Department of Molecular Biology, University of Texas-Southwestern Medical Center, Dallas, TX 75390-9148, USA
* Author for correspondence (e-mail: dennis.mckearin{at}utsouthwestern.edu)
Accepted 3 December 2002
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SUMMARY |
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Key words: Stem cell, Asymmetric division, bam transcription, Drosophila melanogaster
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INTRODUCTION |
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Studies from several laboratories have identified genes required in ovarian
somatic tip cells that are necessary for germline stem cell (GSC) function,
including piwi, dpp and fs(1)Yb
(Cox et al., 2000;
King and Lin, 1999
;
Xie and Spradling, 1998
).
Genes required within the GSCs include nanos (nos),
pumilio (pum) and the Dpp-dependent receptors and
transcription factors (Forbes and Lehmann,
1998
; Lin and Spradling,
1997
; Xie and Spradling,
1998
). Asymmetry of the GSC division produces a specialized
cystoblast that will produce a clone of cells from which the oocyte proper
will form. Mutations in such genes as pum or nos destroy
asymmetry because both daughters differentiate as cystoblasts, causing the
loss of GSCs (Forbes and Lehmann,
1998
; Lin and Spradling,
1997
). The opposite is true when cystoblast differentiation
factors, such as bam or bgcn, are inactivated. In these
cases, asymmetry is disrupted because both GSC daughters retain GSC
characteristics (Gateff, 1982
;
Lavoie et al., 1999
;
McKearin and Ohlstein, 1995
;
McKearin and Spradling,
1990
).
The cystoblast is a differentiated cell because, unlike the GSC, it divides
precisely four times to produce 16 daughter cystocytes, which eventually
produce 15 nurse cells and one oocyte. Also, unlike the GSC, each cystoblast
or cystocyte cell division executes only partial cytokinesis and thus the
cystocytes remain interconnected as a cyst
(de Cuevas et al., 1997).
Fig. 1 presents a schematic
view of germarial Region 1 (the cyst-forming region) on which the pattern of
bam mRNA and protein accumulation is superimposed. Low levels of Bam
protein are expressed in GSCs, where it accumulates in fusomes but
bam mRNA is undetectable by in situ hybridization. These
observations indicate that bam is transcribed at a very low level in
GSCs and that this small amount of transcript produces the protein seen
associated with GSC fusomes. In the cystoblast and young cystocytes,
bam transcripts become readily detectable, reflecting an upregulation
in transcription rate or increased stability of the mRNA. This more abundant
pool of mRNA produces higher levels of Bam protein that begin to accumulate in
the germ cells' cytoplasm and continues to decorate the growing fusome
(McKearin and Ohlstein, 1995).
As cysts mature, the abundance of bam transcripts declines such that
mRNA is again undetectable in eight-cell cysts and Bam protein levels fall
precipitously once the 16-celled cyst forms such that Bam is again restricted
to the fusome (McKearin and Ohlstein,
1995
).
|
Although loss of bam or bgcn function produced
hyperplasia of undifferentiated germ cells, ectopic Bam expression caused GSC
elimination (Ohlstein and McKearin,
1997) similar to that seen when GSC maintenance factors were
inactivated (Xie and Spradling,
1998
). These data are most readily explained by assigning
bam the role of a necessary and sufficient cystoblast differentiation
factor (Ohlstein and McKearin,
1997
). A simple model to account for the experimental observations
is that the microenvironment, or niche
(Spradling et al., 2001
),
created by somatic tip cells maintains the most anterior germ cells as GSCs by
suppressing bam+ expression, thereby preventing them from
differentiating as cystoblasts.
The nature of intrinsic and extrinsic regulatory factors can provide clues
about which aspects of gene expression are targeted by the signaling that
sustains GSCs. The abrupt appearance of bam transcripts in
cystoblasts and their persistence in cystocytes for only a few cell divisions
is consistent with control of either bam transcription or mRNA
stability or both (McKearin and Spradling,
1990). The demonstration by Xie and Spradling
(Xie and Spradling, 1998
) that
GSC maintenance depends on the Dpp signaling cassette suggests that
Smad-dependent transcriptional control could regulate the choice between GSC
or cystoblast fate (Xie and Spradling,
1998
; Xie and Spradling,
2000
). Alternatively, the asymmetric distribution of bam
mRNA could arise if bam transcripts were more stable in cystoblasts
than in GSCs.
