First published online January 16, 2004
doi: 10.1242/10.1242/dev.00939
Development 131, 513-523 (2004)
Published by The Company of Biologists 2004
A bZIP/bRLZ transcription factor required for DIF signaling in Dictyostelium
Christopher R. L. Thompson1,*,
Qing Fu1,
Caroline Buhay1,2,
Robert R. Kay3 and
Gad Shaulsky1,*
1 Department of Molecular and Human Genetics, Baylor College of Medicine, One
Baylor Plaza, Houston, Texas 77030, USA
2 Summer Medical and Research Training (SMART) Program, Baylor College of
Medicine, One Baylor Plaza, Houston, Texas 77030, USA
3 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
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Fig. 1. Isolation of a mutant defective in DIF responses. (A) The 8-Br-cAMP
monolayer assay as an enrichment for DIF-1 non-responsive mutants. (B) The
dimA– mutant shows no DIF-1 response in monolayer
assays; compare with wild type (AX4). DIF-1 responses were measured in
8-Br-cAMP or cAMP removal monolayer assays. Results shown are the mean of
three experiments. (C) The dimA– mutant shows no
DIF-1 response in a dissociated cell assay. Wild-type AX4 or
dimA– mutant cells were harvested at the mound stage
of development and disaggregated. Cells were shaken for 4 hours in buffer
containing 1 mM cAMP, with or without 100 nM DIF-1. Total RNA was extracted
and, following northern transfer, probed with the ecmA and
ecmB (prestalk), and cotB (prespore), markers. Methylene
Blue staining of ribosomal RNA (rRNA) is shown as a loading control. (D)
Prestalk (ecmAO/lacZ and ecmB/lacZ) and prespore
(cotB/lacZ) reporter constructs are DIF-1 non-responsive in the
dimA– mutant in monolayer assays. Results are
averages and standard deviations of two biological replicates, where each
assay was performed in triplicate.
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Fig. 2. Structure and function of the dimA gene. (A) Site of insertion of
the disruption plasmid and structure of the dimA gene. The pBSR1
disruption vector was recovered from the dimA–
mutant by plasmid rescue. The insertion lies in the second exon of a 3846 bp
gene, encoding a putative 1242 amino acid protein. The protein contains long
stretches of asparagine (N) and glutamine (Q) residues as indicated. The
region between amino acids 545-676 shows extensive homology to the DNA-binding
and dimerization domains of bZIP and bRLZ transcription factors. (B) Sequence
alignment of the putative dimA DNA-binding and dimerization domain
with examples of bZIP and bRLZ proteins from human, mouse, Drosophila
and yeast (gi:19745184, gi:10835484, gi:135304, gi:17647933 and gi:135867).
(C) DimA binds DNA. Binding of total soluble protein extracts prepared from
bacteria expressing the putative DimA DNA-binding/dimerization domain
(dimA) fused to GST was compared with extract from cells expressing
GST alone (pGEX). Equal amounts of total protein were assayed and loaded. A 48
bp fragment from the 3' half of the minimal ecmO/lacZ promoter
was used as a probe and poly dAdT was included as a non-specific competitor.
The probe is only retarded when mixed with DimA-expressing extract. The amount
of binding is reduced by the addition of a 10-fold excess of unlabeled
oligonucleotide (CC). (D) The effects of varying non-specific competitor
species on DNA binding. Strongest binding is evident in the absence of
non-specific competitor. The addition of poly dIdC strongly reduces binding,
whereas poly dAdT addition results in a small reduction in binding. Fewer
retarded bands are visible (compared with C), as electrophoresis was performed
at 4°C to stabilize protein DNA interactions.
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Fig. 3. The developing dimA– mutant phenocopies the
dmtA– mutant. (A,D,G)
dimA– and dmtA– fingers
tend to appear long and thin compared with wild type. (B,E,H) Migratory slugs
of both mutants are also long and thin, and have a tendency to break apart
(arrowheads). (C,F,I) After 24 hours the wild type has produced fruiting
bodies, but both mutants produce fewer normal fruiting bodies, although stalks
and spores litter the agar.
