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First published online January 12, 2006
doi: 10.1242/10.1242/dev.02240
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.
* Authors for correspondence (e-mail: christopher.thompson{at}man.ac.uk and gadi{at}bcm.tmc.edu)
Accepted 24 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Dictyostelium, DIF-1, bZIP, DimB
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Individual
cells aggregate using cAMP as a chemoattractant to form a mound of cells.
Within the mound, prestalk and prespore cell types differentiate intermingled
with one another (Thompson et al.,
2004b; Williams et al.,
1989). Each cell type then sorts out into distinct regions, so
that at the slug stage of development, the major prestalk and prespore cell
types are arranged along the anteroposterior axis. For example, the pstA cells
occupy the most anterior region constituting the slug tip, while the pstO
population lies just posterior and abuts the prespore zone
(Early et al., 1993;
Jermyn et al., 1989;
Maeda et al., 2003;
Maruo et al., 2004;
Yamada et al., 2005). Finally,
in response to the appropriate signals, prestalk and prespore cells terminally
differentiate and a fruiting body is formed, consisting of a ball of spores
supported by dead stalk cells (Kessin,
2001).
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). DIF-1
treatment also results in prestalk marker gene induction, together with the
repression of prespore markers and spore cell differentiation
(Kay et al., 1999). These
studies were performed on cells in culture and have been instrumental for our
understanding of DIF-1 action. Furthermore, much progress has now been made in
defining the role of DIF-1 during multicellular development. A consensus has
now emerged in which DIF-1 is synthesized by prespore cells
(Kay and Thompson, 2001) and
plays a role at least in the normal differentiation of the pstO cell
population (Kimmel and Firtel,
2004; Strmecki et al.,
2005). The identification of DIF-1 signalling mutants has been
central to this. For example, in a mutant engineered to be defective in DIF-1
biosynthesis (dmtA-), pstO cell differentiation is
compromised, although pstA and prespore cells differentiate apparently
normally (Maeda et al., 2003;
Maruo et al., 2004;
Thompson and Kay, 2000). The
dmtA- mutant defines hallmark DIF-1 signalling
morphological defects: the slugs are extremely long and thin compared with
wild type, while culmination is clearly aberrant. These defects are due to the
absence of DIF-1 as they can be rescued by addition of exogenous DIF-1.
Further support for these ideas came from the identification of a mutant
(dimA-) that is unable to respond to DIF-1 under all
tested conditions (Thompson et al.,
2004a). For example, when dimA- cells are
treated with DIF-1, prestalk markers are not induced and prespore markers are
not repressed. The dimA- mutant also exhibits
morphological and cell type differentiation defects that phenocopy the
dmtA- mutant. However, unlike the
dmtA- mutant, and consistent with a role in regulating DIF
responses rather than DIF-1 biosynthesis, the defects of the
dimA- mutant are cell-autonomous
(Foster et al., 2004).
To date, only one other DIF signalling component, STATc, has been
identified (Fukuzawa et al.,
2001). Like other DIF signalling mutants, the STATc mutant
exhibits aberrant pstO cell differentiation. In this case, however, it is a
failure to repress pstA markers in this cell type rather than any detectable
defect in pstO marker induction. STATc encodes a member of the STAT family of
transcription factors. Importantly, STATc is also generally accepted to be
directly downstream of the DIF-1 signal because STATc exhibits DIF dependent
tyrosine phosphorylation together with rapid nuclear accumulation in response
to DIF-1 (Fukuzawa et al.,
2001).
The disrupted gene in the dimA- mutant has been cloned
and also encodes a transcription factor, although in this case of the bZIP
family (Thompson et al.,
2004a). It has therefore been proposed that DimA is a direct
regulator of DIF-1 target gene expression. However, one problem with this idea
is paradoxically due to the similarity of the dimA- and
dmtA- developmental phenotypes. DimA would therefore
appear to regulate the expression of most, if not all, DIF-1 target genes.
Furthermore, DimA functions as both an activator of prestalk gene expression
and repressor of prespore gene expression. In order to explain how DimA could
have such diverse activities, two hypotheses have been put forward (1) DimA is
a permissive factor required to set up cellular competence to respond to DIF-1
(Kimmel and Firtel, 2004;
Strmecki et al., 2005). In
this model, it is proposed that DimA is not downstream of the DIF-1 signal.
