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 relatively simple combinatorial rules responsible for establishing the initial metameric expression of sloppy-paired-1 (slp1) in the Drosophila blastoderm embryo make this system an attractive model for investigating the mechanism of regulation by pair rule transcription factors. This investigation of slp1 cis-regulatory architecture identifies two distinct elements, a proximal early stripe element (PESE) and a distal early stripe element (DESE) located from −3.1 kb to −2.5 kb and from −8.1 kb to −7.1 kb upstream of the slp1 promoter, respectively, that mediate this early regulation. The proximal element expresses only even-numbered stripes and mediates repression by Even-skipped (Eve) as well as by the combination of Runt and Fushi-tarazu (Ftz). A 272 basepair sub-element of PESE retains Eve-dependent repression, but is expressed throughout the even-numbered parasegments due to the loss of repression by Runt and Ftz. In contrast, the distal element expresses both odd and even-numbered stripes and also drives inappropriate expression in the anterior half of the odd-numbered parasegments due to an inability to respond to repression by Eve. Importantly, a composite reporter gene containing both early stripe elements recapitulates pair-rule gene-dependent regulation in a manner beyond what is expected from combining their individual patterns. These results indicate interactions involving distinct cis-elements contribute to the proper integration of pair-rule regulatory information. A model fully accounting for these results proposes that metameric slp1 expression is achieved through the Runt-dependent regulation of interactions between these two pair-rule response elements and the slp1 promoter (Prazak, 2010).
This work identifies two distinct CRMs from the slp1 gene that drive early expression in response to pair-rule gene regulation. The observation that a composite reporter gene containing both elements faithfully emulates the initial metameric expression of slp1 in wild-type embryos as well as the response to manipulations in pair-rule activity strongly suggests these two CRMs together account for most of the early regulation of slp1 in response to pair-rule transcription factors. This view is supported by recent ChIP/chip results from the Berkeley Drosophila Transcription Network Project indicating there are two major regions of association for both Runt and Ftz in the 20 kb of DNA flanking the slp1 transcription unit, a distal region spanning from ~8.4 to ~6.7 kb and a proximal region spanning from ~3.7 to ~2.2 kb upstream of the transcription start site. These two intervals correspond extremely well to the minimal intervals defined by the functional analysis of ~8.1 to ~7.1 and ~3.1 to ~2.5 kb, respectively. Although this genome-wide analysis did not include results for either Eve or Opa it is interesting to note that Paired (Prd) and Hairy, the two other pair-rule transcription factors included in this study also show association with these same two regions. This observation further suggests Prd and Hairy may also participate in slp1 regulation, although genetic experiments do not provide any evidence indicating that either of these factors play important direct roles in regulating the early slp1 stripes (Prazak, 2010).
A most conspicuous finding from the work presented in this study is that pair-rule dependent regulation of slp1 transcription involves non-additive interactions between two distinct upstream CRMs. The ability of composite reporters containing both the DESE and PESE enhancers to mimic expression of the endogenous gene cannot be explained solely by the independent regulatory capabilities of the two elements as a simple addition of the two patterns will include inappropriate DESE-driven expression in anterior even-numbered parasegments. This non-additive interaction potentially conflicts with the generally accepted paradigm for the modular and independent action of distinct CRMs, a point that will be discussed further below (Prazak, 2010).
Although the early stripe elements need to be combined in order to fully recapitulate pair-rule regulation, studies on the independent elements provide new insights on the pair-rule to segment polarity gene transition. The homeodomain proteins Eve and Ftz both participate in slp1 repression. Several lines of evidence indicate differences in the cis-regulatory requirements for repression by these two structurally related transcription factors. DESE is insensitive to repression by Eve, but is capable of mediating repression by Ftz. The exact opposite specificity is demonstrated by the PESE:C1+ element, which is repressed by Eve but not by Ftz. The DNA-binding specificities of Eve and Ftz are similar both in vitro and in vivo and their specificity of action is thought to involve co-factor interactions that dictate the manner in which they regulate different targets. An established cofactor for Ftz is the orphan nuclear receptor protein Ftz-F1. Indeed, elimination of maternally provided Ftz-F1 results in alterations in slp1 expression that are identical to those seen in ftz mutants. The Ftz-dependent repression of slp1 also requires Runt, making this a second prospective co-factor for this activity of Ftz. Further studies on slp1 regulation should provide valuable information on the mechanisms that underlie repression by the Eve and Ftz proteins (Prazak, 2010).
These studies on the independent DESE and PESE reporters also provide information on the properties of the unidentified factor(s) that are responsible for slp1 activation in posterior even-numbered parasegments. In the case of PESE, expression of the minimal slp1[PESE:C1+]lacZatt reporter throughout the entire pre-segmental region of eve mutant embryos indicates that a factor(s) capable of activating this element is present in all cells within this region of the embryo. DESE also drives expression of even-numbered stripes, but interestingly fails to generate stripe 0. This difference between DESE and PESE suggests there are differences in the factors responsible for activating these two elements in even-numbered parasegments. DESE also generates the odd-numbered stripes, an aspect of slp1 expression that is normally driven by the combination of Runt and Opa. Runt is normally expressed in the posterior half of only the odd parasegments and not in the posterior half of even-numbered parasegments. However, the observation that transient elimination of Runt does not abrogate DESE-driven expression in odd parasegments suggests Opa may be capable of activation in the absence of Runt. This same proposal could account for the ectopic DESE-driven expression in the anterior half of the odd parasegments as Opa is uniformly expressed in all cells within the pre-segmental region that are posterior to the cephalic furrow. The observation that Opa expression is lost anterior to the head-fold in late blastoderm stage embryos may further account for the failure of DESE to generate stripe 0. Although the only Opa-expressing cells that do not activate the DESE-lacZ reporters are those that express the combination of Runt and Ftz, there are differences in the level of expression in different cells. The increased expression in posterior versus anterior odd-numbered parasegments may reflect a contribution from Runt in potentiating DESE-driven expression (Prazak, 2010).
A central issue raised by the results is to understand how interactions involving two distinct CRMs can account for their ability to faithfully recapitulate the regulation of slp1 in response to the pair-rule transcription factors. A major discrepancy between the expression of the composite [DESE+PESE] reporter and the pattern expected from the independent action of the separate CRMs is repression of DESE-driven expression in anterior odd parasegments. One potentially trivial explanation for inappropriate expression of the DESE-lacZ reporters in these cells is close juxtaposition of binding sites for DESE-interacting activators with the basal promoter region. However, the observations that this inappropriate expression is seen for DESE-lacZ reporters that have slp1 basal promoter segments that extend anywhere from ~71 bp to ~1.8 kb upstream of the transcription start site indicates it is not due to short range interactions between DESE-bound activators and the promoter region (Prazak, 2010).
