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First published online January 12, 2006
doi: 10.1242/10.1242/dev.02240

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bZIP transcription factor interactions regulate DIF responses in Dictyostelium

¹ Graduate Program in Structural Computational Biology and Molecular Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
² Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
³ Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.

View larger version (66K): [in a new window]   Fig. 1. Identification of a DimA-interacting protein. The Dictyostelium genome contains 19 potential bZIP genes. Alignments and phylogenetic trees of the DimA basic DNA-binding domain (A) and leucine zipper dimerisation domain (B) are shown. Identifiers from the Dictyostelium database (www.dictybase.org) are shown with DimB highlighted in blue. Basic residues are shown in red, hydrophobic in blue, small and polar in white, and secondary structure breakers in orange. The basic regions were defined as the first 26 amino acids from the basic DNA-binding domain, and all 19 sequences were aligned using CLUSTALX (default setting) and a phylogenetic neighbour-joining tree was plotted using NJ-Plot. Scale bars above the alignment indicate 0.5 estimated amino acid substitutions per site. Multiple sequence alignment and phylogenetic analysis were performed based on the dimerization domain, represented by the first four hepatads from the leucine zipper region of each protein. The results indicated a greater sequence identity between DimA and DimB bZIP domain. (C) DimA and DimB form homo- and heterodimers in vitro. Fusion proteins were expressed in E. coli and purified by affinity chromatography. DimA or DimB GST fusion proteins were mixed with His tagged versions before incubating in the presence of glutathione sepharose beads. Presence of His fusion proteins was monitored in the supernatant (S, unbound) and pellet (P, bound).

View larger version (19K): [in a new window]   Fig. 2. Developmental regulation and cell type expression of dimB RNA. Cells were developed on agar for the times indicated (hours) and RNA was harvested. After reverse transcription, levels of each transcript were determined by quantitative PCR and normalized to IG7. (A) Developmental regulation of dimB compared with dimA. Expression of the two genes is upregulated during development. (B) RNA from prespore and prestalk cells was analyzed by quantitative RT-PCR with oligonucleotides specific to the dimB mRNA, indicating expression in both cell types with some prespore enrichment. Primers against prestalk (ecmA) and prespore (cotB) specific genes were used as controls. Expression levels are given in arbitrary units.

View larger version (90K): [in a new window]   Fig. 3. The dimB- mutant exhibits DIF-1 signalling morphological defects. (A) Schematic of the dimB gene and disruption vector. The predicted DimB protein contains 1809 amino acids. Regions of low complexity [poly glutamine (Q) and poly asparagine (N) tracts] and the basic-leucine zipper (bZIP) domain are highlighted. The dimB disruption vector was generated by in vitro transposition of sequences containing the tetracycline resistance gene (for selection in bacteria) and the blasticidin resistance gene into the dimB-coding sequence. (B) Developmental morphology of dimB- mutant cells. (C) dimB- and dimAB- double-mutant cells form long thin slugs compared with wild type (AX4).

View larger version (64K): [in a new window]   Fig. 4. dimB- exhibits gene expression and cell-autonomous defects. (A) Expression pattern of representative prestalk and prespore markers at the slug stage of development. The prestalk marker ecmAO/lacZ shows a reduced zone of expression compared with wild type. The prespore marker cotB/lacZ shows an expanded zone of expression, although expression is weaker in the expanded region than in the prespore zone proper. (B) The dimB- mutant exhibits cell-autonomous defects when mixed with wild-type cells. A minority (2%) of wild-type or dimB- mutant cells were labelled by constitutive expression of lacZ and mixed with unlabelled cells. Wild-type and dimB- mutant cells are evenly distributed in homotypic control chimaeras (a and d). dimB- mutant cells preferentially localize to the rear of the prespore zone when mixed with an excess of wild-type cells (b). Wild-type cells localize to the pstO and prespore zone when mixed with dimB- mutant cells (c).

View larger version (26K): [in a new window]   Fig. 5. dimB- mutant cells fail to induce prestalk markers and stalk cells in response to DIF-1. (A) Stalk cell induction was measured in response to DIF-1 in the cAMP removal assay. The assay was repeated at least three times and a representative example is shown. Wild-type cells (AX4) differentiated as stalk cells, whereas mutants defective either in dimA or in dimB, or in both (dimAB-) failed to do so. (B) In the dimB- mutant, a prestalk reporter (ecmAO/lacZ) was non responsive to DIF-1, whereas a prespore marker (cotB/lacZ) was repressed as in the wild type. The results are means of three experiments where each assay was performed in triplicate. (C) The dimB- mutant did not show rapid induction of prestalk gene expression (ecmA or ecmB) in culture, although the prespore gene (cotB) was repressed. After treatment with cAMP for 9 hours, samples were incubated with or without DIF-1 for 3 hours. Expression levels were normalized to IG7 and compared with or without DIF-1. In the case of ecmA and ecmB, fold increase is shown, and for cotB fold decrease. Broken line indicates no change.

View larger version (42K): [in a new window]   Fig. 6. dimB is required for stalk cell induction and repression of non-vacuolar cell death in response to DIF-1. (A) Repression of spore cell formation by DIF-1 was measured in an 8-Br-cAMP assay. In the absence of DIF-1, all strains produced similar numbers of spores. Only dimA- mutant cells produced spore cells in the presence of DIF-1. Each assay was performed at least three times and the results of a representative assay are shown. dimAB- represents the double mutant dimA-dimB- and AX4 is the wild type. (B) dimB- cells were stained with DAPI without DIF-1 treatment (a,b) or after DIF-1 treatment (c,d). Micrographs were taken with phase contrast (a,c) or fluorescence microscopy (b,d). (C) dimB- mutants produce cells with identical characteristics to those that have undergone non-vacuolar cell death. (a) Phase-contrast image showing cell morphology. Cells appear rounded and contain a dense/granular region. (b) Staining of DNA with DAPI. (c) F-actin distribution detected with phalloidin. (D) Model of DIF-1 responses based on the epistatic relationship between DimA and DimB in the 8-Br-cAMP assay.

View larger version (58K): [in a new window]   Fig. 7. DIF-1-dependent nuclear accumulation of DimA and DimB. DimA and DimB rapidly accumulate in the nucleus of DIF-1-treated cells. DimA and DimB were constitutively expressed as GFP fusion proteins in cells starved for 4 hours (A,B) or 10 hours (C,D). After DIF-1 treatment, the number of cells with even staining in cytoplasm and nucleus rapidly decreased with a concomitant increase in the number of cells with stronger staining in the nucleus compared with cytoplasm. No change in distribution was observed in untreated control cells. Nuclear accumulation was verified by comparison with DAPI staining. In the pictures, 10 minutes DIF-1 treatment is shown. In the samples starved for 10 hours (C,D), clumping of cells can be seen. In addition, an increase in the number of cells with increased nuclear fluorescence is observed in control samples, presumably owing to endogenous DIF-1 production at this time point.

View larger version (52K): [in a new window]   Fig. 8. Quantification of the nuclear accumulation kinetics. (A,B) DimA and DimB exhibit rapid nuclear accumulation kinetics. Cells with higher levels of nuclear fluorescence than cytoplasmic fluorescence were scored as positive. (C) Nuclear accumulation of DimA-GFP is dependent on DimB. DimB-GFP was expressed in dimA- cells and DimA-GFP was expressed in dimB- cells. Nuclear accumulation of DimB-GFP was unaffected, whereas no DIF-1-induced nuclear accumulation of DimA-GFP could be detected.