Previously, we had found that a reporter gene driven by a partial
bam promoter could be transcribed in bgcn
cells that could not become cystoblasts, implying that bam
transcriptional activation might precede full cystoblast differentiation
(Lavoie et al., 1999). On this
basis, we set out to determine the role of transcriptional control using
promoter mutagenesis studies in transgenic flies. We did so with the
expectation that, if transcriptional regulation was important for the
GSC-to-cystoblast switch, identification of the essential promoter elements
could provide significant insights into the signaling pathways responsible.
Here we confirm that bam+ transcriptional control is
necessary for proper GSC and cystoblast fate and we define the regions of the
bam promoter that are required and sufficient for the
GSC-low/cystoblast-high pattern of expression. We find that one
transcriptional control element in the 5'-UTR acts as a silencer in the
GSC and bam transcriptional silencing is required for GSC fate.
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MATERIALS AND METHODS |
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Transformation constructs
Constructs testing promoter activity used either green fluorescent protein
(GFP) alone or Bam:GFP recombinant protein. Although Bam:GFP protein could
provide full rescuing activity, the distribution of GFP fluorescence from
fusion protein did not entirely recapitulate native Bam protein localization
because fusomes were GFP-negative even when the Bam:GFP transgene was the only
source of functional Bam protein. The basis for this difference from native
Bam distribution (McKearin and Ohlstein,
1995) is currently the object of study.
Unless stated otherwise, all constructs were made using the CaSpeR4
transformation vector (Pirrotta,
1988). The P[bam promoter-Gal4:VP16] transgene used a
derivative of P[hsp70-CaSpeR] that contained the promoter and
3'UTR from the hsp70 gene (gift of D. Smith). The various
constructs described in this paper were generated using primers listed in
Table 1.
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Constructs for Bam promoter 5' deletion analysis
PCR reactions to recover promoters that were truncated from the
5'-side were anchored on the 3'-end by an antisense primer at
position +133 (Table 1). These
PCR fragments were joined to Bam:GFP or GFP reporter gene with appropriate
restriction enzyme digestions and subsequently linked to the bam
3'UTR in pCaSpeR4.
Constructs for Bam promoter 3' deletion analysis
Fragments truncated from the 3'-end were generated by a set of
antisense primers (Table 1) and
were anchored at the 5' side with oligonucleotides corresponding to
positions 898, 799 or 698
(Table 1). These PCR fragments
were cloned upstream of a GFP reporter and linked to bam 3'UTR
in pCaSpeR4.
Constructs for silencer element sufficiency test
Fragments for sufficiency tests were recovered by PCR or assembled by
annealing synthetic oligonucleotides. Promoters containing small deletions
were synthesized directly by PCR using primers that lacked the targeted
residues. A two-step strategy was used to construct promoters with internal
deletions; separate PCR products were ligated such that the targeted
nucleotides were deleted and the ligation products were used as templates for
subsequent PCR. We used nos and P-element minimal promoter sequences
that had been identified previously (Van
Doren et al., 1998; Rorth,
1998
) for constructing heterologous promoters.
Inverted silencer element transgene
A two-step PCR was performed to amplify the fragment containing the
bam promoter and an inverted silencer element. Fly genomic DNA was
used as template for first round PCR. After the first PCR reaction for 10
cycles, resulting PCR products were used as template for second round PCR. The
final PCR products were subcloned into CaSPeR vector in front of a GFP
reporter.
Two-step PCR method:
Cytological methods
Germline clones were produced by inducing the expression of autosomal
P(hsp70-FLP) transgenes at 37°C for 1 hour. Heat shock was
repeated at least two more times on the first day and the same flies were
occasionally heat-shocked again by the same protocol on the next day to
achieve higher production of clones. Cells made homozygous for brk
mutant alleles were recognized as GFP-negative against a background of
GFP-positive cells.
GFP fluorescent images from reporter transgenes and germline clones were collected on a Zeiss LSM410 confocal microscope. Samples were evaluated as single optical sections in the plane that included the terminal filament to make certain that GSCs were included in the image. The optical files were processed using Adobe Photoshop 6.0 and Deneba Canvas 7.0.
RACE for determining transcriptional start site
Total RNA was isolated from 0-3 h collections of w1118
embryos and digested with RNase-free DNase. cDNA was prepared by reverse
transcription using an antisense bam primer and tagged with a poly(A)
tail using terminal deoxynucleotide transferase according to manufacturer's
instructions (Promega). The ends of tailed cDNA were amplified by PCR with
oligo-dT and bam-specific primers. The resulting PCR products were
cloned into the pGEM T-Easy vector (ProMega) and sequenced by a
UT-Southwestern DNA Sequencing facility.
