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First published online 5 January 2006
doi: 10.1242/dev.02190

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The Dictyostelium bZIP transcription factor DimB regulates prestalk-specific gene expression

Natasha V. Zhukovskaya^*, Masashi Fukuzawa^*, Yoko Yamada, Tsuyoshi Araki and Jeffrey G. Williams

School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK.

View larger version (19K): [in a new window]   Fig. 1. The promoter of the ecmA gene. A representation of the ecmA promoter showing the positions of the regions known to direct pstA and pstO specific expression. The minimal region that directs pstO-specific expression is a 132 bp subfragment, located just over 1 kb upstream of the ecmA cap site (Kawata et al., 1996). The structures of the various mutants mentioned in the text are displayed below (see also Fig. 6B legend). The symbol Act15 indicates the basal promoter elements of the actin 15 gene (Pears et al., 1988). The figure also shows the positions and sequences of R1 and R2, the regulatory elements analysed in the present study.

View larger version (86K): [in a new window]   Fig. 2. Purification of proteins that bind to R1 and R2. These are gels of the proteins that bind to R1 and R2 affinity columns, stained with Coomassie Blue. The DimB and DimB' proteins were identified by mass spectrometry. Several of the other proteins, selected by both R1 and R2, are RNA-binding proteins. The asterisked species is MybE (M.F. and J.G.W., unpublished).

View larger version (53K): [in a new window]   Fig. 3. Amino acid sequence alignments. (A) Alignment of DimB with DimA. The sequences of the entire DimB protein and a C-terminal proximal fragment of DimA, DimA*, were aligned using Clustal W. The arrowed line over part of the sequence shows the positions of the proposed DNA-binding and dimerisation domains. (B) Partial alignment of DimB with DimA and other bZip proteins. The indicated segments of the proteins were aligned using Clustal W and the arrowed lines over the sequences show the positions of the DNA-binding and dimerisation domains. Closed diamonds indicate basic residues and open diamonds indicate leucine residues. The other proteins in the alignment are Dictyostelium DimA and human CREB, AP1 and ATF6A.

View larger version (78K): [in a new window]   Fig. 4. In vitro binding of DimB to R2. Full-length DimB:HIS fusion protein was used in band shift assays. The probe is R2 and the unlabelled competitors are R1, R2, a fragment (gatcCTTTTAATGTTAGAAATAGGAGATGAAA) from the promoter of the ecmB gene and a fragment (gatcAAATCCAAACAAAAAAAAAAAATTGATTGTTTTTT) from the promoter of the discoidin 1 gene. The ecmB oligonucleotide is the activator, that binds to DdSTATa and to an unidentified protein by virtue of repeated GAAA tracts (Ceccarelli et al., 2000). The discoidin 1 promoter fragment, the TTG element, is an activator of early gene expression but the binding protein is not known (Vauti et al., 1990).

View larger version (91K): [in a new window]   Fig. 5. Mutational analysis of the binding of DimB to R2. (A) Binding of DimB to an R2 scanning mutant series. The probe is R2 and the unlabelled competitors are scanning mutants of R2. They are labelled M1 to M9 and each contains a four nucleotide substitution, of GCGC, at the indicated position relative to the unmutated R2 sequence. The position of the mutation that produces a major reduction in competition is shown above the R2 sequence. (B) Proposed consensus sequence for DimB binding and comparison with known bZIP binding sites. This is a manually generated alignment of the R1 and R2 binding sites of DimB with that of several bZIP proteins: human AP1 and CREB, fission yeast GCN4 and the two alternate binding sites of budding yeast Pap1 (Fuji et al., 2000). Residues common to at least four out of the seven sequences are in red.

View larger version (67K): [in a new window]   Fig. 6. Mutational analysis of R2. (A) Band shift analysis of the binding of DimB to point mutated forms of R2. The labelled probe is R2 and the unlabelled competitors are R2 and the three R2 mutants illustrated at the base of the figure. The underlined residues in R2 show the position of the scanning mutation that reduced binding; the lower case letters in the mutant sequences show the changes from the wild-type sequence. (B) In vivo analysis of the effect of multiple point mutations in R2. This was determined by creating the indicated fusion constructs and analysing their expression patterns in Dictyostelium. All published ecmA promoter 5'-3' deletion constructs (Fig. 1) share the same proximal end point at nucleotide +251 (labelled relative to the cap site of ecmA), four nucleotides upstream from the translation initiation codon (Early et al., 1993). All these constructs are fused to the vector A15bam-gal, which provides the basal transcription signals (Pears and Williams, 1988). By contrast, constructs S, R2S and R2pM1S:lacZ fuse immediately downstream of the ATG initiation codon of ecmA to the lacZ-coding region, hence using the basal promoter elements of ecmA (Fig. 1). The distal end point of S lies at nucleotide -493, just one nucleotide downstream of the cap-site proximal end point of R2. The R2 oligonucleotide has a cap-site distal end point just two nucleotides shorter than construct O (Fig. 1). Hence, construct R2S has a structure that, at its distal end, is very similar to that of construct O. Construct R2pM1S has an identical structure to R2S, except that it contains the four point mutations present in R2pM1.

