sloppy paired 1
Genetic analysis suggests that both slp genes contribute to the segmentation
phenotype, that they have characteristics of gap, pair-rule, and segment polarity genes, and that they
interact functionally. The spatial expression patterns of the two transcripts are very similar, suggesting common regulation of the two genes. slp1 and slp2 appear to share an enhancer element situated upstream of slp1 that acts on both the proximal slp1 promoter and the distal slp2 promoter (Grossniklaus, 1992).
Inactivation of either the secreted protein Wingless (Wg)
or the forkhead domain transcription factor Sloppy Paired
(Slp) has been shown to produce similar effects in the
developing Drosophila embryo. In the ectoderm, both gene
products are required for the formation of the segmental
portions marked by naked cuticle. In the mesoderm, Wg
and Slp activities are crucial for the suppression of bagpipe
(bap), and hence visceral mesoderm formation, and the
promotion of somatic muscle and heart formation within
the anterior portion of each parasegment. During these developmental processes, wg and slp act in a common pathway in which slp serves as a
direct target of Wg signals that mediates Wg effects in both
germ layers. Evidence has been found that the induction of
slp by Wg involves binding of the Wg effector Pangolin
(Drosophila Lef-1/TCF) to multiple binding sites within a
Wg-responsive enhancer, located in 5' flanking
regions of the slp1 gene. Based upon genetic and
molecular analysis, it is concluded that Wg signaling induces
striped expression of Slp in the mesoderm. Mesodermal Slp
is then sufficient to abrogate the induction of bagpipe by
Dpp/Tinman, which explains the periodic arrangement of
trunk visceral mesoderm primordia in wild type embryos.
Conversely, mesodermal Slp is positively required,
although not sufficient, for the specification of somatic
muscle and heart progenitors. It is proposed that Wg-induced
slp provides striped mesodermal domains with the
competence to respond to subsequent slp-independent Wg
signals that induce somatic muscle and heart progenitors.
It is also proposed that in wg-expressing ectodermal cells, slp
is an integral component in an autocrine feedback loop of
Wg signaling (Lee, 2000).
Since the progenitors of pericardial cells, cardioblasts and
somatic muscles are largely derived from the segmental areas
between the bap domains, and their formation requires wg and
slp as positive activities, it was asked whether in this regulatory
pathway slp also acts strictly downstream of wg. In wild-type
embryos, eve-expressing progenitors of pericardial cells and
dorsal muscles are located underneath the ectodermal wg
stripes and within the domains of mesodermal slp expression. In wg mutant embryos, these progenitors are not formed. Interestingly, while ectopic slp expression in the mesoderm of wg mutants rescues bap
repression, it fails to rescue the formation of heart
and dorsal muscle progenitors. Identical effects are
observed in slp mutants and in slp
mutant embryos with ectopic mesodermal expression of wg; neither produces any eve-expressing heart and muscle progenitors. The results of ectopic expression of slp or wg in wild-type backgrounds are also consistent with these
observations. Although ectopic expression of slp in the
mesoderm of wild-type embryos causes complete repression
of bap, it does not produce a significant increase
of eve-expressing cells under the same conditions.
Similarly, ectopic expression of wg in the mesoderm (or
ectoderm) of wild-type embryos with the GAL4/UAS system
fails to produce a major increase of eve-expressing cells. By contrast,
when wg and slp are coexpressed in the mesoderm, there is a dramatic increase in eve-expressing cells, which become distributed all along the
dorsal margin of the mesoderm. Analogous
experiments have shown that the formation of slouch-expressing
ventrolateral muscle progenitors and tin-expressing
heart progenitors also requires the joint activities of wg and slp. These combined data show that neither wg nor slp alone is sufficient to allow the formation of various heart and muscle progenitors. Instead, it appears that wg and slp cooperate in parallel or consecutive pathways during the
formation of these cell types (Lee, 2000).
It was next asked whether one of these pathways involves the
regulation of twist expression by mesodermal Slp. Previous
reports have indicated that wg and slp are required to generate
segmentally elevated levels of twist expression during stage 11
and that these higher levels of twist are necessary for normal
somatic muscle specification. The stripes of
elevated Twist coincide with the mesodermal domains of Slp
during stage 11. Moreover, uniform expression of slp in the
mesoderm produces uniform expression of high Twist levels,
even in the absence of Wg activity. Thus, it appears
that one route of wg function in myogenesis involves the
upregulation of twist by Wg-induced slp in the mesoderm.
Additional experiments confirm that, analogous to bap
repression, the mesodermal component of slp is essential for
somatic muscle and heart specification. In these experiments,
mesodermal slp expression is specifically removed by
ectopically expressing en in the mesoderm of wild-type embryos, as described above. Embryos of this genotype show a complete absence of eve-expressing muscle and pericardial progenitors, and an almost complete absence of Mef2-labeled somatic mesoderm. The expression of nautilus in muscle founders is also missing in embryos of this genotype. In addition, tin-expressing heart progenitors fail to be formed and only a few somatic muscle fibers are present in late-stage embryos. In contrast to the loss of derivatives of the mesodermal A (slp) domains, derivatives of the P (eve) domains, including trunk visceral mesoderm and fat body, are present and even expanded in these embryos. In the aggregate, the evidence suggests that Wg-induced slp activity in the mesoderm is required but not sufficient to generate somatic muscle and heart progenitors. It appears that, in addition to this route, wg is required independent of slp for the formation of these cell types (Lee, 2000).
Based upon the breakpoints and phenotypes of several large
deletions in the slp locus, it has been
concluded that the 5' flanking regions of slp1 contain essential
regulatory elements that are shared between the slp1 and slp2
genes. Indeed, reporter gene analysis has identified
three separate enhancer elements within ~5.5 kb of
upstream sequences, each of which reproduces one particular
aspect of endogenous slp expression. The two distal elements
drive head-specific expression during blastoderm stages and
striped expression in the ventral ectoderm following stage 11,
respectively. The third, more proximal element is active in both
the mesoderm and ectoderm between stages 10 and 11,
coinciding with the period when mesodermal genes such as
bap and eve are activated. Its spatial and temporal profile, as
well its wg dependency, are fully consistent with the notion that
this enhancer is targeted by the Wg signaling cascade during
mesoderm induction and autocrine signaling in the ectoderm.
Because it is not active during the period when the pair-rule
genes are expressed and is turned on in every parasegment,
without displaying any transient pair-rule pattern, this enhancer
appears to be exclusively responsive to Wg in terms of its
spatial regulation. Hence, there must be separate pair-rule
enhancer(s), which were not uncovered by this dissection. The
observation that this Wg response element becomes rapidly
inactive at late stage 11, although wg continues to be expressed,
indicates that it requires at least one additional, temporally
restricted regulator in conjunction with the Wg signal (Lee, 2000).
Ubx is one of the few genes known to be a direct target of
Wingless, although there are indications that en may be another
example. An enhancer
element of Ubx, which is activated by Wg after the formation
of the midgut visceral mesoderm, contains at least two
functionally important Pan-binding sites. In
the present study, it has been determined that a Wg-responsive
enhancer of slp contains nine in vitro Pan-binding sites. It is not known whether all of these sites are functional because in vivo tests of mutations of individual
sites or various combinations of them have not yet been carried out. However, the current data from two rounds of mutagenesis show that some of them
are indeed functional in vivo. This analysis also indicates that
the activities of different functional binding sites are additive
and therefore partially redundant (Lee, 2000).
Although most of the known wg downstream genes are
upregulated by Wg signals, there are several examples of
negatively regulated genes, in addition to bap. For instance, the
Ubx midgut enhancer is downregulated by high levels of Wg
signaling through a mechanism that involves Smad-binding
sites. It has been speculated that Wg signals may induce the
transcription of an unknown repressor that competes with
Mad/Medea binding to these sites. By
contrast, the repression of shavenbaby (ovo) in the
epidermis of late stage embryos by Wg signaling has been proposed to involve the direct binding of
hypothetical Pangolin/Armadillo/corepressor complexes to
ovo regulatory elements. The present study
provides strong evidence for an indirect mode of bap
repression by Wg that is more comparable to the one proposed
for Ubx. It appears that during this pathway, Wg signals induce
the transcription of slp, whose encoded protein product
probably acts as a repressor that interferes with Dpp/Tinman
mediated activation of bap transcription (Lee, 2000).
The maternal morphogen Bicoid (Bcd) is distributed in an embryonic gradient
that is critical for patterning the anterior-posterior (AP) body plan in
Drosophila. Previous work identified several target genes that respond directly
to Bcd-dependent activation. Positioning of these targets along the AP axis is
thought to be controlled by cis-regulatory modules (CRMs) that contain clusters
of Bcd-binding sites of different 'strengths.' A combination of
Bcd-site cluster analysis and evolutionary conservation has been used to predict Bcd-dependent CRMs. Tested were 14 predicted CRMs by in vivo reporter gene assays; 11 showed Bcd-dependent activation, which brings the total number of known Bcd target elements to 21. Some CRMs drive expression patterns that are restricted to the
most anterior part of the embryo, whereas others extend into middle and
posterior regions. However, no strong correlation is detected between AP
position of target gene expression and the strength of Bcd site clusters alone.
Rather, binding sites for other activators, including Hunchback and
Caudal correlate with CRM expression in middle and posterior body regions. Also,
many Bcd-dependent CRMs contain clusters of sites for the gap protein Krüppel,
which may limit the posterior extent of activation by the Bcd gradient. It is
proposed that the key design principle in AP patterning is the differential
integration of positive and negative transcriptional information at the level of
individual CRMs for each target gene (Ochoa-Espinosa, 2005).
In reporter gene assays, 11 of the 14 tested fragments directed expression
patterns in wild-type embryos that recapitulate all or part of the endogenous
patterns of the associated genes. These experiments identified several elements that
control segmentation genes, including three new gap gene CRMs. Two CRMs were
found in the genomic region that lies 5' of the gap gene gt. One CRM
(gt23) is initially expressed in a broad anterior domain and then refines
into two stripes. A second CRM (gt1)
is expressed later in a small dorsal domain very near the anterior tip.
Double stain experiments indicated that the timing and spatial regulation of both patterns are
indistinguishable from the anterior expression domains of the endogenous
gt gene. A CRM 3' of the gap gene tll was identified that drives
expression similar to the anterior tll domain (Ochoa-Espinosa, 2005).
Four novel CRMs were identified near known pair rule genes. One CRM was detected
in the 3' region of hairy and drives expression of a small anterior
dorsal domain similar to the hairy 0
stripe of the endogenous gene. Another CRM is located 3' of the paired gene and
directs expression of an early broad domain
that coincides with the later position of the native paired stripes 1 and 2. Two more CRMs
(slpA and slpB) were identified in the slp locus, which contains the
two related genes, slp1 and slp2. Both slpA and slpB faithfully
reproduce parts of the early slp1 and slp2 expression patterns (Ochoa-Espinosa, 2005).
Four other CRMs were identified near the genes bowl, CG9571,
D/fsh, and bl/Mir7. In three cases (bowl, CG9571, and
D/fsh), the newly identified CRMs direct patterns similar to their
associated endogenous genes. The final CRM (bl/Mir7) is located in the
sixth intron of the bl gene and directs a strong anterior domain of
expression. However, the endogenous
bl gene is expressed nearly ubiquitously , which makes it an unlikely target of regulation by this CRM. One
potential target of this element is the microRNA gene (Mir7), which is
located 7 kb downstream in the eighth intron of bl. Four of
the CRMs reported here (gt1, gt23,
slpA, and D/fsh) were also identified in a recent
genome-wide search for new patterning elements based on clusters of combinations
of different binding sites including Bcd. The
fragments used in that study were significantly larger in size but show very
similar patterns to those in this study (Ochoa-Espinosa, 2005).