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RESULTS |
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To begin our studies, it was necessary to define the precise
transcriptional start site. Initial RNAse protection assays had defined the
start site at a sequence `TTCTGGG'
(McKearin and Spradling,
1990), but more precise RACE assays placed the transcriptional
start site at `gccAtcattctt'. This site will be designated as `+1'
in this paper for the purposes of orienting bam promoter
elements.
Previously we found that a genomic fragment of 1008 bp immediately upstream
from the +1-site plus the 5'-UTR could fully rescue bam
phenotype (McKearin and Spradling,
1990), and subsequently had refined essential promoter sequences
to 898 bp plus 5'-UTR (B. Ohlstein and D. M., unpublished). Both
promoters recapitulated the native bam pattern by driving expression
in cystoblasts and young germ cells, but remained quiescent in GSCs. We
concluded that all sequences necessary for bam+
transcriptional control were contained within the sequences spanning from
898 bp 5' to the bam transcriptional start site through
133 bp of the bam 5'-UTR.
We constructed P[UASp-Bam:GFP] transgenes to test the effects of expressing
bam+ from a variety of different promoters. By itself, the
construct produced no detectable GFP expression. When expressed from a
nos promoter in P[UASp-Bam:GFP];
P[nosP-Gal4:VP16-nos3'UTR] flies, adult ovaries from
young females contained only a few maturing eggs but no other germline cells
(not shown). This was the same phenotype obtained when
bam+ was expressed from a heat shock promoter
(Ohlstein and McKearin, 1997)
or when any of several GSC maintenance genes were inactivated by mutation
(Forbes and Lehmann, 1998
;
Lin and Spradling, 1997
;
Xie and Spradling, 1998
;
Xie and Spradling, 2000
). Thus
the nos promoter, which directed bam+ expression
in all PGCs and adult germ cells including GSCs, caused GSC ablation. We also
noted that the testes of [UASp-Bam:GFP];
P[nosP-nos5'-Gal4:VP16-nos3'UTR] males
were shriveled but did not determine the basis of the defect. In contrast to
heterologous transcriptional control of bam+ expression,
oogenesis was normal and GFP was expressed in cystoblasts and young cystocytes
but not GSCs when a native bam promoter controlled transgene
expression in
P[bamP-bam5'-Gal4:VP16-hsp70-3'UTR];
P[UASp-Bam:GFP] flies. This combination of transgenes also fully rescued
bam mutant flies. Because levels of GFP reporters suggested that the
relative `strength' of the nos and bam promoters was similar
in cystoblasts (Van Doren et al.,
1998
), the different results obtained with bam and
nos promoters predicted that the timing of bam transcription
was critical for GSC fate. In addition, as GFP expression in P[UASp-Bam:GFP];
P[nos-Gal4:VP16] animals recapitulated the nos expression
pattern and not the bam pattern, we concluded that the Bam ORF and
3'-UTR were not sufficient for the wild-type bam
expression.
To determine whether the 3'-UTR contained necessary regulatory signals, we examined the GFP expression in P[bamP-bam5'-Gal4:VP16-hsp70-3'UTR]; P[UASp-GFP] flies and found that GFP expression pattern mimicked wild-type bam mRNA accumulation (not shown). Thus the bam 3'-UTR was not required for the bam expression pattern.
Consistent with the conclusion stated above, comparison of the expression
of Bam:GFP and GFP reporters using bam promoters showed that the Bam
ORF did not contain sequences that were necessary or sufficient for the
wild-type bam expression pattern. Inclusion of the Bam ORF in
reporter constructs, however, did affect perdurance of reporter protein in
maturing cysts such that GFP fluorescence was much shorter-lived with Bam:GFP
than GFP alone (for example, see Fig.
2). This probably reflects instability of Bam protein in maturing
cystocytes, as has been postulated previously based on the distribution of
native Bam protein (McKearin and Ohlstein,
1995).
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Sequences controlling cystoblast-specific expression are present
within bam upstream proximal region
A series of deletions from the 5'-end of the bam promoter
defined cis-acting regulatory elements required for bam
expression (Fig. 2). We assayed
four aspects of expression of constructs that fused promoter fragments to a
Bam:GFP chimeric protein: (1) germ cell specificity of GFP fluorescence,
relative GFP levels in (2) GSCs and (3) cystoblasts and (4) rescuing activity
of the tested transgene. The full bam promoter (898) showed no
GFP expression in GSCs and strong expression in cystoblasts and young
cystocytes (Fig. 2A). For
example, Fig. 2B.a shows an
example GFP-negative GSC and a GFP-positive cystoblast within the same
germarium. The GSC can be recognized because it is the anteriormost cell in
the germarium and carries a spherical fusome (arrowhead) at the most anterior
position, whereas the cystoblast is immediately posterior to the GSC and has a
fusome (arrow) that is located more centrally within the cell.
