1 Institute of Molecular Biology and Biotechnology, Foundation for Research and
Technology Hellas, Heraklion, Greece
2 Department of Biology, University of Crete, Heraklion, Greece
* Present address: Department of Anatomy and Developmental Biology, University
College London, London WC1E 6BT, UK
Author for correspondence (e-mail:
delidaki{at}imbb.forth.gr)
Accepted 10 October 2002
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SUMMARY |
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Key words: Basic-helix-loop-helix, Proneural, HES, Transcriptional repression, Neurogenesis, Lateral inhibition, Drosophila, E(spl)
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INTRODUCTION |
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Within the anlagen of the CNS and PNS, proneural genes are initially
expressed in groups of cells termed proneural clusters
(Campuzano and Modolell, 1992).
From these broad domains, only a subset of cells will commit to the neural
fate. These neural precursors transiently upregulate proneural gene expression
and activate a number of neural differentiation genes, such as ase, sens,
dpn and others (Bier et al.,
1992
; Dominguez and Campuzano,
1993
; Jarman et al.,
1993
; Nolo et al.,
2000
), which are direct transcriptional targets of proneural bHLH
activators. The remaining cells of the proneural cluster are inhibited from
embarking into a neural pathway and will either continue proliferation or
differentiate to alternative cell types, such as epidermis
(Artavanis-Tsakonas and Simpson,
1991
). This is the outcome of intercellular signaling within the
proneural cluster, which is mediated by the Notch pathway
(Artavanis-Tsakonas et al.,
1999
) and is termed lateral inhibition. Cells that receive a high
level of Notch signal cannot turn on the proneural target genes (such as
ase, dpn, etc.); this block requires the activity of members of yet
another class of bHLH proteins, named Class VI or HES proteins
(Fisher and Caudy, 1998
). The
seven clustered E(spl) genes in Drosophila, m8, m7, m5, m3, mß,
m
and m
(Delidakis et al., 1991
;
Schrons et al., 1992
), encode
Class VI bHLH proteins and are directly turned on (transcriptionally) by Notch
signaling (Bailey and Posakony,
1995
; Lecourtois and
Schweisguth, 1995
). Their products accumulate in all cells of the
proneural cluster, but are minimal within the neural precursors
(Jennings et al., 1994
); they
can be therefore considered `anti-neural' proteins. Indeed, deletion of the
entire E(spl) locus results in severe overcommitment of neural precursors
(Lehman et al., 1983
).
However, mutations in individual E(spl) genes display no phenotypic
defects, as a result of partial functional redundancy, a fact that prohibits
forward genetic dissection of E(spl) protein function
(Delidakis et al., 1991
;
Ligoxygakis et al., 1999
;
Schrons et al., 1992
).
The link between proneural bHLH proteins, Notch signaling and HES proteins
is evolutionarily conserved, as it is encountered also in vertebrates, where
the cellular events of neurogenesis are very distinct from those in insects
(Kageyama and Nakanishi,
1997). In both phylogenetic groups, allocation of neural versus
non-neural fates is the outcome of two antagonistic bHLH activities: proneural
proteins that promote neurogenesis and HES proteins that inhibit it. As in
Drosophila, in vertebrates some HES genes are direct transcriptional
targets of Notch. Despite the central importance of these bHLH transcription
factors in early neural commitment, there are many gaps in our knowledge of
the regulatory circuits underlying neurogenesis, both in terms of the target
genes of proneural and HES genes, and in terms of the mechanisms of gene
activation and repression by these bHLH proteins. It was originally proposed
that E(spl) proteins might block neurogenesis in Drosophila by
repressing proneural genes (Martin-Bermudo
et al., 1995
; Skeath and
Carroll, 1992
). More recent data suggest that this is true only
for specific enhancers of the proneural genes that are autostimulatory and
sensory organ precursor (SOP) specific
(Culi and Modolell, 1998
),
while the major function of E(spl) proteins is to repress downstream target
genes of the proneural proteins (Culi and
Modolell, 1998
; Nakao and
Campos-Ortega, 1996
). HES proteins are indeed transcriptional
repressors. Key amino acid differences between the basic domains of HES and
Ac/Sc proteins endow these different bHLH factors with distinct target site
specificities: Da-Ac/Sc heterodimers bind the EA box GCAGSTG
(Singson et al., 1994
),
whereas E(spl) homodimers preferentially bind to EB-boxes (CACGTG)
and variants thereof, the C and N boxes (CACGCG and CACNAG, respectively)
(Jennings et al., 1999
;
Oellers et al., 1994
;
Ohsako et al., 1994
;
Tietze et al., 1992
;
Van Doren et al., 1994
).
EA, EB, C and N boxes are encountered clustered in
enhancers of proneural target genes, such as ase and dpn,
which are expressed strongly in the neural precursor and repressed in the
remaining proneural cluster cells. The importance of EA sites in
such enhancers has been confirmed by mutagenesis; ablation of EA
boxes leads to loss of transcriptional activity
(Culi and Modolell, 1998
;
Jarman et al., 1993
). The same
does not hold true, however, for EB/C/N boxes; mutation of these
does not lead to derepression of reporter genes mutant versions of the
sc SMC enhancer lacking all E(spl) binding sites are still expressed
only in the SOPs (Culi and Modolell,
1998
). Furthermore, E(spl) proteins retain residual activity after
disruption of their DNA-binding basic domain
(Giebel and Campos-Ortega,
1997
; Nakao and Campos-Ortega,
1996
; Oellers et al.,
1994
), although this is still somewhat controversial
(Jiménez and Ish-Horowicz,
1997
). As a result, alternative models regarding the mechanism of
target gene repression by E(spl) have been suggested. One proposes that E(spl)
can sequester activator complexes away from DNA
(Alifragis et al., 1997
;
Kageyama and Nakanishi, 1997
).
