even-skipped
The entire functional even-skipped locus of Drosophila
is contained within a 16 kilobase region. As a
transgene, this region is capable of rescuing even-skipped
mutant flies to fertile adulthood. Detailed analysis of the 7.7
kb of regulatory DNA 3' of the transcription unit reveals
ten novel, independently regulated patterns. Most of these
patterns are driven by non-overlapping regulatory
elements, including ones for syncytial blastoderm stage
stripes 1 and 5, while a single element specifies both stripes
4 and 6. Expression analysis in gap gene mutants shows
that stripe 5 is restricted anteriorly by Krüppel and
posteriorly by giant, the same repressors that regulate
stripe 2. Consistent with the coregulation of stripes 4 and
6 by a single cis-element, both the anterior border of stripe
4 and the posterior border of stripe 6 are set by zygotic
hunchback, and the region between the two stripes is
carved out by knirps. Thus the boundaries of stripes 4 and
6 are set through negative regulation by the same gap gene
domains that regulate stripes 3 and 7, but at
different concentrations (Fujioka, 1999).
Based on the regions of overlap of
larger transgenes, these studies suggest that early stripes 1
and 5 might be driven by the region from +4.8 to +8.4 kb, and
stripes 4 and 6 by the +4.8 to +6.6 kb region.
Consistent with a composite element driving stripes 4 and 6,
both the anterior border of stripe 4 and the posterior border of
stripe 6 are determined by zygotic hb expression. In addition,
in a kni mutant, the isolated stripe 4+6 composite element drives
expression throughout the interstripe region. The spatial and
temporal expression patterns of zygotic hb and kni are consistent with the products of these loci
exerting direct repression on the element. Thus, as for stripes 2, 3 and 7, much of the spatial
regulation of stripes 4, 5 and 6 appears to be due to repression
by gap gene products. The sequences of these regulatory
elements contain potential binding sites for the gap gene
products that may directly regulate them. However, further analysis will be
required to determine if these regulatory interactions are indeed
direct (Fujioka, 1999).
The above observations concerning regulation of stripes 4
and 6 include a striking parallel with the regulation of stripes
3 and 7. The stripe 7 element is not separable from that of stripe
3, although full activation of stripe 7 requires sequences
outside of the minimal stripe 3 element.
Like the 4+6 element, a combined stripe 3+7 element directs
expression throughout the interstripe region in a kni mutant,
and both the anterior and posterior borders (of stripes 3 and 7,
respectively) are set by hb-dependent repression. Thus, an intriguing situation exists in which the stripe
4+6 element is repressed by a higher concentration of Knirps
protein than is the stripe 3+7 element and, at the same time, by
a lower concentration of Hunchback protein. The differential
sensitivity of these elements to repressor concentrations might
be due to simple mechanisms, such as differential affinities of
binding sites, or to more complex mechanisms, such as
combinatorial interactions with different cofactors. Whatever
the mechanism, this differential sensitivity is precise enough to
allow three gap protein domains (those of Knirps and the
anterior and posterior Hunchback domains), acting as repressor
gradients, to regulate the positioning of eight distinct
expression boundaries, thus helping to define four of the early
stripes of eve expression.
In a similar vein, stripe 5 is negatively regulated by the same
gap genes that regulate stripe 2. The Kr domain represses both
the posterior border of stripe 2 and the anterior border of stripe
5, while the anterior and posterior domains of giant expression are
involved in setting the anterior and posterior borders of stripes
2 and 5, respectively (Fujioka, 1999).
Recently, several genes were reported to show stripe-specific
effects on eve activation. lacZ expression driven in stripes 4, 5
and 6 by the eve 3' region were weakened in a fish-hook mutant
(fish, also known as Dichaete). It was also shown that the product of
this gene can bind within this large regulatory region, as
determined by gel mobility shift assays. While expression from the minimal elements also shows some
reduction in fish embryos, expression from these elements is
clearly activated by other proteins as well. In a marelle mutant
(encoding D-STAT), lacZ expression from a stripe 3 element
was seen to be weakened. D-STAT is a primary activator of stripe 5,
since expression from the stripe 5 element is absent in this
mutant. Consistent with a direct effect on this element,
several consensus sequences for D-STAT binding were found within the
+7.4 to +8.2 kb region (unpublished
observations).
None of the gap and pair-rule mutants tested have a
strong effect on stripe 1 element expression. hb
mutants weaken reporter gene expression, but not severely.
In a buttonhead mutant (btd), endogenous eve expression in the
stripe 1 region was seen to be reduced.
In beetles, as in Drosophila, eve forms stripes with anterior
borders that coincide with parasegment boundaries but, rather
than forming multiple stripes at once, stripe 1 is formed first,
followed by sequential progression toward the posterior. Further analysis of the regulation of stripe 1 may
reveal regulatory relationships that predate the divergence of
Diptera. Recent analyses of eve stripe elements among
Drosophila species
suggests that many of the regulatory mechanisms are
evolutionarily conserved. The growing body of information
from various species may soon support detailed hypotheses for
how the regulatory mechanisms of segmentation evolved (Fujioka, 1999 and references).
evenskipped is expressed in the nervous system, initially in GMCs 1-
1a, 4-2a and 7-1a, and later in the aCC/pCC, RP2, CQ and EL
neurons. In an analysis of the eve promoter, elements for GMC 1-1a,
its cellular progeny the aCC/pCC neurons, GMC 4-2a and its
progeny neuron RP2 could not be separated. This is surprising, since these cells
originate from different neuroblasts. A single element drives
lacZ expression strongly in these neurons, at least through stage
11. However, by stage 15, transgene expression is reduced,
particularly at the protein level, when both endogenous Eve
expression and expression from rescue constructs (in an eve-
background) remain strong. The eve 3' UTR, which the initial
lacZ transgenes did not contain, appears to affect the efficiency
of translation in these cells. Transgenic lines in which the
standard 3' UTR (from the alpha-tubulin gene) is replaced by that
of eve (while they show reduced mRNA levels at stage 11 and
similar levels at later stages) give lacZ protein levels that
remain high through stage 15 in the RP2 and aCC/pCC
neurons. The eve 3' UTR confers a
rapid turnover rate in early cycle 14
of the blastoderm stage. Thus it appears that the eve 3' UTR
has functions in controlling protein levels in several tissues, at
various stages, and probably through multiple mechanisms.
Elements for EL cells and for GMC 7-1a and its progeny
CQ neurons were also localized. However, the CQ and EL
elements overlap those for posterior region expression and for
even-numbered parasegment expression, respectively, suggesting that common activators may be
utilized in these different tissues (Fujioka, 1999).
An element for muscle precursor cell expression is separable
from those of other tissues. However, its expression at stage 15 becomes
weaker than that of endogenous Eve, as observed
for the RP2+aCC/pCC element. The eve 3' UTR may provide
for a high level of protein expression in this tissue, at a similar
time as that in the nervous system.
