- Research article
- Open Access
Structural and functional studies of a family of Dictyostelium discoideum developmentally regulated, prestalk genes coding for small proteins
© Vicente et al; licensee BioMed Central Ltd. 2008
Received: 31 July 2007
Accepted: 03 January 2008
Published: 03 January 2008
The social amoeba Dictyostelium discoideum executes a multicellular development program upon starvation. This morphogenetic process requires the differential regulation of a large number of genes and is coordinated by extracellular signals. The MADS-box transcription factor SrfA is required for several stages of development, including slug migration and spore terminal differentiation.
Subtractive hybridization allowed the isolation of a gene, sigN (SrfA-induced gene N), that was dependent on the transcription factor SrfA for expression at the slug stage of development. Homology searches detected the existence of a large family of sigN-related genes in the Dictyostelium discoideum genome. The 13 most similar genes are grouped in two regions of chromosome 2 and have been named Group1 and Group2 sigN genes. The putative encoded proteins are 87–89 amino acids long. All these genes have a similar structure, composed of a first exon containing a 13 nucleotides long open reading frame and a second exon comprising the remaining of the putative coding region. The expression of these genes is induced at10 hours of development. Analyses of their promoter regions indicate that these genes are expressed in the prestalk region of developing structures. The addition of antibodies raised against SigN Group 2 proteins induced disintegration of multi-cellular structures at the mound stage of development.
A large family of genes coding for small proteins has been identified in D. discoideum. Two groups of very similar genes from this family have been shown to be specifically expressed in prestalk cells during development. Functional studies using antibodies raised against Group 2 SigN proteins indicate that these genes could play a role during multicellular development.
The social amoeba Dictyostelium discoideum is one of the simplest model systems utilized for the study of multi-cellular development. This organism lives as individual amoeba on forest soils, feeding on other microorganisms. However, when their food source is exhausted, they aggregate in groups of up to 100,000 cells and initiate a multi-cellular developmental program to form a fruiting body that stands on the substrate (reviewed in ). At the top of the fruiting body, inside the sorus, a large proportion of the original amoeba differentiate into resistant forms, called spores, that stay alive for long periods of time. Spores disseminate in the media and germinate to give rise to new amoeba when they reach favourable environmental conditions.
Aggregation of the amoebae is directed by chemotaxis to cAMP, secreted from discrete aggregation centres (reviewed in ). Cells that converge towards aggregation centres adhere among them forming small mounds covered by an extracellular matrix . Cell-cell adhesion is mediated by several membrane proteins, whose expression is induced during development. The first known cell-cell adhesion system to be induced, soon after starvation, is Ca-dependent and is composed of the homophilic protein DdCAD-1 (gp24), encoded by the gene cadA. A second homophilic, EDTA-resistant, adhesion system is induced at the onset of aggregation and is composed by the gp80 protein, encoded by the csaA gene [5, 6]. A third adhesion system is induced later during aggregation being mediated by the gp150 proteins, encoded by the gene lagC . Mutations in some of the genes coding for these adhesion systems or experimental conditions that interfere with their function, compromise the formation or stability of the multicellular structure [8–10].
Cells in the aggregates follow two alternative differentiation programs to become prestalk or prespore cells. At the same time, these cells continue to move towards cAMP secreted from the centre of the mound. Differences in chemotaxis to cAMP and in cell adhesion mediate the segregation of cell types so that prestalk cells migrate centrally and upwards to form a small protrusion, or tip, at the upper part of the structure . This organization is maintained during most of development, including a migratory structure, the slug, that is formed under particular environmental conditions. Coordinated cell movement and differentiation continues during the rest of the morphogenetic process when prestalk cells move downwards to the substrate and differentiate to form the stalk. In the meanwhile, prespore cells migrate upwards to form the sorus where they differentiate to spores.
Morphogenesis is coordinated through the secretion of different factors that regulate cell adhesion, migration and differentiation, cAMP being the most important among them. The chlorinated alkyl phenone DIF-1 is also an important regulator of stalk cell differentiation . Other cell-signalling factors are peptides or small proteins that are secreted by some cells to regulate the activity of neighbouring cells. For example, countin (258 amino acids long) and D11 (284 amino acids, encoded by the gene ampA) are secreted proteins that inhibit cell adhesion and contribute to regulate the size of the structure [13, 14]. Also two secreted peptides, SDF-1 and SDF-2, originated by proteolysis of the AcbA protein, induce spore differentiation at culmination .