A second model proposes that E(spl) proteins may be recruited to target
enhancers indirectly, via interactions with other uncharacterized DNA bound
factors (Culi and Modolell,
1998
).
The simplest explanation for the fact that proneural target enhancers can be repressed by E(spl) in the absence of cognate DNA-binding sites is that E(spl) proteins use a DNA-binding-independent mechanism for proneural target gene repression, instead of, or in addition to, a DNA-binding-dependent one. In the present work, we ask if this is indeed the case. We present in vivo data that strongly support protein-tether-mediated recruitment of some E(spl) repressors onto DNA interestingly, this is achieved via protein-protein interactions with proneural activators. We demonstrate that direct DNA binding also contributes significantly to E(spl) activity, while activator sequestering is unlikely to be used by E(spl) proteins to counteract proneural function.
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MATERIALS AND METHODS |
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Ract-E(spl)m, Ract-E(spl)m
and Ract-E(spl)m7 were constructed
by subcloning the relevant BglII-XhoI fragments from pUAST
constructs (Ligoxygakis et al.,
1999
) into the BamHI/SalI sites of RactHAdh, an
actin5C promoter-containing plasmid
(Swevers et al., 1996
).
pT5-0.9wt/luc (ac proximal promoter luciferase reporter), as well as
its C-box mutated version pT5-0.9mut/luc have been described elsewhere
(Ohsako et al., 1994
). pAc-Da
and pAc-Sc have been described previously
(Van Doren et al., 1992
).
pUC-E(spl)m7KNEQ was constructed by simultaneous ligation of an
EcoRI/BamHI 5'-terminal fragment of E(spl)m7KNEQVP16
[from a pBluescript KSII/EcoRI/XbaI clone the
BamHI site, a naturally occurring site within E(spl)m7 at
codon G142 is the junction between E(spl)m7 and VP16 coding regions
(Jiménez and Ish-Horowicz,
1997)] with a BamHI/SalI C-terminal fragment of
E(spl)m7 (from a pBluescript KSII/EcoRI/XhoI clone) into
pUC18/EcoRI/SalI. Then, it was subcloned in pUASTmod vector
[pUASTmod is a modified pUAST vector that contains a synthetic oligonucleotide
bearing an optimized translation start site just before the cloning sites
(Ligoxygakis et al., 1999
)]
and digested with EcoRI/XhoI.
E(spl)mVP16 was released from pHK3N-E(spl)m
VP16, kindly
provided by B. Jennings and S. Bray, as a BamHI/BglII
fragment and subcloned into pUAST/BglII. The VP16 domain is fused to
amino acid 169.
Ract-E(spl)m was subjected to mutagenesis in order to create a
BamHI site, followed by a stop codon, just after the R154 codon. A
BamHI/SalI VP16 fragment was then inserted at this site to
create Ract-E(spl)m
VP16. E(spl)m
VP16 was released from Ract
E(spl)m
VP16 as an EcoRI/PstI fragment and subcloned
into pBluescipt SKII. pUASTmod E(spl)m
VP16 was then constructed by
inserting E(spl)m
VP16 into pUASTmod/EcoRI/XbaI.
pBluescript KSII-E(spl)mKNEQ was produced by mutagenizing
pBluescript KSII-E(spl)m
[E(spl)m
cloned into the EcoRI
site]. The E(spl)m
KNEQ mutagenesis primer (sequence available upon
request) was based on the E(spl)m7KNEQ construct described elsewhere
(Jiménez and Ish-Horowicz,
1997
); it changes two conserved basic domain amino acids: K17 to N
and E24 to Q. pBS-E(spl)m
KNEQ-VP16 was constructed by replacing an
EcoRV fragment of pBluescript SKII E(spl)m
VP16 with a
SmaI/EcoRV fragment from pBluescriptKSII E(spl)m
KNEQ,
which corresponds to the 5' terminal part of E(spl)m
KNEQ that
bears the desirable point mutations. E(spl)m
KNEQ-VP16 was isolated with
EcoRI and XbaI and cloned into pUASTmod.
All DNA manipulations were carried out using standard techniques. Mutagenesis reactions were performed using the Gene Editor kit by Promega according to the manufacturer's instructions. Every construct was sequenced prior to injection into Drosophila embryos or transfection into S2 cells to verify success of mutagenesis procedures and integrity of the constructs.
Cell culture, transient transfections, luciferase and ß-
galactosidase assays
Drosophila Schneider S2 cells were cultured at 25°C in M3
medium supplemented with 10% heat inactivated fetal bovine serum and
gentamycin. Transient transfections of approx. 2.5x106
cells/2.5 ml were performed with the
Ca3(PO4)2 co-precipitation method. All
plasmids were purified with Qiagen columns, according to the manufacturer's
instructions. Plasmids and amounts used per well are listed in the panels of
Fig. 1; all effectors were
expressed under the actin5C promoter using either the pAc or the
RactHAdh vectors. Additionally 100 ng hs-lacZ plasmid was added for
normalization. Empty vectors were used to bring the total DNA amount per
transfection to 5 µg. ß-Galactosidase assays were conducted in order
to measure the efficiency of the transfections and to normalize luciferase
measurements. Luciferase assays were performed using the luciferase kit
(Promega) according to the manufacturer's instructions. Luminescence was
measured using a Turner TD-20/20 luminometer.
|
Drosophila strains and crosses
All transformants were obtained in a yw67c23
background. All crosses were kept at 25°C, unless otherwise stated.