The response kinetics of known and
putative target genes of Ftz has been examined in order to distinguish between direct and indirect Ftz targets. This kinetic analysis was achieved by providing a brief
pulse of Ftz expression and measuring the time required
for genes to respond. The time required for Ftz to bind and
regulate its own enhancer, a well-documented interaction,
is used as a standard for other direct interactions.
Surprisingly, both positively and negatively
regulated target genes respond to Ftz with the same kinetics
as autoregulation. The rate-limiting step between
successive interactions (<10 minutes) is the time required
for regulatory proteins to either enter or be cleared from
the nucleus, indicating that protein synthesis and
degradation rates are closely matched for all of the proteins
studied. The matching of these two processes is likely to be
important for the rapid and synchronous progression from
one class of segmentation genes to the next. In total, 11
putative Ftz target genes have been analyzed, and the data provide
a substantially revised view of Ftz roles and activities
within the segmentation hierarchy (Nasiadka, 1999).
To determine the regulatory relationships between Ftz and the
other non-primary pair-rule genes, the expression
of odd-skipped (odd) and sloppy-paired (slp) was examined in HSFtz
embryos fixed 20 and 35 minutes post Ftz induction.
Expression patterns of these genes were also examined in ftz
mutant embryos to obtain genetic confirmation of the
interactions observed.
Like ftz, en and prd, odd appears to be directly activated by
Ftz. In stage 5 embryos, ectopic expression of Ftz causes rapid
expansion of odd, from its initiating pattern of six stripes, to near homogeneous expression across the germband. In stage 6 embryos, odd is normally expressed in 14
evenly expressed stripes. Ectopic Ftz causes an
intensification of the primary odd stripes at this stage.
These stripes are derived from the original 7 stripes that overlap
ftz stripes. In stage 7 embryos, these primary stripes are not
only intensified, but expand anteriorly as well (from about 1
cell wide to 2 cells wide). The percentage of
embryos responding to ectopic Ftz, at all stages tested, is
about the same as the percentage of embryos that show ftz
autoregulation, en and prd activation and wg repression. Thus,
Ftz appears to be an activator of odd at all stages tested. This
positive relationship between Ftz and odd is consistent with the
differences in odd expression observed in ftz mutant embryos.
Stripes of odd appear to be diminished in intensity in stage 5
embryos, and primary stripes are weak or missing in
stage 6 and 7 embryos (Nasiadka, 1999).
Unlike prd and odd, the pair-rule gene slp is negatively
regulated by Ftz: ectopic expression of Ftz results in the
differential repression of secondary slp stripes.
Again, the penetrance of repression at the 20 minute recovery
time was about 60%, as has been measured for the other genes
exhibiting direct responses. As might be expected, slp stripes
expand in ftz mutant embryos, filling the regions where Ftz is
normally expressed. Thus, as with wg, Ftz appears to
act as a direct repressor of slp. This effect is likely exerted
through the response elements or trans-acting factors that
regulate secondary stripe expression (Nasiadka, 1999).
sloppy paired, in addition to its roles as a segment polarity and as a pair rule gene, acts like a gap gene in the head. All three maternal systems that are active in the cephalic region are required for
proper slp expression. High levels of the terminal system (torso) inhibit slp through Bicoid. Low levels of
terminal system activity seem to potentiate BCD as an activator of slp in more posterior positions. Dorsal, the morphogen of the
dorsoventral system, and the head-specific gap protein Empty spiracles, act as repressor and
corepressor in the regulation of slp (Grossnicklaus, 1994) .
The early bell-shaped gradient of even-skipped expression is sufficient for generating
stable parasegment borders. The anterior
portion of each early stripe has morphogenic activity, repressing different target genes at different
concentrations. These distinct repression thresholds serve to both limit and subdivide a narrow
zone of paired expression. Within this zone, single cell rows express either engrailed, where runt
and sloppy-paired are repressed, or wingless, where they are not (Fujioka, 1995).
Groucho acts as a co-repressor for several Drosophila DNA
binding transcriptional repressors. Several of these proteins
have been found to contain both Groucho-dependent and -independent
repression domains, but the extent to which this
distinction has functional consequences for the regulation of
different target genes is not known. The product of the pair-rule
gene even skipped contains a Groucho-independent repression activity. In the Eve, outside the Groucho-independent repression domain, a conserved C-terminal
motif (LFKPY), similar to motifs that mediate Groucho interaction in Hairy, Runt and Hückebein, has been identified. Eve interacts with Groucho in yeast and in vitro, and groucho and even skipped genetically interact in vivo. Eve with a mutated Groucho interaction motif, which abolishes binding to Groucho, shows a significantly reduced ability to rescue the eve null phenotype
when driven by the complete eve regulatory region. Replacing this motif with a heterologous Groucho interaction motif restores the rescuing function of Eve in segmentation. Further functional assays demonstrate that the Eve C terminus acts as a
Groucho-dependent repression domain in early Drosophila embryos. This novel repression domain is active on two target genes that are normally repressed by Eve at
different concentrations: paired and sloppy paired. When the
Groucho interaction motif is mutated, repression of each target gene is reduced to a similar extent, with some activity remaining. Thus, the ability of Eve to repress
different target genes at different concentrations does not appear to involve differential recruitment or function of Groucho. The accumulation of multiple domains of similar function within a single protein may be a common
evolutionary mechanism that fine-tunes the level of activity
for different regulatory functions (Kobayashi, 2001).
What is the significance of the two distinct Eve repression
activities, only one of which is dependent on Gro? Gro could
be required to repress a subset of Eve targets, whereas
repression of other target genes might be Gro independent.
Alternatively, the two repression activities might function
cooperatively, in which case both activities might be required
for repression of each target gene. Extensive molecular and
genetic studies have identified several target genes that are
likely to be directly repressed by Eve. The best characterized
of these genes are sloppy paired (slp), paired (prd) and odd skipped (odd). The posterior boundaries of expression of slp
and prd correspond to the anterior and posterior borders,
respectively, of the odd-numbered en stripes. As these en
stripes shift posteriorly, both when the dose of gro is reduced
and when the Groucho interaction domain (GID) is mutated, the boundaries of slp and prd
may be coordinately shifted. slp and prd
expression were examined in embryos rescued with a GID-mutated transgene.
Both slp and prd expression were
expanded in the eve domains, relative to wild-type embryos. The degree of expansion of each gene correlates with the shift of en stripes. Furthermore, both the width of individual en stripes and their spacing are very
similar to those in eve hypomorphs. Thus, the removal of the GID has an effect that is similar to that of a general reduction of eve activity on both targets, slp and prd. This expansion of slp and prd expression is reversed, in each case, when the Eve GID motif is replaced
by that of Hairy. These results suggest that Gro is
required by Eve to a similar degree for its repression activity
on each of these genes (Kobayashi, 2001).
Repression of another Eve target gene (odd) is required for
the establishment of the even-numbered (ftz-dependent) en
stripes. Intriguingly, these are
established more or less normally in embryos rescued by the
GID-mutated transgene, and examination of odd
expression in those embryos has showen it to be normal in the
even-numbered parasegments. However,
repression of odd and the establishment of even-numbered en
stripes are also normal when eve function is reduced in other
ways (e.g. in the hypomorph),
suggesting that a lower threshold of Eve activity is required for
this eve function than for proper repression of slp and prd.
Therefore, this assay did not allow for a full assessment of the
contribution of Gro to odd repression by Eve (Kobayashi, 2001).
Engrailed is a key transcriptional regulator in the nervous system and in the maintenance of developmental boundaries in Drosophila, and its vertebrate homologs regulate brain and limb development. The functions of both of the Hox cofactors Extradenticle and Homothorax play essential roles in repression by Engrailed. Mutations that remove either of them abrogate the ability of Engrailed to repress its target genes in embryos, both cofactors interact directly with Engrailed, and both stimulate repression by Engrailed in cultured cells. A model is suggested in which Engrailed, Extradenticle and Homothorax function as a complex to repress Engrailed target genes. These studies expand the functional requirements for Extradenticle and Homothorax beyond the Hox proteins to a larger family of non-Hox homeodomain proteins (Kobayashi, 2003).
Exd cooperates with En to repress target genes and to pattern embryos.
Loss of exd function has been shown to result in a loss of en expression at later embryonic stages. Because en function is required to maintain its own expression, the loss of en expression could be a downstream effect of a loss of en function, or it could be due to some other consequence of the lack of exd. This ambiguity concerning the role of exd in en function led to an investigation of whether the activities of ectopically expressed En are dependent on exd function. En was ectopically expressed in two ways: from a heat-shock promoter and using a patterned Gal4 'driver' transgene. An advantage of the former approach is that one can often distinguish between immediate and secondary downstream effects based on how rapidly they occur following heat induction. Advantages of the second approach include having normal and altered expression in parts of the same embryo, providing a rigorous internal control. Both of these approaches led to similar conclusions, that exd function is important for the repression by En of its direct target gene slp, that wg also shows a strong dependence on exd function for its repression by En and that the ability of En to alter the pattern of embryonic cuticles is sensitive to the gene dosage of exd. Further, in each set of experiments, the observed dependence of repression on exd was accompanied by a residual repression activity when exd function was removed both maternally and zygotically. This residual exd-independent repression activity might be due to the ability of En to bind to target sites independently of exd but with a reduced affinity, or it could be accounted for by the existence of two classes of binding sites, one exd dependent and the other exd independent. This possibility is paralleled by the relationship of Exd with Ubx, which has been shown to function either co-operatively with Exd or alone on multiple binding sites in target genes. Alternatively, exd might be exerting an indirect effect on repression by En. However, because Exd forms complexes with En in yeast and in vitro, and because it appears to facilitate repression by En directly in cultured cells, it seems likely that the dependence of En on exd function in vivo is due at least in part to the direct action of En-Exd complexes. Confirmation of this model will require the analysis of specific regulatory sites, which have not yet been identified, in target genes such as slp. If this model is correct then these results suggest that the repression activity of Exd-En complexes might come exclusively from En repression domains, because Exd has been shown to act as a cofactor in the activation of target genes in vivo in conjunction with Hox proteins (Kobayashi, 2003).
The identification of slp as a direct target gene of En has implications for the mechanism by which En helps to maintain the activity of its own and other genes, including hedgehog, within its domains of expression, the posterior compartments. The slp locus produces two closely related, coordinately regulated gene products (Slp1 and Slp2), which have essentially indistinguishable functions. They are forkhead-domain transcription factors that repress en expression, and both contain a conserved motif (homology region II) that is similar to the Groucho-binding domain of En. Slp1 has also been shown to bind the Groucho co-repressor in vitro, suggesting that it is a repressor and therefore that its action on the en gene is likely to be direct. Thus, the mechanism of en autoregulation, as well as the ability of En to activate other target genes, is likely to be due, at least in part, to an indirect effect of repression of slp expression. In addition, En might activate target genes indirectly by repressing other repressors that are also normally excluded from its expression domain, such as Odd-skipped and the repressor form of Cubitus interruptus (Kobayashi, 2003).