Deletions of sequences between 898 to 198 showed no effect on the timing, germ cell specificity or relative intensity of reporter activity (Fig. 2A,B.b). In addition, bam/bam flies expressing Bam:GFP protein from these truncated promoters showed complete rescue because females developed wild-type ovaries and retained an active GSC population (Fig. 2A). Expression from a promoter fragment that contained sequences starting at 96 was very low or, in approximately half the ovaries assayed, was not detectable at all. Consistent with its apparent weak promoter activity, this construct was also unable to rescue bam/bam flies (Fig. 2A). Nevertheless, when Bam:GFP protein was produced at detectable levels, its accumulation was restricted to cystoblasts and the cystocytes of developing cysts (Fig. 2B.c). Thus the sequence between 198 and 96 provided element(s) that enhanced transcriptional output but did not effect the timing or cell specificity of transcription. Additional deletions refined the limits of quantitative element(s) to between 168 and 140 (Fig. 2B.e,f).
When deletions from the 5'-end extended to the 47 position, all GFP expression was eliminated (Fig. 2A,B.d). Thus sequences between 96 and 47 contained elements that could direct germ cell-specific transcription and were essential for bam promoter activity. The 96 to 47 region could include germ cell enhancer sites or elements required for basal transcription or both, but the 5'-deletion series could not distinguish between these possibilities. Strikingly, the data from the 5'-deletion constructs could also not explain bam transcriptional asymmetry in GSCs and cystoblasts.
A silencer element is present in the 5'UTR
A series of deletions from the 3'-end of the bam promoter +
5'-UTR fragment complemented the data obtained from the constructs
described above. Promoters contained sequences as far upstream as 799
and were truncated at various positions within or immediately outside of the
5'-UTR to test for elements that might be included within the UTR region
(Fig. 3A). Promoters that
started 799 bp upstream of the transcriptional start site and included at
least 55 bp of the 5'-UTR reproduced the GSC-negative,
cystoblast-positive pattern of bam expression
(Fig. 3A,B.a and
Fig. 3B.b). Note that, because
these constructs included only the GFP reporter protein and were therefore not
targets of Bam-dependent degradation, cystocytes in fully formed and
developing cysts were GFP-positive.
|
Additional deletions in this series revealed a new feature of bam cis-regulatory sequences. When the promoter fragment extended only to +4, reporter activity expanded to include GSCs as well as germ cells in cysts and beyond (Fig. 3A,B.c). Although other deletions from the 3'-side that reached as far as 20 continued to express GFP in all germ cells including GSCs (Fig. 3A,B.d), GFP expression was eliminated when the 3' deletion series reached the 47 position (Fig. 3A,B.e). Thus sequences between 46 and 20 were essential for any transcriptional activity, whereas sequences between +5 and +55 were necessary to silence bam expression in GSCs. Combining these observations with the results of deletions from the 5'-end of the bam promoter fragment, we concluded that sequences between 96 and 20 provided positively acting elements sufficient for young cystocyte-specific bam transcription, and that critical components of a silencer element that blocked bam expression specifically in GSCs lay between +5 and +55.
Bam promoter without inhibitor element causes GSC loss
Previous studies had shown that bam misexpression from a
hsp70 (Ohlstein and McKearin,
1997) or nos promoter
(Fig. 4A,B) that extended the
domain of bam expression could cause specific GSC loss. The most
attractive model to explain these effects was that inappropriate temporal
control of bam transcription, specifically misexpression in GSCs, was
responsible for GSC ablation. Transgenes that expressed Bam:GFP from promoter
fragments that lacked the silencing element but retained other native promoter
elements would provide a powerful test of this hypothesis. In fact, young
females that expressed a functional Bam:GFP protein controlled by a promoter
fragment from 799 to +4 showed the expected `few eggs no
germline' phenotype associated with GSC elimination
(Fig. 4C,D). The effect was
fully penetrant but showed variable expressivity when the transgene was
present in one copy; two copies increased expressivity to 100%. These
observations indicated that bam transcriptional quiescence was
necessary for GSC fate and that regulation of the silencer element was the key
component of the switch between GSC and cystoblast fate.