Instead of directly regulating the expression of DIF-1 target genes, DimA
would be required for the activation of genes that permit cells to respond to
DIF-1, such as the DIF-1 signal transduction machinery. (2) DimA activity is
regulated by heterodimerisation with other factors
(Thompson et al., 2004a). As
bZIP transcription factors not only bind DNA as homodimers, but also as
heterodimeric complexes, the formation of heterodimers with other bZIP family
members can greatly expand their regulatory potential
(Hurst, 1995).
Heterodimerisation can even turn an activating factor into a repressor
(Chinenov and Kerppola, 2001).
In this way, heterodimerisation could explain the ability of DimA to regulate
diverse DIF-1 responses.
Most lines of evidence indicate that DimA plays a key role in the
regulation of DIF responses. Conflicting views of its mode of action
illustrate that in order to understand the DIF-1 signal transduction pathway,
it will be vital to determine whether DimA plays an active or permissive role
in its regulation. We have therefore set out to investigate how DimA activity
is regulated. First, we have examined the possible role of heterodimerisation
in DimA-regulated gene expression. We report the identification of a second
bZIP transcription factor (DimB) that can directly interact with DimA in
vitro. Consistent with the idea that DimB also regulates DIF responses,
dimB- mutant cells exhibit similar but not identical
phenotypes to the dimA- mutant. These observations provide
support for the idea that interactions between DimA and DimB serve to regulate
their activity. Second, we have investigated whether DimA and DimB are
directly downstream of the DIF-1 signal or are required to set up the
conditions that would allow DIF-1 responses. One way to distinguish between
these possibilities would be to establish whether DIF-1 treatment elicits
rapid changes in the activity of DimA or DimB, as has been reported for STATc
(Fukuzawa et al., 2001). Here,
we report that upon DIF-1 stimulation, DimA and DimB rapidly accumulate in the
nucleus. Finally, we find that nuclear accumulation of DimA is dependent on
DimB activity, which we interpret as further evidence of an in vivo
interaction between these transcription factors.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Transformants
were selected in 10 µg/ml blasticidin or 10 µg/ml G418. For development,
cells in exponential growth phase were harvested and washed free of medium
before plating at a density of 6.4x105 cells/cm2
on KK2 (16.1 mM KH2PO4, 3.7 mM
K2HPO4) plates in 1.5% purified agar. Whole-mount
lacZ staining was performed as described
(Dingermann et al., 1989).
Monolayer assays
Stalk and spore monolayer assays, in addition to lacZ activity
measurements were performed as described
(Thompson et al., 2004a). For
induction of prestalk and prespore markers in monolayers, AX4 and mutant cells
were resuspended in stalk medium containing cAMP (2 mM NaCl, 10 mM KCl, 1 mM
CaCl2, 10 mM MES (pH 6.2), 10 µg/ml streptomycin sulphate, 10
unit/ml penicillin, 37.5 µM cerulenin and 5 mM cAMP) at a density of
2.5x106/ml. Cell suspensions (10 ml) were incubated in 10 cm
tissue culture dishes at 22°C for 9 hours. Each strain was further
incubated with or without the addition of 100 nM DIF-1, for 1-3 hours. Samples
were harvested and total RNA extracted using TRI-reagent (Sigma). Genomic DNA
was removed by DNAseI (Roche) treatment followed by phenol extraction and
ethanol precipitation.
mRNA measurement by quantitative PCR
Total RNA was reverse transcribed using Mu-MLV reverse transcriptase
(Eurogentec). Primers were designed to flank short genomic sequences (100-200
bp) of the ecmA, ecmB and cotB genes. IG7 was used as a
normalizing gene to eliminate variation in cDNA concentration between the
samples. Standard PCR reactions, including addition of the nucleic acid dye
SYBR Green I (Molecular Probes) at 1x concentration, were then set up in
96-well plates and cycled in an Opticon 2 Quantitative PCR machine (MJ
Research). Cycle threshold (Ct) values were then obtained and differences in
gene transcript levels between DIF-1 treated, and untreated samples were
calculated. Values were normalised using the IG7 Ct values for each sample to
give 
Ct values, using the following calculation:
Ct= (1+Etarget)ct
target/(1+Enorm)ct norm where Ct is
the difference in Ct values between the control and DIF treated samples, and E
is the efficiency of the reaction, normally taken as 1.