Genetic analysis shows that Engrailed has both negative and positive targets. Negative regulation is expected from a factor that has a well-defined repressor domain but activation is harder to comprehend. VP16En, a form of En that has its repressor domain replaced by the activation domain of VP16, has been used to show that En activates targets using two parallel routes, by repressing a repressor and by being a bona fide activator. The intermediate repressor activity has been identified as being encoded by sloppy paired 1 and 2 and bona fide activation is dramatically enhanced by Wingless signaling. Thus, En is a bifunctional transcription factor and the recruitment of additional cofactors presumably specifies which function prevails on an individual promoter. Extradenticle (Exd) is a cofactor thought to be required for activation by Hox proteins. However, in thoracic segments, Exd is required for repression (as well as activation) by En. This is consistent with in vitro results showing that Exd is involved in recognition of positive and negative targets. Moreover, genetic evidence is provided that, in abdominal segments, Ubx and Abd-A, two homeotic proteins not previously thought to participate in the segmentation cascade, are also involved in the repression of target genes by En. It is suggested that, like Exd, Ubx and Abd-A could help En recognize target genes or activate the expression of factors that do so (Alexandre, 2003).
slp1 and slp2 are repressed by
En and their products repress en expression. Importantly, Slp1 and Slp2 are the only dominant repressors that stand between En and its positive targets, hh and en --
at least in the paired-Gal4 domain. If another such repressor
existed, it would prevent VP16En from activating the expression of hh
(or en) in a slp mutant. Expression of slp at the
anterior, and of en at the posterior, of prospective parasegment
boundaries is initiated by the activity of pair-rule genes.
Mutual transcriptional repression ensures that neither factor can subsequently
'invade' the other's domain of expression after pair-rule genes have ceased to
function and when cell communication starts to dominate segmental patterning
and thus contributes to the stability of parasegment boundaries. Note that
slp is expressed only at the anterior of each stripe of en
expression (not at the posterior). It may be that no analogous repressive
function is needed at the posterior because the Wg pathway, which contributes
to activation by En, is not active there. Indeed, in otherwise wild-type
embryos, ectopic activation of Wg signaling is sufficient to cause posterior
expansion of en stripes (Alexandre, 2003).
The key evidence for En being a bona fide activator is that, in the absence
of slp, both En and VP16En activate hh transcription. Either En activates hh directly or it activates an intermediate activator of hh transcription. Either
way, it is suggested that En must be capable of transcriptional activation (in
addition to repression). Note that in otherwise wild-type embryos, VP16En
formally represses the expression of hh and en. This led
initially to the belief that wild-type En acts solely via an intermediate repressor
since no positive effect of VP16En on the expression of
en or hh could be observed. As is know now, however, this was masked by the presence of Slp. It was therefore essential to identify the intermediate repressor and
assess the effect of removing its activity in order to infer the true
activation function of En (Alexandre, 2003).
A crucial step in generating the segmented body plan in Drosophila
is establishing stripes of expression of several key segment-polarity genes, one stripe for each parasegment, in the blastoderm stage embryo. It is well established that these patterns are generated in response to regulation by the transcription factors encoded by the pair-rule segmentation genes. However, the full set of positional cues that drive expression in either the odd- or even-numbered parasegments has not been defined for any of the segment-polarity genes. Among the complications for dissecting the pair-rule to segment-polarity transition are the regulatory interactions between the different pair-rule genes. An ectopic expression system that allows for quantitative manipulation of expression levels was used to probe the role of the primary pair-rule transcription factor Runt in segment-polarity gene regulation. These experiments identify sloppy paired 1 (slp1), most appropriately classified as segment polarity genes, as a gene that is activated and repressed by Runt in a simple combinatorial parasegment-dependent manner. The combination of Runt and Odd-paired (Opa) is both necessary and sufficient for slp1 activation in all somatic blastoderm nuclei that do not express the Fushi tarazu (Ftz) transcription factor. By contrast, the specific combination of Runt + Ftz is
sufficient for slp1 repression in all blastoderm nuclei. Furthermore Ftz is found to modulate the Runt-dependent regulation of the
segment-polarity genes wingless (wg) and engrailed
(en). However, in the case of en the combination of Runt +
Ftz gives activation. The contrasting responses of different downstream
targets to Runt in the presence or absence of Ftz is thus central to the
combinatorial logic of the pair-rule to segment-polarity transition. The
unique and simple rules for slp1 regulation make this an attractive target for dissecting the molecular mechanisms of Runt-dependent regulation (Swantek, 2004).
The role of Runt as a primary pair-rule gene complicates interpreting the alterations in segment-polarity gene expression that are observed in
run mutants. Recent experiments utilizing a GAL4-based
NGT-expression system [the transgene construct used to express GAL4 maternally contains the nanos promoter and the 3' untranslated region of an alpha-tubulin mRNA and is thus referred to as an NGT transgene (nanos-GAL4-tubulin)] to manipulate expression in the blastoderm embryo have demonstrated that low levels of Runt repress en in odd-numbered parasegments without altering expression of the pair-rule genes eve and ftz. This observation suggested that this approach might provide a useful tool for defining the role of Runt in regulating other segment-polarity genes. A systematic survey was undertaken of the response of other segmentation genes to increasing levels of NGT-driven Runt expression. These experiments revealed significant differences in sensitivity as well as interesting differences in the nature of the response of different genes to ectopic Runt. The
odd-numbered en stripes are repressed at both intermediate and high levels of ectopic runt. After en, the second most sensitive target is slp1. This gene shows a partially penetrant and subtle defect in the spacing of the segmentally repeated stripes in embryos with low levels of NGT-driven Runt. A more pronounced alteration is
obtained in embryos with intermediate levels of Runt. In these embryos the slp1 pattern is converted from a segment-polarity-like, 14-stripe pattern to a pair-rule-like, seven-stripe pattern. At this level, expression of other segmentation genes is normal although there are subtle changes in the spacing of the wg stripes and a
partial loss of the odd-numbered hh stripes. All three of these
genes show clearer alterations at higher levels of NGT-driven Runt, with wg responding in a manner similar to slp1 and hh responding in a manner similar to en. High Runt levels also produce spacing defects in the expression of odd and gsb, as well as a more subtle effect on prd. Several of the changes observed at high levels of ectopic
Runt are likely to be indirect and due to alterations in the expression of eve, ftz and hairy. The
response of slp1 to ectopic Runt is notable both because of its
sensitivity and apparent simplicity, thus suggesting that Runt plays a pivotal role in regulating slp1 transcription (Swantek, 2004).
The differential combinatorial effects of Runt and Ftz on segment-polarity
gene regulation emerged as a result of analyzing the sensitive
and relatively simple response of slp1 to ectopic Runt. The
slp1 transcription unit is one of two redundant genes that comprise the slp locus. This locus was initially characterized as having a pair-rule function in the segmentation gene hierarchy based on a weak pair-rule phenotype associated with loss of slp1 function. The slp1 and slp2 genes are expressed in
similar patterns during early embryogenesis. Embryos deficient for both
slp1 and slp2 have an unsegmented lawn cuticle phenotype
similar to that produced by wg mutations.
This raises the question of whether it is most appropriate to consider
slp as a pair-rule or segment-polarity locus. In the most
straightforward interpretation of the segmentation hierarchy, the role of the pair-rule genes is to establish the initial metameric expression patterns of the segment-polarity genes. The initial expression of the key segment polarity genes en and wg is normal in gastrula stage embryos that are deleted for both slp1 and slp2. The expression of wg begins to become abnormal and is lost during early germband extension. These observations are consistent with the proposal that slp expression identifies cells that are competent to maintain wg expression subsequent to the blastoderm stage. Based on these observations, it is concluded that slp1 and slp2 are most appropriately classified as segment polarity genes, not pair-rule genes (Swantek, 2004).
The expression of slp1 (and slp2) differs from several
other segment-polarity genes in that the metameric pattern is comprised of two-cell wide, rather than single-cell wide stripes. These two cell-wide stripes comprise the posterior half of each parasegment. slp1 activation in odd-numbered parasegments requires the cooperative
action of Runt and Opa, whereas in even-numbered parasegments Runt works
together with Ftz to repress slp1 expression. The simple rules
involving these three factors fully account for slp1 regulation in
all of the Runt-expressing cells in the blastoderm embryo but also raise a question regarding the positional cues that regulate slp1 expression in cells that do not express Runt (Swantek, 2004).
There are four other pair-rule transcription factors that could be involved in slp1 regulation: Eve, Hairy, Odd and Prd. Expression of both Odd and Prd overlaps the slp1 stripes in a manner that suggests that neither of these factors provides positional information crucial for slp1 regulation. Consistent with this, there are no substantial changes in the early 14-striped slp1 pattern in embryos mutant for either odd or prd. By contrast, elimination
of either Eve or Hairy leads to changes in both the number and spacing of the slp1 stripes. However, as these are both primary pair-rule genes some of these changes are certainly indirect and due to alterations in Runt and Ftz expression (Swantek, 2004).
Several lines of evidence indicate that Eve has a direct role in
slp1 repression. Experiments with the temperature-sensitive
eve[ID19] mutation indicate that transient elimination of Eve at the cellular blastoderm stage leads to expanded six cell-wide slp1
stripes because of de-repression in the anterior two cells of each
odd-numbered parasegment. These two are the cells with the
highest level of Eve, indicating that the primary role of Eve at this stage is to repress slp1 expression. Complementary experiments with an inducible hs-Eve transgene reveal that ectopic Eve blocks slp1 activation in both odd- and even-numbered parasegments. This result not only confirms
Eve's role as a repressor, but also reveals a crucial difference between
Eve and Ftz-dependent repression. Ftz-dependent repression is
restricted to odd-numbered parasegments unless Runt is also ectopically
expressed. This same
restriction is observed in experiments with hs-Ftz transgenes, indicating that the difference between Eve and Ftz is not due to
the mode of ectopic expression. Taken altogether these results indicate that
Eve and Ftz normally have comparable roles in repressing slp1
transcription in the anterior half of the odd- and even-numbered parasegments, respectively, in late blastoderm stage embryos. The key distinction
in the regulation of slp1 by these two homeodomain transcription
factors is the critical role that Runt plays in Ftz-dependent repression (Swantek, 2004).
One aspect of slp1 expression not accounted for by the above rules is the factor (or combination of factors), referred to here as factor X, that is responsible for slp1 activation in the posterior half of the even-numbered parasegments. Activation in these cells is blocked either by the combination of Runt+Ftz or by
ectopic Eve. Runt and Ftz are co-expressed anterior to these
even-numbered stripes and presumably both play a role in defining the anterior
margin of these stripes. Conversely, Eve is expressed posterior to these cells and probably has a role in defining the posterior margins of these stripes. The sole pair-rule transcription factor that remains as a candidate for Factor X is Hairy, which is expressed in the posterior half of even-numbered parasegments. However, it is not thought that factor X is Hairy for several reasons. All of the evidence to date indicates that Hairy functions as a repressor. Furthermore, NGT-driven expression of Hairy does not lead to slp1 activation in anterior blastoderm cells similar to that produced by the co-expression of Runt and Opa.
Identification of factor X is clearly important for a complete understanding
of slp1 regulation (Swantek, 2004).
Previous studies have indicated that Runt has roles in both activating and
repressing transcription of different target genes in the Drosophila. The
current results provide additional compelling evidence for this dual
activity and also provide insight on factors that contribute to this
context-dependent regulation. The dramatic effects of Ftz on Runt-dependent slp1 regulation clearly demonstrate that one important component of context is the specific combination of other transcription factors that are present in a cell. Indeed, the unique and relatively simple rules for slp1 regulation make this an especially attractive target for dissecting the molecular mechanisms whereby Ftz converts Runt from an activator to a repressor of transcription. It seems likely that the rules
governing the Runt-dependent regulation of slp1 will provide a
foundation for understanding the regulation of wg and gsb,
two segment-polarity genes that are expressed in a subset of
slp-expressing cells and that respond to Runt in a manner similar,
but not identical to slp1 (Swantek, 2004).
