage was between 6 and 9 hrs. of development in this experiment, whe the Ddpl-20 tNsfoiants reached this stage between 8 and I11 hrs. The RNAs were size-fr-actionated on denaturing formaldehyde gels, transferred to nylon filters, and probed with random primer probe made from the Hindlll fragment internal to the dS coding region (see Materials and Methods for details). B. Induction of d5 expreasimn by high levels of cAMP in NC4 cells. Cells were starved for 6 hrs. in shaking culture. Some cells were then given cAMP to 3O00AM every 2 hrs. (10). Total cellular RNA was extracted from vegetative cells, cells that had starved for 6 hrs., and cells that had starved for I1I hrs. with (+) or without (-) cAMP. RNA extracted from NC4 cells after 18 hrs. of development is shown as a control.

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Nucleic Acids Research, Vol. 20, No. 11 2797

KpnI and XbaI and were ligated into the KpnI and XbaI sites upstream of the promoter in C2KRR. cAMP induction of gene expression Cells were harvested as described above and resuspended at 3 x 106 cells/ml in PDF. The cells were starved for 6 hrs. in fast-shake conditions (17) for 6 hrs. and then exposed to high levels of cAMP for 5 hrs.

dried, and exposed to X-ray film. Prehybidization, hybridization, and wash solutions were as previously described (18). DNA was isolated and analyzed by Southern blot analysis as

described previously (1,22). Linear molecular weight standards were run on all gels, and bulk uncut chromosomal D. discoideum DNA always sized greater than 25 kb. S1 nuclease mapping A Ddpl fragment containing d5 upstream as well as some coding region (-375 bp to 218 bp relative to the endogenous transcriptional start) was cloned into M13. Single-stranded DNA from this construct was used as a template to synthesize continuously-labeled [32p] probes that were complementary to the d5 niRNA. The reaction was size-fractionated, and the probe was eluted from urea-containing polyacrylamide 'sequencing' gels (25). The 'antisense' probe was coprecipitated with 20%g total cellular RNA and was dissolved in hybridization solution (80% formamide, 40mM Na+Pipes, 1mM EDTA, 400mM NaCl). The reactions were heated to 70°C and incubated at 42°C for 6-8 hrs. and then 6-8 hrs. at 37°C. Following the hybridization, the unprotected probe was digested with 200 U/ml SI nuclease (Pharmacia) in 50mM NaOAc, pH4.7, 280mM

RNA and DNA analysis RNA was extracted from cells by lysing them in 50mM Tris pH 8.4 containing 1 % diethylpyrocarbonate by addition of sodium dodecyl sulfate to 1 % and then extracting 3 times with 1:1 phenolchloroform solution. A 0.1 volume of 3M sodium acetate pH 4.7 was added prior to the last extraction. The RNA was then precipitated once with ethanol, once by the addition of LiCl to 4M, and twice more with ethanol (24) RNA was size fractionated on 1.2% agarose gels containing formaldehyde and transferred to nylon membranes (Magnagraph, MSI) with 20 x SSC. Filters were baked and then prehybridized and hybridized at 37°C with [32P]-labeled random primer probes or nick-translated probes. They were then washed, air-

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Figure 2. Map of Ddpl-20 and summary of deletion vector data. The diagram at top is a map of Ddpl-20 (1) in which the dS coding region is represented as a clear box and the endogenous transcriptional start site is indicated with an arrow and + 1. The restriction sites shown are HindIII (H), KpnI (K), Pstl (P), and EcoRI (R). The HindIII sites are not indicated on the smaller maps in the table. The reference table shows the Ddpl-derived sequence that is in a given vector and how much of the sequence 3' to the dS stop codon is included. The table also lists whether each vector replicates extrachromosomally (E) in D. discoideum or integrates into the genome (I), and it indicates whether the transformants transcribe the gene from the endogenous start site (+ 1) and/or the aberrant sites. The asterisks indicate that these constructs include the Ddp2 origin.

2798 Nucleic Acids Research, Vol. 20, No. 11 NaCl, 5mM ZnS04 at 20°C for 45 minutes. The reactions were run on urea-containing polyacrylamide gels, and dideoxy sequencing ladders were run as markers.