GFP construct generation
A 3.8 kb dimA genomic fragment and a 1.8 kb dimB genomic
fragment were amplified by PCR (dimA primers,
5'-CGCGGATCCATGGACTCAGATAATTGG-3 and
5'-CGCCTCGAGAATATTAGGGGTCTTATAACT-3'; dimB primers,
5'-CGCGGATCCATGAATCAATTTTATCAATCTACC-3' and
5'-CGCCTCGAGTTATTGTCTCGAAGGTTGTTG-3'. Each fragment was cloned as
a translational fusion into the BamHI and XhoI sites of
pTX-GFP (Levi et al., 2000).
Clonal transgenic Dictyostelium lines with equally strong GFP
fluorescence were selected for further analysis. For simultaneous detection of
DAPI and GFP, cells were fixed with 3.7% paraformaldehyde in PBS and
permeabilised with 0.1% NP-40 before mounting in Vectashield containing DAPI
(Molecular Probes).
In vitro protein interaction
Expression vectors for Glutathione S-transferase (GST) fusion proteins of
DimA and DimB were constructed by cloning a 393 nucleotide fragment of the
dimA or dimB gene encoding the DNA-binding domain and the
dimerization domain into the pGEX4T2 (DimA) or pGEX4T1 (DimB) vectors
(Amersham Biosciences). His-tag fusion proteins were generated by cloning the
same fragments into pET-DEST42 (Invitrogen). All recombinant proteins were
expressed in E. coli BL21 star cells. GST fusion proteins were
purified with Glutathione Sepharose beads (Amersham Biosciences) according to
the manufacturer's instructions. His-tag fusion proteins were purified with
Talon metal affinity resins (BD Biosciences). Eluted proteins were dialyzed
against 20 mM Tris-HCl (pH 7.4), 50 mM NaCl and 10% glycerol and protein
concentrations were estimated using the Bradford reagent (BioRad). For GST
pull-down assay, 3 µg of His-tag fusion proteins were mixed with equal
amount of GST alone or GST fusion proteins in binding buffer [20 mM Tris-HCl,
pH 8.0, 150 mM NaCl and 0.2% NP40 supplemented with a cocktail of protease
inhibitors (Roche)]. The mixture was kept at 4°C for 1 hour before 100
µl of 50% glutathione-Sepharose beads slurry was added. After 18 hours of
incubation at 4°C on a rocker, the beads were washed three times in 20 mM
Tris-HCl (pH 8.0), 150 mM NaCl and 0.2% Triton X-100. Both the supernatants
and the pellets were mixed with equal volumes of 2xSDS sample buffer,
boiled for 5 minutes and separated by SDS-12%PAGE prior to transfer to BA85
nitrocellulose membrane (Schleicher and Schuell). The filter was first probed
with rabbit anti-V5 antibody (Novus Biologicals) followed by goat
anti-Rabbit-IgG antibody conjugated to alkaline phosphatase (AP).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Hurst, 1995).
We investigated whether this could also represent a mode of regulation of the
Dictyostelium bZIP transcription factor DimA. We made four
predictions for a putative DimA heterodimerisation partner. It should (1)
exhibit significant sequence homology to DimA within the leucine zipper
dimerisation domain; (2) directly interact with DimA in vitro; (3) be
expressed at the same time as DimA; and (4) be expressed in the same cells as
DimA during development.
The Dictyostelium genome contains 18 additional putative bZIP
transcription factor encoding genes (Fig.
1A) (Huang and Shaulsky,
2005). We used the computational method of Fong et al. to predict
which of the putative bZIP transcription factors might dimerise with DimA
(Fong et al., 2004). We found
eight bZIPs, including one we named DimB, that were as likely to form
heterodimers with DimA as DimA was to homodimerise (data not shown). We then
aligned all 19 putative leucine zipper domains and found that DimB was the
most similar to DimA (Fig. 1B).