The results also point to a second important component of context-dependent regulation by Runt. The specific combination of Runt + Ftz, which represses slp1, does not always give repression, since these same two factors work together to activate en in some of these same cells at the same stage of development. Thus, cellular context alone cannot fully account for the regulatory differences and there must be a target-gene specific component of context-dependent regulation. A similar gene-specific example of context-dependent regulation has recently been described for the Runx protein Lozenge. In this case, the presence of binding sites for the Cut homeodomain protein helps to stabilize a complex that leads to repression of deadpan transcription in the same cells in which Lozenge is responsible for activation of Drosophila Pax2. In a strict parallel of this model, it would be speculated that the slp1 regulatory region contains binding sites for some factor that helps to stabilize a repressor complex that includes the Runt and Ftz proteins. In a reciprocal, and not mutually exclusive model, perhaps there are binding sites for a factor in the en regulatory region that helps to stabilize a Runt- and Ftz-dependent transcriptional activation complex. Further studies on the en and slp1 cis-regulatory regions are needed in order to address these questions at the molecular level. This future work is crucial for understanding the context-dependent activity of Runt and thus the molecular logic of the control system that underlies the pair-rule to segment-polarity transition in Drosophila segmentation (Swantek, 2004).
The simple combinatorial rules for regulation of the sloppy-paired-1 (slp1) gene by the pair-rule transcription factors during early Drosophila embryogenesis offer a unique opportunity to investigate the molecular mechanisms of developmentally regulated transcription repression. Initial repression of slp1 in response to Runt and Fushi-tarazu (Ftz) does not involve chromatin remodeling, or histone modification. Chromatin immunoprecipitation and in vivo footprinting experiments indicate RNA polymerase II (Pol II) initiates transcription in slp1-repressed cells and pauses downstream from the promoter in a complex that includes the negative elongation factor NELF. The finding that Negative elongation factor E also associates with the promoter regions of wingless (wg) and engrailed (en), two other pivotal targets of the pair-rule transcription factors, strongly suggests that developmentally regulated transcriptional elongation is central to the process of cell fate specification during this critical stage of embryonic development (Wang, 2007).
DNase I hypersensitivity was used to probe the chromatin structure of the slp1 locus. These assays revealed the presence of a DNase I-hypersensitive site near to the 5'-end of the slp1 transcription unit. Chromatin immunoprecipitation (ChIP) experiments with antiserum against histone H3 provide an explanation for this DNase I hypersensitivity. There is significantly reduced association of H3 with the slp1 promoter region compared with both the structural gene as well as sequences upstream of the promoter. These observations strongly suggest that the promoter region is nucleosome free. Importantly, matched collections of wild-type and Runt + Ftz (R+F) embryos show both the same pattern of DNase I hypersensitivity and histone H3 association. These results indicate that the 40-fold decrease in mRNA expression in slp1-repressed embryos is not due to gross changes in the accessibility of the slp1 promoter region (Wang, 2007).
Histone acetylation and deacetylation are important for transcriptional regulation with a general correlation between histone acetylation and active transcription. Indeed, prior work has demonstrated that the Rpd3 histone deacetylase is important for maintaining the Runt-dependent repression of the segment-polarity gene en. ChIP experiments reveal no significant difference in the H3 acetylation pattern of slp1 chromatin from wild-type versus R+F embryos. Although no differences were detected in H3 or Ac-H3 association that correlate with slp1 repression, there are interesting differences in the H3 acetylation levels at different genomic locations. The slp1 structural gene shows stronger Ac-H3 association than the upstream region. This difference is not observed for the association of H3 with these same intervals, suggesting that H3 acetylation marks genomic regions that are permissive for transcription. The relative levels of H3 and Ac-H3 association with Brother (Bro), a gene that is not transcribed in the early embryo (as measured by RT-PCR), provide additional evidence for this trend. Although H3 association with the Bro gene is greater than for any region of the slp1 locus, the level of Ac-H3 association with Bro is lower than for any region of slp1. Based on the observation that differences in H3 and Ac-H3 association can be detected that correlate with transcriptional potential and yet no differences was detected between wild-type and slp1-repressed embryos, it is concluded that H3 acetylation plays a negligible role in the establishment of slp1 repression (Wang, 2007).
The above observations led to a characterization the interactions of the transcriptional machinery with slp1. Association of the TATA-box-binding protein (TBP) is a first step in assembly of the transcriptional machinery on a promoter. As expected, TBP association is detected with a promoter-proximal interval centered 6 base pairs (bp) upstream of the slp1 transcript initiation site in chromatin from wild-type embryos. A weaker signal is detected for an interval within the 5' untranslated region (UTR), centered 124 bp downstream from the start site, whereas all other intervals give background level signals. Very similar levels of TBP association were found in chromatin from R+F embryos. More surprising is the finding that there is almost no difference in the level of Pol II association with the slp1 promoter-proximal interval in chromatin from wild-type and R+F embryos. Pol II is also associated with the slp1 structural gene in wild-type embryos, but at lower levels than at the promoter. In contrast, Pol II association with the slp1 structural gene is markedly reduced in R+F embryos and near to background levels for regions downstream from the 5'-UTR. Based on these results, it is concluded that promoter recruitment of Pol II is not blocked in slp1-repressed embryos. slp1-associated Pol II was further characterized using an antibody that recognizes the Phospho-Ser-5 form of the heptad repeats that comprise the C-terminal domain (CTD) of the largest Pol II subunit. Phospho-Ser-5 modification of the CTD is associated with transcription initiation. This antiserum also gives the strongest signals with the slp1 promoter-proximal interval in wild-type chromatin, and this signal is not reduced in chromatin from R+F embryos. This result indicates that slp1 repression occurs at a step downstream from transcription initiation (Wang, 2007).
The Drosophila hsp70a promoter is an extensively studied example of regulated transcriptional elongation. Pol II initiates transcription at the hsp70a promoter, and then, in the absence of a heat shock, pauses immediately downstream from the promoter. All somatic cells in 3-4-h-AED embryos are capable of activating the hsp70a gene, and as expected, Phospho-Ser-5-modified Pol II is readily detected on the hsp70a promoter in chromatin preparations from non-heat-shocked embryos. The paused Pol II complex on the hsp70a promoter is also readily detected using permanganate footprinting due to the increased sensitivity of thymine residues in single-stranded regions. This same technique was used to carry out footprinting studies on the slp1 promoter region. The results reveal strong hyperreactivity of thymine residues at +15, +28, +30, +38, and +50 downstream from the transcription start site in blastoderm. This interval is similar, though perhaps somewhat larger than the interval detected for hsp70a, within which the most prominent increases in reactivity are at residues +22 and +30. The pattern of reactivity on slp1 is extremely similar in both wild-type and slp1-repressed embryos, indicating that the hyperreactivity is not due to active transcription of the slp1 gene. Importantly, this pattern is not observed in nuclei from Drosophila tissue culture cells. Thus, unlike hsp70a, the footprint on the slp1 5'-UTR is developmentally regulated (Wang, 2007).
The negative elongation factor NELF is thought to play a key role in establishing the paused Pol II complex on the hsp70a promoter. Indeed, NELF association provides a marker for the paused complex as heat-shock-induced transcriptional elongation involves release of NELF (Wu, 2005). In agreement with the results of footprinting studies, ChIP experiments reveal the NELF-D and NELF-E subunits are associated with the slp1 promoter region in chromatin from wild-type embryos, but not in chromatin from Drosophila tissue culture cells. Strong signals are obtained in chromatin from embryos with both the promoter-proximal and 5'-UTR intervals, whereas background level signals are obtained with other intervals of the slp1 locus. It is notable that the promoter-proximal signal is less than or equal to the signal detected for the 5'-UTR interval. This pattern of association contrasts that obtained with TBP, which shows a threefold stronger signal with the promoter-proximal primer pair. These association patterns suggest is that NELF is bound downstream from the slp1 transcription start site, presumably as a component of the paused Pol II complex. Consistent with this interpretation, a similar differential pattern was found of TBP and NELF association with promoter-proximal and 5'-UTR intervals of hsp70a. These results strongly suggest that NELF plays a key role in regulating slp1 elongation in the blastoderm-stage Drosophila embryo (Wang, 2007).
The initial indications that slp1 expression was regulated at a step downstream from transcription initiation came from ChIP experiments on chromatin from a homogeneous population of embryos that uniformly repress slp1. Localized association of NELF in a region downstream from the transcription start site is a hallmark of promoter-proximal pausing. Importantly, this association provides a method for detecting paused Pol II complexes in chromatin from embryos that contain a mixture of cells, some of which are expressing full-length mRNA transcripts. ChIP assays were used to determine whether NELF associates with the promoter regions of wg and en, two pivotal segment-polarity gene targets of the pair-rule transcription factors. The results reveal specific association of NELF with the promoter-proximal and 5'-UTR regions of both genes in 3-4-h-AED embryos. Furthermore, the differential association pattern of TBP and NELF with these two intervals indicates that NELF is localized to a region immediately downstream from the initiation sites for both genes. These findings indicate that regulation of transcriptional elongation is likely to be central in generating the initial patterns of segment-polarity gene expression in the Drosophila embryo (Wang, 2007).
Regulation of transcriptional elongation has been described for several genes in addition to the Drosophila heat-shock genes, including human c-myc, c-myb, c-fos, junB, and p21. A feature shared by these previously characterized examples is rapid induction of gene expression in response to external stimuli. The initial establishment of segment-polarity gene-expression patterns in response to the pair-rule transcription factors occurs within a relatively brief developmental window of ~30 min, spanning the completion of cellularization and the beginning of germ band extension. The temporal advantages offered by regulating these genes at a transcriptional elongation step as compared with chromatin remodeling and/or Pol II initiation complex assembly may be essential for the timely establishment of differing gene expression programs during cell fate specification in the Drosophila blastoderm embryo. The observations that Pol II molecules are enriched at the 5'-ends of a number of genes, coupled with findings that defects in transcriptional elongation factors produce specific developmental defects, strongly suggest that regulation of transcriptional elongation is a hitherto overlooked, but potentially widespread strategy for controlling gene expression during development (Wang, 2007).
Sloppy paired regulates both wingless and engrailed. Removal of slp gene
function causes embryos to exhibit a severe pair-rule/segment polarity phenotype.
The en stripes expand anteriorly in slp mutant embryos. slp activity is an absolute
requirement for maintenance of wg expression at the same time that wg transcription is dependent
on hh. The SLP proteins are expressed in broad stripes, just anterior of the en-positive cells and
overlapping the narrow wg stripes. By virtue of their ability to activate wg and
repress en expression, the distribution of the SLP proteins defines the wg-competent and
en-competent compartments. Thus SLP works either in or parallel with the ptc/hh signal transduction pathway to regulate wg transcription (Cadigan, 1994a).
An effect on the early stripe of Goosecoid expression is observed in sloppy-paired, orthodenticle, tailless and decapentaplegic mutants. Both slp and otd affect Gsc in a similar way: the early stripe of Gsc appears normally but at the end of the cellularization stage, there is no reinforcement of its expression and it is prematurely lost. dpp is necessary to bring aboud Gsc repression in the dorsal-most region of the embryo, while tll is required to promote Gsc expression in the lateral region, or to prevent its repression by the dorsoventral patterning system (Goriely, 1996).