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Fig. 4. Gene expression profiles in the dmtA– mutant. (A)
Developmental time course of gene expression. The ecmA and
ecmB (prestalk), cotB (prespore) and cprD
(growth/differentiation transition) markers are expressed with comparable
levels and timing in dimA– and wild-type cells, from
0-24 hours. The  5 kb dimA transcript is also developmentally
regulated in wild-type cells but is absent in the mutant. However, a larger
transcript (>7 kb), due to a readthrough transcription into the blasticidin
resistance cassette, is detectable. Methylene Blue staining of ribosomal RNA
(rRNA) is shown as a loading control. (B) dimA transcripts are
expressed in both prespore and prestalk cells at the slug stage of
development, although the highest levels are detectable in prespore cells. The
purity of the isolated cell populations is indicated by the great enrichment
of ecmA and ecmB in prestalk cells, and cotB in
prespore cells.
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Fig. 5. The dimA– mutant exhibits pstO patterning
defects. (A-D) Expression pattern of the prestalk specific ecmAO/lacZ
reporter at the late finger stage of development. Mutant slugs show a greatly
reduced zone of expression. (A,B) Several representative samples are shown.
(C,D) Higher magnification highlights the shortening of the prestalk zone in
the mutant (brackets). (E-H) Expression pattern of the prespore specific
pspA/lacZ reporter at the late finger stage of development. (E,F)
Representative samples show that the marker is expressed throughout the
prespore zone of wild-type and mutant slugs. (G,H) Higher magnification
highlights the small size of the unstained prestalk zone in mutant structures
(brackets).
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Fig. 6. Measurement of DIF biosynthesis in dimA–, and DIF
response in dmtA–. (A) dimA produces normal
levels of cell-associated DIF-1. Cells were developed for the indicated times
(hours) on agar containing 36Cl– and labeled
compounds extracted with organic solvents, resolved by TLC and detected using
a phosphorimager. The inclusion of the P450 inhibitor ancymidol (ancy) in the
agar at the time points indicated results in the build-up of the DIF-1
breakdown product DIF-3. LCCs are late chlorinated compounds produced by stalk
cells. (B,C) Stalk cell induction and spore cell repression in
dmtA–. The efficiency of stalk cell induction (B) or
spore cell repression (C) by DIF-1 was measured in 8-Br-cAMP monolayers. No
difference was observed between dmtA– and wild type.
Results shown are from a representative experiment. The experiment was
performed at least three times.
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Fig. 7. Cell autonomous defects of the dimA– mutant. (A)
Development on DIF-agar. 100 nM DIF-1 slightly slows the development of the
wild type as some tip mounds are still visible up to 15 hours (a,b), although
all structures ultimately fruit normally (c). dimA–
development is unaffected by the addition of exogenous DIF-1, as slugs remain
long and thin (d,e) with a tendency to break (arrowhead), and fruiting bodies
still lie on the surface of the agar (f). By contrast, 100 nM DIF-1 is
sufficient to rescue the phenotype of the dmtA–
mutant, as both slugs (g,h) and fruiting bodies (i) appear normal. (B)
dimA– cell-autonomous defects in chimeras with
wild-type cells. Wild-type or dimA– mutant cells
were transformed with the constitutively expressed
actin15/lacZ marker and mixed with unlabeled cells. (a,b)
Control samples illustrate that expression of the marker itself does not
affect cell fate or position (c) Labeled AX4 cells localize to the pstO and
anterior prespore zones in chimeras with unlabeled
dimA– mutant cells. (d) Labeled
dimA– mutant cells are strongly enriched in the
posterior prespore zone in chimeras with unlabeled wild-type cells. (C)
Expression of the cotB/lacZ prespore marker in chimeric slugs. (a)
cotB/lacZ-expressing AX4 cells are scattered throughout the
prespore zone of chimeras with unmarked dimA– cells.
(b) dimA– cells that express the
cotB/lacZ prespore marker are predominantly found at the
rear of the prespore zone in chimeras with unmarked wild-type cells.
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© The Company of Biologists Ltd 2004