These findings implicate DimB as a potential heterodimerisation partner for
DimA. We therefore tested whether DimA and DimB could interact in vitro. The
DNA-binding domains of DimA or DimB were expressed as either 6-His or GST
fusion proteins. Pull-down assays with purified proteins showed that DimA and
DimB can form homodimeric and heterodimeric complexes in vitro
(Fig. 1C).
In order for two proteins to interact in vivo, their expression must
overlap spatially and temporally. We tested whether the expression of DimB was
consistent with the possibility of heterodimerisation with DimA. The levels of
dimA and dimB transcripts were determined throughout
development by quantitative RT-PCR. Consistent with previous northern blots
(Thompson et al., 2004a), we
found that dimA transcription was developmentally regulated with
levels peaking at culmination (Fig.
2A). Similarly, dimB transcription was also
developmentally regulated and exhibited an overlapping profile of
developmental regulation (Fig.
2A). Most importantly, both genes were expressed at about 8 hours
of development, the time of cell type divergence when DIF-1 is believed to
act. We also tested the cell-type specificity of dimB expression by
quantitative RT-PCR from RNA samples made from separated prespore and prestalk
cells (Van Driessche et al.,
2002). We found that dimB was present in both prespore
and prestalk cells with some enrichment in the prespore cells
(Fig. 2B). This pattern is
essentially identical to that of dimA
(Thompson et al., 2004a). The
overlap in gene expression suggests that the DimA and DimB proteins are also
likely to be co-expressed, allowing heterodimerisation.
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) (Fig.
3A). Mutants in the dimB locus were generated by
homologous recombination and verified by PCR (not shown).
dimB- mutant cells grew normally in axenic medium.
However, following starvation, development proceeded normally only until the
finger stage, when the dimB- mutant produced extremely
long and thin fingers/slugs compared with the wild type
(Fig. 3B,C). When these slugs
were allowed to migrate, they were also often found to break up.
dimB- mutant slugs therefore show indistinguishable
morphological defects from those previously described in the
dimA- and dmtA- mutants
(Thompson et al., 2004a). One
simple explanation for this behaviour would be that dimA is not
expressed in the dimB- mutant or vice versa. This is not
so because dimA and dimB transcripts were detected in the
dimB- mutant and dimA- mutant,
respectively (not shown).
The dimB- allele we generated did not delete any of the
coding region, so it might not be a null allele
(Fig. 3A). Nevertheless, the
phenotypes we found were almost identical to those described in the
accompanying paper (Zhukovskaya et al.,
2006). In addition, both insertions are in similar positions
(nucleotide 600 and 696 with respect to the ATG). These researchers used
antibodies against DimB to show that the protein was undetectable in their
insertional dimB- mutant strain. We therefore conclude
that our dimB- allele is essentially null. A double mutant
where both dimA and dimB were disrupted, had an identical
phenotype to the single gene mutations
(Fig. 3C). It is therefore also
unlikely that DimA and DimB play functionally redundant roles in
Dictyostelium development. These observations suggest that DimB is
also required for DIF-1 signalling.
The dimA- and dmtA- mutants exhibit
similar defects in developmental gene expression in addition to the
morphological defects (Thompson et al.,
2004a). Most notably, both mutants show a reduced zone of
expression of the prestalk marker ecmAO-lacZ and an expanded zone of
expression of prespore-lacZ markers. Similarly, we found that in
dimB- mutant slugs the relative length of the ecmAO-lacZ
labelled prestalk zone was considerably shorter than in wild type
(Fig. 4A). In addition, we
found that the prespore zone of dimB- mutant slugs was
expanded (especially when stained for long periods)
(Fig. 4A). However, this was
generally not seen to the same extent as in dimA- mutant
slugs.