Expression in the posterior region of the embryo is
apparently a highly conserved feature of eve function, since it
is shared by eve homologs in C. elegans, zebrafish and mice. While it was reported that posterior
structures are not affected in certain eve mutants at the non-permissive
temperature, the possibility remains that eve has some function in this region. eve
homologs have been shown to have important functions in
specifying posterior cell fates in C. elegans and zebrafish. The regulation of eve
expression in the posterior region is complex. Initially, the late
stripe element is responsible for expression in this region,
which appears as an 8th stripe corresponding to parasegment
15. Later, expression is driven in a ring near the
posterior end of the embryo by two separable elements, one
active through germband retraction and the other after dorsal
closure. The latter expression corresponds to the anal plate
ring.
Just downstream of the eve-coding region (+1.5 kb to +2.6 kb)
lies an element that, when assayed by itself, drives lacZ
expression strongly in the even-numbered parasegments, where
only very weak eve expression is normally observed. As
suggested previously, the upstream late element may be
responsible for long-range repression of these ftz-like stripes in the endogenous eve gene. The
biological function of this element, if any, is unclear, although
eve expression does extend into this region, where it is required
to clear odd-skipped expression from the anterior ftz domain,
allowing activation of engrailed. This
element may serve a function in this context (Fujioka, 1999 and references).
The regulatory DNA that was characterized downstream
of the transcription unit, in combination with upstream regions
described above, is sufficient to functionally rescue eve null
mutants. In most cases, a single copy of the rescue transgene
is not sufficient for full rescue. Many mutant embryos
exhibit a weak eve hypomorphic phenotype when they
carry only one copy of the transgene, suggesting that
transgene expression is below that of the endogenous gene
when inserted at most chromosomal locations. This might
indicate that the transgene is missing a general enhancer of
early eve expression. Alternatively, sequences within the P-element
vector may repress eve expression at early stages. It is
also possible that a chromosomal environment exists around
the eve locus that is required for full activity which most
insertion sites do not provide. The PSR element described
below might participate in providing such an environment.
The genomic region downstream of the RP2+aCC/pCC
element causes strong pairing-sensitive repression (PSR) of
the mini-white gene. Similar PSR is observed when Polycomb-group
gene responsive elements are introduced into the
genome with mini-white. Recently, it was reported that a
region from the engrailed gene that exhibits PSR is bound
directly by the Drosophila YY1 homolog, encoded by
the pleiohomeotic gene. Consensus sites
for YY1/Pho binding, as well as for GAGA factor, which are
also seen in the engrailed element, exist within this region. Consistent
with chromatin-based regulation of eve, a Polycomb-group
protein, Polyhomeotic, was found to bind to polytene
chromosomes in the region of the eve locus, and eve expression in the NB4-2 lineage is affected by Polycomb-group activity. Nonetheless, the function of the eve PSR
element is unclear, since rescue transgenes that lack it do not
show abnormal eve expression, and since including it does
appear to enhance either expression or rescue. It
is possible that the PSR element is only required in the context
of the eve locus, perhaps to prevent inappropriate activation
of eve by enhancers from a neighboring gene, or of a
neighboring gene by eve enhancers. Other regions of the eve locus are also capable of
repressing mini-white expression, since the -6.4 to +8.4 kb
transgenes consistently gives transformants with very weak
eye color. The utilization of Glass activator binding sites to
enhance mini-white expression facilitates the identification of
transformants, but these also show weak eye color relative
to other Glass-mini-white transgenes. Although this repression
is not consistently pairing sensitive, it may represent a
function that is redundant with that of the PSR region in some
aspect of eve regulation (Fujioka, 1999 and references).
Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and
Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting
both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects
of three signal-activated transcription factors (dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and
Tinman) on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman
determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).
Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial
signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess
information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and
founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).
Previous genetic experiments have defined multiple intercellular signaling events that govern the progressive determination of the Eve progenitors. Signaling from both the Wnt family member Wingless (Wg) and the TGF family member Decapentaplegic (Dpp) prepatterns the
mesoderm and renders cells competent to respond to Ras/MAPK activation. Localized Ras activation within the competence domain determined by the
intersection of Wg and Dpp expression occurs through the action of two RTKs: the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl)
fibroblast growth factor receptor. This RTK signaling induces two distinct equivalence groups, each of which expresses Eve. Lateral inhibition mediated by
Notch then selects a single progenitor from each equivalence group (Halfon, 2000).
The present study explores how the prepattern genes wg and dpp establish competence for mesodermal cells both to activate and to respond to the
Ras/MAPK cascade; how multiple intercellular signals are integrated to establish Eve progenitor fates, and how muscle- and cardiac-specific responses to Ras
signaling are generated. Wg provides competence for the generation of the Ras/MAPK inductive signal by regulating the expression of key
proximal components of the Egfr and Htl RTK pathways. Wg and Dpp then create competence for a specific response to the inductive signal both through their
own respective downstream transcriptional effectors, dTCF and Mothers against
dpp (Mad), and through their regulation of the mesoderm-specific transcription factors Tinman (Tin) and Twist (Twi). Specificity of the Ras/MAPK response is achieved though the
integration of these signal-activated and tissue-restricted transcription factors, along with the Ras/MAPK-activated Ets domain transcription factor PointedP2
(Pnt), at a single transcriptional enhancer. These results provide a direct link between the initial axis patterning processes in the early embryo and the subsequent
combinatorial signaling events that lead to the progressive determination of muscle and cardiac progenitors (Halfon, 2000).
The Eve progenitors in each mesodermal hemisegment arise during embryonic stage 11 in a dorsal region demarcated by the intersecting domains of Wg and Dpp expression. The cells exposed to both Wg and Dpp are competent to respond to localized Ras signaling, which induces the initial expression of Eve in two clusters of equipotent cells. In each of these equivalence groups, activity of the Notch pathway leads to the rapid refinement of Eve expression to a single muscle or cardiac progenitor. The two Eve equivalence groups arise sequentially. Cluster C2, from which progenitor P2 derives, is first to form. P2 divides asymmetrically, with one daughter maintaining Eve expression and becoming the founder of the two EPCs (F2EPC), and the other losing Eve expression and becoming the founder of muscle DO2. The second Eve-expressing cluster, C15, forms slightly later and produces the progenitor P15, which in turn divides to yield the founder of the Eve-expressing muscle, DA1, and an Eve-negative cell of as-yet-undetermined identity. Activation of the Ras/MAPK pathway in C15 depends on both the DER and Htl RTKs, but only Htl signaling is required for C2 formation (Halfon, 2000).
The progressive determination of Eve mesodermal progenitors requires that Wg prepattern the mesoderm, rendering cells competent to respond to inductive RTK/Ras signaling. To further investigate the basis of this competence, whether or not the Ras pathway is active in the absence of Wg signaling was examined by monitoring the expression of the activated, diphosphorylated form of MAPK in wg mutant embryos. Diphospho-MAPK is expressed in progenitor P2 in early stage 11 wild-type embryos. Not only is this progenitor missing from wg mutant embryos, but activation of MAPK in the C2 equivalence group, which is dependent on Htl, fails to occur. Similarly, Wg is essential both for P15 formation and for the DER- and Htl-dependent activation of MAPK in the equivalence group from which this progenitor is derived (Halfon, 2000).