EE4-lacZ and UAS-sc transgenic flies have been described
previously (Culi and Modolell,
1998). UAS-E(spl)m7VP16 and UAS-E(spl)m7KNEQVP16
flies have been described previously
(Jiménez and Ish-Horowicz,
1997
). Gbe-B1-lacZ flies have been described previously
(Jennings et al., 1999
).
Df(1)sc10-1 (abbreviated as
sc10-1), groE48,
Df(3R)grob32.2 (deletion of the entire
E(spl) locus), Df(3R)P709 and Df(3R)Espl22 are
described in FlyBase
(flybase.bio.indiana.edu).
We use the following abbreviations for Gal4 lines: omb-Gal4 for P[Gal4]biomb-Gal4, pnr-Gal4 for P[GawB]pnrMD237 and ap-Gal4 for P[GawB]apmd544, all described in FlyBase (flybase.bio.indiana.edu).
In mosaic analysis experiments, clones were induced by heat shocking larvae (1 hour at 38°C) 48-96 hours after egg laying (AEL) of the following genotypes:
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Larvae were picked at wandering third instar, heat shocked again for 90
minutes (38°C) to induce Myc expression and then allowed to recover at
25°C for 90 minutes before dissection.
X-gal staining and immunocytochemistry
For X-gal staining, larvae were dissected in phosphate buffer and fixed in
1% glutaraldehyde/1xPBS for 9 minutes at room temperature. After
extensive washing with 1xPBS, they were placed in colorization buffer
[10 mM Na-PO4 buffer (pH 7.2), 150 mM NaCl, 1 mM MgCl2,
3 mM K4[FeII(CN)6], 3 mM
K4[FeIII(CN)6], 0.3% Triton X-100], prewarmed
at 65°C, containing 0.2% X-gal and they were incubated at 37°C in a
humid chamber for 15 minutes to overnight. For the experiment in
Fig. 2K,L, larvae from the
following cross were used:
EE4-lacZ/EE4-lacZ; TM6B, Tb/ Df(3R)P709 x P[gro+]/P[gro+]; TM6B, Tb/ Df(3R)Espl22.
Tb+ larvae have the viable deficiency combination
P[gro+]; Df(3R)E(spl)22 /Df(3R)P709, which is
null for E(spl)m7 and m8
(Delidakis et al., 1991),
whereas TM6B (Tb-) carry a wild-type copy of the E(spl)-C.
To measure
-galactosidase activity semi-quantitatively, both Tb and
Tb+ larvae were fixed and incubated in the same test tubes. They
were distinguished by leaving a piece of gut on one of the genotypic classes
at dissection. X-gal development lasted only 20 minutes to avoid
saturation.
Immunocytochemistry was performed as described previously
(Pavlopoulos et al., 2001).
Antibodies were from the Developmental Studies Hybridoma Bank (developed under
the auspices of the NICHD and maintained by the University of Iowa, Department
of Biological Sciences, Iowa City), Cappel, Jackson Immunochemicals and
Molecular Probes. Special conditions were used for the anti-Ac antibody:
Dissected larvae were fixed in 1xPEM [100 mM PIPES, 1 mM EGTA, 1 mM
MgCl2 (pH 6.9) corrected with KOH] 1% Triton X-100, 1% PFA, for 1
hour at 8°C. Subsequent washes and incubations were carried out in a 50 mM
Tris-Cl (pH 6.8), 150 mM NaCl, 0.5% NP40 buffer, supplemented, where needed,
with 1-5% normal goat serum. Specimens were observed either on a Leica Diaplan
microscope or on a Leica SP confocal microscope (University of Crete).
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RESULTS |
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To gain more insight into this novel repression mechanism of E(spl)m7 and
m, we turned into an in vivo system. We decided to study an artificial
reporter gene in the fly driven solely by EA boxes to avoid the
possibility of E(spl) proteins binding to atypical sites, a behavior for which
there is ample precedent (Chen et al.,
1997
; Culi and Modolell,
1998
; Yang et al.,
2001
), and may have been the cause of repression of T5m-luc in our
transfection experiments. The EE4-lacZ reporter, consisting of eight
tandem EA boxes in front of a minimal promoter
(Table 1), was shown by Culi
and Modolell (Culi and Modolell,
1998
) to respond to proneural proteins by turning on in all
proneural cluster cells in the wing disk. We assayed the response of
EE4-lacZ in larval imaginal disks in response to E(spl) proteins
expressed using the Gal4/UAS system. Overexpression of
E(spl)m7 abolished EE4-lacZ activity, whereas
E(spl)m
only moderately reduced expression
(Fig. 2A-C). This was somewhat
surprising, given that E(spl) proteins do not recognize the EA
target site (Culi and Modolell,
1998
; Jennings et al.,
1999
; Oellers et al.,
1994
). Thus, we entertained the possibility that the repression by
E(spl) was not a direct effect on the EE4 enhancer, rather it could
have arisen from the fact that overexpression of E(spl) repressed endogenous
proneural genes, which in turn are needed to activate EE4. We
therefore visualized Ac protein in wing disks overexpressing E(spl)m7
(Fig. 2I,J). The overall
proneural pattern of Ac was not altered, but expression levels were variably
reduced within the overexpression domain. Strongly expressing SOP cells within
proneural clusters were never seen (Fig. 2J
arrow), in agreement with the well-established sensory-organ
suppressive activity of E(spl) proteins
(Culi and Modolell, 1998
;
Giebel and Campos-Ortega,
1997
; Ligoxygakis et al.,
1999
; Nakao and Campos-Ortega,
1996
; Tata and Hartley,
1995
).