In addition to sharing morphological and gene expression defects, if DimB
is required to integrate responses to DIF-1, any defects would be predicted to
be cell-autonomous. Consistent with this idea, we found that the morphological
defects of the dimB- mutant could not be rescued by
addition of DIF-1 to the agar (not shown). Second, dimB-
mutant cells exhibited cell-autonomous defects in chimaeras. Wild-type or
dimB- mutant cells were labelled by constitutive
expression of lacZ and their position followed in chimaeric slugs
when mixed with an excess of unlabelled cells. In homotypic control chimaerae,
lacZ-labelled cells were distributed evenly
(Fig. 4B). However, when
labelled dimB- mutant cells were mixed with wild type, the
mutant cells were enriched in the prespore zone. It is also noteworthy that
their predominance within the rear of the prespore zone was reminiscent of the
distribution of dimA- cells in similar chimaerae
(Foster et al., 2004).
Furthermore, in the reciprocal experiment, labelled wild-type cells were
enriched in the prestalk zone and anterior prespore zone of chimaeric slugs.
Taken together, the similarities between the phenotypes displayed by the
dimA- and dimB- mutants strongly
suggest that DimB plays a similar role to that of DimA in the regulation of
DIF-1 responses.
DimB is required for normal responses to DIF-1
In order to directly test whether DimB is required for DIF-1 responses, we
examined the behaviour of dimB- mutant cells in the cAMP
removal monolayer assay. In this assay, cells are initially induced by cAMP to
become competent to respond to DIF-1. After removal of cAMP and addition of
DIF-1, wild-type cells differentiate as stalk cells. The DIF-1 non-responsive
mutant, dimA-, does not produce stalk cells in response to
DIF-1 in this assay. Similarly, we found that dimB- and
dimA-/dimB- mutant cells also failed to respond
to DIF-1 (Fig. 5A). Instead,
the mutants cells remained as amoebae, demonstrating that DimB, like DimA, is
required for normal responses to DIF-1.
The cAMP removal assay uses a terminal differentiation phenotype (stalk
cell formation) as a measure of DIF-1 responsiveness. We therefore employed
another monolayer assay that provides a more direct transcriptional readout in
order to further characterize the defective DIF-1 response in the
dimB- mutant. In this assay, cells do not undergo terminal
differentiation owing to the continued presence of cAMP. Previously, we have
demonstrated that DimA is required for both the induction of prestalk markers
and repression of prespore markers under these conditions
(Thompson et al., 2004a). We
compared the behaviour of dimB- cells using strains
transformed with the prestalk marker ecmAO-lacZ and the prespore marker
cotB-lacZ. We found that dimB- mutant cells showed little
or no induction of ecmAO-lacZ over a 24-hour period in the presence of DIF-1
when compared with wild type (Fig.
5B). Despite this, we were surprised to find that repression of
the prespore marker cotB-lacZ by DIF-1 was unaffected
(Fig. 5B). This is in marked
contrast to the behaviour of dimA- mutant cells and
suggests that, despite other similarities, DimA and DimB do not play identical
roles. To confirm these findings, we used a second assay that gives a more
rapid measure of DIF responses. In this assay, cells are initially brought to
competence to respond to DIF-1 by cAMP treatment followed by a short DIF-1
treatment. Expression levels of representative prestalk and prespore
transcripts were measured by quantitative real time RT-PCR. Using this assay,
the dimA- mutant showed no responses to DIF-1. By
contrast, although dimB- cells also exhibited defects,
they were distinct from those observed in the dimA-
mutant. No induction of the prestalk markers ecmA and ecmB
was detected after 3 hours of DIF treatment
(Fig. 5C). Despite this defect
in prestalk marker induction, dimB- mutant cells exhibited
clear DIF-1 dependent repression of the prespore marker cotB,
although the magnitude was less than that seen in wild type
(Fig. 5C).
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DimB is required to repress autophagy independent cell death
As DIF-1 treatment in cAMP monolayer assays results in the repression of
prespore gene expression in the dimB- mutant, this
suggested that DimB might not play a role in DIF-dependent repression of the
prespore/spore fate. To further test this idea, we employed the 8-Br-cAMP
monolayer assay, in which wild-type cells efficiently differentiate into
viable spores in the absence of DIF-1
(Kay, 1989). However, when
DIF-1 is added, spore formation is repressed and wild-type cells differentiate
as dead stalk cells or remain as detergent sensitive amoebae. This response
was exploited to identify the dimA- mutant, which fails to
respond to DIF-1 and therefore forms spores in the presence of DIF-1
(Thompson et al., 2004a). We
examined the behaviour of dimB- cells in this assay and
found that dimB- mutant cells efficiently make spores in
the absence of DIF-1, illustrating that they are able to undergo terminal
differentiation (Fig. 6A). As
predicted from the cAMP assays, spore cell formation was repressed by DIF-1 in
dimB- mutant cells as efficiently as in the wild type
(Fig. 6A). This finding further
supports the idea that DimB is not required for the repression of the
prespore/spore fate in response to DIF-1.