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Defining the limits of the silencer element
In order to delineate the silencer element more precisely, we tested the
ability of various fragments to suppress activity of a promoter containing the
germ cell enhancer and basal elements. We chose the (799 to 20)
promoter fragment as the germ cell `constitutive promoter' because it directed
GFP expression in GSCs and remaining germ cells but not ovarian somatic cells
(Fig. 3B.d). When an
oligonucleotide containing (+5 to +55) was fused to this basal promoter,
cystoblasts and cystocytes were GFP-positive but GSCs were negative (not
shown), indicating that this fragment carried full silencer activity.
One of the 3'-deletion constructs (799 to +34) had caused reproducible intermediate silencer activity (Fig. 5B). We suspected that this truncation might leave some of the silencer element intact and noted that the truncation breakpoint fell within an imperfect palindromic sequence (+27-CGCaGaCaGCG to +37; Site A). We probed the structure of the silencer element by constructing (799 to +55) promoters that precisely lacked the palindromic sequence. The GFP reporter from this construct was active in GSCs as well cystoblasts and cystocytes (Fig. 5C), indicating that residues +27 to +37 were essential for full silencer activity. The palindromic element was not sufficient for silencer activity, however, because promoters constructed by fusing an oligonucleotide encoding only Site A to a (799 to 20) basal promoter permitted GFP expression in both cystoblasts and GSCs (Fig. 5D). These assays demonstrated that the palindromic sequences were an essential part of the silencer element but were insufficient to block bam transcription in GSCs.
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We discovered that the (+27 to +37) oligonucleotide used in this experiment carried the sequence `CGCAGACTGCG' instead of the native bam sequence `CGCAGACAGCG'. Although it seemed unlikely, we could not eliminate the possibility that silencer activity of this element might have been destroyed by the mutation that generated an even more symmetrical palindrome. We subsequently constructed a D. yakuba bam promoter-GFP reporter that allowed us to test the efficiency of this silencer sequence because the D. yakuba Site A sequence corresponds to the perfect palindrome. We found that the yakuba promoter showed the same GFP expression pattern as wild-type melanogaster bam and concluded that `CGCAGACAGCG' and `CGCAGACTGCG' palindromes were both efficient silencers in the context of the bam promoter sequences.
Although promoters that included only to +37 permitted GSC expression,
promoters that extended to +55 did not, indicating that a fully active
silencer must include sequences between +37 and +55. We noted that the eight
base pairs directly adjacent to the palindromic element closely matched the
consensus binding site (Site B) for a known sequence-specific transcriptional
repressor, Brinker (Campbell and Tomlinson,
1999; Jazwinska et al.,
1999
; Minami et al.,
1999
; Rushlow et al.,
2001
; Saller and Bienz,
2001
; Kirkpatrick et al.,
2001
; Zhang et al.,
2001
). We therefore tested whether Site B was part of the silencer
element sequences. A (799 to +55) construct that contained a deletion
of base pairs +37 to +39 expressed GFP in GSCs
(Fig. 5E), suggesting that Site
B was important for full silencer activity. Note that this deletion altered
Site B but reconstituted a complete Site A sequence. To determine whether
Sites A and B were sufficient for the silencer element, we joined an
oligonucleotide encoding (+25 to +45) (Materials and Methods) to the
(799 to 20) constitutive germ cell promoter. GFP expression from
this construct was strong in cystoblast and cystocytes but was undetectable
above background levels in GSCs (Fig.
5F), demonstrating that full silencer activity requires only
sequences contained in Sites A and B.
Despite the fact that Site B matched a Brinker consensus binding element,
the results of subsequent experiments made Brinker a poor candidate for an in
vivo regulator of bam transcription. Ovarian ß-galactosidase
expression in brk[XA]/+ females that carry a P[lacZ]
enhancer-trap transposon in the brk gene
(Campbell and Tomlinson, 1999)
was restricted to cap, terminal filament and inner sheath cells, but, notably,
was not expressed in any germline cells (not shown). Furthermore, germline
clones of brk[M68] loss-of-function allele produced wild-type egg
chambers and GSCs were stable for at least two weeks after clone induction
(Fig. 5G). Thus, germline cells
neither express nor require Brinker for GSC maintenance or cyst
development.
Is transcription the target of the silencer?