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In order to test more rigorously the mechanism of E(spl)-mediated
repression of EE4-lacZ and to avoid the fluctuation of endogenous
proneural protein levels caused by E(spl) overexpression, we decided to bypass
the need for endogenous proneural proteins altogether by providing excess Sc
exogenously. A UAS-sc transgene was expressed alone
(Fig. 2E) or together with
UAS-E(spl) transgenes (Fig.
2F,G). Ectopic Sc gave the expected broad, yet patchy, ectopic
activation of EE4-lacZ. Patchy activation of proneural target genes
has been observed before (Hinz et al.,
1994) and apparently reflects stochastic damping of Sc activity,
at least partly because of induction of endogenous E(spl) genes
(Cooper et al., 2000
;
Nellesen et al., 1999
), which
inhibit Sc activity (Giebel and
Campos-Ortega, 1997
; Hinz et
al., 1994
) (this work). Co-expression of E(spl)m7
resulted in strong repression of the EE4 enhancer
(Fig. 2E,F), whereas
E(spl)m
did not affect activation by UAS-sc
(Fig. 2E,G). We observed the
same effects using two different GAL4 lines, pnr-GAL4
(Fig. 2) and omb-GAL4
(data not shown), which drive expression in a central wing pouch region
(visualized in Fig. 5). It thus
appears that E(spl)m7, but not m
, can repress transcription of
EE4-lacZ without directly binding to DNA, consistent with the
different behavior of these proteins in transfection assays. m
still
weakly represses EE4-lacZ transcription
(Fig. 2C), most probably
through repression of activators, such as sc. Another
UAS-E(spl) transgene, E(spl)m
, was able to repress
UAS-sc-driven activation of EE4-lacZ, similar to
E(spl)m7 (data not shown).
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If direct DNA binding is dispensable for the repression by E(spl)m7 and
m of EE4-lacZ, mutant versions that lack the DNA-binding basic
domain should be functional. We therefore generated E(spl)m7KNEQ, a double
point mutation in the basic domain, which abolishes DNA binding
(Jiménez and Ish-Horowicz,
1997
), and tested it in transgenic flies.
UAS-E(spl)m7KNEQ had strong repressive activity on EE4-lacZ
when expressed either alone or together with UAS-sc
(Fig. 2D,H), confirming the
dispensability of the basic domain in this assay. UAS-m
KNEQ,
which bears the same basic domain inactivating mutations as m7KNEQ,
was also capable of repressing EE4-lacZ, even in the presence of exogenous
UAS-sc (data not shown).
In a converse experiment, we examined the activity of the EE4-lacZ
reporter in loss-of-function backgrounds for E(spl). EE4-lacZ was
consistently more active in a mutant background lacking E(spl)m7 and
m8 (see Materials and Methods) compared with wild type (12 disks of
each genotype scored in three repeats of the experiment;
Fig. 2K,L). This happens even
though the number and pattern of SOPs in this mutant background is identical
to wild type, presumably owing to the activity of the remaining
E(spl) genes (Delidakis et al.,
1991). We conclude that activity of E(spl)m7 and
m8, the two most highly expressed E(spl) genes in wing disk
proneural clusters (de Celis et al.,
1996
), attenuates EE4-lacZ expression. As E(spl)
genes other than m7 and m8 were still present in the above
genetic background, we tested the response of EE4-lacZ in homozygous
clones of a deficiency removing the entire E(spl) locus
(Fig. 2M). Increased levels of
ß-galactosidase expression were again observed within mutant patches,
confirming the response of EE4-lacZ to E(spl) activity, despite the
absence of E(spl) binding sites on this reporter. A caveat in interpreting
these experiments is that E(spl) loss-of-function may increase
sc expression, which would then act on the EE4-lacZ
reporter.
Ectopic expression of sc in flies is known to induce formation of
supernumerary chetae (Hinz et al.,
1994; Rodriguez et al.,
1990
), reflecting induction of endogenous Sc target genes. We
tested individual UAS-E(spl) transgenes for their ability to block
ectopic cheta production by sc. When expressed alone by
pnr-Gal4, all UAS-E(spl) genes inhibited formation of both
macro- and mirco-chetae, resulting in a bald stripe in the center of the
thorax (Fig. 3A,C,E), in
agreement with previous findings (Culi and
Modolell, 1998
; Giebel and
Campos-Ortega, 1997
;
Ligoxygakis et al., 1999
;
Nakao and Campos-Ortega, 1996
;
Tata and Hartley, 1995
). This
was even true for UAS-E(spl)m7KNEQ
(Fig. 3G) and
E(spl)m
KNEQ (data not shown), suggesting that, under the
conditions of this assay, direct DNA binding (to presumably natural target
genes controlling SOP fate) is dispensable. When co-expressed with UAS-sc,
UAS-E(spl)m7 and m
, as well as E(spl)m7KNEQ and
m
KNEQ, still produced completely bald thoracic stripes
(Fig. 3B,D,H), indicating that
these proteins can inhibit the activity of both endogenous and overexpressed
Sc on (endogenous) target genes very effectively. By contrast,
UAS-E(spl)m
only partially suppressed the ectopic bristle
phenotype of UAS-sc (Fig.