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RESULTS Developmental and cAMP-induced expression of the d5 gene To establish an accurate time course of dS expression in wildtype NC4 cells, RNA was isolated from this strain at different times through development, blotted, and probed with a fragment internal to the d5 coding sequence. As shown in Figure IA, dS is not detectably expressed until 9 hours of development (loose mound stage), and the level of mRNA increases until 18 hours (early culmination). These results are similar to earlier observations (3). Using SI nuclease protection, we have mapped the transcriptional start site of d5 to a cluster of nucleotides 127 bp 5' to the putative ATG translation initiation site (Figure 6), in agreement with the findings of Gurniak et. al. (14). Many Dictyostelium genes that are expressed late in development are induced by cAMP interacting with cell-surface cAMP receptors (5,26), and G-rich regulatory sequences (G boxes) have been implicated in this induction (27). The dS promoter includes a G-rich element 5SObp 5' to the endogenous transcriptional start sites that is similar to the G-boxes required for the cAMP induction of the CP2/pst-cathepsin and CP1 genes (13,14,24,28). To determine whether extraceilular cAMP induces d5 transcription, NC4 cells were starved in shaking culture for 6 hours to allow the cells to express the necessary components of the signal transduction system (5,29) and then exposed to high, constant levels of cAMP, conditions known to induce cAMPinduced prestalk and prespore genes (17). As seen in Figure iB, dS was expressed at extremely low levels in the absense of cAMP. However, the level of d5 mRNA was substantially higher in cAMP-treated cells, although the levels were still low relative to its expression during multicellular development. These results suggest that extracellular cAMP plays a role in regulating the expression of this gene, but other developmental factors may also be essential for maximal expression. While Ddpl is endogenous to the wild-type strain NC4, the plasmid was lost in the formation of the axenic strains KAx-3 and Ax-2. We have previously described the construction of Ddpl-20 in which Ddpl was cloned into the Sphl site of pBR322 (2). This vector also carries an actin-neomycin gene fusion that confers G418 resistance to D. discoideum and acts as a dominant drug-selectable marker (see map in Figure 2). As shown in Figure IA, KAx-3 cells transformed with Ddpl-20 express d5 in the same developmental pattern as NC4 cells, except that it is delayed by 2-3 hours. due to the 2-3 hour slower development of these cells. In both cases, dS mRNA was first detected at the loose mound stage of development.

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Analysis of dS expression in Ddpl-20 deletion constructs To identify the elements required to regulate dS expression, we examined dS expression patterns in a series of Ddpl-20 deletion constructs. The vector C1KSBB contains the dS gene, 2.2 kb of 5' flanking sequence, and 1955 bp of 3' flanking sequence (1; see map in Figure 2). Ahern et al. determined that the 2.2kb HindHI fragment including the 3' end of the dS gene contained all Ddpl sequences required for extrachromosomal replication (1). The C1KSBB vector includes this entire region. As seen in Figure 4, C1KSBB DNA isolated from transformed cells runs as both super-coiled plasmid (higher mobility band) and nicked-

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Figure 3. d5 expression in transformed cell lines. A. Developmental kinetics of d5 expression in transformants. Total cellular RNA was isolated from KAx-3 cells stably transformed with the indicated constructs at the time points shown. The RNA was then blotted and probed as described in the legend to Figure 1. RNA was extracted from NC4 cells after 18 hrs. of development and is shown as a control. The band corresponding to the endogenous transcript is indicated with an 'A'. and the larger, aberrant transcripts are labelled 'B/C'. SI nuclease experiments show that all of the dS mRNA in the CIKSBB transformants is transcribed from the proper start site (data not shown). B. RNA blot illustrating dS expression pattern in transformants carrying plasmids with deletions in the dS 3' flanking region.

circles (slower mobility band). Vectors such as BIOSX (12) that integrate into the genome in tandem arrays co-migrate with the bulk chromosomal DNA (see Figure 4); no ClKSBB is seen to have this mobility. Developmental RNA blot analysis shows that dS is expressed properly in CIKSBB transformed cells (Figure 3A and summary of expression data in Figure 2). The only RNA transcribed from the Ddpl-derived regions of ClKSBB is d5, and thus, no other Ddpl-encoded gene products are required for the regulation of d5 expression. Untransformed KAx-3 cells express no dS-complementary transcripts (data not shown). The sequences of the dS coding region and of 290 bp of 5' and 3300 bp of 3' flanking region has been published previously by other groups (13,14). Our sequence analysis (not shown) of this region is in agreement with that of Gurniak etal. (13). The CIKSBB plasmid includes the entire 2033 bp HindlII fragment that includes the 3' end of the dS gene (14) and the

Nucleic Acids Research, Vol. 20, No. 11 2799

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A negative transcriptional control region of a developmentally-regulated gene co-localizes with the origin of replication of an endogenous plasmid in Dictyostelium.

The endogenous nuclear plasmid Ddp1 from the wild-type Dictyostelium discoideum strain NC4 has been cloned, its origin of replication has been localiz...
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