Dictyostelium multi-cellular development is also dependent on the co-ordinated regulation of gene expression. Multiple genes are either inhibited or induced at different stages of development . This regulation is obtained by the activity of several transcription factors whose expression, or activity, is regulated during development . One of the best-known transcription factors that regulates development is GBF (G-box binding factor), that is required for induction of many prestalk and prespore genes in the mound. For example, expression of the gene lagC, coding for the adhesion protein gp150, is dependent on GBF .
The MADS-box transcription factor SrfA is also involved in the regulation of multi-cellular development. Strains where the srfA gene has been knocked out showed defects in slug migration, morphogenesis and spore differentiation [19, 20]. The identification of genes regulated by SrfA and involved in these processes can be an interesting approach to the study of development. Twenty four SrfA-dependent genes specifically expressed during spore differentiation have been described previously [21, 22]. This article describes the isolation of SrfA-dependent genes expressed at the slug stage of development and focused on one of them, sigN (SrfA-induced gene N). This gene belongs to an extensive family of genes coding for small proteins, containing less than 100 amino acids, which are expressed in the prestalk region and seem to be involved in maintaining the integrity of the cell aggregates.
Cell culture, transformation and development
Dictyostelium cells were grown axenically in HL5 medium . Transformations were carried out as described by Kuspa and Loomis . Transformed cells were selected by treatment with blasticidine  or neomycin (GP418). Transformants were grown on SM plates in association with Klebsiella aerogenes for clonal isolation . Development on nitrocellulose filters was performed as described by Shaulsky and Loomis .
Subtractive library construction
RNA was isolated from slug structures of Wild Type (AX4) and srfA- strains using Trizol reagent (Gibco-BRL). Four micrograms of poly(A)+RNA, purified using a mRNA purification kit (GEHealthcare), were used for synthesis of a cDNA subtraction library using a PCR-Select cDNA subtraction kit (Clontech), as previously described . Wild-type cDNA was used as the tester cDNA, and srfA- cDNA as the driver cDNA. Subtracted cDNAs were cloned in the pGEMT-Easy vector (Promega).
Analyses of the promoter regions
Putative promoter regions of the sigN2 and sigN9 genes were amplified by PCR and cloned in the PsA-ialphagal vector , in substitution of the PsA promoter (excised as a XbaI-BglII fragment). Reporter vectors were transfected in AX4 cells by electroporation and the transformed colonies selected by geneticin (GP418) resistance. β-galactosidase activity was detected on developing structures as previously described .
Generation of plasmid vectors
The plasmid vector used for RNA interference was based on exon 2 of sigN3. This exon was amplified by PCR and introduced in a pGEMT-Easy vector (Promega). This sequence was used to amplify the reverse complement. Forward and reverse fragments were cloned flanking a stuffer DNA  and the whole construct introduced in a Dictyostelium expression vector: pDXA-HC .
For generation of the sigN over-expression vector, a 300 bp fragment of the sigN4 gene was amplified by PCR and introduced in pGEMT-Easy vector. The fragment was taken out through a HindIII/XhoI digestion, and introduced in the expression vector pDXA-HC, under the control of the actin 15 promoter.
The sigN Group1 KO construct was generated from two regions of 460 and 1300 bp respectively, flanking the genomic region to be deleted. These fragments were amplified by PCR and introduced in the pBlueScript vector, flanking a blasticidin-resistance cassette. Oligonucleotides 5'-CGAATTCCCTTCTAATTGGTCTAATC-3' and 5'-CGCGGCCGCGGAGTTGGTTCCATTTTAACTGG-3' were used for amplification of the 460 bp fragment and oligonucleotides 5'-CGGATCCCGCCGTGGCGGCATTAGCATTAGC-3' and 5'-GAATTCGTGAGAACAGCACTGACTTACCTCC-3' for that of the 1300 bp fragment. The sigN Group 2 KO vector was constructed in a similar way from two fragments of 1000 and 730 bp, flanking the genomic region to be eliminated. These fragments were generated by PCR and cloned in pBlueScript at both sites of the Blasticidin-resistance cassette. Oligonucleotides 5'-CGAATTCCTACCGATGTTTCAGCAAGAGG-3' and 5'-CGGATCCCACAGTTGGTACCATTACAAATCC-3' were used for amplification of the 1000 bp fragment and 5'-CCGGTACCCTGTCAATGGAGTTGTTGGAGG-3' and 5'-CCCTGCAGGAATCAAGAGGATCTGGTCAATTGG-3' for that of the 730 bp fragment. The double-KO of Group 1 and Group 2 genes was made by co-transfection of the plasmid vector used for generation of the Group 1 deletion and the reporter vector ialphagal, carrying the neomycin resistance gene , in Group 2 mutant cells. Transformed cells were selected by treatment with GP418, grown on SM plates in association with Klebsiella aerogenes and individual colonies analysed for deletion of Group 1 genes by PCR. Colonies that presented deletion of Group 1 and Group 2 genes were isolated.