3F). This behavior was essentially the same as that documented
above using EE4-lacZ and was further confirmed by assaying the
expression of two target genes, SMC-lacZ and ase (data not
shown). The sole difference was that E(spl)m
could partially decrease
the number of ectopic bristles (Fig.
3F), while having no effect on EE4-lacZ activation
(Fig. 2G). We attribute the
bristle/SOP suppressive activity of E(spl)m
to DNA-binding-dependent
repression of proneural target genes (see Discussion). Taken together,
reporter and bristle repression assays demonstrated that E(spl)m7 and
m
, but not m
, can repress an EA-driven artificial
reporter gene, as well as endogenous target genes, despite the overexpression
of sc. Based on the fact that basic domain mutated versions of
E(spl)m7 and m
are much more potent repressors than m
, we
conclude that in this assay some activity of E(spl) proteins other than their
direct DNA binding ability is most important in target gene repression.
|
m7 is tethered to EA-boxes via proneural protein
complexes
We have previously shown that E(spl) proteins interact selectively with
proneural ones in a yeast two-hybrid assay
(Alifragis et al., 1997);
E(spl)m7 and m
interact with Ac, Sc and Da, whereas m
interacts
with none. In the light of results presented in the previous section, the
interesting possibility arose that the ability of E(spl) proteins to interact
with activator bHLH proteins might underlie the ability of the former to
repress target genes in the absence of direct DNA binding and enhance their
potency in neural fate suppression. The question arises as to how interaction
with proneural proteins might help realize this potent repressive activity: do
E(spl) proteins sequester proneural activators off the target DNA or do they
use the proneural complexes as tethers to bind to DNA? A way to approach the
question of whether a repressor works on or off DNA has been devised by
Jiménez and Ish-Horowicz
(Jiménez and Ish-Horowicz,
1997
), whereby a fusion of a strong transcriptional activation
domain (VP16) to a repressor is tested for its ability to activate
transcription, which can only happen if the VP16 domain is tethered to the
DNA. If, however, the repressor works by sequestering activators off DNA, the
VP16-tagged repressor should still be able to repress (rather than activate)
target genes. We expressed a hybrid E(spl)m7VP16 protein
(Jiménez and Ish-Horowicz,
1997
) in wing disks and assayed its effect on EE4-lacZ.
In both pnr-Gal4 and omb-Gal4 expression domains, we
observed strong activation of EE4-lacZ
(Fig. 4A,B,D), suggesting that
E(spl)m7VP16 is somehow tethered to this artificial enhancer. Rather than
being ubiquitous, activation by E(spl)m7VP16 was patterned in a way that
strongly resembled the proneural pattern, suggesting that E(spl)m7VP16 was
tethered to EE4-lacZ via proneural complexes. To demonstrate this we
assayed the same effector-reporter combination in both loss-of-function and
gain-of-function backgrounds for proneural genes. sc10-1
is a null allele for both ac and sc, the only proneural
proteins expressed in the wing disk. In sc10-1 wing disks,
EE4-lacZ was not expressed and could not be activated by E(spl)m7VP16
(Fig. 4E,F). In the converse
experiment, we supplied ectopic Sc by co-expressing UAS-sc with
UAS-m7VP16 (Fig.
4G-I); in this case, the pattern of EE4-lacZ activation
was broadened to encompass the whole expression domain and was not restricted
to proneural clusters (compare Fig. 4B with
4I). It therefore appears that it is the availability and spatial
distribution of proneural proteins, which determines the pattern of activation
of EE4-lacZ by E(spl)m7VP16. The simplest way to account for this
finding is to propose that E(spl)m7VP16 is recruited onto DNA using the
proneural complexes (and not some other DNA-bound factor) as tethers. This was
confirmed by testing the ability of two other E(spl)VP16 variants:
E(spl)m
VP16 and m
VP16. Whereas the former behaved identically to
E(spl)m7VP16 (data not shown), E(spl)m
VP16 had no effect on
EE4-lacZ expression (Fig.
4C). We attribute the inability of E(spl)m
VP16 to become
recruited onto EE4-lacZ to its inability to interact with the
proneural protein-tethering factors.
|
It is possible that proneural cluster restriction of EE4-lacZ
activation by E(spl)m7VP16 and mVP16 was due to some regional
inactivation (by protein modification) of the VP16 effector itself, and not to
its recruitment onto DNA via proneural complexes. We therefore asked whether
the E(spl)-VP16 variants were inherently capable of transcriptional activation
in all cells by assaying their ability to activate another artificial enhancer
[Gbe-B1-lacZ; Table 1
(Jennings et al., 1999
)] that
bears three EB boxes (recognized by HES-family proteins) in
addition to binding sites for Grh, an activator ubiquitously present in wing
disk cells. In a wild-type background, Gbe-B1-lacZ is expressed very
weakly and cannot be activated by UAS-sc [as Sc only weakly binds the
B1 EB box (Jennings et al.,
1999
); Fig. 5A,B].