A second potential explanation is that repression in these cells involves interactions that allow the Eve-sensitivity of PESE to be transmitted to DESE. In this version of the model, Eve-interacting PESE is acting as an insulator element that prevents DESE from communicating with the slp1 promoter by blocking propagation of signals that track along the chromosome. It is also possible to imagine insulator models involving looping, such as sequestration of DESE by Eve-interacting PESE, thus preventing DESE-dependent activation at the promoter. However, in both of these insulator models ectopic Eve expression would be expected to block expression of both the odd- and even-numbered stripes, an effect that is not observed for slp1 or for the composite [DESE+PESE] reporter (Prazak, 2010).
A second discrepancy between the single elements and the composite [DESE+PESE] reporter is the Runt-independent activation by DESE in odd parasegments. This observation strongly suggests that Runt’s role in activating the composite reporter involves enabling DESE-dependent activation. This could be due to Runt-dependent antagonism of the insulator-like activities of PESE depicted in. However, there is a perhaps more straightforward explanation that does not invoke Runt-dependent regulation of PESE insulator activity, but that instead involves regulating competition between the two upstream enhancers and the slp1 promoter. In this model it is proposed that Runt plays a role in switching the promoter from interacting with PESE to interacting with the further upstream DESE. This proposed promoter-targeting role of Runt is bypassed in the DESE-lacZ reporter due to the lack of competition from PESE, thus accounting for the expression of this reporter in all cells within the segmented region of the embryo except for those that express both Runt and Ftz. Importantly, this model fully accounts for expression of both slp1 and the composite [DESE+PESE] reporters in both wild-type and mutant embryos. All expression in anterior odd-parasegments of wild-type embryos is restricted to PESE-dependent regulation as these cells do not express Runt and thus will not reveal the activating potential of DESE. The role that Eve normally plays in repressing both slp1 and the [DESE+PESE] reporter in anterior odd parasegments is also clearly accounted for by Eve’s activity as a repressor of PESE. Further work is needed to determine whether an insulator or enhancer competition model more readily accounts for the non-additive interaction between these two cis-elements, but in either event the regulatory output would appear to be due to functional attributes of the Runt transcription factor (Prazak, 2010).
There are two aspects of the current results that have widespread implications for studies on cis-regulatory DNA elements and their role in developmentally regulated gene expression. The first point is that cis-elements (such as DESE) that have broad activities when tested as autonomous single elements can have more restricted roles in regulating gene expression in the context of their normal chromosomal environment. A second, and perhaps even more crucial point is that transcription factors (such as Runt) that are not essential for activation by a single autonomous enhancer, even when tested in a physiologically relevant context, may have critical roles in enabling the activity of this enhancer in a developmental setting. Further studies on the functional interplay between the slp1 early stripe elements during Drosophila segmentation should provide insights on a phenomenon of potentially far-reaching importance in understanding the developmental regulation of gene expression (Prazak, 2010).
In order to investigate regulation and redundancy within the sloppy paired (slp) locus, 30 kilobases of DNA encompassing the tandem, coordinately regulated slp1 and slp2 transcription units were analyzed. A remarkable array of stripe enhancers was found with overlapping activities surrounding the slp1 transcription unit, and, unexpectedly, glial cell enhancers were found surrounding slp2. The slp stripe regulatory region generates 7 stripes at blastoderm, and later 14 stripes that persist throughout embryogenesis. Phylogenetic analysis among drosophilids suggests that the multiplicity of stripe enhancers did not evolve through recent duplication. Most of the direct integration among cis-regulatory modules appears to be simply additive, with one notable exception. Despite the apparent redundancy among stripe enhancers, transgenic rescue suggests that most are required for full function, to maintain wingless expression and parasegment boundaries throughout embryogenesis. Transgenic rescue also reveals indirect positive autoregulation by the 7 early stripes, without which alternate stripes within the 14-stripe pattern are lost, leading to embryos with a pair-rule phenotype (Fujioka, 2012a).
No CRMs were identified that drive individual slp stripes in the germ band at any stage, consistent with the slp locus acting strictly downstream of the primary pair-rule genes, which are responsible for converting non-periodic patterns of the maternal and gap gene products into periodic 7- or 8-stripe patterns. However, a head stripe is driven by several separable CRMs. The early slp head stripe is regulated by the maternal gradient-generating gene bicoid, among other genes, and some of these CRMs contain previously identified clusters of Bicoid binding sites (Fujioka, 2012a).
The slp1, but not the slp2, transcription unit is surrounded by stripe CRMs. This situation may have arisen following a chromosomal duplication that gave rise to these twin transcription units. However, a simple duplication within an array of unique CRMs cannot explain the current regulatory landscape of this locus. In stark contrast to slp1, slp2 is surrounded by nervous system CRMs. These drive expression mostly, if not exclusively, in glial cells. Consistent with this expression being dispensable for viability, an earlier study indicated that slp2 and the flanking region are not required for viability. However, flies can survive in the laboratory with clear CNS defects. A previous study suggested negative regulation of glial cell specification by slp. The data suggest that slp is expressed in glial cells alongside Repo. Because 4 non-overlapping CRMs drive expression in glial cells, it is suggested that slp has a separate, positive function in glial cells following their specification. Previous analysis of slp-related protein-coding sequences in non-drosophilid insects and basally branching arthropods suggested that the common ancestral coding sequence of slp1 and slp2 was more similar to slp2. It was found that this is also true in the mosquito A. gambiae. A conserved nervous system function for slp2 might help to explain why the slp2 coding region has diverged more slowly than slp1 from their common ancestral sequence (Fujioka, 2012a).
Some of the stripe CRMs surrounding slp1 also drive embryonic CNS expression, possibly in neuroblasts. Previous studies showed that slp is involved in specifying neuroblast identity. These CRMs are good candidates for providing this function. In addition, multiple CRMs drive expression in the larval brain and in eye discs (Fujioka, 2012a).
Standard P-element transgenesis revealed that many of the CRMs surrounding slp1 can cause pairing-sensitive silencing of mini-white in some transgenic lines, a rare phenomenon that is usually associated with Polycomb-response elements (PREs). This may indicate a set of dispersed PREs in this region that facilitate the association of Polycomb with the locus, and maintain a chromatin domain enriched in histone H3 tri-methylated at lysine 27, which was found to be present throughout the slp locus in embryos (Fujioka, 2012a).
Recent studies of genes with apparently redundant enhancers suggest that true redundancy may be rare, and that distinct enhancers with overlapping activities contribute to phenotypic robustness that is likely to be maintained by natural selection. The current results are consistent with this, although they suggest that there is some redundancy among the stripe CRMs, as those downstream of slp1 do not noticeably contribute to patterning the cuticle when all the upstream ones are present. However, the sequences within these elements appear to be conserved, suggesting they contribute to function, at least in the wild. The results further suggest that even following a genomic duplication that generates partially redundant coding regions, redundant CRMs may be rapidly lost during subsequent evolution (Fujioka, 2012a).