Generation of antibodies against Group 1 and Group 2 proteins
The differences in amino acid sequence between group 1 and group 2 proteins were used to design group-specific peptides, CGSVLHGVGSILTGG (Group 1) and CGTVVGTVNGVVGGL (Group 2), used as antigens. Antibodies were generated by Genosphere Biotechnologies (Paris, France)
Immunohistochemistry and Western blotting
Cells were washed free of liquid medium spread on cover slips and fixed with 4% PBS-paraformaldehyde during 30 minutes at room temperature. Mound structures were developed on Nitrocellulose Filters and transferred to cover slips before fixation. Cells and structures were washed twice with PBS, permeabilized with chilled methanol during 2 minutes, washed again with PBS and blocked with PBS+0,2% BSA for 20 minutes. Incubation with the first antibody was made in 150 μl of blocking buffer for 1 hour. Preparations were washed 6 times (5 minutes each) with PBS+BSA before incubation with150 μl of Alexa 568-coupled secondary antibody (dilution 1/1000) for 30 minutes. Preparations were washed twice with PBS+BSA and mounted with Prolong. Images were taken with a confocal microscopy (Olympus Fluoview 1000 confocal microscope) and processed with Adobe Photoshop software.
Western blot analyses of total protein extracts from vegetative cells were made as previously described .
Nucleotide and amino acid sequence alignments were performed with the ClustalW program at the San Diego Supercomputer Centre server. The alignments obtained were checked out in a local machine with the ClustalX program. Aligned sequences were used for the generation of phylogenetic trees using the CLC Free Workbench software.
Identification of a large family of genes related to sigN, a srfA-dependent gene
Strains mutant for the srfA gene showed abnormal slug migration , suggesting that genes whose expression is regulated by the transcription factor SrfA could be directly involved in proper function of the slugs. The isolation of SrfA-dependent genes was approached by the synthesis of a differential cDNA library using RNA isolated from WT slugs subtracted with RNA from srfA- slugs. Several cDNA clones were obtained whose expression was higher in WT than in srfA- slugs. One of them, named sigN (SRF-induced gene N), is described in this article.
Dictyostelium discoideum genes highly similar to sigN1.
Exons number – size (bp)
Intron size (bp)
Identity with sigN1 protein (%)
1 – 13//2 – 256
1 – 13//2 – 256
1 – 13//2 – 256
1 – 13//2 – 256
1 – 13//2 – 253
1 – 13//2 – 250
1 – 13//2 – 253
1 – 13//2 – 253
1 – 13//2 – 256
1 – 13//2 – 256
1 – 13//2 – 256
1 – 13//2 – 256
1 – 13//2 – 253
1 – 13//2 – 256
1 – 13//2 – 214
1 – 13//2 – 268
1 – 13//2 – 268
1 – 13//2 – 250
Analysis of the complete sequence of Dictyostelium genome at the DictyBase database allowed determination of the location of all these genes. The gene coding for the cDNA initially isolated and the closest homologous genes were grouped in a 7 kb long region of chromosome 2 (Fig 1A and Table 1). All these genes code for highly similar proteins (Fig 1B, Fig 2) and have been named sigN1 (the gene isolated from the subtractive library), sigN2, 3, 4, 5 and 25 (Group 1). Another group of genes with high identity to sigN1 was found in a different 10 kb long region of chromosome 2 (Fig 1A). The members of this second group showed lower identity with sigN1, between 60–70% of the amino acids of the encoded proteins are identical to SigN1 (Table 1). However, the identity among the proteins encoded by this second group of genes was about 95% (Fig 1B), which made them group together in the phylogenetic tree (Fig 2). These genes have been named sigN6, 7, 8, 9, 11, 12 and 14 (Group 2). The identity of the proteins encoded by the two groups of genes was very high in their N-terminal regions. However, there were some group-specific differences at the C-terminal region (Fig 1B). Genes DDB0217094 (sigN105) and DDB0168488 (sigN10) are located in the chromosomal region of group 1 and 2 genes, respectively. They have not been included in these groups because of their lower identity with the other genes, as shown in the phylogenetic tree (Fig 2). The identity of the hypothetical protein encoded by sigN105 with group 1 proteins was about 62% and that encoded by sigN10 showed an identity with the members of group 2 of about 64%.