The exact positioning of neuroblasts in the neuroectodermal region that gives rise to the CNS is regulated by a combination of pair-rule genes. Proneural achaete-scute genes are controlled by combinations of axis-patterning genes through a common intergenic control region. Specifically, in embryos mutant for sloppy-paired the second and fourth rows of achaete expression fuse in odd-numbered segments. (Skeath, 1992)
At least two pair-rule genes, paired (prd) and sloppy paired, and all
segment-polarity genes analysed to date are under the control of Tenascin major, the extracellular protein related to tenascin.
Tenascin major initiates a signal transduction cascade which acts in concert with odd-paired or via opa, on downstream
targets such as prd, slp, gooseberry, engrailed and wingless, leading to an opa-like phenotype (Baumgartner, 1994).
Regulatory genes directing embryonic development are expressed in complex patterns. The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a striped pattern that is controlled by several discrete and large cis- regulatory elements. One key cis-element is the ftz proximal enhancer, which is required for stripe establishment and which mediates autoregulation by direct binding of Ftz protein. To identify the trans-acting factors that regulate ftz expression and autoregulation, a modified yeast two hybrid screen, the Double Interaction Screen (DIS), was developed. The DIS was designed to isolate both DNA binding transcriptional regulators that interact with the proximal enhancer and proteins that interact with Ftz itself when it is bound to the enhancer. The screen identified two candidate Ftz protein cofactors as well as activators and repressors of ftz transcription that bind directly to the enhancer. One of these [Tramtrack (Ttk)] is known to bind to at least five sites in the proximal enhancer; genetic studies suggest that Ttk acts as a repressor of ftz in the embryo. In yeast cells, Ttk protein strongly activates transcription, suggesting that yeast may be missing a necessary co-repressor that is present in Drosophila embryos. Also characterized was the activity of a second candidate ftz repressor isolated in the screen: the product of the pair-rule gene sloppy paired, a member of the forkhead family. Slp1 is shown in this study to be a DNA binding protein. A high affinity binding site for Slp1 in the ftz proximal enhancer was identified. Slp1 represses transcription via this binding site in yeast cells, consistent with its role as a direct repressor of ftz stripes in interstripe regions during late stages of embryogenesis. The DIS should be a generally useful method used to identify DNA binding transcriptional regulators and protein partners of previously characterized DNA binding proteins (Yu, 1999).
The striped expression pattern of the pair-rule gene even skipped
(eve) is established by five stripe-specific enhancers, each of which
responds in a unique way to gradients of positional information in the early
Drosophila embryo. The enhancer for eve stripe 2
(eve 2) is directly activated by the morphogens Bicoid (Bcd) and
Hunchback (Hb). Since these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. A well-conserved sequence
repeat, (GTTT)4 has been identified that is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior
factors. Further genetic analysis suggests that the
(GTTT)4-mediated mechanism is independent of the Gt-mediated
mechanism that sets the anterior stripe border, and suggests that a third
mechanism, downregulation of Bcd activity by Torso, prevents activation near
the anterior tip. Thus, three distinct mechanisms are required for anterior
repression of a single eve enhancer, each in a specific position.
Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees,
and the eve 1 and eve 3+7 enhancers each contain GTTT
repeats similar to the site in the eve 2 enhancer. These results
suggest a common mechanism for preventing anterior activation of three
different eve enhancers (Andrioli, 2002).
Previous experiments suggested that the gap gene gt and the
Gt-binding sites are required for the correct positioning of the anterior
eve 2 border. To test the relationship between the
(GTTT)4-binding activity and Gt-mediated repression, the
eve2Delta(GTTT)4-lacZ construct was crossed
into a gt mutant background. If the two repression mechanisms are
independent, an additive effect would be expected from combining these two
perturbations. If, however, Gt-mediated repression is partially redundant with
the (GTTT)4-binding activity, removing both might cause a more
severe derepression. In this cross there is an anterior
shift and slight expansion of stripe 2 that is similar to the effects on the
wild-type eve 2 transgene in gt mutants. No new effect is
detected on the band of derepression created by deleting the
(GTTT)4 site, and a small repressed area is still maintained
between the two parts of the pattern. This result is consistent with an additive effect, and
suggests that the (GTTT)4-binding activity functions independently
of Gt-mediated repression. The failure to derepress in the region between the
two parts of the pattern probably reflects the activity of the unknown protein
X, which normally participates with Gt in repression (Andrioli, 2002).
Because of the peculiar sequence of the (GTTT)4 site, gel shift experiments were performed using nuclear extracts from 0- to 12-hour-old
wild-type embryos.
These experiments showed the formation of several specific protein-DNA
complexes. To identify specific proteins that bind the (GTTT)4
sequence, a yeast one-hybrid assay was constructed with constructs containing
four tandem copies of the intact site. From an initial screen of ~500,000
clones, 66 true positives were obtained. These clones were then
transformed into a yeast strain containing identical reporters except for base
pair substitutions in the (GTTT)4 sequence. Forty-nine clones also
activated one of the mutant constructs, leaving only 16 that activated the
(GTTT)4 constructs, but not the negative controls. Among these 16 were
two clones that encode histone H1 and a single clone that encodes the forkhead
domain (FD) protein Slp1. slp was originally classified as a
pair-rule mutation, but the slp locus contains two tightly linked
genes, slp1 and slp2.
These genes are related in their primary structure, and their expression
patterns overlap significantly. However, slp1 is expressed much
earlier in an anterior 'gap gene-like' domain, which first appears as an
anterior cap, and then evolves into a broad stripe at approximately 80% egg
length. Double staining experiments with gt show that both
genes are expressed at the same time, and that slp1 expression
overlaps the anterior part of the gt expression domain. Thus, the temporal and spatial expression patterns of slp1 are consistent with a role in anterior repression of eve 2 (Andrioli, 2002).
The (GTTT)4 sequence bears little resemblance to the only
previously defined Slp1-binding site (TCTTCGATGTCAACACACC). Thus,
tests were performed to see whether bacterially expressed Slp1 can bind directly to the
(GTTT)4 sequence in vitro. These experiments show that Slp1 binds specifically to this sequence, suggesting that it may directly interact with this sequence in vivo. Similar results were obtained using a fragment of the Slp1 protein that contains only the forkhead domain (Andrioli, 2002).
If Slp1 acts as an anterior repressor of eve 2, genetically
removing it might cause an anterior derepression of the
eve2-lacZ expression pattern. To test this, the
reporter was crosssed into a slp deletion mutant that completely removes the slp1-coding region and disrupts slp2.
No anterior expansion was detected in this experiment. Endogenous eve expression was also analyzed in this mutant background,
and slight anterior shifts of stripes 1 and 2 were detected, but
no significant derepression in anterior regions. To test whether Slp1-mediated
repression requires gt, eve and the
eve2-lacZ reporter gene were both examined in gt; slp double mutant embryos. The double mutant shows no increase in the anterior derepression over that caused by removal of gt alone. These results
argue against a role for Slp1 in anterior repression of eve, but do
not rule out the possibility that Slp1 is one of several redundant proteins
that repress through the (GTTT)4 site. Two other FD proteins, Fkh
and Crocodile (Croc), are expressed in anterior regions of the embryo. However, the expression domains of both proteins are located very near the anterior pole, making it unlikely that either gene is involved in this repression mechanism. To make sure, eve and eve
2-lacZ expression was examined in each mutant; neither shows an anterior derepression. Thus these two genes are unlikely to play important roles in this repression mechanism (Andrioli, 2002).
The repressive effects of ectopic Slp1 on the three anterior eve
stripes suggest a common mechanism for repression in anterior regions of the
embryo. To test this, the eve locus was examined for the presence of binding sites similar to the (GTTT)4 site in the eve 2 enhancer. Interestingly, there are only two other such sites in the eve locus; these are located within the boundaries of the stripe 1 and stripe 3+7
enhancers. Whether repression by ectopic Slp1 is mediated through these enhancers and the eve 2 MSE was examined. In these experiments, ectopic Slp1 causes a ventral repression of stripe 1 in the context of an
eve1+5-lacZ transgene. A similar repression of stripe 3 was observed in the context of an eve3+7-lacZ transgene. These results are consistent with the idea of a common mechanism. By contrast, no ventral repression of the eve2-lacZ transgene was detected, which is surprising in light of the fact that eve 2 is the most strongly affected stripe in the context of the endogenous gene (Andrioli, 2002).
The reason for the discrepancy between the reporter and the endogenous gene
is not clear. It is possible that Slp1-mediated repression of eve 2
requires eve sequences outside the minimal enhancer. For example, the
late element (LE) mediates the refinement of all seven eve stripes after they are initially positioned. Perhaps interactions between the LE and/or other cis sequences are required for effective repression by Slp1. To test this, a larger reporter gene (-7.8 eve-lacZ) was used that contains all native sequences from the 5' border of the locus to the
transcription start site. This transgene contains the LE, the eve 2
and eve 3+7 enhancers, and all native sequences that lie between
these elements, and drives expression of stripes 2, 3 and 7, and a single line
of nuclei located within the normal position of stripe 1. Expression from this
reporter is effectively repressed at the position of stripes 1 and 3 in
embryos containing ventrally expressed Slp1, but there is still no effect on
the stripe 2 response. Thus, the addition of these extra sequences does not restore the sensitivity of eve 2 to Slp1-mediated repression. This suggests that undefined properties of the endogenous eve locus are required for Slp1-mediated repression of eve 2 (Andrioli, 2002).
The results presented here indicate that three distinct mechanisms are
required for anterior repression of eve 2, with each activity
functioning within a specific subregion (Andrioli, 2002).
In subregion III, the Gt-binding sites
are crucial for
repression -- deletion of these sites leads to an anterior expansion of
the stripe. However, it is clear that Gt does not act alone, and that at least
one other factor (X) must be involved in repressing through these sites. The
identity of X is not clear, but genetic studies have localized a Gt-like
patterning activity to the left arm of chromosome II.
Segmental aneuploids that remove this arm show an expansion similar to that
seen in gt mutants (Andrioli, 2002).
In subregion II, repression of eve 2 is mediated by the
(GTTT)4 site described in this paper. A
candidate protein, Slp1, has been identified that is expressed at the right time and place for the repression activity and binds specifically to this site in the yeast 1-hybrid experiment and in vitro. The (GTTT)4 site shows little similarity to the other known Slp1-binding site, but is
quite similar to sites bound by other members of the FD protein family. For
example, a 115 amino acid FD fragment of Fkh binds specifically to the site
CTTTGTAAA, which bears some resemblance to the (GTTT)4
site. Also, the hepatocyte FD protein HNF-3 binds to a site (TGTTTGTTTTAGTT)
that contains two perfect GTTT repeats. Ventrally
expressed Slp1 specifically represses eve 2, strongly supporting a
role in regulation of the endogenous eve gene. However, there is no
effect on eve 2 in slp mutants, suggesting that Slp1 is
redundant with at least one other protein (Y), which also mediates repression
through the (GTTT)4 site. The existence of multiple complexes in
gel shifts with embryo extracts is consistent with this, but the identity of Y
is still unknown (Andrioli, 2002).
In subregion I, eve 2 repression is controlled by Tor, which may
act by downregulating Bcd-dependent activation. This is consistent with the
previous demonstration that Tor interferes with Bcd-dependent activation of
hb, otd and slp1 (Andrioli, 2002).
In summary, at least five different protein activities are involved in
three distinct mechanisms that repress eve 2 in anterior regions.