In almost all cases, larger elements drive expression in all the places where expression is driven by smaller CRMs that they contain. As an example, the 2.1 kb u8766 drives expression both in the larval brain and in 14 stripes, consistent with the fact that it spans the 600 bp u8781 and the 900 bp u8172, which drive expression in the brain and in 14 stripes, respectively. Furthermore, most of the differences among partially redundant CRMs are consistent with their activities combining additively to generate endogenous slp expression. For example, while the regions u8172, u4734, and u3225 each drive a 14-stripe pattern in the ectoderm beginning at embryonic stage 7 or 8, the regions u5547, u2316, and i1523 are expressed later, at stages 9–11. Thus, while there is considerable overlap among the striped patterns driven by these elements, they are not all redundant, and each may be important to produce the robust slp striped pattern in the endogenous context (Fujioka, 2012a).
In contrast, some negative positional cues depend on more complex CRM interactions. A recent study (Prazak, 2010) described a detailed analysis of the u8172 region (whose 14-stripe pattern includes some cells outside the normal slp expression domain). That study showed that u3125 (which drives a 7-stripe pattern with no ectopic expression), and derivatives of it, can suppress ectopic expression from u8172 when combined in the same construct. Rescue data show that ectopic expression driven by the upstream CRM disrupts normal function, and so must be suppressed within the endogenous locus. Thus, non-additive interactions among individual CRMs have important roles in regulating slp expression, even though the general trend is for the activities of slp CRMs to combine additively (Fujioka, 2012a).
Another kind of interaction among CRMs is revealed in slp mutants that are rescued using the stripe CRMs located downstream of slp1, which do not drive an early 7-stripe pattern. Although i1530 drives a regular 14-stripe pattern in wild-type embryos, in a slp mutant the longer i1539 drives expression strongly in only 7 stripes, and weakly in the other 7. This difference is explained by positive autoregulation, in that the early slp stripes are required for functional levels of later slp expression in the same cells. This is reminiscent of the positive autoregulation of eve stripes, which is indirect. The late loss of slp expression in the absence of early slp stripes can be explained by expanded odd expression, which apparently represses later slp expression in every other stripe of the 14 stripe pattern. The site of action of this odd-dependent repression has not been localized; it could be either in the stripe CRM region downstream of slp1, or within the slp1 promoter region, both of which are contained within the rescue construct. In either case, it is interesting to note that the 14-stripe pattern driven by these CRMs is regulated, at least initially, in a pair-rule fashion, with independent inputs to two interdigitated sets of 7 stripes (Fujioka, 2012a).
The fact that there is such an indirect autoregulatory requirement for only half of the slp stripes highlights the pair-rule character of slp function in its intimate relationship with eve and odd, even though it is clearly also required in 14 stripes at later stages, where it has a similar mutual repressive relationship with engrailed. This example illustrates that the pair-rule genes are difficult to neatly classify into early and late classes because of the complexity of their interactions both with gap genes and with each other. A recent study (Schroeder, 2011) placed odd, which had traditionally been classified as a secondary pair-rule gene, into the 'early' class, while slp was assigned to the 'late' class. Despite the fact that odd participates directly in translating non-periodic pattern information into periodic pattern, while slp does not, slp nonetheless regulates odd after periodic pair-rule patterns have been established. This secondary cross-regulation, which formally goes 'backwards' in the hierarchy, is essential for the correct transition to segment polarity gene control. Specifically, without early 7-stripe slp expression, half of the wg stripes are not established (those that coincide with the 'missing' slp stripes), and the adjacent parasegment borders decay, resulting in pair-rule defects. Thus, complex regulatory interactions occur at both the early pair-rule stage and the late pair-rule stage, and may be the norm for developmental processes (Fujioka, 2012a).
The 7- and 14-stripe slp patterns occur at different stages, and are driven in part by separable elements. Among the 14-stripe CRMs, some drive earlier expression, which overlaps in time with expression driven by the later-acting CRMs. This suggests that different combinations of activators, and possibly different repressors, may be responsible for activating, and restricting the activity of, these elements at different stages. This, in turn, provides a rationale for the existence of multiple regulatory elements with temporally overlapping patterns. As the expression of activators change during development, maintenance of expression within a given cell is subject to changing constraints on the relevant CRMs. In particular, the need to maintain both the on state and the off state in the appropriate cells may limit the ability of a single CRM to respond properly at all stages, making it advantageous to utilize different CRMs as the milieu of trans-acting factors changes within the nucleus (Fujioka, 2012a).
BLAST searches were used to map sequence similarities for each stripe CRM among the sequenced drosophilid genomes, all of which contain both slp1 and slp2 coding regions, in a similar arrangement to that in D. melanogaster. The highest-stringency similarity was found between two CRMs expressed in stripes in the presumptive mesoderm, u1609 and i2330. Analysis of likely transcription factor binding to this 12 bp sequence based on known specificities did not reveal any specific factors with a pattern of expression suggesting regulation of these CRMs. However, the arrangements of best-match sequences to each stripe CRM in the most distantly related drosophilids suggest that ancestral sequences for each stripe CRM existed separately in their common ancestor. However, whether these apparently conserved sequences represent distinct, ancestral CRMs with functions similar to those in D. melanogaster remains an open question (Fujioka, 2012a).
Attempts were made, without success, to find clear evidence of homologies to stripe CRMs in the next-most closely related sequenced genome, that of A. gambiae, which might indicate an ancestral element from which more than one drosophilid CRM evolved. Although numerous short sequence similarities were found, their arrangements did not suggest any specific relationship to a drosophilid CRM. Presumably, future analysis will reveal how the locus evolved, when sequenced genomes become available for species that diverged from the drosophilids more recently than mosquitoes (Fujioka, 2012a).
Fujioka, M., Gebelein, B., Cofer, Z. C., Mann, R. S. and Jaynes, J. B. (2012). Engrailed cooperates directly with Extradenticle and Homothorax on a distinct class of homeodomain binding sites to repress sloppy paired. Dev. Biol. 366(2): 382-92. PubMed Citation: 22537495
Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1. Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. Cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), were located within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function (Fujioka, 2012b).
Consistent with the fact that Eve is expressed earlier than En, with some overlap at embryonic stages 8–9, slp CRMs tended to respond to ectopically expressed Eve at earlier stages than to En. Transgenic dissections further showed that they have distinct responsive regions within CRMs, suggesting that many of their binding sites are distinct. This is somewhat surprising because they are both homeodomain-containing repressors that set the posterior borders of slp stripes, and they have been seen to have similar in vitro binding specificities. A possible explanation is that they cooperate in DNA binding with different cofactors, making their functional sites distinct. Despite detailed analyses of Eve function in segmentation, no candidate co-factors for specifying target genes have emerged (Fujioka, 2012b).
A recent study showed that CRM u8172 drives ectopic expression within odd-numbered parasegments in cells that normally do not express detectable levels of slp RNA. However, when combined with the promoter-proximal CRM u3125, which drives properly restricted expression within even-numbered parasegments, ectopic expression is repressed, suggesting that an Eve-responsive element resides within this region. Consistent with these findings, transgenes containing this region responded to ectopically expressed Eve, and rescue-type transgenes carrying u8172 without this region drove ectopic Slp, causing embryonic defects (Fujioka, 2012b).