If dimB- mutant cells do not make spores in the
presence of DIF-1, then what do they become? As predicted by the cAMP
monolayer assays, dimB- cells still failed to make stalk
cells (Fig. 6B). Surprisingly,
however, despite the fact that DIF-1-treated dimB- cells
made neither spores nor stalk cells, they did not remain as amoebae. Instead
the dimB- mutant cells exhibited an unusual morphology,
rarely seen in monolayer assays (Fig.
6B). These cells appeared to exhibit a similar morphology to
DIF-1-treated atg1- cells, which are unable to undergo
autophagy and do not make stalk cells in response to DIF-1
(Kosta et al., 2004). Instead,
these cells exhibit non-vacuolar cell death (NVCD). We found that this
similarity extended beyond morphology. Other shared features include loss of
cell viability, the collapse of the cytoplasm and the organelles
(mitochondria) into the perinuclear compartment
(Fig. 6B), and the
concentration of F-actin to the cell periphery, surrounding the central
condensation of organelles (Fig.
6C).
One explanation for these observations is that stalk cell formation is a dependent sequence of events. The first step would require DimA to trigger NVCD, whereas subsequent vacuolisation requires DimB. This scheme predicts that a dimA-/dimB- double mutant should behave like a dimA- mutant in a 8-Br-cAMP monolayer assay. Instead, we found that the double mutant phenocopied the dimB- mutant, suggesting that dimB is epistatic to dimA (Fig. 6A). In order to explain this observation, we propose that there are at least three distinct routes that can be taken in response to DIF-1 in an 8-Br-cAMP assay (Fig. 6D): (1) vacuolised stalk cell formation requires both DimA and DimB; (2) NVCD is repressed by DimB; and (3) spore cell formation is repressed by DimA. These results further illustrate that DimA and DimB participate in distinct and overlapping pathways.
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).
Interestingly, it has also been reported that a variety of stimuli regulate
the subcellular localization of bZIP transcription factors in other organisms
(Kircher et al., 1999;
Kuge et al., 1997). We
therefore tested whether DIF-1 treatment affects the localization of the bZIP
transcription factors DimA or DimB. DimA:GFP and DimB:GFP fusion proteins were
expressed under the control of a constitutive promoter. Both fusion proteins
were functional because DimA:GFP expression in the dimA-
mutant or DimB-GFP expression in the dimB- mutant rescued
stalk cell formation (not shown). In order to follow subcellular localization,
cells were shaken in buffer for 4 hours before the addition of 50 nM DIF-1. In
the absence of DIF-1, GFP was fairly uniformly distributed throughout the
cells. However, after DIF-1 treatment, both DimA:GFP and DimB:GFP exhibited
nuclear accumulation (Fig.
7A,B). In mock-treated control cells, no change in distribution
was observed. DIF-1 normally accumulates to measurable levels after cell
aggregation. We therefore tested whether the nuclear accumulation of the
proteins could also be observed in cells that have been developed for 10
hours. The results in Fig. 7C,D
show DIF-1 induced nuclear accumulation of both bZIPs. The similarity between
the responses at 4 hours and 10 hours of development suggests that DimA and
DimB are the initial components in the cellular response to DIF-1.
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).
Nuclear accumulation of DimA:GFP is dependent on DimB
We also tested whether nuclear accumulation of DimA:GFP or DimB:GFP was
dependent on DimB or DimA, respectively. In control experiments, normal
accumulation was observed. When DimA:GFP was expressed in
dimA- cells, DIF-1 treatment triggered nuclear
accumulation. Nuclear accumulation of DimB:GFP was also observed when
expressed in dimB- cells (not shown). Similarly, when
DimB:GFP was expressed in the dimA- mutant, DIF-1 induced
nuclear accumulation of DimB:GFP with normal kinetics
(Fig. 8C). By contrast, nuclear
accumulation of DimA-GFP was not induced by DIF-1 when expressed in the
dimB- mutant (Fig.