Next to be determined was at what level in the RTK/Ras pathway Wg is required for MAPK activation. In wg mutant embryos, there is loss of (1) the P2-specific expression of Htl; (2) its specific downstream signaling component, Heartbroken (Hbr, also known as Dof and Stumps), and (3) Rhomboid (Rho), a protein involved in the presentation of the Egfr ligand Spitz. Conversely, constitutive Wg signaling, achieved by ectopic expression of Wg or an activated form of the downstream Wg pathway component Armadillo (Arm), induces Htl, Hbr, and Rho expression in more dorsal mesodermal cells than the single P2 progenitor found at a comparable developmental stage. This effect is less prominent for Rho than for Htl and Hbr, which may reflect different threshold responses to Wg. Alternatively, the effect on Htl and Hbr may be more pronounced because ectopic Wg signaling prolongs their earlier expression in the entire C2 cluster; Rho, in contrast, is normally expressed in P2 but not in C2, possibly making it more refractory to a prepattern factor such as Wg. Expanded expression of these RTK pathway components is associated with increased MAPK activation and Eve expression. However, these effects of Wg hyperactivation are transient, with a normal number of Eve progenitors eventually segregating. Moreover, activated Arm is able to fully rescue Htl, Hbr, Rho, diphospho-MAPK, and Eve expression in wg mutant embryos. Htl, Hbr, and Rho expression, as well as MAPK activation, are also Dpp dependent. In summary, Wg and Dpp regulate the production of several key proximal components of the DER and Htl signal transduction pathways (Halfon, 2000).
Given the involvement of Wg in the expression of Htl, Hbr, and Rho, it was reasoned that a constitutively activated form of Ras1 might bypass the requirement of Wg for MAPK activation. Constitutively activated Ras1, when targeted to the mesoderm of wild-type embryos, leads to an overproduction of Eve progenitors, as well as to the expected hyperactivation of MAPK in these cells. In the absence of Wg signaling, diphospho-MAPK expression is restored by activated Ras1. However, despite this recovery of MAPK activation, constitutive Ras1 does not rescue Eve progenitor formation in a wg mutant background. This is in marked contrast to the ability of activated Arm to fully rescue RTK signaling and Eve progenitor specification in a wg mutant. These results suggest that, in addition to enabling activation of Ras/MAPK signaling as a result of the induction of Htl, Hbr, and Rho expression, Wg signaling must contribute other factors that are essential for the specification of mesodermal Eve progenitors (Halfon, 2000).
Given the importance of Ras/MAPK signaling in Eve progenitor determination, a determination was made of whether Pnt, an Ets domain transcriptional activator that functions downstream of MAPK, is also involved in this process. In pnt mutant embryos, there is a severe reduction in the number of both Eve progenitors, although this loss is more pronounced for the P15 lineage. Since mesoderm migration is normal in pnt embryos, Pnt must only be required for the progenitor specification function of Htl. Consistent with this conclusion, an activated form of Pnt induces extra Eve progenitors (Halfon, 2000).
In embryos mutant for yan, which encodes a MAPK-regulated Ets-domain transcriptional repressor, there is an increase in the number of Eve progenitors and their differentiated derivatives. Conversely, a constitutively activated form of Yan inhibits Eve progenitor formation. Thus, two MAPK-regulated transcription factors are involved in the development of Eve progenitors (Halfon, 2000).
One mechanism that would ensure the convergence of the multiple regulatory inputs required for the formation of P2 and P15 is integration by a transcriptional enhancer. Since Eve expression is the feature that uniquely identifies these progenitors, an investigation was made of whether eve itself is a direct target for regulation by both signal-activated and tissue-specific transcription factors. Regulatory sequences responsible for mesodermal eve expression are located approximately 6 kb downstream of the transcription start site. Deletions of this region were generated and a 312 bp minimal enhancer was defined that has been termed the eve Muscle and Heart Enhancer (MHE). When fused to a nuclear-lacZ reporter gene, the MHE drives expression in a mesodermal pattern identical to that of the endogenous eve gene. Reporter expression initiates at early stage 11, coincident with the onset of Eve expression in the equivalence group C2. Following formation of P2, MHE activity is observed in P15 and in the P2 daughters, F2EPC and F2DO2, then in the EPCs and the F15 daughters of P15, and finally in muscle fiber DA1. Colocalization of MHE-driven ß-galactosidase expression with Runt, which marks the F2DO2 founder and muscle DO2, establishes that the reporter gene expression present in Eve-negative sibling cells is a result of ß-galactosidase perdurance. Of note, the MHE mimics endogenous Eve expression despite its lack of a consensus binding site for the transcription factor Zfh-1 that had previously been proposed to play a role in mesodermal eve regulation (Halfon, 2000).
Strikingly, the MHE is only active in a single nucleus of the mature DA1 and DO2 muscles. It is inferred that these are the original nuclei of the F15DA1 and F2DO2 founders based on prior reporter expression in those cells. Similar results were obtained when DNA flanking the MHE by several hundred base pairs on either side (+4.96 to +7.36 kb), including the previously described Zfh-1 site, was included in the reporter construct, or when the MHE was placed 3' to a reporter gene fused to the endogenous eve promoter. Thus, additional sequences are required for eve expression in non-founder myofiber nuclei. Of critical importance to the present study, the MHE fully recapitulates mesodermal Eve expression during the signal-dependent induction of progenitor and founder cells (Halfon, 2000).
Genetic manipulation of the Wg, Dpp, and RTK/Ras signaling pathways causes predictable alterations of endogenous mesodermal Eve expression. A determination was made of whether the isolated MHE responds appropriately to these signals. In all genetic backgrounds, reporter gene expression corresponds precisely to that of endogenous eve. For example, constitutively activated Arm transiently increases the expression of both genes. However, Wg hyperactivation does not have a stable effect on MHE function. In contrast, both endogenous eve and the MHE-driven reporter are induced throughout the initial competence domain by constitutively activated Pnt, and expression of both markers extends laterally in the presence of activated Arm plus Pnt. Ectopic Dpp leads to both endogenous Eve and MHE-driven reporter expression in the ventral mesoderm, while coexpression of Dpp and activated Ras1 induces expression of both genes in a dorsal-ventral stripe. These results demonstrate that the isolated MHE is responsive to all of the known signals that are essential for the specification of Eve progenitors (Halfon, 2000).
Given that the MHE recapitulates early mesodermal Eve expression, a determination was made of whether this enhancer contains binding sites for candidate signal-dependent and mesoderm-specific transcription factors. Focus was placed on two mesoderm-specific factors, Tin and Twi, as well as the nuclear factors that act downstream of Wg (dTCF), Dpp (Mad) and Ras (Pnt, Yan). A computer-based search of the MHE sequence has suggested the presence of potential binding sites for each of these transcription factors. Gel-shift assays confirm that these putative sites actually bind the relevant factors. This analysis establishes the existence of one binding site for dTCF, six for Mad, two for Twist, and four each for Tin and Pnt. Since Yan binds to each of the Pnt sites, these are referred to as Ets sites (Halfon, 2000).