Recently, a striking number of distinct CRMs surrounding the slp1 transcription unit were found to drive expression that overlaps in both space and time. Extensive dissection of this regulatory region and rescue of slp mutants with various transgenes suggested that apparent redundancy may be necessary to provide fully functional levels of expression across the various stages of slp expression. This study shows that there are functionally redundant En/Exd/Hth binding sites within CRM u1523. In vitro binding analysis identified a strongly cooperative binding site and a weaker, but still highly cooperative site. Despite the apparent difference in in vitro binding affinity, either site is sufficient to confer repression in the En domain, and both sites must be mutated to cause significant derepression. Thus, apparent redundancy exists at multiple levels in slp regulation. Whether apparent redundancy at this level has a function in increasing the robustness of functional gene expression within the organism, as does apparent redundancy among multiple enhancers regulating the same gene, remains to be determined. Furthermore, cooperativity with cofactors in vitro seems to be a significant indicator of function in vivo, in addition to affinity. It was found that while the B1b site has the same apparent affinity as A2a, A2a confers considerably stronger repression activity, and shows greater cooperativity in binding by En with Exd/Hth. The discrepancy between relative affinity and functionality may be attributed to the challenge of reproducing functional binding conditions in vitro, where protein–protein interactions leading to cooperativity may be less sensitive to the differences in conditions than are protein–DNA interactions. Relatedly, competition with a variety of DNA binding proteins in vivo for sites on the DNA may lead to a greater reliance on cooperativity in vivo for occupancy of functional sites (Fujioka, 2012b).
Previous studies indicated that En requires the Hox co-factors Exd and Hth to efficiently repressslp, especially in the anterior half of the embryo, and En was found to act cooperatively on target sites in the distalless gene with both Exd/Hth and posteriorly-expressed Hox gene products. Although it remains possible that the relatively weak, yet functional binding site (A2a) within i1523 might bind En with other cofactors in addition to Exd/Hth, dissection and construction experiments with this and other sites have not revealed any clear anterior–posterior differences in their activity that might suggest a functional interaction with cofactors such as Hox proteins that are restricted in expression along the anterior–posterior axis. Nonetheless, previous studies suggested that regulation of slp by En might utilize posterior-specific factors. Further analysis will be required to more fully explore this possibility (Fujioka, 2012b).
The relative arrangement of consensus En and Exd sites that facilitate cooperative binding appears to be quite flexible. For example, the A2a site contains no canonical consensus core for En binding (ATTA), while for the other two functional sites, the distance between the centers of the En and Exd sites is 10–12 bp for A1a and only 2 bp for B1a. The latter is reminiscent of En–Exd/Hth binding in distalless, where simultaneous Hox binding occurs, although the position of the En site is on the opposite side of the Exd core consensus ATCA. This relative arrangement of En and Exd sites (En binding 5′ of the Exd core ATCA) is seen for all of the functional sites analyzed in this study. This arrangement is similar to the relative positions of Hox and Exd binding to sites where there is no En involvement. The flexibility overall is consistent with that seen for Exd/Hth binding in conjunction with the Hox gene products, and suggests that while homeodomain family transcription factors are able to function combinatorially in vivo on a wide variety of binding sites, there are significant constraints on the positions of contact by the individual homeodomains. A full understanding of the similarities and differences between En binding in conjunction with Exd and Hth, and Hox binding with these cofactors, will require further investigation (Fujioka, 2012b).
The highly cooperative, strong En/Exd/Hth binding site B1a was both necessary and sufficient for repression of u4734 in the En domain. However, it did not fully substitute for the entire repression element that contains it, located between −3.9 and −3.4 kb from the slp1 TSS. This finding suggests that there may be other functional En binding sites in this region. Consistent with this, in vitro binding suggested that other subregions (B2 and/or B3) harbor some binding activity. Thus, like i1523, there may be partial redundancy in En complex binding within u4734, despite the existence of a single essential binding site (Fujioka, 2012b).
This study has established the functional significance of three cooperative En/Exd/Hth binding sites within slp. Interestingly, two of them are well conserved among the 12 species of Drosophila whose genomes have been sequenced, and the other site is conserved within the more closely related species. The duplication that generated the twin slp transcription units apparently took place before the divergence of these 12 species, as all drosophilids (but not mosquitoes) contain two tandem slp-related protein coding regions. This might suggest that the two conserved En/Exd/Hth sites were duplicated along with the locus as a whole. It has been shown that Drosophila enhancers contain clusters of conserved sequences blocks, and the two CRMs analyzed in this study contain such conserved sequence clusters. However, the patterns of conservation in the regions surrounding the conserved En/Exd/Hth sites do not suggest that they are directly related to each other. Furthermore, both CRMs are more closely linked to slp1 than to slp2. Clearly, there have been other chromosomal rearrangements in the history of the slp locus, precluding a simple description of its evolution (Fujioka, 2012b).
A recent study investigating the genome-wide distribution of En binding showed a peak on i1523, but not on u4734. The data were derived from 7–24 h-old embryos, which were mostly at later stages than those at which these CRMs are active. In addition, the data show peaks where our analysis has not identified functional CRMs. Such sites may function to assist those within the core enhancer regions, or they might be functional during larval or adult stages to keep slp in the off state. Alternatively, they might not be functionally important. Further study will be required to address these issues (Fujioka, 2012b).
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).
The generation of metameric body plans is a key process in development. In Drosophila segmentation, periodicity is established rapidly through the complex transcriptional regulation of the pair-rule genes. The 'primary' pair-rule genes generate their 7-stripe expression through stripe-specific cis-regulatory elements controlled by the preceding non-periodic maternal and gap gene patterns, whereas 'secondary' pair-rule genes are thought to rely on 7-stripe elements that read off the already periodic primary pair-rule patterns. Using a combination of computational and experimental approaches, a comprehensive systems-level examination was conducted of the regulatory architecture underlying pair-rule stripe formation. runt (run), fushi tarazu (ftz) and odd skipped (odd) were found to establish most of their pattern through stripe-specific elements, arguing for a reclassification of ftz and odd as primary pair-rule genes. In the case of run, long-range cis-regulation was observed across multiple intervening genes. The 7-stripe elements of run, ftz and odd are active concurrently with the stripe-specific elements, indicating that maternal/gap-mediated control and pair-rule gene cross-regulation are closely integrated. Stripe-specific elements fall into three distinct classes based on their principal repressive gap factor input; stripe positions along the gap gradients correlate with the strength of predicted input. The prevalence of cis-elements that generate two stripes and their genomic organization suggest that single-stripe elements arose by splitting and subfunctionalization of ancestral dual-stripe elements. Overall, this study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo (Schroeder, 2011).