Temporal expression of sigN genes
The probe used to determine the differential expression of sigN covered 190 nt in the coding sequence of this gene. It is very likely that the probe can detect Group 1 and Group 2 mRNAs, due to the high identity that exists among the genes. Specific oligonucleotides were designed to study the expression of the two groups of genes independently, based on the differences in their C-terminal-coding regions. RT-PCR studies showed that expression of Group 1 genes was first detected at 2 hours of development with a rise at 12 hours, when the mound is completely formed (Fig 3B). Group 2 genes showed no expression until 10 hours of development with maximal induction obtained at 14 hours. In this case, the lower bands obtained in the RT-PCR reactions were sequenced and did not correspond to any specific amplification product.
Analyses of sigN promoters
Intergenic regions of the Group 1 genes sigN1-sigN3 and sigN2-sigN4 are very similar between them but do not have the palindromic structure described for Group 2 genes. Therefore, the putative promoter region of the sigN1 gene is very similar to that of sigN2 and the same hold true for sigN3 and sigN4 regions. However, the sigN1 promoter region does not show similarity to that of the contiguous sigN3 gene. Similarly, the sigN2 putative promoter region does not show similarity to that of sigN4. These regions do not show similarity with the sigN5 and sigN25 putative promoter regions, either.
The comparison of the nucleotide sequence of the putative promoter regions of Group2 genes detected the existence of four repetition of a conserved G-rich sequence (boxed in Fig 4A), also present in Group 1 promoters. These sequences are very similar to the GBF response element: (T/G)G (T/G)G (T/G)G (T/G) . Group 2 promoter regions contain two pairs of closely locate GBF-binding sites, labelled as regions A and B in Figures 4A and 4C, that could regulate the expression of the contiguous opposite genes.
Function of sigN genes
To further study the possible function of these proteins, antibodies were added in vivo to developing cells to try interfering some of the developmental processes. Pre-immune antiserum was used as control. Specific antibodies against Group 1 proteins did not show any obvious interference with the developmental process in comparison with pre-immune and phosphate-based buffer (PDF) controls.
The study of the gene sigN1, partially dependent on the transcription factor SrfA for expression, has uncovered the existence of a family of genes coding for very similar small proteins in D. discoideum. The family is large with, at least, 96 genes, although the present study has been centred on 13 closely related genes located in two regions of chromosome 2. These genes have in common their structure and developmental pattern of expression, in addition to the high similarity of their nucleotide sequences. These genes have been divided in two groups based on the sequence of a small divergent region that code for the C-terminal region of the protein, and their chromosomal location. Phylogenetic analyses of the coding and intergenic regions suggest that the genes of each group might have originated by duplication of an original tandem of two genes, oriented in opposite directions.
All the genes are constituted by an exon, containing a 13 nt long open reading frame, an intron and a second exon, coding for the rest or the protein. The same structure is share by most of the 96 similar genes mentioned above. The conserved structure of these genes might be indicative of their common evolutionary origin.
Northern and RT-PCR studies have shown that Group1 and Group2 sigN genes expression is induced at 10–12 hours of multi-cellular development. Analyses of the promoter regions of one gene of each group indicate that sigN genes are expressed in the prestalk region. The sequence of the putative promoter regions, very similar among Group 2 genes, contain several copies of a conserved G/C rich motif similar to the GBF transcription factor binding site . GBF expression is developmentally regulated and necessary for the expression of numerous prestalk and prespore genes . Microarray analysis of GBF-dependent genes identified sigN12 as one of them , in agreement with the presence of putative GBF-binding sites in the promoter region. Two other sigN related genes, DDB0230164 (sigN103) and DDB0231563 (sigN107), coding for proteins that are 41% and 35% identical to SigN1, respectively, are also dependent on GBF for their expression The developmental pattern of expression of these genes was also very similar to that of sigN genes. Besides, Microarray and in situ hybridization analyses of the expression of these genes have shown that these three GBF-dependent genes are expressed in the prestalk region, including sigN12 [18, 33]. These data are in agreement with the results presented in this article and suggest that, since all these genes are very similar in sequence and present similar temporal and spatial patterns of expression, might accomplish similar, and perhaps redundant, biological functions.