To ascertain whether these in vitro binding sites have in vivo functional significance, the sites were mutated, both singly and in combination, within the context of the entire MHE. All mutagenesis was by base substitution so as not to affect the spacing between other potential cis-regulatory elements. The ability of the mutated MHEs to drive reporter gene expression was tested in transgenic embryos and this expression was compared to that of endogenous Eve. Of the six Mad sites, only Mad4, 5, and 6 are critical for MHE function when inactivated singly or in combination. Mutation of the single dTCF site or of individual binding sites for Twi, Tin, or the Ets factors also lead to loss of reporter gene expression in some, but not all, Eve-expressing cells, with some mutant sites associated with a more severe loss than others. Of note, both the EPC and DA1 lineages are affected equally by all of the mutations. In addition, the activity level in those Eve-expressing cells that do maintain reporter gene expression is on average lower than that seen with the wild-type MHE. In contrast to the single site mutants, mutation of the two Twi, all four Tin, or all four Ets sites completely eliminate MHE activity. It is concluded that binding sites for two tissue-specific and three signal-responsive transcription factors are required for full activity of the MHE in both the muscle and the heart lineages (Halfon, 2000).
The finding that the three Wg-dependent factors, dTCF, Twi, and Tin, that directly regulate eve could explain why activated Ras is incapable of bypassing Wg in the induction of Eve progenitors. Therefore attempts were made to rescue Eve expression in wg mutant embryos by ectopically expressing Twi and Tin together with activated Ras. However, Eve progenitors were not recovered by this manipulation, perhaps due to the direct requirement of dTCF for eve MHE activity. While activated Arm can supply the missing downstream Wg transcription factor in this rescue experiment, Arm alone is capable of fully rescuing not only the Eve progenitors but also all of the Wg-dependent factors that regulate the MHE, including Twi, Tin, and the RTK/Ras pathway components. Thus, the combined effects of the MHE transcription factors could not be further evaluated in the absence of Wg signaling. Nevertheless, the rescue and enhancer mutagenesis data strongly support the involvement of Wg as a mesodermal competence determinant both upstream of the Ras pathway and directly (via dTCF) as well as indirectly (via Twi and Tin) in the transcriptional response to inductive RTK signaling (Halfon, 2000).
Since mutation of any single transcription factor binding site in the MHE causes only a partial loss of enhancer activity, it was considered whether different sites might function together synergistically. To test this possibility, binding site mutations for two different activators were combined. Simultaneous mutation of the dTCF and Twi1 sites led to reporter gene expression in approximately 5-fold fewer cells than would be expected from the additive independent effects of each mutation. A similar, though slightly less robust, synergy was observed when the dTCF and Ets3 mutations were combined (Halfon, 2000).
An assessment was made of whether ectopic coexpression of individual transcription factors or upstream signals would lead to cooperative effects on endogenous Eve expression. As previously reported, ectopic Wg has no effect on Eve expression at late stage 11, activated Ras1 induces extra Eve progenitors, and ectopic Wg plus activated Ras1 cause a lateral expansion of the progenitor clusters. When Twi is expressed using a twi-Gal4 driver, a few Eve-positive cells develop at ectopic positions. The magnitude of this effect is increased by coexpression of Wg and Twi, and even more so by coexpression of Twi with activated Ras1. The latter effect strikingly resembles that of Wg plus activated Ras1. With the simultaneous ectopic expression of Wg, Twi, and activated Ras1, Eve progenitors form an almost continuous anteroposterior stripe confined to the dorsal mesoderm. These results demonstrate a synergistic induction of Eve progenitors by various combinations of Wg, Twi, and activated Ras1 that parallels the synergistic loss of MHE activity seen by mutating the dTCF, Twi, and Ets binding sites. Taken together, these loss- and gain-of-function findings suggest that dTCF, Twi, and Pnt cooperate at the MHE to synergistically regulate Eve transcription and, by extension, to induce the specification of Eve progenitor fates (Halfon, 2000).
It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).
Individual somatic muscles and heart progenitors are specified at defined positions within the mesodermal layer of Drosophila. The expression of the homeobox gene even-skipped (eve) identifies one specific subset of cells in the dorsal mesoderm, which give rise to particular pericardial cells and dorsal body wall muscles. Genetic analysis has shown that the induction of eve in these cells involves the combined activities of genes encoding mesoderm-intrinsic factors, such as Tinman (Tin), and spatially restricted signaling activities that are largely derived from the ectoderm, particularly those encoded by wingless and decapentaplegic. A Dpp-activated Smad protein, phosphorylated Mad, is colocalized in eve-expressing cells during an extended developmental period. A mesodermally active enhancer of eve contains several Smad and Tin binding sites that are essential for enhancer activity in vivo. This enhancer also contains a number of binding sites for the Wg-effector Pangolin (Pan/Lef-1), that are required for full levels of enhancer activity. However, the main function of these sites is to prevent ectopic enhancer activity in the dorsal mesoderm. This suggests that, in the absence of Wg signaling, Pan binding serves to abrogate the synergistic activities of Smads and Tin in eve activation, while in cells that receive Wg signals, Pan is converted into a coactivator that promotes eve induction. Together, these data show that the eve enhancer integrates several regulatory pathways via the combinatorial binding of the mesoderm-intrinsic regulator Tin and the effectors of the Dpp and Wg signals (Knirr, 2001).
Functional dissection of eve flanking regions has identified a
900-bp fragment, located about 5.7-6.6 kb downstream of
the eve transcription start site and named Eve Mesodermal
Enhancer (EME) 0.9SS: this fragment is able to reproduce the full mesodermal pattern of endogenous eve expression between embryonic early stage 11 and stage 15.
Between stages 11 and 12, reporter gene expression occurs
exclusively in the 11 (plus one posterior) bilateral clusters
of mesodermal eve progenitors. Perduring
ßGalactosidase (ßGal) protein at stage 16 shows that these
clusters include the progenitors of the eve pericardial cells
and muscle 1 (DA1). Because the long half life of
ßGal protein precludes accurate determination of the temporal
dynamics of reporter gene expression,
transgenic embryos were stained for both lacZ mRNA, which is less
stable, and Eve. This analysis confirms that the EME 0.9SS
enhancer is active in all pericardial cells and muscle 1
precursors until stage 15 and continues to be
active in the eve pericardial cells, predominantly those
surrounding the posterior portion of the dorsal vessel (the
heart proper), until the end of embryogenesis. This pattern is similar to that of eve mRNA, except that eve continues to be expressed evenly in pericardial cells along the entire length of the dorsal vessel. A second difference is ectopic reporter gene expression, which is observed following stage 15 in muscle 2, several ventral muscles, and in ectodermal muscle attachment
sites. From this it is inferred that EME 0.9SS lacks site(s) for negative
regulators, which normally prevent eve activation by late-acting
regulators outside of eve's normal domain of expression (Knirr, 2001).