The fact that the silencer was in the 5'-UTR raised the possibility
that it acted at a post-transcriptional level. We tested this possibility by
making bam transcripts that included the silencer element but were
transcribed from the nos promoter that is active in all germline
cells including GSCs (Van Doren et al.,
1998). Transgenic flies carrying the construct
[nosP-bam5'-GFP-bam3']
(Fig. 6A) expressed GFP in
GSCs, cystoblasts and other cystocytes at approximately equivalent levels
(Fig. 6B). We examined the
structure of the [nosP-bam5'-GFP-bam3']
mRNA to verify that these transcripts included the silencer element motif
using RT-PCR and primers that would bracket potential start sites
(Fig. 6A). RT-PCR of RNA from
[nosP-bam5'-GFP-bam3'] flies showed
that the transcript produced from the transgene started very close to or
precisely at the native bam gene transcription start site
(Fig. 6C). Therefore, the
bam silencer element did not act post-transcriptionally because the
reporter mRNA carrying the element was stably expressed in GSCs.
|
We confirmed the transcriptional role of the silencer element by constructing a [bamP-SErev-GFP] transgene that carried an inverted silencer (Materials and Methods). This transgene expressed GFP in the same pattern as a wild-type construct (not shown), demonstrating that the silencer element was active in either orientation. Because the inverted silencer would encode a sequence that was the complement of wild-type in the [bamP-SErev-GFP] mRNA, we concluded that the silencer acts as a DNA, not RNA, element.
The silencer element shows specificity for the bam promoter
Although transcripts from the
[nosP-bam5'-GFP-bam3'] transgene showed
that the silencer did not act as an RNA cis-element, GFP expression
in GSCs also revealed that the silencer element did not block transcription
from the nos promoter. This observation suggested that effective
transcriptional silencing by the bam element was sensitive to
context. As an additional test of silencer properties, a bam fragment
containing a fully active silencer (16 to +133) was placed upstream of
the basal bam promoter (799 to 20). GFP was expressed
in GSCs, cystoblasts and cystocytes in flies carrying these constructs,
indicating the silencer was inactive in this position
(Fig. 7A).
|
Refining enhancer elements
Previous experiments had shown that the fragment from (96 to
20) was sufficient for promoter activity in germline cells and that
fragments (96 to 47) and (46 to 20) were necessary
(Fig. 3). We reasoned that
these regions contained enhancer element(s) that directed germ cell-specific
transcription and a core promoter element, respectively.
We sought to identify essential enhancer sequences experimentally by
joining bam fragments to a previously identified core promoter from
the Drosophila P-element (Rorth,
1998). The transcriptionally inactive (799 to 47)
fragment (Fig. 4E) was joined
to the P-element core promoter driving the GFP reporter gene
(Fig. 7B). Flies carrying this
construct expressed GFP in GSCs, cystoblasts and cystocytes just as had been
observed for the (799 to 20) bam promoter fragment
(Fig. 4D). Thus the (47
to 20) region could be replaced by a heterologous minimal promoter
element and at least one germ cell-specific enhancer lies in the region
between 96 and 47.
An additional deletion series refined the position of the enhancer by
revealing that promoters lacking nucleotides from 86 to 61
deletion were inactive (compare Fig. 7C and
D). Deletion of a GC-rich block of nucleotides within the required
region (68 to 61) also extinguished germ cell expression of the
GFP reporter (Fig. 7E). Thus,
`gcgacggc' block of nucleotides was essential for the bam enhancer
element. Although the `gcgacggc' element matches the consensus binding site
for the Mad protein (Kim et al.,
1997), we note that Xie and Spradling
(Xie and Spradling, 1998
) have
shown previously that mad/ cysts develop
normally. It is therefore unlikely that this essential bam promoter
element binds Mad in germ cells.
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DISCUSSION |
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Our collection of transgenes allowed us to address the role of post-transcriptional control in regulating GSC to cystoblast differentiation. Transgenic reporters driven by bam promoters but in which GFP replaced the bam ORF and hsp70 3'-UTR replaced the bam 3'-UTR were expressed in cystoblasts and cystocytes but not in GSCs, demonstrating that sequences in the bam ORF and 3'-UTR were dispensable for proper regulation. Animals that produced a bam5'-GFP-bam3' reporter from a nos promoter, however, expressed GFP in GSCs as well as other germ cells. RT-PCR showed that the transcript produced from the P[nosP-bam5'-GFP-bam3'] transgene included all of the bam 5'-UTR sequences and would therefore contain all elements that could direct post-transcriptional regulation. Because GSCs were GFP-negative when the same reporter was expressed from fully active fragments of the bam promoter, we concluded that cell-specific transcriptional activation was the first and most significant level of control for regulating differential bam expression in stem cells and cystoblasts.