Interestingly, it seems that all aspects of eve 2 regulation are
controlled, directly or indirectly, by the Bcd morphogen gradient. The eve 2
enhancer is directly activated by Bcd, but activation is prevented near the
anterior pole by Tor. The anterior expression patterns of the defined
repressors of eve 2 (Slp1 and Gt) are also activated by Bcd. It is
proposed that the relative positions of these domains, and the ultimate
position of eve 2, are controlled by differential sensitivity to the
Bcd concentration gradient. Future experiments on the cis-elements that
regulate slp1 and gt transcription will be required to test
this (Andrioli, 2002).
slp was originally classified as a pair-rule gene based on its
cuticular phenotype, and has been shown to function at both the level of the
pair-rule genes and the segment polarity genes.
However, specific patterning functions for the early anterior Slp1 expression domain
have remained unclear, although strong alleles of slp1
exhibit severe defects in the mandibular lobe. The results presented in this paper suggest that Slp1
acts at the level of the gap genes by repressing enhancer elements that
control the initial eve stripes. Thus, Slp1 function is required at
three different levels of the segmentation hierarchy (Andrioli, 2002).
The mechanism involved in Slp1-mediated repression of eve is
unknown, but may involve an interaction with the co-repressor Groucho (Gro). The Slp1 protein sequence contains a motif (FSIDAIL),
which is very similar to the EH1 Gro-binding consensus (FSIDNIL) and
Slp1 has been shown to bind Gro in vitro (Andrioli, 2002).
It has been proposed that Gro mediates repression by creating a direct
physical link between DNA-bound proteins and components of the basal
transcription machinery. As such, repressors that act through Gro can function over very long distances, and have been classified as long-range repressors. The ability of Slp1 to interact with Gro suggests such a long-range mechanism, but several considerations are not consistent with this model. For example,
ventral expression of a long-range repressor that 'locks' the basal
transcription machinery should repress all seven eve stripes, not
just the anterior three. Also, the three (GTTT)4 sites described
in this study are all located within minimal enhancer elements that control specific stripes. In a long-range mechanism, these sites could be located anywhere in the promoter, and need not be associated with specific enhancers (Andrioli, 2002).
One of the most intriguing findings of this study is that ectopic Slp1
represses eve 2 in the endogenous gene, but not in the context of
several lacZ reporter genes. Despite much effort, this discrepancy has not been resolved, but it is informative to compare the structural
differences between the endogenous gene and the tested transgenes. One obvious
difference between the lacZ reporter genes tested in these
experiments and the endogenous eve gene is copy number. Perhaps
Slp1-mediated repression requires two copies of the enhancer in a homozygous
situation. Consistent with this hypothesis, a pairing-sensitive element (PSE) that reduces marker gene
expression in homozygotes has been identified in the far 3' region of
the eve gene. Two experiments
argue against this hypothesis: (1) ectopic Slp1 still represses endogenous
eve in Df (eve)/+ heterozygotes and (2) Slp1 fails to repress eve 2-containing transgenes when they are homozygosed (Andrioli, 2002).
Another difference between the endogenous gene and the reporters is that
the endogenous gene contains genomic regions outside those tested in the
reporter genes, and is located in a different genomic position. Perhaps
control sequences in the 3' region of the gene, or further 5' are
required for this repression mechanism. As mentioned above, the eve 1
enhancer, which is located in the 3' region, contains a
(GTTT)4-binding site. Perhaps effective repression of eve
2 requires all three sites contained in the three different enhancers. This
will be tested in future experiments (Andrioli, 2002).
Finally, it is possible that the native eve locus is organized in
a specific chromatin conformation that permits repression by Slp1, and this
configuration is not maintained when eve 2 transgenes are inserted
into ectopic genomic locations. The fact that Slp1 protein contains an FD
DNA-binding domain is interesting in this regard. Structural studies suggest
that FD domains form a 'winged-helix' are very similar to the globular DNA-binding
domain of the linker histone H1. Furthermore, it has been shown that the mammalian FD protein hepatic nuclear factor 3 (HNF3) competes with H1 for binding to specific sites, and that this competition is critical for the in vivo regulation of the albumin liver-specific enhancer. Such a mechanism may be involved in Slp1-mediated repression of eve 2. Consistent with this, two clones have been isolated that encode histone H1 in the one-hybrid experiment with the (GTTT)4 site. This suggests that both proteins can bind to this site, and supports the idea that regulation of chromatin structure may be an important part of Slp1-mediated repression of eve 2. More experiments will be required to test this hypothesis (Andrioli, 2002).
The eve 2 enhancer is one of the best-characterized patterning
elements in Drosophila development. Proteins involved in activation
and repression have been identified, and a simple model has emerged that
explains the basic activity of the enhancer. Anterior repression of this element requires at least three position-specific mechanisms -- this fact significantly extends understanding of this aspect of
enhancer function. These results also suggest that the current model for
activation of this enhancer is also incomplete. The deletion analysis identified four
regions that are required for efficient activation of the enhancer. The
effects of these deletions may be caused by changing the spacing between known
activator and/or repressor sites within the enhancer. However, it possible
that these regions contain specific binding sites required for activation.
Consistent with this, there are several well-conserved sequence blocks that
might represent specific sites required for activation. Base-pair
substitutions that disrupt the conserved sequences without changing site
spacing will be used to initially test this (Andrioli, 2002).
Segmentation of the Drosophila embryo is a classic paradigm for pattern formation during development. The Wnt-1 homolog Wingless (Wg) is a key player in the establishment of a segmentally reiterated pattern of cell type specification. The intrasegmental polarity of this pattern depends on the precise positioning of the Wg signaling source anterior to the Engrailed (En)/Hedgehog (Hh) domain. Proper polarity of epidermal segments requires an asymmetric response to the bidirectional Hh signal: wg is activated in cells anterior to the Hh signaling source and is restricted from cells posterior to this signaling source. This study reports that Midline (Mid) and H15, two highly related T box proteins representing the orthologs of zebrafish hrT and mouse Tbx20, are novel negative regulators of wg transcription and act to break the symmetry of Hh signaling. Loss of mid and H15 results in the symmetric outcome of Hh signaling: the establishment of wg domains anterior and posterior to the signaling source predominantly, but not exclusively, in odd-numbered segments. Accordingly, loss of mid and H15 produces defects that mimic a wg gain-of-function phenotype. Misexpression of mid represses wg and produces a weak/moderate wg loss-of-function phenocopy. Furthermore, it has been shown that loss of mid and H15 results in an anterior expansion of the expression of serrate (ser) in every segment, representing a second instance of target gene repression downstream of Hh signaling in the establishment of segment polarity. The data presented indicate that mid and H15 are important components in pattern formation in the ventral epidermis. In odd-numbered abdominal segments, Mid/H15 activity plays an important role in restricting the expression of Wg to a single domain (Buescher, 2004).
Previous work has suggested that Slp permits the Hh-dependent activation of Wg anterior to the En/Hh stripe by antagonizing a repressor of Wg. Based on the data presented above, Mid/H15 appear to be such repressors. To determine if Slp is a negative regulator of mid expression, the effect of slp loss-of-function and slp misexpression was studied on the distribution of mid RNA. In slp mutant embryos the early mid expression is normal. However from early stage 9 onward the mid stripes broaden to approximately twice their normal width. Using mid-positive neuroblasts as a landmark (these remain unchanged in slp mutant embryos), it was possible to characterize the increase in mid expression as an anterior expansion. This aberrant mid expression pattern is unstable; from stage 11 onward mid decays in odd-numbered segments. Conversely, misexpression of slp in the ventral ectoderm from early stage 9 onward led to a complete loss of ectodermal mid expression. These data show that Slp functions as a repressor of mid expression. Taken together with the observation that misexpression of mid in otherwise wild-type embryos results in the loss of Wg expression, these results lead to the conclusion that the Slp-mediated repression of mid anterior to the En/Hh stripe is an important component of wg competence (Buescher, 2004).
As a further test of the relationship between slp and mid, the effect was compared of expressing mid and slp, alone or in combination, on Wg expression. Ectopic expression of mid results in a rapid and almost complete loss of Wg expression, whereas ectopic expression of slp results in weak ectopic expression of Wg posterior to the En/Hh stripe. This slp-induced phenotype resembles that of the loss of mid, except that ectopic Wg expression is weaker and appears randomly in even- and odd-numbered segments. The ectopic Wg expression is blocked when mid and slp are expressed together, suggesting that in this context mid acts downstream of slp. The Wg expression anterior to the En/Hh stripe still decays in UAS-mid/UAS-slp embryos, albeit more slowly and variably than in UAS-mid alone. This result may reflect that Wg expression is sensitive to the amounts of available Mid and Slp. It may also indicate that anterior to the En/Hh stripe, Slp function is required for more than just repression of mid and may possibly have independent activating functions. An analysis of slp1,slp2;mid/H15 quadruple mutants would be highly helpful in clarifying the relationship between slp genes and mid/H15. Unfortunately, the generation of such a quadruple mutant by genetic recombination is impossible because the slp deletion that removes these genes (Δ34B) is on a balancer chromosome that precludes recombination (Buescher, 2004).
Integration of positive Dpp signals, antagonistic Wg inputs and mesodermal competence factors and thir impact of Bagpipe expression during Drosophila visceral mesoderm induction
Tissue induction during embryonic development relies to a significant
degree on the integration of combinatorial regulatory inputs at the enhancer level of target genes. During mesodermal tissue induction in
Drosophila, various combinations of inductive signals and
mesoderm-intrinsic transcription factors cooperate to induce the progenitors of different types of muscle and heart precursors at precisely defined positions within the mesoderm layer. Dpp signals are required in cooperation with the mesoderm-specific NK homeodomain transcription factor Tinman (Tin) to induce all dorsal mesodermal tissue derivatives, which include dorsal somatic muscles (the dorsal vessel and visceral muscles of the midgut). Wingless (Wg)
signals modulate the responses to Dpp/Tin along anteroposterior positions by
cooperating with Dpp/Tin during dorsal vessel and somatic muscle induction
while antagonizing Dpp/Tin during visceral mesoderm induction. As a result,
dorsal muscle and cardiac progenitors form in a pattern that is reciprocal to
that of visceral muscle precursors along the anteroposterior axis. The present
study addresses how positive Dpp signals and antagonistic Wg inputs are
integrated at the enhancer level of bagpipe (bap), a NK
homeobox gene that serves as an early regulator of visceral mesoderm
development. An evolutionarily conserved bap enhancer element requires combinatorial binding sites for Tin and Dpp-activated Smad proteins for its activity. Adjacent binding sites for the FoxG transcription factors encoded by the Sloppy paired genes (slp1 and slp2), which are direct targets of the Wg signaling cascade, serve to block the synergistic activity of Tin and activated Smads during bap induction. In addition, binding sites for yet unknown repressors are essential to prevent the induction of the bap enhancer by Dpp in the dorsal ectoderm. These data illustrate how the same signal combinations can have opposite effects on different targets in the same cells during tissue induction (Lee, 2005).
Unlike Dpp, Wg signals act indirectly upon the early bap enhancer. Previous genetic and molecular data have shown that Wg induces the expression of the forkhead domain-encoding gene slp via crucial dTCF/Lef-1 binding sites in both mesoderm and ectoderm. slp, in turn, functions as a repressor of bap. The present data show that slp products exert this function by direct binding to the Dpp-responsive bap enhancer, which obviously results in a suppression of the synergistic activity of bound Tin and Smad complexes. Slp proteins contain eh1 motifs that can potentially bind the Groucho co-repressor and Slp has known repressor activities in other contexts. In addition, the vertebrate counterpart of Slp, FoxG (BF-1), is known to interact with Groucho and histone deacetylases (Yao, 2001). Thus, it is proposed that Slp overrides nuclear Dpp signaling activities by dominantly establishing an inactive state of the chromatin at the bap locus (Lee, 2005).