The spatial and temporal activity of a subfragment comprising
709 bp of the 59 portion of EME 0.9SS, EME A, is identical to the full-length fragment. Both the 394-bp fragment EME B, from the center of EME
0.9SS, and the longer EME C have activities similar to that
of EME 0.9SS, although their lacZ expression already terminates at stage 14 in muscle 1 precursors and there is little ectopic expression (Knirr, 2001).
To define minimally active enhancer fragments, EME B
was further subdivided into two overlapping portions, EME
B59 (232 bp) and EME B39 (361 bp). Both subfragments
drive expression in mesodermal eve progenitors,
although the levels of EME B59 activity are lower than those
of the parental EME B fragment. Similar to EME B, but unlike EME 0.9SS, EME B59
and EME B39 exhibit low levels of ectopic activity in dorsal
mesodermal cells outside of the endogenous early Eve
clusters, while other embryonic regions show very little
ectopic activity. Enhancer activity of EME B59 persists in eve pericardial cells until stage 16 (more strongly posteriorly), while dorsal muscle founders have already become negative at mid stage 12 (Knirr, 2001).
EME B39 enhancer activity ceases prematurely at early
stage 13 in both pericardial and dorsal muscle progenitors. The identical spatial activity of EME B59 and EME B39 at stages 11 and 12 indicates that both fragments
include a similar set of regulatory sequences that are
necessary for normal early activation of mesodermal eve
expression. Either all of these sequences are contained
within the ~90-bp overlap between the two fragments or
there is redundancy of individual regulatory sites within
EME B. By contrast, only EME B59 contains regulatory
sequences that are sufficient for late pericardial expression,
and both EME B59 and EME B39 have to cooperate to
drive expression in syncytial muscle 1 precursors at later stages (Knirr, 2001).
The expression of eve identifies a small subset of pericardial
and somatic muscle progenitors and its onset coincides
with the processes that determine their developmental
fates. Although eve itself cannot be sufficient to specify the
distinct identities of these cells, it may fulfill such a
function in specific combinations with additional regulators,
such as Krüppel (in muscle 1 progenitors) and Runt (in muscle 10 progenitors). Therefore, eve activation can serve as a paradigm
for studying the genetic and molecular processes that determine
the identities of individual muscle and heart progenitors (Knirr, 2001).
Previous work has provided insight into the regulatory
cascades and some of their components that are critical for
eve expression. These studies show that the combined
activities of tin and slp, which themselves are induced in
the mesoderm by Dpp and Wg, respectively, are required
but not sufficient for eve activation. Further, ectopic expression
of slp and wg in combination, but not of either
component alone, results in uniform eve expression along
the dorsal margin of the mesoderm. These and other data indicate that wg and possibly also dpp are required during multiple steps in the regulatory
cascade of eve induction. The results of this study confirm that
there is a renewed requirement for Wg and Dpp signaling at
the level of eve activation. More generally, this means that
these two signaling molecules first induce the spatially
restricted and overlapping expression of prepatterning genes
in the mesoderm and subsequently act again, this time in conjunction with the products of prepatterning genes, to induce genes that determine the identities of heart and muscle progenitors. During this downstream step, the restricted areas of overlap between prepatterning gene expression patterns determine the domains in which cells are competent to respond to signals (Knirr, 2001).
The integration of the regulators of eve involves the direct interaction of Dpp and Wg effectors as well as Tin (but not Slp) with a mesodermal eve enhancer. Tin and Dpp-activated Smads appear to synergize to allow
eve induction, analogous to the situation that has been described
for the broad induction of tin in the dorsal mesoderm. However, this raises the question of why, unlike the case of tin, the combined activities of Smads and Tin are unable to induce eve in the whole dorsal mesoderm. A
likely explanation is that negative regulators bind to the eve
enhancer and abrogate the synergistic activities of Smads
and Tin. Based on this analysis, these negative regulators
include Pangolin (Lef1), and the ETS protein Yan has also been
identified as a negative factor in this
process. In the current view, the role of Wg and RTK
signaling would be to neutralize these negative regulators
and convert them into positive ones, which would then
enable Smads and Tin to activate eve exclusively in the
cells that receive these additional signals (Knirr, 2001).
The POZ domain is a conserved protein-protein interaction motif present in a variety of transcription factors involved in
development, chromatin remodeling and human cancers. The role of the POZ domain of the GAGA
transcription factor (Trithorax-like) in promoter recognition has been examined. Natural target promoters for GAGA factor typically contain multiple
GAGA-binding elements. The POZ domain mediates strong co-operative binding to multiple sites
but inhibits binding to single sites. Promoters regulated by GAGA have been identified by in vivo as well as in vitro studies. The Ultrabithorax (Ubx), fushi tarazu (ftz), hsp70 and evenskipped (eve) promoters were used to
compare the binding of GAGA polypeptides. All these promoters are
characterized by the presence of multiple GAGA-binding sites. DNase I footprinting
experiments reveal a dramatic difference in DNA-binding properties between full-length
GAGA and the polypeptides lacking the POZ domain. The GAGA elements on the
natural promoters are bound efficiently by full-length GAGA but not by equal molar amounts
of either deltaPOZ (lacking the POZ domain) or a construct possessing only the DNA binding domain (DBD). The amount of GAGA required to bind the multiple promoter
elements is significantly lower (>4- to 12-fold, depending on the promoter) than that required
to bind a single site, indicative of co-operative DNA binding. The spacing of the GAGA
elements in these different promoters varies considerably. However, GAGA
appears to be quite flexible and able to bind co-operatively to GAGA sites located at variable
distances from each other. The hsp70 promoter is generally GA rich and, at increasing
GAGA concentrations, the footprints start to spread and most of the promoter DNA is
protected against digestion (Katsani, 1999).
In contrast to full-length GAGA, equal molar amounts of the deltaPOZ or DBD polypeptides
fail to bind the GAGA target promoters significantly. On the Ubx, ftz and eve promoters,
protection of a single GAGA site by deltaPOZ and DBD can be observed. As expected, these
sites are the ones that most closely resemble the optimal GAGA-binding sequence. In these
experiments, deltaPOZ and DBD fail to bind to the weaker GAGA sites. This indicates that
POZ-mediated co-operativity increases the binding affinity for these sites by at least one order
of magnitude. Together, these DNase I footprinting experiments demonstrate that efficient
binding of GAGA to its natural target promoters depends critically on the presence of the
POZ domain, in addition to the DBD (Katsani, 1999).
Thus, GAGA oligomerization increases binding specificity by selecting only promoters with multiple sites. Electron microscopy reveals that GAGA binds to multiple sites as a large oligomer and induces bending of the promoter DNA. These results indicate a novel DNA binding mode by GAGA, in which a large GAGA complex binds multiple GAGA elements that are spread out over a region of a few hundred base pairs. A model is proposed in which the promoter DNA is wrapped around a GAGA multimer in a conformation that may exclude normal nucleosome formation. Since the GAGA DBD clamps almost one turn of the DNA, GAGA binding to multiple sites within a nucleosome repeat length is expected to severely compromise histone-DNA contacts. These contacts might be hampered further by DNA bending and wrapping around a GAGA oligomer. However, it is not clear whether GAGA binding leads to complete displacement of the histone core or whether some
histone-DNA contacts are preserved. In summary, after transient chromatin remodelling by NURF to allow for GAGA binding, GAGA may function as an architectural factor that reorganizes the promoter DNA
and maintains it in an open conformation (Katsani, 1999).
Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. This study investigated the regulatory 'grammar' of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid-Bicoid, Hunchback-Hunchback, Bicoid-Dorsal, Bicoid-Caudal and Dorsal-Twist. Direct evidence is presented for the importance of Bicoid-Dorsal linkage in the integration of the anterior-posterior and dorsal-ventral patterning systems. Hunchback-Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. Evidence is presented that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. It is concluded that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo (Papatsenko, 2009).
Nearly 100 characterized enhancers and ~30 associated binding motifs control the patterning of the early Drosophila embryo, probably the best understood developmental process. These enhancers and sequence-specific TFs regulate the expression of ~50 genes controlling AP and DV patterning, including segmentation and gastrulation. The known TFs controlling embryogenesis represent less than ~10% of all TFs in the Drosophila genome. Thus, this analysis of regulatory grammar was restricted to the ~100 AP and DV enhancers and their ~30 TF inputs (31) (Papatsenko, 2009).
The recent completion of whole-genome sequence assemblies for 12 divergent Drosophila species has created an unprecedented opportunity for analyzing enhancer evolution. In this study 96 selected enhancer sequences from D. melanogaster were mapped to all 12 Drosophila genomes, using the UCSC Browser. The resulting collection combined 1420 kb of genomic sequence data in 1127 sequences, representing 60 enhancers in 23 AP genes and 36 enhancers in 31 DV genes. The entire collection of sequences and binding motifs is available at the Berkeley on-line resource (Papatsenko, 2009).
Inspection of aligned enhancer sequences among all 12 Drosophila species revealed strong conservation within the D. melanogaster subgroup (D. melanogaster, D. simulans, D. seichellia, D. yakuba and D. erecta) and also within the D. obscura group (D. pseudoobscura and D. persimilis). In order to focus on evolutionary changes in these enhancers the seven most divergent Drosophilids were analyzed: D. melanogaster, D. ananassae, D. pseudoobscura, D. willistoni, D. mojavensis, D. virilis and D. grimshawi. The remaining five species contain conservation patterns that are similar to those present in D. melanogaster or D. pseudoobscura (Papatsenko, 2009).
Short-range TF-binding linkages (0-80 bp) were examined in the collection of 96 enhancers from seven species for homo- and heterotypic pairs of binding motifs. Binding sites for the 30 most reliable TF motifs (see the Berkeley online resource) were mapped in enhancers using position weight matrices with match probability cutoff values set to ~2E-04. Distance histograms were generated for distances smaller than 80 bp, measured between the putative centers of each pair of neighboring site matches. Periodic signals were identified in the distance histograms using Fourier analysis, and statistical significance was estimated by bootstrapping positions of site matches in each enhancer sequence (Papatsenko, 2009).
Fourier analysis has identified helical phasing (~11 bp spacing) for several different homotypic activator-activator motif pairs. Such periodic signals were found in the distributions of Bcd-binding sites. Weaker helical-phasing signals were also identified for Caudal (Cad) and Dl-binding sites. Periodic signals close to two DNA turns (~20-22 bp) were found for Twi, Hb and Kruppel. Such helical phasing raises the possibility of direct protein-protein interactions (Papatsenko, 2009).
A weaker, ~11.4-bp periodic signal was detected in the distribution of heterotypic activator-activator site pairs, including Dl-Twi and Bcd-Cad. In contrast, there is a significant reduction in helical phasing signatures for activator-repressor motif pairs, and in fact, an over-representation of site pairs with 'anti-helical' spacing (15.2 bp). A similar 15.2 bp anti-helical signal was detected in distributions of all possible pair-wise combinations of the 30 binding motifs examined in this study. Thus, it would appear that any two randomly chosen binding sites are more likely to occupy the opposite sides of the DNA duplex as compared with helical phasing. This observation raises the possibility that most TFs function either additively or antagonistically to one another and just a special subset of TFs function in a synergistic fashion as reflected by helical phasing of the associated binding sites (Papatsenko, 2009).
The preceding analysis considered 'short-range' organizational constraints, involving linked binding sites separated by <25-30 bp. The possibility of 'long-range' constraints were also considered. The 96 enhancers under study possess characteristic 'unit lengths' of ~500 bp to 1.5 kb (300 bp minimum). The minimal/maximal sizes of the functional enhancers and the 'optimal' site densities can be determined by the amount of encoded information (pattern complexity), mechanisms of TF-DNA recognition such as lateral diffusion, or structural chromatin features like nucleosome positioning (Papatsenko, 2009).
Differential distance histograms reveal an over-representation of short-range linkages (<50 bp), but a depletion in mid-range distances (100-500 bp). These observations raise the possibility that TFs are distributed in a non-uniform manner across the length of the enhancer. That is, there may be sub-clusters, or 'hotspots', of binding sites within a typical enhancer. Such hotspots are observed in the prototypic eve stripe 2 enhancer, whereby 8 of the 12 critical binding sites are observed within two ~50-bp fragments located at either end of the minimal 480 bp enhancer. Homotypic motifs display the greatest propensity for such sub-clustering. Homotypic clusters (38) usually contain 3-5-binding sites distributed over 50-100 bp. Heterotypic activator-activator motif pairs also demonstrate sub-clustering, but these clusters are smaller (<25-30 bp) and usually contain just a pair of heterotypic sites. Heterotypic activator-repressor pairs show moderate enrichment over a distance of 50-70 bp, which is in agreement with the well-documented phenomenon of 'short-range repression'. Depletion of mid-range spacing constraints (around ~200 bp) is especially striking in the case of heterotypic motif pairs. Thus, activator synergy is like short-range repression: it appears to depend on closely linked binding sites (Papatsenko, 2009).
A possible explanation for this depletion of mid-range spacing is the occurrence of positioned nucleosomes, which might separate functionally distinct regions within an enhancer, and also separate neighboring enhancers. To test this hypothesis, nucleosome formation potential was compared with the distributions of TF-binding motifs in enhancers using the 'Recon' program. Three of the four eve enhancers that were examined (eve 1+5, eve 2 and eve 4+6) display a clear negative correlation between potential nucleosome formation and the distribution of TF-binding sites. This observation is consistent with the depletion of nucleosomes near TF-binding sites in vertebrates. This anti-correlation is especially striking in the case of the bipartite eve stripe 1+5 enhancer, where two enhancer regions (stripe 1 and stripe 5) are separated by a 400 bp 'spacer' DNA (in positions 600-1000), which might promote positioning of two nucleosomes and associated linker sequences (Papatsenko, 2009).