In the presence of UAS-E(spl)m7VP16, m
VP16 or
m
VP16, strong ubiquitous activation was observed
(Fig. 5C-E), indicating that
all three E(spl)VP16 variants are strong activators when directly tethered to
DNA and their activity does not seem to be spatially modulated. We therefore
favor that the variable activation of EE4-lacZ
(Fig. 4) reflects selective
recruitment of the VP16 proteins onto the EE4 enhancer and is not a result of
post-translational modulation of their transactivation ability. This result
also strengthens our conclusion from Fig.
4C that E(spl)m
VP16 cannot become recruited onto
EE4-lacZ.
An E(spl)m7VP16 variant with mutated basic region should behave in a manner
complementary to E(spl)mVP16, as it should lack direct DNA-binding
activity but should retain the ability to be indirectly tethered to targets
via proneural proteins. The behavior of a UAS-E(spl)m7KNEQ-VP16
transgene showed that this was indeed the case. First, this effector was
unable to activate the Gbe-B1-lacZ reporter, confirming disruption of
its basic region (Fig. 5F). By
contrast, it was able to activate the EE4-lacZ reporter to the same
extent as wild type E(spl)m7VP16 (Fig.
6A-C). One interesting difference was that the activity of
E(spl)m7KNEQ-VP16 was restricted to proneural clusters (where ac and
sc are expressed), whereas E(spl)m7VP16 gave additional patchy
activation of EE4-lacZ in non-proneural cells of the
pnr-Gal4 domain. This was accompanied by marked ectopic accumulation
of the Ac proneural protein, something not seen with E(spl)m7KNEQ-VP16
(Fig. 6G-I). Ectopic activation
of endogenous proneural genes by E(spl)m7VP16 is probably achieved by directly
binding to enhancers that contain EB/C/N boxes (such as the
autoregulatory ones), because it is abolished by mutation of the basic region.
The resulting ectopic proneural protein is subsequently used as a tether to
bring E(spl)m7VP16 onto the EE4-lacZ reporter. To bypass this
feedback loop involving endogenous proneural genes, we supplied Sc via
co-expression of a UAS-sc transgene. As shown before, UAS-sc
alone resulted in patchy activation of EE4-lacZ
(Fig. 6D). However, in the
presence of E(spl)m7VP16 or m7KNEQ-VP16 activation became ubiquitous and much
stronger (compare Fig. 6D with
6E,F), reflecting ubiquitous tethering of the E(spl)m7VP16
effector regardless of the integrity of its basic domain.
|
Direct versus protein-mediated binding to target genes by E(spl)
proteins
The data presented so far have highlighted a novel mechanism of target gene
repression by E(spl), one that requires recruitment on DNA via protein-protein
interactions with proneural proteins. What role, if any, does direct DNA
binding play in the activity of E(spl) proteins? We addressed this question by
assaying the ability of E(spl)VP16 variants to activate endogenous target
genes in the absence of ac and sc
(Fig. 7A,F), which eliminates
the possibility of proneural-protein-mediated recruitment. All E(spl)m7VP16,
mVP16 and m
VP16 induced bristles when driven by
pnr-Gal4 in a sc10-1 background
(Fig. 7C,E,H,J and data not
shown). This suggests that these E(spl)VP16 variants can bypass the
requirement for endogenous proneural genes and trigger the sensory organ
pathway, presumably by directly activating one or more proneural target genes.
Indeed direct binding of target genes must be involved, since cheta production
in a sc10-1 background was abolished by mutating the basic
domain of E(spl)m7VP16 (Fig.
7D,I). In a wild-type background, E(spl)m7KNEQ-VP16 induces fewer
ectopic bristles than its wild-type counterpart
(Fig. 7C,D), which suggests a
lower activity, consistent with its ability to activate target genes only via
protein-mediated recruitment, whereas E(spl)m7VP16 can also directly bind to
its target genes. m
KNEQ-VP16 behaved identically to m7KNEQ-VP16 (data
not shown). Therefore, both mechanisms, direct DNA contact and interaction
with the pre-bound proneural activators, seem to play a role in the
recruitment of E(spl) proteins to their target genes. It should be noted that
in a wild-type background both E(spl)m7- and m
-VP16 variants produced a
larger number of excess bristles than that produced in a
sc10-1 background (Fig.
7C,E,H,J), indicating synergy between the hybrid E(spl) activators
and the proneural ones, which is in part due to protein-mediated recruitment
of the former onto the latter (see Discussion).
|
Proneural-mediated repression by E(spl)m7 involves an active
repression mechanism
E(spl) proteins are known to recruit the co-repressor Groucho in order to
silence target genes (Fisher and Caudy,
1998). It is conceivable that when E(spl) exert their repressive
effect by interacting with proneural proteins, a different mechanism might be
at play, such as occlusion of the transcriptional activation domain of
proneural activators. We therefore wanted to address whether Gro is needed to
mediate repression when E(spl) proteins are indirectly bound to DNA. To this
end, we drove expression of UAS-sc together with
UAS-E(spl)m7 in a mosaic background containing patches homozygous for
the severe groE48 allele and assayed the response of the
EE4-lacZ reporter. As described in a previous section, this reporter
is repressed by E(spl)m7 exclusively via protein-mediated recruitment. Indeed
in gro+ territory little or no expression was observed, as
expected (Fig. 8A), cells
stained green; Fig. 8B);
however, within mutant clones EE4-lacZ was strongly expressed
(Fig. 8A, cells lacking green).