It is likely that additional components are involved in the antagonistic interaction of Slp with Tin/Smad complexes. The Slp-binding site includes sequences that are also required positively for the mesodermal response to Dpp, although not for ectopic responses in the ectoderm. In a genome-wide expression analysis, any forkhead domain genes other than bin were found that are mesoderm specific. However, the function of an essential co-activator in the mesoderm interacting with this site could be fulfilled by a ubiquitously expressed forkhead domain protein, and in part by Bin, which is required for the prolongation of the Dpp response. In the yeast one-hybrid screens with this site that yielded Slp clones, a clone of fd68A, a uniformly expressed ortholog of vertebrate FoxK1 (Myocyte Nuclear Factor) was isolated, but genetic confirmation of its involvement in bap induction is currently lacking. Regardless of the identity of this factor, Slp could either compete with this protein and with Bin for DNA binding, or it could disrupt their productive functional interactions with the Tin/Smad complexes. Interestingly, the latter type of mechanism has been proposed to operate during the interference of the slp ortholog BF-1 with TGFß signaling in the vertebrate cerebral cortex (Lee, 2005).
On top of this basic arrangement that allows the enhancer to be active in the dorsal mesoderm, the enhancers from bap and eve, but not tin, include binding sites that make them respond to Wg inputs in an opposite fashion. In the case of bap, Wg-induced Slp binds and dominantly suppresses the activity of bound Smad effectors. For the eve enhancer it has been proposed that there is an analogous repressive activity; however, in this case, it is exerted by bound Wg signal effectors, i.e., dTCF/Lef-1, in the absence of Wg signals. In the domains with active Wg signaling, the repressive activity of dTCF/Lef-1 is neutralized by the Wg signaling cascade, which allows the Dpp effectors to be active at the eve enhancer (since it lacks Slp binding sites). Through these switches, the bap and eve enhancers become induced in reciprocal AP patterns. In addition, the eve enhancer includes binding sites for activators and repressors downstream of receptor tyrosine kinases and Notch, respectively, which serve to restrict eve activity to specific subsets of cells within the domains of overlapping Dpp and Wg activities. Clearly, many of the molecular details still need to be clarified. Nevertheless, the basic principles of how differential inputs from inductive signals and tissue-specific activities can be integrated at the enhancer level to achieve distinct patterns of target gene expression during early tissue induction in the Drosophila mesoderm are now beginning to be understood (Lee, 2005).
During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. This study shows tha abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. The results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes (Gebelein, 2004).
The segregation of groups of cells into compartments is fundamental to animal development. Originally defined in Drosophila, compartments are critical for providing cells with their unique positional address. The first compartments to form during Drosophila development are the anterior and posterior compartments and the key step to defining them is the activation of the gene engrailed (en). Expression of en, which encodes a homeodomain transcription factor, results in a posterior compartment fate, and the absence of en expression results in an anterior compartment fate. Once activated by gap and pair-rule genes, en expression and, consequently, the anterior-posterior compartment boundary later become dependent upon the protein Wingless (Wg), which is secreted from adjacent anterior compartment cells. Concurrently with anterior-posterior compartmentalization and segmentation, the expression of the eight Drosophila Hox genes is also initially established by the gap and pair-rule genes. The Hox genes, however, which also encode homeodomain transcription factors, do not contribute to the formation or number of segments but instead specify their unique identities along the anterior-posterior axis (Gebelein, 2004).
This flow of genetic information during Drosophila embryogenesis has led to the idea that anterior-posterior compartmentalization and segment identity specification are independent processes. In contrast to this view, this study shows that these two pathways are interconnected in previously unrecognized ways. Evidence is provided that Hox factors directly interact with segmentation proteins such as En to control gene expression. Moreover, Hox proteins collaborate with two different segmentation proteins in anterior and posterior cell types to regulate the same Hox target gene, revealing a previously unknown use of compartments to control gene expression by Hox proteins (Gebelein, 2004).
Distalless (Dll) is a Hox target gene that is required for leg development in Drosophila. In each thoracic hemisegment, wg, expressed by anterior cells adjacent to the anterior-posterior compartment boundary, activates Dll in a group of cells that straddle this boundary. A cis-regulatory element derived from Dll, called DMX, drives accurate Dll-like expression in the thorax. The abdominal Hox genes Ultrabithorax (Ubx) and abdominalA (abdA) directly repress Dll and DMX-lacZ in both compartments, thereby blocking leg development in the abdomen. DMX is composed of a large activator element (DMXact) and a 57-base-pair (bp) repressor element referred to here as DMX-R. Previous work demonstrated that Ubx and AbdA cooperatively bind to DMX-R with two homeodomain cofactors, Extradenticle (Exd) and Homothorax (Hth). In contrast, the thoracic Hox protein Antennapedia (Antp) does not repress Dll and does not bind DMX-R with high affinity in the presence or absence of Exd and Hth. Thus, repression of Dll in the abdomen depends in part on the ability of these cofactors to selectively enhance the binding of the abdominal Hox proteins to DMX-R (Gebelein, 2004).
Exd and Hth, as well as their vertebrate counterparts, are used as Hox
cofactors at many target genes. Moreover, Hox/Exd/Hth complexes are used for both gene activation and repression, raising the question of how the decision to activate or repress is determined. One view posits that these complexes do not directly recruit co-activators or co-repressors, but instead are required for target gene selection. Accordingly, other DNA sequences present at Hox/Exd/Hth-targeted elements would determine whether a target gene is activated or repressed. Consistent with this notion, DMX-R sequences isolated from six Drosophila species show extensive conservation outside the previously identified Hox (referred to here as Hox1) Exd and Hth binding sites, suggesting that they also play a role in Dll regulation (Gebelein, 2004).
To test a role for these conserved sequences, a thorough mutagenesis of DMX-R was performed. Each mutant DMX-R was cloned into an otherwise wild-type, full-length DMX and tested for activity in a standard reporter gene assay in transgenic embryos. Thoracic expression was normal in all cases. However, surprisingly, many of the DMX-R mutations, such as X5, resulted in abdominal de-repression only in En-positive posterior compartment cells, whereas other mutations, such as X2, resulted in abdominal de-repression only in En-negative anterior compartment cells. Single mutations in the Hox1, Exd, or Hth sites also resulted in de-repression predominantly in posterior cells. In contrast, deletion of the entire DMX-R (DMXact-lacZ), or mutations in both the X2 and X5 sites (DMX[X2 + X5]-lacZ), resulted in de-repression in both compartments. These results suggest that distinct repression complexes bind to the DMX-R in the anterior and posterior compartments and that segmentation genes play a role in Dll repression (Gebelein, 2004).
One clue to the identity of the proteins in these repression complexes is that the sequence around the Hth site is nearly identical to a Hth/Hox binding site that had been identified previously by a systematic evolution of ligands by exponential enrichment (SELEX) approach using vertebrate Hox and Meis proteins. This similarity suggested the presence of a second, potentially redundant Hox binding site, Hox2. In agreement with this idea, mutations in both the Hox1 and Hox2 binding sites resulted in de-repression in both the anterior and posterior compartments of the abdominal segments. Similarly, although individual mutations in the Exd and Hth binding sites lead predominantly to de-repression in the posterior compartment, mutation of both sites resulted in de-repression in both compartments. These results suggest that a Hox/Exd/Hth/Hox complex may be used for repression in both compartments. Furthermore, they suggest that although single mutations in these binding sites are sufficient to disrupt the activity of this complex in the posterior compartment, double mutations are required to disrupt its activity in the anterior compartment (Gebelein, 2004).
To provide biochemical evidence for a Hox/Exd/Hth/Hox tetramer, DNA binding experiments were performed using DMX-R probes and proteins expressed and purified from E. coli. Previous experiments demonstrated that a Hox/Exd/Hth trimer cooperatively binds to the Hox1, Exd and Hth sites. The function of the Hox2 site was tested in two ways. First, binding was measured to a probe, DMX-R2, that includes the Exd, Hth and Hox2 sites, but not the Hox1 site. It was found that Exd/Hth/AbdA and Exd/Hth/Ubx trimers cooperatively bind to this probe and that mutations in the Hth, Exd or Hox2 binding sites reduced or eliminated complex formation (Gebelein, 2004).
Second, if both the Hox1 and Hox2 sites are functional, the full-length DMX-R may promote the assembly of Hox/Exd/Hth/Hox tetramers. Using a probe containing all four binding sites (DMX-R1 + 2), the formation of such complexes was observed. Mutation of any of the four binding sites reduced the amount of tetramer binding whereas mutation of both Hox sites or both the Exd and Hth sites eliminated tetramer binding. Furthermore, Antp, which does not repress Dll, formed tetramers with Exd and Hth that were approximately tenfold weaker than with Ubx or AbdA, but bound well to a consensus Hox/Exd/Hth trimer binding site. Because mutation of both Hox sites or both the Exd and Hth sites resulted in de-repression in both compartments, these experiments correlate the binding of a Hox/Exd/Hth/Hox complex on the DMX-R with the ability of this element to mediate repression in both compartments (Gebelein, 2004).
Although binding of a Hox/Exd/Hth/Hox tetramer is sufficient to account for the necessary abdominal Hox-input into Dll repression, it does not explain the compartment-specific de-repression exhibited by some DMX-R mutations. The X2 and X5 mutations, for example, result in abdominal de-repression but do not prevent the formation of the Hox/Exd/Hth/Hox tetramer. Sequence inspection of the DMX-R revealed that the X2 mutation, which resulted in de-repression specifically in the anterior compartment, disrupts two partially overlapping matches to a consensus binding site for Forkhead (Fkh) domain proteins. With this in mind, the expression pattern of Sloppy paired 1 (Slp1), a Fkh domain factor encoded by one of two partially redundant segmentation genes, slp1 and slp2, was examined. The two slp genes are expressed in anterior compartment cells adjacent and anterior to En-expressing posterior compartment cells. In the thorax, cells expressing Dll and DMX-lacZ co-express either Slp or En at the time Dll is initially expressed. In the abdomen, the homologous group of cells, which express DMXact-lacZ (a reporter lacking the DMX-R), co-express either Slp in the anterior compartment or En in the posterior compartment. The expression patterns of Slp and En were compared with Ubx and AbdA. Ubx levels are highest in anterior, Slp-expressing cells whereas AbdA levels are elevated in posterior, En-expressing cells. In contrast, both Exd and Hth are present at similar levels in both compartments throughout the abdomen (Gebelein, 2004).
On the basis of these data, a model is presented for Hox-mediated repression of Dll in both the anterior and posterior compartments of the abdominal segments. In the anterior compartment it is proposed that Slp binds to DMX-R directly with a Ubx/Exd/Hth/Ubx tetramer. In the posterior compartment it is suggested that En binds to DMX-R directly with an AbdA/Exd/Hth/AbdA tetramer. One important feature of this model is that Antp/Exd/Hth/Antp complexes fail to form on this DNA, thereby accounting for the lack of repression in the thorax. Furthermore, the model proposes that Slp and En should, on their own, have only weak affinity for DMX-R sequences because repression does not occur in the thorax, despite the presence of these factors. The Hox/Exd/Hth/Hox complex, perhaps in conjunction with additional factors, is required to recruit or stabilize Slp and En binding to the DMX-R. Both Slp and En are known repressor proteins that directly bind the co-repressor Groucho. Thus, the proposed complexes in both compartments provide a direct link to this co-repressor and, therefore, a mechanism for repression. DNA binding and genetic experiments are presented that test and support this model (Gebelein, 2004).