The transition from non-periodic to periodic gene expression patterns is a key step in the establishment of the segmented body plan of the Drosophila embryo. The pair-rule genes that lie at the heart of this process have been the subject of much investigation, but important questions, in particular regarding the organization of cis-regulation, have remained unresolved. We have revisited the issue in a comprehensive fashion by combining computational and experimental cis-dissection with mutant analysis and a detailed characterization of expression dynamics, both of endogenous genes and of cis-regulatory elements. This systems-level analysis under uniform conditions reveals important insights and gives rise to a refined and in some respects substantially revised view of how periodic patterns are generated (Schroeder, 2011).
The most important finding of this study is that ftz and odd, which had been regarded as secondary pair-rule genes, closely resemble eve, h and run in nearly all respects and should thus be co-classified with them as primary pair-rule genes. Expression dynamics clearly subdivide the pair-rule genes into two distinct groups, with eve, h, run, ftz and odd all showing an early and non-synchronous appearance of most stripes, whereas the 7-stripe patterns of prd and slp1 arise late and synchronously. The early expression of stripes is associated with the existence of stripe-specific cis-elements that respond to positional cues provided by the maternal and gap genes. Unlike previously thought, maternal and gap input is used pervasively in the initial patterning not only of eve, h and run, but also of ftz and odd. Aided by computational predictions, eight new stripe-specific cis-elements were identifed for ftz, odd, and run. Although molecular epistasis experiments reveal a stronger role for eve, h and run in pair-rule gene cross-regulation, odd clearly affects the blastoderm patterning of other primary factors as well and ftz affects the patterning of odd (Schroeder, 2011).
The revised grouping brings the classification of pair-rule genes in line with their role in transmitting positional information to the subsequent tiers in the segmentation hierarchy. By the end of cellularization, the expression patterns of h, eve, run and ftz/odd are offset against each other to produce neatly tiled overlaps of four-nuclei-wide stripes. In combination, these patterns define a unique expression code for each nucleus within the two-segment unit that specifies both position and polarity in the segment and is read off by the segment-polarity genes. ftz (as an activator of odd) and odd together represent the fourth essential component of this positional code and are thus functionally equivalent to h, eve and run. Placing all components of this code under the same direct control of the preceding tier of regulators presumably increases both the speed and the robustness of the process (Schroeder, 2011).
Although the results support a subdivision between primary and secondary pair-rule genes, they also reveal surprising complexities in the regulatory architecture that are not captured by any rigid dichotomy. The five primary pair-rule genes all have different cis-regulatory repertoires: stripe formation in h appears to rely solely on stripe-specific cis-elements, whereas eve employs a handoff from stripe-specific elements to a late-acting 7-stripe element. In run, ftz and odd, by contrast, the emergence of the full 7-stripe pattern is the result of joint action by stripe-specific elements and early-acting 7-stripe elements, with ftz and odd requiring the 7-stripe element to generate a subset of stripes. This difference in the importance of 7-stripe elements in stripe formation is supported by the results of rescue experiments: whereas the 7-stripe elements of both run and ftz provide partial rescue of the blastoderm expression pattern when the stripe-specific elements are missing, this is not the case for eve. Interestingly, run appears to occupy a unique and particularly crucial position among the five genes: it has both a full complement of stripe-specific elements and an early-acting 7-stripe element, and thus serves as an early integration point of maternal/gap input and pair-rule cross-regulation. Its regulatory region is particularly large and complex, with cis-elements acting over long distances across intervening genes and with partial redundancy between elements. Moreover, the removal of run has the most severe effects on pair-rule stripe positioning among the five genes. Another complexity lies in the fact that the early anterior expression of slp1 and prd, which otherwise exhibit all the characteristics of secondary pair-rule genes, has a clear role in patterning the anteriormost stripes of the primary pair-rule genes. In fact, evidence is provided that the most severe defect observed in the primary pair-rule gene mutants, namely the loss of pair-rule gene stripes 1 or 2 under eve loss-of-function conditions, is an indirect effect attributable to the regulation of these stripes by slp1 (Schroeder, 2011).
This investigation provides important new insight regarding the relative roles of maternal/gap input and pair-rule gene cross-regulation in stripe formation. As described, nearly all stripes of the primary pair-rule genes are initially formed through stripe-specific cis-elements. Cross-regulatory interactions between the pair-rule genes then refine these patterns by ensuring the proper spacing, width and intensity of stripes, as revealed by mutant analysis. However, these two aspects or layers of regulation are not as clearly separated as has often been thought. For example, 7-stripe elements have been regarded primarily as conduits of pair-rule cross-regulation; by contrast, this study found that the 7-stripe elements of run, ftz and odd are activefrom phase 1 onwards and contain significant input from the maternal and gap genes, resulting in modulated or partial 7-stripe expression early. In the case of the KR binding sites predicted within the run 7-stripe element, mutational analysis showed that this input is indeed functional. Conversely, stripe-specific cis-elements receive significant regulatory input from pair-rule genes. This is particularly clear in the case of eve and h: expression dynamics indicate that their 7-stripe patterns become properly resolved without the involvement of 7-stripe elements, and the defects in the h and eve expression patterns that were observe in pair-rule gene mutants occur at a time when only stripe-specific elements are active. In a few cases, pair-rule input into stripe-specific elements has been demonstrated directly, but the comparative analysis of expression dynamics underscores that it is indeed a pervasive feature of pair-rule cis-regulation (Schroeder, 2011).
An important question arising from these findings is how the different types of regulatory input are integrated, both at the level of individual cis-elements and at the level of the locus as a whole. Binding sites for maternal and gap factors typically form tight clusters, supporting a modular organization of cis-regulation. Such modularity is crucial for the 'regional' expression of individual stripes, which can be achieved only if repressive gap inputs can be properly separated between cis-elements. By contrast, binding sites for the pair-rule factors appear to be more dispersed across the cis-regulatory regions and not tightly co-clustered with the maternal and gap inputs. Unlike the gap factors, which are thought to act as short-range repressors, with a range of roughly 100 bp, most pair-rule genes act as long-range repressors, with an effective range of at least 2 kb. Pair-rule factor inputs may therefore influence expression outcomes at greater distances along the DNA, which would allow a single cluster of binding sites to affect multiple cis-elements. Remarkably, the 7-stripe elements of ftz, run, eve, prd and odd all combine inputs from promoter-proximal and more distal sequence over distances of at least 5 kb, and, is seen in the case of odd, the combination can involve non-additive effects. Thus, the refinement of expression patterns through pair-rule cross-regulation might rely on interactions over greater distances, consistent with the 'global' role of pair-rule genes as regulators of the entire 7-stripe pattern. How such interactions are realized at the molecular level and how the inputs from stripe-specific and 7-stripe elements are combined to produce a defined transcriptional outcome is currently unknown (Schroeder, 2011).