Therefore, E(spl) proteins employ a Gro-dependent repression mechanism
regardless of mode of recruitment on target genes.
The requirement for Gro was corroborated by cuticle phenotype:
groE48 clones produce tufts of bristles on the notum
(Fig. 8C), a result of the
breakdown of lateral inhibition during SOP commitment. Although ubiquitous
expression of E(spl)m7 abolishes bristles
(Fig. 3C,D), when we induced
groE48 clones in an ap-Gal4; UAS-E(spl)m7
background (which abolishes bristles throughout the notum), we recovered
patches of high bristle density in a bald notum
(Fig. 8D). This suggests that
ectopic (as well as normally expressed) E(spl)m7 cannot repress endogenous
target genes in the absence of Gro, just as it cannot repress the artificial
EE4-lacZ target (Fig.
8A). Finally, a UAS-E(spl)m7W transgene, which
lacks the C-terminal tryptophane of the Gro-binding WRPW motif, was completely
inactive in both bristle suppression and reporter gene repression (results not
shown). A corollary from these experiments is that E(spl)m7 does not function
by sequestering proneural activators off DNA. The latter activity should have
no requirement for a co-repressor like Gro, as physical removal of activators
should suffice to turn target genes off.
![]() |
DISCUSSION |
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Repression targets of E(spl) proteins
It is sometimes assumed that E(spl) proteins suppress neurogenesis solely
by repressing proneural gene transcription. We have shown this not to be the
case, as E(spl)m7 and m can completely block sensory organ commitment
in a background of exogenously (transgenically) provided high levels of Sc.
Target genes (genuine and artificial) that are activated by Da/Sc are still
repressed by E(spl)m7 and m
in the above genetic background. This is
consistent with the earlier observation that E(spl) overexpression has only a
moderate effect on ac expression, whereas it completely represses
downstream targets, such as SMC-lacZ
(Culi and Modolell, 1998
) (see
below), ase or EE4-lacZ (this study). Even though
ac and sc are not the main targets of E(spl), some of their
enhancers are repressible by E(spl). ac and sc genes
elaborate expression pattern is dependent on a number of prepattern enhancers,
which are controlled by patterning systems and are weakly, if at all,
repressible by E(spl) (Gomez-Skarmeta et
al., 1995
). One enhancer each of ac [the proximal 900bp,
used in the experiments whose results are shown in
Fig. 1
(Martinez et al., 1993
)] and
sc (the SMC enhancer)
(Culi and Modolell, 1998
) has
been described that is repressible by E(spl). Both of these are autoregulatory
inasmuch as they contain EA boxes and are activated by Da/Sc or Ac,
hence they act to boost ac/sc levels after transcription has been
initiated via the prepattern enhancers; in this context the SMC and
ac-proximal enhancers can be viewed as `target genes' of the
proneural proteins.
Another piece of evidence in favor of regulation of proneural target genes
(rather than proneural genes themselves) by E(spl) is that E(spl)m7VP16 can
activate the neural pathway in genetic backgrounds mutant for ac and
sc. Other than displaying aberrant spacing, bristles produced in such
a background are normal, at least in external appearance. This is consistent
with E(spl)m7VP16 binding and activating many, perhaps all, target genes of
Ac/Sc (not just the autoregulatory ac/sc enhancers), bypassing the
need for proneural proteins. One should be aware, however, that there are
other bHLH proneural genes, besides ac and sc, in the fly
genome; e.g. l'sc is not affected by the sc10-1
allele used in the experiments whose results are shown in
Fig. 7. Although l'sc
is not normally expressed in the larval wing disk, it is conceivable that it
is turned on by E(spl)VP16 activators and then takes over the task of
activating the panel of downstream genes. Another potential candidate that
might single-handedly mediate the sensory-organ promoting activity of
E(spl)VP16 is ase, a SOP-specific gene that bears homology to the
proneural genes of the ac/sc family and can act as a proneural gene
itself (Dominguez and Campuzano,
1993). Thus, it is a matter of further research whether the
bristle-induction ability of E(spl)VP16 in a sc10-1
background is channeled through activation of a single E(spl) target gene or
of a number of target genes.
Dual mechanism of E(spl) recruitment onto enhancers
All proneural target genes contain EA boxes, via which the
Da/proneural activators exert their effect. Our analysis of the
EE4-lacZ enhancer has revealed that the same EA boxes are
sufficient for E(spl)-mediated repression, even though the latter bind a
different class of target sites, the EB/C/N-boxes. Based on the
data presented in this work, we propose that this is achieved by enhancer
recruitment of E(spl) proteins via protein-protein interactions with proneural
activators. We focused on three E(spl) proteins. Two, m7 and m, have
been shown to interact with both Da and Ac/Sc
(Alifragis et al., 1997
) and in
the present study displayed equivalent ability to be indirectly recruited onto
DNA by Da/Sc. The third, E(spl)m
, showed no proneural-mediated
recruitment activity, apparently because of its inability to interact with
either Da or Sc. Perhaps this Da/Sc-binding activity of some of the E(spl)
proteins has evolved to enable them to repress all proneural target genes
effectively without the need for direct DNA binding. Ac and Sc seem to play a
central role in this repression mechanism, as the ubiquitous Da was not
sufficient to recruit E(spl)-VP16 proteins to EE4-lacZ and other
proneural target enhancers (e.g. Fig.
