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First published online 29 March 2006
doi: 10.1242/dev.02327

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Regulation of Dictyostelium prestalk-specific gene expression by a SHAQKY family MYB transcription factor

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

View larger version (14K): [in a new window]   Fig. 1. Regulatory elements within the promoter of the ecmA gene. The domains of ecmA expression within the slug and the regions of the promoter that direct this expression are depicted. The distal region, which directs pstO-specific expression, is also active in many of the anterior-like cells (ALCs).

View larger version (40K): [in a new window]   Fig. 2. Deletion analysis of the ecmO region. (A) Structure of the minimal ecmO region and expression patterns for two deletion mutants. The structure of the minimal 94 nucleotide ecmO promoter region (see Fig. 1) and of two 5'-3' deletion mutants is shown. The STATa-binding site is underlined, as are the MybE dyad and the adjacent C-rich region. These three promoter fragments were cloned upstream of basal promoter elements in a lacZ reporter construct and their spatial expression patterns determined at the indicated stages of development. (B) Analysis of DIF inducibility of the 94-mer and the two deletion mutants. Transformants of the three constructs analysed in A were rendered DIF-competent and either induced or not induced with DIF. The lacZ expression level is presented without normalisation, so that absolute expression levels can be compared between the three constructs.

View larger version (36K): [in a new window]   Fig. 3. Analysis of the multimerised 22-mer lacZ construct. (A) Structure of the 4x22-mer lacZ construct. A fourfold repeated version of a 22-mer sequence, derived from within the 30-mer (Fig. 2A), was synthesised as a single oligonucleotide with BamHI cohesive ends. This was cloned into the vector Actin15Bam:gal, which provides basal transcription sequences. (B) Expression patterns for the 4x22-mer lacZ construct. Stable transformant cells of the 4x22-mer lacZ construct in Ax2 were developed to the indicated stages of development and stained for ß-galactosidase activity. (C,D) Analysis of DIF inducibility of the 4x22-mer lacZ construct in Ax2 and dimA– cells. Stable transformants of the 4x22-mer:lacZ construct in Ax2 and in the DIF insensitive, dimA-null strain were rendered DIF-competent and then either induced or not induced with DIF. The lacZ expression level for Ax2 is presented as OD575 (C) and as raw data (D), with duplicate micro-titre wells displayed for each experimental point.

View larger version (47K): [in a new window]   Fig. 4. Analysis of point mutations within the 22-mer region determined within the context of a 94-mer construct. (A) Expression patterns for the mutant 94-mer constructs. The control construct contains the minimal, 94 nucleotide ecmO promoter region, upstream of basal promoter elements in a lacZ reporter construct. Three analogous constructs were made, bearing the point mutations indicated by lower case letters and described in the text. The spatial expression patterns of the four constructs were determined at the slug stage. (B) Analysis of DIF-1 inducibility of the mutant 94-mer constructs. The four constructs analysed in A were rendered DIF-competent and either induced or not induced with DIF-1. The lacZ expression level was determined and the ß-galactosidase activity with DIF-1 is shown normalised to the activity without DIF-1.

View larger version (74K): [in a new window]   Fig. 5. Detection and purification of specific DNA-binding activities directed against the 30-mer. (A) Gel retardation assay using the 30-mer as probe. A partially purified nuclear extract from slug stage cells was bound to radioactively labelled, 30-mer oligonucleotide (Fig. 2A) with: (1) no competitor (0); (2) the 30-mer wild-type (wt); (3) three mutant forms of the 30-mer (mut 1-3, Fig. 4A and as indicated); or (4) a wild type and a mutant form of a G box oligonucleotide. The major complex (indicated by an arrow) is efficiently competed by the wild-type oligonucleotide but not by the three mutant forms or the G boxes. There are also two minor complexes (indicated by arrowheads) that show the same behaviour as the major complex. (B) Purification of proteins that bind to a 22-mer DNA affinity column. Slug nuclear extracts were purified as shown schematically and the twice affinity-purified proteins were separated by SDS gel electrophoresis. The bands indicated by letters were excised and the proteins identified by mass spectrometry.