Alternative explanations for differential bam expression are that
positively acting elements first become active in cystoblasts, that negatively
acting elements function specifically in GSCs to repress bam
transcription or a combination of these mechanisms. The fact that bam
promoters that lacked the silencer element could direct reporter expression in
germ cells, including GSCs, demonstrated that all germ cells contained the
factors necessary to activate the bam promoter but that only GSCs
contained factors to repress bam transcription. Because Bam is
necessary and sufficient for cystoblast differentiation
(Ohlstein and McKearin, 1997),
the choice between GSC or cystoblast fate can be reduced to understanding the
cis- and trans-acting factors that activate and repress
bam transcription.
Germ cell specificity
The native pattern of bam mRNA expression alerted us to at least
two features of bam transcription; it was active only in germ cells
and it was undetectable in GSCs. The sum of control elements should explain
activity in this pattern and we therefore examined putative promoter deletions
for their capacity to (1) limit bam expression to germ cells and (2)
limit bam expression to cystoblasts and very early cystocytes.
The 5'-deletion series showed that the minimal bam promoter was small because a fragment containing as few as 168 bp upstream of the transcriptional start site was sufficient to reproduce all aspects of native bam expression. Additional deletions demonstrated that sequences between 168 and 140 accounted for quantitative effects, but additional functional assays are necessary to identify the specific elements that regulate transcriptional output.
Even fragments that could not produce wild-type levels of transcription, however, retained cell specificity of the remaining transcriptional activity. At a minimum, these elements (Fig. 8) include (1) an enhancer element between 86 and 61 that directs bam transcription in GSCs, cystoblast and germline cyst cells and (2) a silencer between +27 and +44 that blocks enhancer activity in GSCs, (3) sequences between 47 and 20 that are essential for transcriptional activation and can be replaced by a P-element core promoter.
|
Promoter elements that act positively
The 3' series of deletions showed that promoters that retained at
least to 20 could reproduce the wild-type bam expression
profile but promoters that retained only to 46 could not. Because
fusion of the (799 to 46) fragment to the P-element minimal
promoter could recapitulate bam-like expression, we concluded that
46 to 20 contained the `basal elements' that would interact with
the core RNA polymerase subunits
(Kadonaga, 1998;
Reinberg et al., 1998
). We
noted that the bam promoter did not comply with the rules for a
TATA-dependent promoter because none of the sequences within the essential
regions matched the TATA element consensus
(Zenzie-Gregory et al., 1992
).
Residues 24 to 17 (TTAACAA)
(Fig. 8) could represent a
functional variation on the TATA motif
(Singer et al., 1990
), but the
fact that promoters that included only to 20 were active means that the
full motif is not required for normal transcriptional activity. `TATA'-less
promoters that lack the `TATAA' box and bind core RNA polymerase proteins with
Initiator (Inr) cis-acting sites have also been characterized
(Smale, 1997
). The efficiency
and accuracy of Inr elements is increased in the presence of two other
sequence motifs, the BRE and DPE (Kutach
and Kadonaga, 2000
). The sequences surrounding the bam
transcriptional start site, however, do not match the consensus for Inr, BRE
or DPE elements. Thus, other elements between 46 and 20 might
function as `TATA-less' RNA polymerase recruitment sites in the bam
promoter or `TTAACAA' at 24 to 17 functions as a `TATAA' box
in vivo and can be adequately replaced in transgenic constructs.
The same transgenes that identified the core element(s) of the bam
promoter revealed that a germ cell-specific enhancer(s) was located upstream
of 47. Deletions from the 5'-side showed that the upstream border
of this enhancer region was located at 96. Additional deletions showed
that essential enhancer sequences lay between 86 and 61. The
sequence `gcgacggc' (Fig. 8)
matches the binding consensus site for the Dpp-activated transcription factor
Mad (Kim et al., 1997) and
specific deletion of that eight-base pair element inactivated the bam
promoter (Fig. 7). Genetic
studies, however, have shown that Mad is not required for cystoblast and cyst
differentiation (Xie and Spradling,
1998
) and is therefore not essential for bam
transcription. We suspect, therefore, that another activating transcription
factor with a Mad-like recognition sequence binds to this site in
bam. The significance of other sequences within this enhancer region
will be determined only by additional transgenic studies with site-directed
mutant constructs.