8C). This failure was not due to altered accumulation kinetics. No
accumulation could be detected at any time point between 1 minute and 2 hours
after DIF-1 addition (not shown). These results demonstrate that nuclear
accumulation of DimA in response to DIF-1 is dependent on DimB. It should be
noted, however, that nuclear DimA:GFP is still detectable in
dimB- mutant cells, because the protein is uniformly
distributed throughout the cell. As any nuclear DimA could be considered
functional, it is therefore possible that some DimA activity remains. This
provides a likely explanation for how DimA-dependent repression of prespore
markers can take place in the DimB mutant
(Fig. 5B,C).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(1) The dimB- mutant exhibits hallmark DIF signalling morphological defects when developed. The long, thin slugs produced by the dimB- mutant are indistinguishable from those seen in the DIF-1 signalling mutants dimA- or dmtA-. These defects are due to a failure to respond normally to DIF-1, rather than DIF-1 production, as they are cell autonomous.
(2) dimB- mutant cells exhibit defective responses to DIF-1. DIF-1 treatment of dimB- mutant cells in monolayer assays does not induce prestalk marker gene expression and dimB- mutant cells fail to produce stalk cells in response to DIF-1.
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DimA and DimB also play independent roles
Some DIF-1 responses, such as the induction of prestalk marker gene
expression, require the activity of both DimA and DimB. As they readily
interact in vitro, they may function as a heterodimeric complex to regulate
these responses. However, a number of DIF responses also appear to occur
independently of either DimA or DimB. For example, DIF-1-mediated prespore
gene repression is dependent on DimA but independent of DimB. By contrast,
repression of NVCD is dependent on DimB but independent of DimA. The most
simple explanation is that DimA and DimB operate as homodimers in these
processes. However, as 17 other bZIP transcription factors are probably
encoded by the Dictyostelium genome, the formation of additional
heterodimeric complexes could play regulatory roles. For example, in mammalian
cells, although Fos and Jun can form a heterodimeric complex, a network of
interactions with other bZIPs vastly increase their regulatory repertoire
(Chinenov and Kerppola, 2001).
In order to test this possibility, we have knocked out eight of the remaining
bZIPs and begun to compare their phenotypes to those displayed by the
dimA- and dimB- mutants (E.H, G.S. and
C.R.L.T., unpublished). Interestingly, although developmental and DIF-1
response defects have been detected, none is identical to those seen in the
dimA- or dimB- mutants. It is
therefore possible that other bZIPs could play a role in regulating the DimA-
or DimB-specific responses reported here, or even in the regulation of
currently unidentified responses to DIF-1.
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DimA and DimB are downstream of the DIF-1 response pathway
The results described here strongly suggest that DimA and DimB are direct
regulators of gene expression in response to DIF rather than required to set
up DIF responses. First, both DimA and DimB exhibit rapid nuclear accumulation
in response to DIF-1. As significant accumulation occurs after as little as 2
minutes, it is probably due to direct post-translational modification of DimA
or DimB, or indirect modification of a regulatory factor. Second, if DimA or
DimB were required for the activation of permissive factors for DIF-1
responsiveness, the mutants would be expected to be completely indifferent to
DIF-1, but this is not the case. For example, DIF-1 treatment induced nuclear
accumulation of DimB in the dimA- mutant while prespore
gene repression and induction of NVCD still occurred in the
dimB- mutant. Finally, DIF-1 responses could even be
measured in the dimA-/dimB- mutant, resulting
in NVCD in 8-Br-cAMP monolayer assays. These results therefore demonstrate
that DimA and DimB play an active role in the regulation of both overlapping
and distinct aspects of DIF-1 signal transduction and gene regulation.
Understanding how each factor is regulated in response to DIF-1 and the nature
of genes regulated by each factor provides a challenge for future studies.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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