4B,D).
Even though E(spl) proteins can be recruited onto their target genes via
proneural complexes, all characterized proneural target enhancers (e.g.
SMC, ac-proximal, ase, dpn, neur) do bear
EB/C/N-boxes in addition to EA-sites
(Culi and Modolell, 1998;
Emery and Bier, 1995
;
Jarman et al., 1993
;
Ohsako et al., 1994
;
Van Doren et al., 1994
) (M.
Monastirioti and C. D., unpublished). Likewise, all E(spl) proteins possess
well-conserved DNA-contacting basic domains. Two observations from our work
strongly suggest that direct DNA binding is also used in the repression of
target genes by E(spl). First, we observed a significant suppression of
bristle formation by E(spl)m
upon co-expression with Sc
(Fig. 3F). This can only be
interpreted as repression of Sc targets by E(spl)m
by direct binding to
their EB/C/N-boxes, as we have established that E(spl)m
is
incapable of proneural-mediated enhancer binding. Second, E(spl)m7VP16, but
not a basic region mutant version, turned on bristle commitment in the absence
of proneural genes (Fig. 7H,I),
pointing towards DNA-binding-dependent recruitment onto proneural target
genes.
The realization that some E(spl) proteins can act as both repressors and
co-repressors of the proneurals prompts reconsideration of the proneural
proteins as dedicated transcriptional activators; they seem to be equally
important in effecting repression of their target genes. Other transcriptional
activators, such as Dorsal and HNF4 can act as repressors in certain contexts
(Dubnicoff et al., 1997;
Ktistaki and Talianidis,
1997
), suggesting that this may be quite a widespread
mechanism.
Implications for lateral inhibition
We have used a transgenic approach to establish the ability of E(spl)
proteins to be recruited onto target genes by the two mechanisms discussed
above. We cannot predict from our results whether in a wild-type background
the two mechanisms are used exclusively of one another or simultaneously. The
presence of EB/C/N-boxes in close proximity to EA-boxes
in enhancers of proneural target genes favors the latter possibility, namely
that proneural and E(spl) proteins each bind their cognate target sites and
subsequently also interact at the protein level. Protein-protein interaction
concomitant with DNA binding may enable cooperative enhancer binding, which
would ensure a rapid response of target genes to changes in concentration of
proneural and E(spl) proteins.
Having realized the plausibility for two (alternative or simultaneous)
modes of E(spl) recruitment onto target enhancers, we still do not have a
complete picture of what it takes (in terms of transcriptional regulation) to
achieve a robustly laterally inhibited response to proneural activity; in
other words, to turn on a proneural target gene solely in the neural
precursor. The artificial EE4-lacZ enhancer, though responsive to
wild-type levels of E(spl) (Fig.
2K-M) is still not fully repressed, and is expressed in most cells
of a wild-type proneural cluster. By contrast, another enhancer that also
lacks EB/C/N boxes has been reported to be fully repressible by
wild-type levels of E(spl); SMCN-147-181 is a mutant version
of the SMC enhancer lacking all E(spl)-binding sites, but containing
two EA-boxes; this enhancer expresses solely in the neural
precursor (SOP) and not in surrounding proneural cluster cells
(Culi and Modolell, 1998
). One
can hypothesize that additional factors binding SMCN-
-147-181
favor the formation of a repressive DNA-protein complex in the
E(spl)-containing non-SOPs. Indeed this enhancer contains two copies each of
conserved
and ß boxes (bound by unknown factors) interspersed
with the EA boxes (Culi and
Modolell, 1998
). One or both of these factors may cooperate with
low (wild-type) levels of E(spl) (bound to EA via interaction with
the proneural complex) to stabilize Gro binding to this enhancer; indeed Gro
often has to simultaneously interact with more than one DNA bound factors to
gain access to an enhancer (Valentine et
al., 1998
).
Natural proneural target enhancers contain EA, EB, C,
N, and ß boxes, in addition to binding sites for other factors,
such as the Zn-finger protein Senseless
(Nolo et al., 2000
). Some of
these enhancers (e.g. SMC, ase, dpn, neur) are expressed solely in
the neural precursor, whereas others [ac proximal, sca,
various E(spl) enhancers] are expressed more widely within the
proneural cluster (Cooper et al.,
2000
; Nellesen et al.,
1999
; Singson et al.,
1994
), apparently not responding (or less responsive) to lateral
inhibition. Yet, the two types of enhancer are not obviously different with
respect to types of target sites contained. Perhaps it is the exact number and
arrangement of the various target sites and DNA-bound factors that defines the
threshold level of lateral inhibition that each enhancer is responsive to.
Seen in this light, it is conceivable that interaction of E(spl) with
proneural factors (and perhaps other factors within a large protein-DNA
complex) may bring about conformational changes, which are needed to fine-tune
crosstalk of these transcription factors with co-activators, co-repressors and
other components of the transcriptional machinery. Characterizing these
regulatory interactions will improve our insight on the transcriptional
mechanisms that mediate neural fate acquisition and will be a major challenge
for the future.
![]() |
ACKNOWLEDGMENTS |
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