The study of the function of sigN genes was approached by their over-expression, under-expression and by deletion. However, even the deletion of the 13 Group 1 and Group 2 genes seemed to have no effect on development. These data could be explained by the redundant functions of this family of genes, as previously suggested on the bases of their structure and expression.
Only the addition of specific antibodies raised against Group 2 sigN genes had some effect on development, inducing disaggregration of mound structures. This effect was specific since antibodies raised against Group 1 peptides or pre-immune serum had no effect on this process. With the exception of the sigN4 over-expressing strain, the antibodies raised against SigN peptides did not recognize these proteins in Western blot, which precluded further studies on their specificity. With this caveat in mind, the simplest explanation for these results obtained would be that Group 2 SigN proteins could participate in cell-cell adhesion and that the presence of the antibody could block interactions of SigN with other proteins, required for cell adhesion. As mentioned in the Results section, many proteins containing cysteine knot domains are extracellular, which would be in agreement with the function proposed for SigN Group 2 proteins. Antibodies raised against Group 1 and Group 2 proteins stained small vesicles in cells at the mound stage or development, which would be also in agreement with their proposed secretion. The expression pattern was similar for both antibodies, as expected from the similar structural domains predicted for the proteins of both groups.
According to the hypothesis suggested above, sigN Group 2 mutants would be expected to show defective aggregation, which was not the case. As mentioned above, functional redundancy between sigN genes could explain these results since the sig N Group 2 mutants could still maintain cell adhesion through the interaction of other related proteins. A search for proteins containing sequences similar to the one of the peptide used to raise Group 2 specific antibodies showed that at least two other proteins of the sigN family, encoded by genes DDB0230164 (sigN103) and DDB0168566 (sigN110), presented sequences very similar to the peptide. One of these genes, DDB0230164 (sigN103), was also dependent on GBF for expression. It is, therefore, possible that these proteins could mediate cell adhesion in sigN Group 2 mutants. Antibodies would bind to all the proteins that contain this sequence, including Group 2 and other proteins, blocking all protein interactions. In agreement with this hypothesis, the antibody is able to disintegrate mounds formed by the Group 2 mutant strain (data not shown). These results are similar to those previously described for other cell-cell adhesion proteins. For example, a peptide from the N-terminal region of the gp80 protein prevented cell adhesion  while a mutant lacking the encoding gene (csA) had no obvious phenotype in experimental conditions similar to the ones used in the present study . Antibodies specific for the cell adhesion protein DdCAD-1 also arrested development at aggregation .
SigN Group 2 proteins could be also involved in the formation of the extracellular matrix that surrounds multi-cellular structures, isolating them from the environment and providing a substrate for cell adhesion and migration. The extracellular matrix is composed of cellulose and proteins, synthesized by prestalk cells . Early studies on the composition of the extra-cellular matrix identified a population of urea-insoluble proteins smaller than 15 kDa in size. These proteins had an higher content in serine, cysteine, glycine, valine and aspartic acid/asparagine and a lower content in lysine and arginine than whole-cell proteins . SigN proteins had a similarly high proportion of cysteine, glycine and serine and a low proportion of lysine and arginine. Besides, they are expressed in prestalk cells, which would be compatible with their presence in the extra-cellular matrix.
Immunocytochemical studies detected the presence of these proteins in small vesicles of mound cells. However, no staining was found either at the plasma membrane, as expected if they were involved in cell-cell interactions, or at the extracellular matrix, making impossible to discern between the alternative possibilities discussed above. The lack of reactivity could be due to conformational changes in the extra-cellular medium or to their incorporation into structures where they would not be accessible to the antibodies used in immunocytochemistry. Therefore, while a function in cell adhesion or extracellular matrix formation is suggested, the lack of information on the specificity of the antibodies and a inability to detect membrane or external antibody binding necessitates further experiments to support this conclusion.
A large family of genes coding for small proteins has been identified. Most of the genes have a similar structure containing a first exon, coding for a 13 nucleotide long open reading frame region, an intron, and a second exon coding for the rest of the protein. Two groups of these genes, coding for very similar proteins and closely assembled in two different regions of chromosome 2, have been studied in more detail. These genes are specifically expressed at the prestalk region during development of multicellular structures. Immunochemical studies indicated that these proteins could be secreted. The addition of antibodies raised against group 2 proteins avoided formation of cellular aggregates, which suggest a possible role for these proteins in development.
This work was supported by grants from the Spanish Ministerio de Educación y Ciencia, Dirección General de Investigación (BMC2002-01501 and BFU2005-00138)
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