To test the idea that En is playing a direct role in Dll repression, the ability of En and Hox proteins to bind to DMX-R probes was examined. On its own, En binds to DMX-R very poorly. Surprisingly, it was found that En binds DMX-R with the abdominal Hox proteins Ubx or AbdA in a highly cooperative manner. The thoracic Hox protein Antp does not bind cooperatively with En to this probe. Mutations in the Hox1 or X5 binding sites block AbdA/En binding in vitro, consistent with these mutations showing posterior compartment de-repression in vivo. In contrast, the X6, X7 and Hth mutations do not affect AbdA/En complex formation (Gebelein, 2004).
On the basis of DMX-R's ability to assemble a Hox/Exd/Hth/Hox tetramer, whether En could bind together with an AbdA/Exd/Hth/AbdA complex was tested. Addition of En to reactions containing AbdA, Exd and Hth resulted in the formation of a putative En/AbdA/Exd/Hth/AbdA complex. This complex contains En because its formation is inhibited by an anti-En antibody. A weak antibody-induced supershift is also observed. Moreover, this complex fails to form on the X5 mutant, which causes posterior compartment-specific de-repression. It is noted that En/Exd/Hth complexes also bind to the DMX-R and that it cannot be excluded that an En/Exd/Hth/AbdA complex may be important for Dll repression. The model emphasizes a role for an En/AbdA/Exd/Hth/AbdA complex because it better accommodates the cooperative binding observed between En and AbdA on the DMX-R (Gebelein, 2004).
Repression in the anterior compartments of the abdominal segments requires the sequence defined by the X2 mutation, which is similar to a Fkh domain consensus binding site. The model predicts that this sequence is bound by Slp. Consistent with this view, Slp1 binds weakly to wild type, but not to X2 mutant DMX-R probes. However, in contrast to En, no cooperative binding was observed between Slp and Hox or Hox/Exd/Hth/Hox complexes, suggesting that additional factors may be required to mediate interactions between Slp and the abdominal Hox factors (Gebelein, 2004).
Together, these results suggest that En and Slp play a direct role in DMX-lacZ and Dll repression. However, these experiments do not unambiguously determine the stoichiometry of binding by these factors. Furthermore, in vivo, additional factors may enhance the interaction between these segmentation proteins and Hox complexes, thereby increasing the stability and/or activity of the repression complexes (Gebelein, 2004).
The model for Dll repression is supported by previous genetic experiments that examined the effect of Ubx and abdA mutants on Dll expression in the abdomen. Ubx abdA double mutants de-repress Dll in both compartments of all abdominal segments. In contrast, Ubx mutants de-repress Dll in the anterior compartment of only the first abdominal segment, which lacks AbdA. abdA mutant embryos de-repress Dll in the posterior compartments of all abdominal segments, where Ubx levels are low (Gebelein, 2004).
Several genetic experiments were performed to provide in vivo support for the idea that Slp and En work directly with Ubx and AbdA to repress Dll. The design of these experiments had to take into consideration that the activation of Dll in the thorax depends on wg, and that wg expression depends on both slp and en. Consequently, Dll expression is mostly absent in en or slp mutants, making it impossible to characterize the role that these genes play in Dll repression from examining en or slp loss-of-function mutants. However, some of the mutant DMX-Rs described here provide the opportunity to test the model in alternative ways (Gebelein, 2004).
According to the model, DMX[X5]-lacZ is de-repressed in the posterior compartments of the abdominal segments because it fails to assemble the posterior, En-containing complex. Repression of DMX[X5]-lacZ in the anterior compartments still occurs because it is able to assemble the anterior, Slp-containing complex. According to this model, DMX[X5]-lacZ should be fully repressed if Slp is provided in posterior cells. A negative control for this experiment is that ectopic Slp should be unable to repress DMX[X2]-lacZ because this reporter gene does not have a functional Slp binding site. To mis-express Slp, paired-Gal4 (prd-Gal4), which overlaps both the Slp and En stripes in the odd-numbered abdominal segments, was used. As predicted, ectopic Slp repressed DMX[X5]-lacZ but not DMX[X2]-lacZ, providing strong in vivo support for Slp's direct role in Dll repression in the anterior compartments (Gebelein, 2004).
Conversely, the model posits that DMX[X2]-lacZ is de-repressed in the anterior compartment because it cannot bind Slp, but remains repressed in the posterior compartment because it is able to assemble the En-containing posterior complex. Thus, providing En in the anterior compartment should repress DMX[X2]-lacZ. A complication with this experiment is that En is a repressor of Ubx, which is the predominant abdominal Hox protein in the anterior compartment. It was confirmed that prd-Gal4-driven expression of En represses Ubx and that AbdA levels remain low at the time Dll is activated in the thorax. Consequently, ectopic En expression is not sufficient to repress DMX[X2]-lacZ, consistent with the observation that low levels of abdominal Hox proteins are present. Therefore, to promote the assembly of the posterior complex in anterior cells, En was co-expressed with AbdA using prd-Gal4. As predicted, this combination of factors repressed DMX[X2]-lacZ but not DMX[X5]-lacZ, providing strong in vivo evidence for En playing an essential role in Dll repression in the posterior compartments (Gebelein, 2004).
Several observations provide additional support for the model. First, ectopic expression of AbdA or Ubx in the second thoracic segment (T2) represses DMX[X5]-lacZ in the anterior compartment, but not in the posterior compartment. Conversely, expression of AbdA or Ubx in T2 represses DMX[X2]-lacZ only in posterior compartment cells. Second, co-expression of Slp with Ubx completely represses DMX[X5]-lacZ in T2 but does not repress DMX[X2]-lacZ in T2. Third, in those cases where repression is incomplete (for example, En + AbdA repression of DMX[X2]-lacZ in the abdomen), cells that escape repression have low levels of either an abdominal Hox protein or Slp/En. Together, these data provide additional evidence that the abdominal Hox proteins work together with Slp and En to repress Dll (Gebelein, 2004).
The segregation of cells into anterior and posterior compartments during Drosophila embryogenesis is essential for many aspects of fly development. The results presented in this study reveal an unanticipated intersection between anterior-posterior compartmentalization by segmentation genes and segment identity specification by Hox genes. Specifically, it is suggested that the abdominal Hox proteins collaborate with two different segmentation proteins, Slp and En, to mediate repression of a Hox target gene (Dll) in the anterior and posterior compartments of the abdomen, respectively. This mechanism of transcriptional repression suggests a previously unknown use of compartments in Drosophila development. The mechanism proposed here contrasts with the alternative and simpler hypothesis in which the abdominal Hox proteins would have used the same set of cofactors to repress Dll in all abdominal cells, regardless of their compartmental origin (Gebelein, 2004).
These results provide further support for the view that Hox/Exd/Hth complexes do not directly bind co-activators or co-repressors but instead indirectly recruit them to regulatory elements. Consistent with previous analyses, it is suggested that Hox/Exd/Hth complexes are important for the Hox specificity of target gene selection. Additional factors, such as Slp or En in the case of Dll repression, are required to determine whether the target gene will be repressed or activated. In the future, it will be important to dissect in similar detail other Hox-regulated elements, to assess the generality of this mechanism (Gebelein, 2004).
These results also broaden the spectrum of cofactors used by Hox proteins to regulate gene expression. Although the analysis of Exd/Hth in Drosophila and Pbx/Meis in vertebrates has provided some insights into how Hox specificity is achieved, there are examples of tissues in which these proteins are not available to be Hox cofactors and of Hox targets in which Exd and Hth seem not to play a direct role. This study shows that En, a homeodomain segmentation protein, is used as a Hox cofactor to repress Dll in the abdomen. Although the complex defined at the DMX-R includes Exd and Hth, the DNA binding studies demonstrate that Hox and En proteins can bind cooperatively to DNA in the absence of Exd and Hth. These findings suggest that En may function with Ubx and/or AbdA to regulate target genes other than Dll, and perhaps independently of Exd and Hth. Consistent with this idea are genetic experiments showing that, in the absence of Exd, En can repress slp and this repression requires abdominal Hox activity. Although these experiments were unable to distinguish whether the Hox input was direct or indirect, the results suggest that En may bind directly with Ubx and AbdA to repress slp, and perhaps other target genes (Gebelein, 2004).
Finally, these results raise the question of why a compartment-specific mechanism is used by Hox factors to repress Dll. The activation of Dll at the compartment boundary by wg may be important for accurately positioning the leg primordia within each thoracic hemisegment, but this mode of activation requires that Dll is repressed in both compartments in each abdominal segment. The utilization of segmentation proteins such as En and Slp may be the simplest solution to this problem. Compartment-specific mechanisms may also provide additional flexibility in the regulation of target genes by Hox proteins by allowing them to turn genes on or off specifically in anterior or posterior cell types. For these reasons, compartment-dependent mechanisms of gene regulation may turn out to be the general rule instead of the exception (Gebelein, 2004).
Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined: (1) WRPW and related tetrapeptide motifs have been well characterized in several repressors; (2) a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. A yeast two-hybrid screen is described that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. One of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation (Goldstein, 2005).
The eh1 Gro recruitment domain was originally defined as a heptapeptide motif that is conserved in members of the En family of homeodomain proteins and their vertebrate homologues. More recently, eh1-dependent binding to Gro has also been demonstrated in vitro for various other Drosophila and mammalian proteins, nearly all of which contain homeodomains. Given that Bowl and Odd, two non-homeodomain ZnF transcription factors, contain this motif and interact with Gro, the possibility was explored that eh1 motifs are prevalent among additional non-homeodomain transcription factor families. Indeed, an unbiased yeast screen for Gro-interacting proteins selected two additional transcriptional regulators that contain eh1-like motifs, namely, Sloppy-paired (Slp; Forkhead related) and Dorsocross (Doc; T box). Alignment of the eh1-like sequences of Bowl, Odd, Slp, and Doc with those of En and Gsc revealed three conserved amino acids: phenylalanine-x-isoleucine-x-x-isoleucine (Phe-x-Ile-x-x-Ile, where x is any amino acid). Subsequent database searches for presumptive Drosophila transcription factors containing this minimal peptide sequence identified a wide range of potential negative regulators belonging to different superfamilies as classified by their distinct DNA-binding domain types. Remarkably, eh1-related motifs have been preserved in many human homologues of these fly proteins, indicating that the ability to bind Gro/TLE has been evolutionarily conserved in human transcriptional regulators and that this sequence may have been widely adopted throughout the proteome as a Gro recruitment domain (Goldstein, 2005).
Several representatives, corresponding to different transcription factor families, were tested for the ability to bind Gro in biochemical assays. Where possible, full-length expressed sequence tags encoding these proteins were obtained; otherwise, single exons containing the eh1-like sequence were PCR amplified from genomic DNA. Each polypeptide was assessed for the ability to pull down radiolabeled Gro in vitro. GST-tagged Slp and Doc (amino acids 254 to 391) readily retain Gro, as do Eyes absent (Eya) and the homeodomain proteins Ventral nervous system defective (Vnd, 1 to 465), Bagpipe (Bap, 1 to 129), BarH1, and Empty spiracles (Ems, 1 to 360), as well as the orphan nuclear hormone receptor DHR96. To confirm that these interactions rely on intact eh1-related sequences, the eh1 motif of one of these, BarH1, was mutated by substituting glutamic acid for Phe at position 1, finding that its binding to Gro is reduced by >60% (Goldstein, 2005).
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