To investigate nucleosome positioning further, nucleosome-forming potential was measured in two sets of sequences, previously identified based on clustering of Dl sites and tested in vivo for enhancer activity. One set of sequences functioned as bona fide enhancers and produced localized patterns of gene expression across the DV axis of early embryos. The other set produced no expression in transgenic embryos, despite the presence of the same quality Dl-binding site clusters. The nucleosome-forming potential of the enhancers (true positives) was lower than that of the non-functional sequences (false-positives). These observations raise the possibility that the false Dl-binding clusters fail to function due to the formation of inactive nucleosomal structures (Papatsenko, 2009).
All 465 possible pairwise motif combinations for the 30 relevant binding motifs were tested for conservation in divergent drosophilids. Only linked binding sites, separated by a distance with small variations (max. distance bin = five bases) were considered. In the case of motif pairs, statistical significance was evaluated by bootstrapping columns in the binding motif alignments, thus preserving patterns of conservation. Pairs of homotypic motifs strongly prevailed in this type of analysis (28% of total pairs versus 6.5% expected), suggesting that homotypic interactions are important and pervasive in embryonic patterning. The strongest linkages were found for Bcd, Cad and Hb homotypic pairs. Each of these pairs was shared by five to six different enhancers and conserved in four to seven species. Among the identified heterotypic motif pairs, the most interesting were Bcd-Dl, Bcd-Cad and Dl-Twi (Papatsenko, 2009).
To identify cases of binding site pairs organized in a more flexible fashion, significant motif combinations were extracted using large distance bins or large distance variations. Along with the previously identified motif pairs, this analysis revealed several additional combinations, mainly involving the 'TAG-team' sequence motif, which is recognized by Zelda, a ubiquitous zinc finger TF. Zelda participates in the activation of the early zygotic genome and regulates a wide range of critical patterning genes. Indeed, significant combinations were identified for the TAG motif and Bcd, Dl and Hb. However, all of these TAG-X combinations exhibit spacing variability in different Drosophilids (Papatsenko, 2009).
It is conceivable that these results represent an underestimate of significantly linked motif combinations since very conservative cutoff values were used for statistical evaluation. A database of shared and/or conserved motif pairs, including those below the selected significance cutoff P = 0.03 is available from the Berkeley online resource (Papatsenko, 2009).
Conserved Bcd-Dl-binding site pairs were identified in the enhancers of several AP- and DV-patterning genes, including sal (AP), brk and sog (DV). The sites were found at similar distances, in the same orientation and were conserved in all seven species. It was suggested that the Bcd sites in the brk enhancer might augment gene expression in anterior regions, but this possibility was not directly tested. In wild-type embryos, both brk and sog exhibit significantly broader patterns of gene expression in anterior regions. This expanded pattern is lost in bcd mutants (Papatsenko, 2009).
Highly conserved Hb tandem repeats were detected in the regulatory regions of pair-rule genes, in the gap gene Kruppel, and in the Notch-signaling gene nubbin. Most of the homotypic Hb-Hb site pairs fall into two major groups, separated by either 6-8 or 13-15 bases. Some of the pair-rule enhancers selectively conserve either the 'short' or 'long' arrangement. For example, the eve stripe 4 + 6 enhancer contains two short Hb elements, while the stripe 3 + 7 enhancer contains a single long element. The odd 3 + 6 enhancer contains both short and long elements with various degrees of conservation. The hairy stripe 2,6,7 enhancer contains a single short element. Among the known gap genes, the long and short Hb elements were widely present in the enhancers of Kruppel, and in the blastoderm enhancer of nubbin, but not in any of the known knirps enhancers. It is conceivable that the distinct Hb site arrangements are important for the differential regulation of pair-rule genes by the Hb gradient (Papatsenko, 2009).
In conclusion, the systematic analysis of TF-binding sites in AP and DV patterning enhancers suggests a much higher degree of grammar, or fixed arrangements of binding sites, than is commonly believed. Developmental enhancers are thought to be highly flexible, with randomly distributed binding sites sufficing for the integration of multiple TFs. The results suggest that a large number of enhancers contain conserved short-range arrangements of pairs of binding sites. For instance, virtually all of the enhancers that respond to intermediate and low levels of the Dl gradient contain conserved arrangements of Dl-binding sites along with recognition sequences for other critical DV determinants, such as Twist and Zelda. Cooperating pairs of Bcd sites are found in enhancers responding to low Bcd concentrations, such as Knirps. Finally, distinctive arrangements of Hb-binding sites might influence whether the associated target genes are activated or repressed by high or low levels of the Hb gradient (Papatsenko, 2009).
Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. This study analyzed an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. The maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and evidence is presented that Zld binds to multiple non-canonical sites. A combination of in vitro binding experiments and bioinformatics analysis was used to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo (Struffi, 2011).
The experiments described in this study significantly refine
understanding of how the eve 3+7 enhancer functions in the early
embryo. In particular, it was shown that the maternal zinc-finger
protein Zld is absolutely required for STAT-mediated enhancer
activation, and that the gap proteins Kni and Hb establish stripe
boundaries by directly binding to multiple sites within the enhancer (Struffi, 2011).
When first activated in late nuclear cycle 13, the minimal eve 3+7
enhancer drives weak stochastic expression in a broad central
pattern, which refines in cycle 14 to a stripe that is about
four nuclei wide. By contrast, stripe 7 expression, which
is visible by enzymatic staining methods, is nearly undetectable using fluorescence in situ hybridization (Struffi, 2011).
Previous work showed that stripe 7 shares regulatory information
with stripe 3 but is also controlled by sequences located between
the minimal stripe 3+7 and stripe 2 enhancers, and possibly by sequences within and downstream of the stripe 2 enhancer. Thus, stripe 7 is unique among the
eve stripes in that it is not regulated by a discrete modular element (Struffi, 2011).
Previous work showed that the terminal gap gene tailless (tll) is required for activation of eve 7. However, since the Tll protein
probably functions as a dedicated repressor,
it is likely that activation of eve 7 by Tll occurs indirectly, through
repression of one or more repressors (Struffi, 2011).
The ubiquitous maternal protein Zld is required for the in vivo function of both the eve 3+7 and eve 2 enhancers, which are activated by the JAK-STAT pathway and Bicoid (Bcd), respectively. Zld was previously shown to bind to five sequence motifs that are over-represented in the regulatory regions of early developmental genes. Mutations of the single TAGteam site in the eve 3+7 enhancer caused a reduction in expression, but zld M- embryos, mutant for maternal zld expression, showed complete abolishment of eve 3+7-lacZ reporter gene expression. Also, the eve 2 enhancer, which does not contain any canonical TAGteam sites, is nonetheless inactive in zld M- embryos. This study showed that this enhancer contains at least four variants of the TAGteam sites, which suggests that Zld binding to non-canonical sites is crucial for its function in embryogenesis. ChIP-Chip data show that Zld binding extends throughout much of the eve 5' and 3' regulatory regions (Struffi, 2011).