View larger version (43K): [in a new window]   Fig. 7. Developmental time course of DimB accumulation. Ax2 cells were allowed to develop to the indicated stages and harvested: t6=streaming; t1011=tight aggregate; t1213=tipped aggregate/first finger; t14-16=slug; t20=mid culminant. Western transfer was performed with the C terminus-specific DimB antibody and with an antibody to GSK3: a gene that is semi-constitutively expressed during development.

View larger version (56K): [in a new window]   Fig. 8 Genetic analysis of DimB function. (A) Isolation of dimB-mutants. A mixture of putative dimB disruptants (marked with an asterisk) and random integrants (i.e. clones where the disruption vector integrated at a position other than the dimB locus) was analysed by western transfer with the C terminus-specific DimB antibody. The lane on the left contains DimB:HIS fusion protein and those on the right were loaded with Ax2 total protein isolated at the slug stage. (B) Morphological and behavioural phenotypes of the dimB- mutant. (Top) A dimB- and a control (random integrant) strain were developed on water agar to the slug stage. (Bottom) A dimB- and a control (random integrant) strain were developed on water agar in a darkened chamber with a narrow slit for 48 hours. The slugs were then lifted, with their adherent trails, onto a clear plastic sheet which was stained with Coomassie Blue.

View larger version (71K): [in a new window]   Fig. 9. Whole-mount staining of control and dimB- mutant standing slugs. dimB- and control (random integrant) strains were created containing ecmAO:lacZ. After development to the standing slug stage, slugs were stained with a blue ß-galactosidase chromogen. Quantitative analysis of additional such images was performed to measure the areas occupied by the prestalk and prespore zones. These measurements were taken from photographic images, using the `magic wand' tool in `Adobe Photoshop' to select areas with similar signal densities. This revealed more heterogeneity in the apparent fraction of prestalk cells in the mutant but, on average, there was no major difference from the control (control=22±4.4%, n=14; dimB null=19±8.9%, n=15). The greater heterogeneity of the dimB-null slugs probably results from their failure to migrate away from their point of origin and from their tendency to break up into fragments. For double staining analysis, dimB- and control (random integrant) strains were created containing a pspA:glucoronidase reporter and one of the three prestalk lacZ reporter fusions indicated at the left. The strains were developed on water agar to the standing slug stage and the slugs were fixed and stained sequentially for ß-glucoronidase (blue) and ß-galactosidase (red).

View larger version (86K): [in a new window]   Fig. 10. Single chromogen whole-mount staining of ecmAO: lacZ expression in migrating slugs. dimB- and a control (random integrant) strain expressing the ecmAO:lacZ reporter fusion were developed for 24 hours in a slug migration chamber. The slugs were then fixed and stained to detect ß-galactosidase. The staining times were controlled, so as to produce approximately equal intensity staining in the extreme tip of the control and the null mutant. However, the same difference in staining patterns was observed at a variety of staining times up to 30 minutes, at which time the reactions started to plateau.

View larger version (23K): [in a new window]   Fig. 11. Analysis of DIF induction of ecmA expression in a control and a dimB- mutant. A dimB-null and a control (random integrant) strain were plated at 105 cells/cm2, in the presence of 50 µM cerulenin, and treated with the indicated amounts of DIF-1. Total cellular RNA was extracted and the relative abundances of the ecmA and Ig7 RNAs (a constitutively expressed gene, used as a loading control) in the two strains determined by semi-quantitative RT-PCR.

View larger version (80K): [in a new window]   Fig. 12. DIF-1 induction of DimB nuclear accumulation. DmtA-null cells at the tight aggregate stage were dissociated, treated with or without 100nM DIF-1 for 3 minutes in shaken suspension and stained using the anti-DimB antibody.

View larger version (30K): [in a new window]   Fig. 13. ChIP analysis of DimB. Ax2 cells or dimB- cells were induced in the manner indicated, and their chromatin was incubated with or without anti-DimB (-DimB) or with anti-CudA antibody (-cudA). PCR was performed on the immunoprecipitated DNA and the data were analysed as described in the text. The asterisk indicates the sample against which all other fluorescence intensities were normalized. The ecmA primers used (see Materials and methods) derive from upstream of R2 but the extent of sonication is such that the R2 region will register in the PCR. In addition, two other primer combinations, which derive from sequences more proximal to R2 were used, in other experiments and they also showed enrichment.