Taken together with previous studies, these data support a coherent and conceptually straightforward model of how stripes are made in theDrosophila blastoderm. In the trunk region of the embryo, the maternal activators BCD and CAD form two overlapping but anti-correlated gradients; they coarsely specify the expression domains of region-specific gap genes, which become refined by cross-repressive interactions among the gap factors. The result is a tiled array of overlapping gap factor gradients, centered around the bilaterally symmetric domains of KR and KNI, which are flanked on either side by the bimodal domains of GT and HB. The same basic principles are used again in the next step: following the initial positioning of stripes by maternal and gap factor input, cross-repressive interactions among the primary pair-rule genes serve to refine the pattern and ensure the uniform spacing and width of expression domains. The significant correlations that were observe between the strength of KR/KNI input and the position of the resulting stripes relative to the respective gradients supports the notion that these factors function as repressive morphogens in defining the proximal borders of the nascent pair-rule stripes. Importantly, owing to the symmetry of the KR and KNI gradients, combined with the largely symmetric positioning of the flanking GT and HB gradients, the same regulatory input can be used to specify two distinct positions, one on either slope of the gradient; this is exploited in a systematic fashion by dual-stripe cis-elements, which account for the majority of stripe-generating elements. Thus, the key to stripe formation is the translation of transcription factor gradients into an array of narrower, partially overlapping expression domains that are stabilized by cross-repressive interactions; the iteration of this process, combined with the duplication of position through bilateral gradient symmetry, creates a periodic array of stripes that is sufficient to impart a unique identity to each nucleus in the trunk region of the embryo (Schroeder, 2011).
With the exception of the anteriormost pair-rule gene stripes, which are subject to more complex regulation (as evidenced by the crucial role of slp1), this model accounts for most of the stripe formation process. Particularly striking is the similarity between the central gap factors and the primary pair-rule factors with respect to regulatory interactions and expression domain positions. The offset arrangement of two pairs of mutually exclusive gap domains, KNI/HB and KR/GT, is mirrored at the pair-rule gene level, with the anti-correlated and mutually repressive stripes of HAIRY and RUN phase-shifted against the similarly anti-correlated expression patterns of EVE and ODD. The parallel suggests that this regulatory geometry is particularly suited to robustly specify a multiplicity of positions. However, such a circuitry of cross-repressive relationships is compatible with a range of potentially stable expression states and thus is insufficient by itself to uniquely define position along the anterior-posterior axis. Therefore, the initial priming of position by the preceding tier of the regulatory hierarchy is crucial. At the level of the pair-rule genes, the extensive repertoire of maternal/gap-driven cis-elements that initiate stripe expression for all four components of the array is thus necessary to ensure that the cross-regulatory dynamics will drive the correct overall pattern. Finally, to achieve a smooth transition between the tasks of transmitting spatial information from the preceding tier of the hierarchy and refinement/stabilization of the pattern, the two layers of regulation need to be closely integrated. This is likely to be facilitated by the fact that in each of the mutually repressive pairs, one gene generates stripes purely through stripe-specific elements (h, eve), whereas the other has an early-acting 7-stripe element that mediates pair-rule cross-repression and acts concurrently with stripe-specific elements (run, odd) (Schroeder, 2011).
Stripe positioning is not always symmetric around the central gap factor gradients. In such cases, the conflicting needs for appropriate regulatory input into the relevant cis-elements appear to have driven the separation of ancestral dual-stripe elements into more specialized elements optimized for generating a single stripe. The co-localization of the cis-elements that generate stripes 1 and 5 within all pair-rule gene regulatory regions, be it in the form of dual-stripe elements or of adjacent but separable single-stripe elements, provides strong evidence that this is indeed a key mechanism underlying the emergence of single-stripe cis-elements. Given the evolutionary plasticity of regulatory sequence, it is not difficult to envision such a separation and subfunctionalization of cis-elements. How the crucial transition from a non-periodic to periodic pattern is achieved in other insects is a fascinating question. Intriguingly, most of the molecular players and general features of their expression patterns are well conserved beyond Diptera, and it is tempting to speculate that dual-stripe cis-regulation might have arisen through co-option as the blastoderm fate map shifted to include more posterior positions. It will be interesting to see to what extent the mechanisms and principles of stripe formation that we have outlined here apply in other species (Schroeder, 2011).
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).
Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. This study demonstrates that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this 'dormant' TF binding signature may represent a molecular footprint of these cells' developmental lineage (Junion, 2012).
Dissecting transcriptional networks in the context of embryonic
development is inherently difficult due to the multicellularity of
the system and the fact that most essential developmental
regulators have pleiotropic effects, acting in separate and
sometimes interconnected networks. This study presents a
comprehensive systematic dissection of the cis-regulatory properties
leading to cardiac specification within the context of
a developing embryo. The resulting compendium of TF binding
signatures, in addition to extensive in vivo and in vitro analysis
of enhancer activity, revealed a number of insights into the
regulatory complexity of developmental programs (Junion, 2012).
Nkx (Tinman in Drosophila), GATA (Pannier in Drosophila), and T box factors (Doc in Drosophila) regulate each others expression
in both flies and mice, where they form
a recursively wired transcriptional circuit that acts
cooperatively at a genetic level to regulate heart development
across a broad range of organisms. The data demonstrate that
this cooperative regulation extends beyond the ability of these
TFs to regulate each others expression. All five cardiogenic
TFs (including dTCF and pMad) converge as a collective unit on
a very extensive set of mesodermal enhancer elements in vivo
(Tin-bound regions) and also in vitro (in DmD8 cells). Importantly,
this TF co-occupancy occurs in cis, rather than being mediated
via crosslinking of DNA-looping interactions bringing together
distant sites. Examining enhancer activity out of context, for
example, in transgenic experiments and luciferase assays, revealed
that the TF collective activity is preserved in situations in which these regions are removed from their native genomic 'looping' context (Junion, 2012).
In keeping with the conserved essential role of these factors
for heart development, the integration of their activity at shared
enhancer elements may also be conserved. Recent analyses of
the mouse homologs of these TFs (with the exception of the
inductive signals from Wg and Dpp signaling) in a cardiomyocyte
cell line support this, revealing a signifcant overlap in their
binding signatures (He, 2011; Schlesinger, 2011),
although interestingly not in the collective 'all-or-none' fashion
observed in Drosophila embryos. This difference may result
from the partial overlap of the TFs examined, interspecies differences,
or the inherent differences between the in vivo versus
in vitro models. Examining enhancer output for a large number
of regions indicates that this collective TF occupancy signature
is generally predictive of enhancer activity in cardiac mesoderm
or its neighboring cell population, the visceral mesodermexpression patterns that cannot be obtained from any one of these TFs alone (Junion, 2012).