Elements that act negatively
Deletions that caused the spreading of reporter expression into GSCs and
constructs that tested the sufficiency of specific bam promoter
fragments revealed that an 18 bp silencer element (SE;
Fig. 8) regulated bam
expression. Although the SE was located in the 5'UTR, experiments that
distinguished between transcriptional and post-transcriptional effects
demonstrated that the bam SE repressed transcription. Chimeric
promoters that joined heterologous sequences to bam fragments
revealed several other features of the silencer element. The bam SE
could not block GSC transcription from a nos enhancer/promoter
fragment nor when the SE was placed upstream of the bam
enhancer/basal region. Both of these observations can be explained if the SE's
range was limited because either construct would place the silencer much
further from the relevant enhancer elements. Such short-range repressive
effects have been described for the dCtBP family of transcriptional repressors
(Chinnadurai, 2002;
Mannervik et al., 1999
). Other
explanations are equally possible, however, and resolution of the silencer's
mechanism of action must await biochemical studies with silencer-binding
proteins.
The structure of silencer, as revealed by complementary sets of necessity
and sufficiency tests, suggests that at least two proteins bind to the site.
The palindromic part of the silencer element (Site A) was essential for
activity but it does not correspond to any known transcriptional regulatory
protein-binding site. The more 3' part of the silencer (Site B)
corresponds to a consensus site for Brinker binding
(Sivasankaran et al., 2000;
Zhang et al., 2001
). Brinker
has been identified previously as a transcriptional repressor of Dpp target
genes and was therefore a reasonable candidate for a silencer binding protein.
Brinker is an antagonist of the Dpp-signaling because it prevents the
transcription of a subset of Mad activated genes
(Kirkpatrick et al., 2001
;
Rushlow et al., 2001
;
Saller and Bienz, 2001
), but
Brinker is itself a Dpp target (Minami et
al., 1999
) because Mad and Schnurri heterodimers can block
brk transcription in dpp-responsive cells
(Marty et al., 2000
). The
paradigm established from these studies for regulation of brk
expression predicted that the gene would be transcriptionally repressed in
cells with activated Dpp signaling, such as GSCs. Indeed, previous studies
demonstrated that both Mad and Shn are active in GSCs
(Xie and Spradling, 1998
;
Xie and Spradling, 2000
) and
are therefore in place to repress brk expression. In addition,
enhancer trap expression and germline clonal analyses did not support a role
for Brk in germ cells, making it unlikely that Brk could play a significant
role in keeping bam transcriptionally quiescent in GSCs. Perhaps
another repressor protein that recognizes Brk-like elements binds to
bam silencer Site B and forms a complex with the palindrome-binding
protein to block bam transcription in GSCs.
bam transcriptional control and GSC-to-cystoblast
differentiation
The discovery of the silencer element permitted refinement of a model
explaining the GSC-to-cystoblast switch. Our dissection of the bam
promoter shows that bam can be expressed in GSCs because promoters
lacking the silencer element are transcribed in all germ cells. For the same
reason, the silencer does not work by blocking an overlapping enhancer site
because silencer sequences can be deleted without disrupting
enhancer-dependent transcription (Fig.
3C). Our current working model
(Fig. 9) is that the silencer
is occupied in GSCs and bam is transcriptionally quiescent because
the germ cell-specific enhancer(s) cannot be engaged. When the presumptive
cystoblast is born, silencer element-dependent repression can be relieved,
allowing enhancer(s) in bam promoter to activate transcription and
promote cystoblast differentiation. The clear implication of this model is
that bam silencer occupancy is the basis of the asymmetric GSC
division and that identification of silencer binding proteins can provide a
mechanism for the asymmetry.
|
Previous studies showing that somatic cell signaling maintained GSCs by
suppressing cystoblast formation suggested that bam might be the
target of these signals (King and Lin,
1999; Xie and Spradling,
1998
). We propose that occupancy of the bam silencer
element in the germ cells adjacent to cap cells (i.e. GSCs) is signal
dependent and is based on asymmetric activation or distribution of silencer
element-binding proteins (SEBPs). For example, with the division of the
neuroblast mother cell as a paradigm (Doe,
2001
; Jan and Jan,
2001
), perhaps asymmetric partitioning of one or multiple SEBPs
accounts for cystoblast-specific bam transcriptional activation.
Alternatively, differences in extrinsic signaling could produce
post-transcriptional modifications of SEBPs to account for differential
activity in GSCs and cystoblasts. Distinguishing between these and other
possibilities to explain the molecular details of the GSC to cystoblast switch
requires the identification of SEBPs and understanding the signaling circuits
that control the occupancy of the bam silencer element.
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ACKNOWLEDGMENTS |
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