View larger version (38K): [in a new window]   Fig. 6. Alignment of the proposed MybE DNA-binding domain. (A) Alignment to plant SHAQKY proteins. ClustalW was used to align the MYB domain of MybE to four plant MYBs: Arabidopsis CCA1 and three others of unknown function, identified by their Accession Numbers. The asterisks indicate two tryptophan residues that are highly conserved in all Myb domains. MybE and the plant proteins lack a third, otherwise conserved tryptophan residue and this is replaced by a `SHAQKY'-related sequence. (B) Alignment of Dictyostelium SHAQKY proteins The Dictyostelium proteins were identified in genome database and aligned using ClustalW.

View larger version (123K): [in a new window]   Fig. 7. Analysis of the DNA binding properties of the MYB domain of MybE. A C terminus-proximal segment of MybE containing the MYB domain was expressed in E. coli and purified using a C terminal GST tag. Recombinant protein (500 ng) and various competitors (5 pmole) were used for gel retardation assay with a 32P-labeled 30-mer probe. Extracts were pre-incubated in the presence of: (1) no competitor; (2) the 30-mer wild-type (wt); (3) three mutant forms of the 30-mer (mut 1-3, Fig. 4A and as indicated); (4) duplicated forms of the 30-mer, the dyad region and the C-rich region. The arrow indicates the major complex and we assume that the higher mobility forms, which mirror the behaviour of the major complex, contain degraded forms of the C terminal region of MybE.

View larger version (31K): [in a new window]   Fig. 8. Developmental time course of mybE expression and identification of a mybE– strain. (A) RT-PCR analysis of a developmental time course. Cells were subjected to development on water agar and total cellular RNA was isolated at the indicated stages. The morphological stages reached at the selected times indicated were: 6 hours streaming, 8 hours loose aggregates, 10 hours tight aggregates, 12 hours standing slugs, 14 hours slugs, 16 hours early-mid culminants and 20 hours mature culminants. The samples were analysed by RT-PCR for mybE mRNA. As a positive control the same samples were analysed for expression of the constitutively active Ig7 gene. (B) Analysis of the mybE gene disruptant by western transfer. Total cellular proteins from a putative mybE– clone and Ax2 were analysed by western transfer using the MybE antibody.

View larger version (91K): [in a new window]   Fig. 9. Analysis of expression directed by the complete ecmA promoter in control cells, mybE– cells and mybE– cells expressing a MybE:GFP fusion gene. (A) The ecmAO-lacZ construct and the pspA gus constructs were co-transformed into Ax2 cells and mybE– cells, and their spatial expression patterns were determined, by double staining at the slug stage. (B) Cells transformed as in A were analysed at the slug stage for lacZ expression alone, i.e. for the activity of the ecmAO promoter fragment. (C) MybE– cells were co-transformed with a MybE:GFP fusion construct under the transcriptional control of a semi-consitutive promoter and with ecmAO-lacZ. The panels on the right show a phase-contrast image of growing cells juxtaposed with the GFP fluorescence image of the same field. The panel on the left shows slugs, derived from the same population, and stained for lacZ expression.

View larger version (107K): [in a new window]   Fig. 10. Comparison of the expression of prestalk markers in parental and mybE– cells. The 4x22-mer-lacZ,ecmA-lacZ, ecmO-lacZ and ecmB:lacZ constructs were transformed into Ax2 cells and mybE– cells, and their spatial expression patterns were determined at the slug stage.

View larger version (67K): [in a new window]   Fig. 11. Analysis of DIF induction of ecmA in a control and a mybE– mutant. (A) Expression of promoter constructs. The mybE– strain and a control random integrant strain, both transformed with the indicated ecmA promoter fusion constructs, were assayed for DIF inducibility in the monolayer assay. Quadruplicate wells were analysed for each assay condition and this result is typical of four separate biological experiments. (B) RT-PCR analysis of the ecmAO:lacZ gene and the endogenous ecmA gene. Control and mybE– cells, transformed with ecmAO:lacZ, were induced with DIF-1 in a monolayer assay using a cell density of 105/cm2 and a cerulenin concentration of 50 µM. Total cellular RNA was extracted and the abundance of the lacZ, ecmA and Ig7 RNAs (a constitutively expressed gene, used as a loading control) in the two strains was determined by semi-quantitative RT-PCR.