There are currently two prevailing models of how enhancers
function. The enhanceosome model suggests that TFs bind to
enhancers in a cooperative manner directed by a specific
arrangement of motifs, often having a very rigid motif grammar. An alternative, the billboard model, suggests that each TF (or submodule) is recruited independently via its own sequence motif, and therefore the motif spacing and relative
orientation have little importance. The results of this study indicate that cardiogenic TFs are corecruited and activate enhancers in a cooperative manner, but this cooperativity occurs with little or no apparent motif grammar to such an
extent that the motifs for some factors do not always need to
be present. This is at odds with either the enhanceosome (cooperative
binding; rigid grammar) or billboard (independent
binding; little grammar) models and represents an alternative
mode of enhancer activity, which was termed a 'TF collective' (cooperative binding; no grammar), and likely constitutes a common principle in other systems (Junion, 2012).
The data suggest that the TF collective operates via the
cooperative recruitment of a large number of TFs (in this case,
at least five), which is mediated by the presence of high-affinity
TF motifs for a subset of factors initiating the recruitment of all
TFs. The occupancy of any remaining factor(s) is most likely facilitated
via protein-protein interactions or cooperativity at a higher
level such as, for example, via the chromatin activators CBP/
p300, which interact with mammalian GATA and Mad homologs. This model allows for
extensive motif turnover without any obvious effect on enhancer
activity, consistent with what has been observed in vivo for the
Drosophila spa enhancer and mouse heart enhancers (Junion, 2012).
Integrating the TF occupancy data for all seven major TFs
involved in dorsal mesoderm specification (the five cardiogenic
factors together with Biniou and Slp) revealed a very striking
observation: the developmental history of cardiac cells is reflected
in their TF occupancy patterns. Visceral mesoderm (VM) and cardiac mesoderm (CM) are both derived from precursor cells within the dorsal mesoderm. Once
specified, these cell types express divergent sets of TFs: Slp,
activated dTCF, Doc, and Pnr function in cardiac cells, whereas
Biniou and Bagpipe are active in the VM. Despite these mutually exclusive expression patterns, the cardiogenic TFs are recruited to the same enhancers as VM
TFs in the juxtaposed cardiac mesoderm. Moreover,
dependent on the removal of a transcriptional repressor, these
combined binding signatures have the capacity to drive expression
in either cell type. This finding provides the exciting possibility
that dormant TF occupancy could be used to trace the
developmental origins of a cell lineage. It also explains why
active repression in cis is required for correct lineage specification,
which is a frequent observation from genetic studies.
At the molecular level, it remains an open question why the
VM-specific enhancers are occupied by the cardiac TF collective.
It is hypothesized that this may occur through chromatin
remodeling in the precursor cell population. An 'open' (accessible)
chromatin state at these loci in dorsal mesoderm cells,
which is most likely mediated or maintained by Tin binding prior
to specification, could facilitate the occupancy of cell type-specific
TFs in both CM and VM cells. Such early 'chromatin
priming' of regulatory regions active at later stages has been
observed during ES cell differentiation. The current data provide evidence that this also holds true for TF occupancy and not just chromatin marks. On a more
speculative level, this developmental footprint of TF occupancy
may reflect the evolutionary ancestry of these two organs. Visceral and cardiogenic tissues are derived from the splanchnic mesoderm in both flies and
vertebrates. These complex VM-heart enhancers may represent
evolutionary relics containing functional binding sites that reflect
enhancer activity in an ancestral cell type (Junion, 2012).
Taken together, the collective TF occupancy on enhancers
during dorsal mesoderm specification illustrates how the regulatory
input of cooperative TFs is integrated in cis, in the absence
of any strict motif grammar. This more flexible mode of cooperative cis regulation is expected to be present in many other complex developmental systems (Junion, 2012).
The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated in this study, led to a hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. This study investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods deviations from the wild-type patterns of the anterior-most pair-rule stripes were detected in different genetic backgrounds, consistent with Hkb-mediated repression. Moreover, an image processing tool was developed that, for the most part, confirmed the assumptions. Using an hkb misexpression system, specific repression on anterior stripes was detected. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1 when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes (Andrioli, 2012).
The aim of this study was to understand the mechanisms underlying the regulation of the anterior pair-rule stripes. The model tested was first proposed for eve 2 regulation. Transcriptional activators do not give enough patterning information, and the presence of repressors is instructive for determining the precise positioning of a particular stripe. The hypothesis was that transcription repressors could be working in a combinatorial manner to determine the correct positioning of the anterior stripes and prevent, in a spatial and temporal manner, the expression of stripe CRMs in the more anterior regions of the embryo by counteracting the activity of activators. There is plenty of evidence supporting this hypothesis, which was further confirmed in this study (Andrioli, 2012).
Regarding activators, computational analysis predicted Bcd, Hb and Btd binding sites are part of significant clusters in the anterior-most stripe CRM. These predictions agree well with previous genetic data and in vivo DNA binding data from ChIP/chip experiments. Thus, Btd, and above all the widely spread maternal factors Bcd and Hb, might activate anterior stripe CRMs early in the anterior blastoderm. Alternatively, the early broad expression patterns of pair-rule genes could be under the control of dedicated CRMs, although no such elements have yet been reported. It is possible that other regulatory elements could contribute to the expression detected early in the anterior blastoderm, for instance, the CRM responsible for the expression of h head patch or the CRMs responsible for eve 3, eve 5 and h 5, which were proposed to be activated by the maternal factor DSTAT (Drosophila Signal Transducer and Activator of Transcription), which is ubiquitously expressed in the embryo (Andrioli, 2012).
The expression of several gap domains covering all of the anterior regions of the embryo ahead of the seven-striped patterns is consistent with the expected subsequent local repression of pair-rule CRMs activated in the head region. Of these gap domains, Slp1 is a common repressor for anterior pair-rule stripes, but other repressors besides Slp1 were predicted to be necessary for correctly determining the borders of the anterior-most stripes. This study investigated hkb, which, in addition to tll, is the other major gap gene target of the Torso signaling regulation in the terminal system. In the anterior region, hkb is required for the proper formation of the foregut and midgut. Its domain at the anterior tip coincides with the region where the diffused early expression patterns of pair-rule genes first fade. These observations are consistent with local repression roles of Hkb. However, it was not possible to detect derepression of pair-rule genes in the anterior pole of hkb- embryos. One possibility is that the progressive non-detection of the expression of pair-rule genes might correspond to a failure in activation. In fact, Bcd activation was shown to be down-regulated by the Torso-signaling cascade at the anterior tip. Nevertheless, other data suggest that the Torso pathway might induce a repression mechanism at the anterior tip that would be parallel and redundant with Torso-induced inhibition of Bcd. Thus, one might predict that another repressor might still able to act on Hkb targets in the absence of Hkb protein (Andrioli, 2012).
Although no pair-rule derepression was detected in the anterior pole, it was possible to detect subtle deviations in the positioning of eve 1 in hkb- embryos, which was confirmed by morphological measurements using the image processing tool. Enhanced derepression effects were also detected for all anterior-most stripes investigated in slp-;hkb- double-mutant embryos compared to the effects observed in slp- embryos; these results were statistically significant. With the hkb misexpression system, repression effects were detected for h 1, eve 1, run 1 and ftz 1. With the exception of gt repression, no other gap domain disruption was detected in these assays. These results strongly suggest direct repression by Hkb on the CRMs of these stripes. In vivo binding data confirms this possibility. Moreover, with the bioinformatics analysis it was verified that Hkb, along with putative activators, increased the already high significance values of predicted clusters for activators that match these stripe CRMs. Therefore, the combined data suggest that Hkb acts as a repressor for a specific group of anterior pair-rule stripes (Andrioli, 2012).
These data also suggest that there is another possible mechanism underlying the repression that involves the activity of repressors further away from their original sources. One example of this mechanism is expression detected for the ectopic hkb domain, demonstrating that target CRMs are sensitive to Hkb-mediated repression even in the presence of low expression levels of Hkb. The prediction is that low concentrations of Hkb that have diffused away from its endogenous domain could still repress these CRMs. For this mechanism, repressors could fulfill additive repression roles at different anterior subdomains or even contribute to the definition of the anterior borders of stripes that are distantly positioned from where gap domains are detected. Thus, the increased derepression observed in slp-;hkb- embryos would be expected if a combinatorial additive mechanism existed in which each repressor had a small contribution to the overall repression. Following the same rationale, one can predict that at least one other repressor is still responsible for setting anterior border stripes in slp-;hkb- embryos (Andrioli, 2012).
The complexity of the regulation of genes involved in early patterning was postulated to be a condition that is necessary for sensing relatively small differences in the concentrations and combinations of many regulatory factors, which is likely the environment found in the syncytial blastoderm. In agreement with that hypothesis, recent studies revealed that the protein gradients of factors such as Bcd and Dorsal alone are not sufficient to determine all of the spatial limits of target gene expression and that these gradients might combine with other factors to pattern the early embryo. In the head region, it has been suggested that Bcd and the terminal system-mediated activities interact at the level of the target CRMs to generate the proper patterning for the head region of the embryo. In contrast to these studies that focused on gap genes, the current data shed light on a mechanism that is involved in the regulation of the anterior stripe CRMs, with the putative participation of hkb (Andrioli, 2012).
The correct positioning of the anterior pair-rule stripes must be a critical issue in the early developmental patterning of the fly. Even a slightly incorrect positioning of the anterior stripes, for instance, results in the non-formation of the mandibular segment in the slp null mutant. Thus, a complex repression mechanism is necessary to shape the stripes and to avoid inappropriate expression of their CRMs. Therefore, Hkb, Slp1 and other repressors are likely involved in a combinatorial repressive activity in the CRMs of the anterior stripes. Other experiments are necessary to test this hypothesis further and to reveal the underlying molecular mechanisms involved in this regulation (Andrioli, 2012).
Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. This study shows that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of NotchOFF or NotchON neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their NotchON progeny systematically die through Reaper activity, whereas later, their NotchOFF progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. It is concluded that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype (Bertet, 2014).
Although apoptosis is a common feature of neurogenesis in both vertebrates and Drosophila, the mechanisms controlling this process are still poorly understood. For instance, several studies in Drosophila have shown that, depending on the context, Notch can either induce neurons to die or allow them to survive during binary cell fate decisions. This is the case in the antennal lobes where Notch induces apoptosis in the antero-dorsal projecting neurons lineage (adpn), whereas it promotes survival in the ventral projecting neurons lineage (vPN). In both of these cases, the entire lineage makes the same decision whether the NotchON or NotchOFF cells survive or die. This suggests that, in this system, Notch integrates spatial signals to specify neuronal survival or apoptosis (Bertet, 2014).
This study shows that, during tOPC neurogenesis, neuronal survival is determined by the interplay between Notch and temporal patterning of progenitors. Indeed, within the same lineage, Notch signaling leads to two different fates: it first induces neurons to die, whereas later, it allows them to survive. This switch is due to the sequential expression of three highly conserved transcription factors-Dll/Dlx, Ey/Pax-6, and Slp/Fkh-in neural progenitors. These three factors have distinct functions, with Dll promoting survival of NotchOFF neurons, Ey inducing apoptosis of NotchOFF neurons, and Slp promoting survival of NotchON neurons. These data suggest that Ey induces death of NotchOFF neurons by activating the proapoptotic factor hid. Thus, Dll probably antagonizes Ey activity by preventing Ey from activating hid. The data also suggest that Notch signaling induces neuronal death by activating the proapoptotic gene rpr. Thus, Slp might promote survival of NotchON neurons by directly repressing rpr expression or by preventing Notch from activating it. In both cases, the interplay between Notch and Slp modifies the default fate of NotchON neurons, allowing them to survive. Further investigations will test these hypotheses and determine how Dll, Ey, Slp, and Notch differentially activate/repress hid and rpr (Bertet, 2014).
Although the tOPC and the main OPC have related temporal sequences, their neurogenesis is very different. This difference is in part due to the fact that newly specified tOPC neuroblasts express Dll, which controls neuronal survival, instead of Hth. Why do tOPC neuroblasts express Dll? The tOPC, which is defined by Wg expression in the neuroepithelium, is flanked by a region expressing Dpp. Previous studies have shown that high levels of Wg and Dpp activate Dll expression in the distal cells of the Drosophila leg disc. Wg and Dpp could therefore also activate Dll in the neuroepithelium and at the beginning of the temporal series in tOPC progenitors. Another difference between the main OPC and tOPC neurogenesis is that Ey and Slp have completely different functions in these regions. Indeed, unlike in the main OPC, Ey and Slp control the survival of tOPC neurons. This suggests that autonomous and/or nonautonomous signals interact with these temporal factors and modify their function in the tOPC (Bertet, 2014).
Finally, tOPC neuroblasts produce neurons for three different neuropils of the adult visual system, the medulla, the lobula, and the lobula plate. This ability could be due to the particular location of this region in the larval optic lobes. Indeed, the tOPC is very close to the two larval structures giving rise to the lobula and lobula plate neuropils-Dll-expressing neuroblasts are located next to the lobula plug, whereas D-expressing neuroblasts are close to the IPC. Interestingly, Dll and D neuroblasts specifically produce lobula plate neurons. This raises the possibility that these neuroblasts and/or the neurons produced by these neuroblasts receive signals from the lobula plug and the IPC, which instruct them to specifically produce lobula plate neurons. These nonautonomous signals could also modify the function of Ey and Slp in the tOPC (Bertet, 2014).
In summary, this study demonstrates that temporal patterning of progenitors, a well-conserved mechanism from Drosophila to vertebrates, generates neural cell diversity by controlling multiple aspects of neurogenesis, including neuronal identity, Notch-mediated cell survival decisions, and the mode of intermediate precursor division. In the tOPC temporal series, some factors control two of these aspects (Ey), whereas others have a specialized function (Dll, Slp, and D). This suggests that temporal patterning does not consist of a unique series of transcription factors controlling all aspects of neurogenesis but instead consists of multiple superimposed series, each with distinct functions (Bertet, 2014).
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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