The implication of such broad binding and the requirement for
Zld for activation of two eve enhancers are consistent with its
proposed role as a global activator of zygotic transcription. How might this work? One possibility is that there are cooperative interactions between Zld and the other activators of these stripes. A non-exclusive alternative is that Zld binding creates
a permissive environment in broad regions of the genome, possibly
by changing the chromatin configuration and making it more likely
that the other activator proteins can bind. However, it is important
to note that eve expression is not completely abolished in zld M-
embryos, so at least some eve regulatory elements could function
in the absence of Zld. Future experiments will be required to
further characterize the role of Zld in the regulation of the entire
eve locus (Struffi, 2011).
The genetic removal of kni causes a broad expansion of eve 3+7-
lacZ expression in posterior regions of the embryo, and ectopic Kni causes a strong repression of both stripes. Interestingly, the posterior
boundary of eve stripe 3 is positioned in regions with extremely
low levels of Kni protein. If the stripe 3 posterior
boundary is solely formed by Kni, the enhancer must be exquisitely
sensitive to its repression, possibly through the high number of sites
in the eve 3+7 enhancer. Previous attempts to mutate sites based on
computational predictions failed to mimic the genetic loss of kni,
so this study used a biochemical approach to identify Kni sites in an
unbiased manner. EMSA analyses identified 11 Kni sites, and
the PWM derived from these sites alone is very similar to the Kni
matrix derived in a bacterial one-hybrid study. Thus, these studies provide biochemical support for the bacterial one-hybrid method as an accurate predictor of the DNA-binding activity of this particular protein (Struffi, 2011).
It was further shown that specific point mutations abolish binding
to nine of the 11 sites, and when these mutations were tested in a
reporter gene they caused an expansion that is indistinguishable
from that detected in kni mutants. This result
strongly suggests that Kni-mediated repression involves direct
binding to the eve 3+7 enhancer, and that Kni alone can account
for all repressive activity in nuclei that lie in the region between
stripes 3 and 7. However, this work does not address the exact
mechanism of Kni-mediated repression. The simplest possibility is
that Kni competes with activator proteins for binding to overlapping or adjacent sites. This mechanism is considered unlikely because only one of the 11 Kni
sites overlaps with an activator site. Also, the in vivo misexpression
of a truncated Kni protein (Kni 1-105) that contains only the DNA-binding
domain and the nuclear localization signal has no
discernible effect on the endogenous eve expression pattern,
whereas a similar misexpression of Kni 1-330 or Kni 1-429
strongly represses eve 3+7 (Struffi et al., 2004) (Struffi, 2011).
Whereas Kni-mediated repression forms the inside boundaries
of the eve 3+7 pattern, forming the outside boundaries is dependent
on Hb, which abuts the anterior boundary of stripe 3 and overlaps
with stripe 7. Both stripes expand towards the poles of
the embryo in zygotic hb mutants, and these
expansions are mimicked by mutations in four or all nine Hb sites
within the eve 3+7 enhancer. Further anterior
expansions of the pattern are prevented by an unknown Bcd-dependent
repressor (X) and the Torso (Tor)-dependent terminal
system. Indeed, eve 3+7-lacZ expression expands all the way to the anterior tip in mutants that remove bcd and the terminal system (Struffi, 2011).
The mutational analyses suggest that Hb is a dedicated
repressor of the eve 3+7 enhancer, and argue against a dual role
in which high Hb levels repress, whereas lower concentrations
activate, transcription. One caveat is that activation of the stripe might occur via maternal Hb in the absence of zygotic expression. However, triple mutants
that remove zygotic hb, kni and tor, a terminal system
component, show eve 3+7 enhancer expression that extends from
~75% embryo length (100% is the anterior pole) to the posterior
pole. It is extremely unlikely that the maternal Hb gradient, which is not perturbed in this mutant combination, could activate expression throughout the posterior
region. It is proposed that any activating role for Hb on this
enhancer is indirect and might occur by repressing kni, which
helps to define a space where the concentrations of both
repressors are sufficiently low for activation to occur. kni
expands anteriorly in hb mutants and is very sensitive to
repression by ectopic Hb, consistent with an indirect role in activation. A similar
mechanism has been shown to be important for the correct
positioning of eve stripe 2. In this case, the
anterior Giant (Gt) domain appears to be required for eve 2
activation, but it does so by strongly repressing Kr, thus creating
space for activation in the region between Gt and Kr (Struffi, 2011).
The correct ordering of gene expression boundaries along the AP
axis is crucial for establishing the Drosophila body plan. All gap
genes analyzed so far seem to function as repressors that
differentially position multiple boundaries. However, it is still unclear how
differential sensitivity is achieved at the molecular level. Simple
correlations of binding site number and affinity with boundary
positioning cannot explain the exquisite differences in the sensitivity
of individual enhancers, suggesting that they do more than 'count'
binding sites and that specific arrangements of repressor and
activator sites might control this process. The experiments described
here better define the binding characteristics of both Hb and Kni and
provide a firm foundation for future experiments designed to decipher the regulatory logic that controls differential sensitivity (Struffi, 2011).
The Drosophila osa gene, like yeast SWI1, encodes
an AT-rich interaction (ARID) domain protein. Genetic and biochemical evidence is presented that Osa is a component of the Brahma
complex, the Drosophila homolog of SWI/SNF. To determine whether Osa is associated with the high molecular weight Brm complex, Schneider cell nuclear extracts were fractionated through a glycerol gradient
and immunoblotted with antibodies against the various proteins. Osa, Brm and Snr1 co-sediment in the bottom third of the gradient, suggesting
that they are part of a large protein complex. Thus, in vivo, Osa is found in a large complex with Brm and
Snr1, but does not bind to proteins in other chromatin remodeling complexes.
The ARID domain of Osa binds DNA without sequence specificity in vitro, but it is
sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation
of some of these genes. Brm-related complexes are thought to promote transcription by altering the architecture of nucleosomal DNA, thus generating a conformation that is more favorable to binding by transcription factors and the basal transcriptional machinery. Some genes, such as even-skipped, show reduced levels of expression in osa mutant embryos, supporting the role of Osa as an activator of gene expression. However, other genes, such as engrailed, show expanded domains of expression in osa mutants. These genes could be directly activated or repressed by Osa, or their changes in expression level could be secondarily due to the regulation of other transcription factors by Osa. Some Brahma-containing complexes do not contain Osa and Osa is not required to localize Brahma to
chromatin. These data suggest that Osa modulates the function of the Brahma complex (Collins, 1999).
Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. The type 1 ftz promoter
contains TATA but lacks the downstream promoter element (Dpe),
whereas type 2 promoters contain initiator (Inr) and/or Dpe sequences but lack TATA. Some enhancers,
such as ftz
autoregulatory enhancer (AE1) , preferentially activate type 1 promoters when given a choice between linked type 1 and type
2 promoters. Others, such as the rhomboid (rho) neuroectoderm enhancer (NEE), promiscuously
activate both classes of promoters (Ohtsuki, 1998 and references).
A transgenic embryo assay was used to
obtain evidence that the Drosophila eve promoter possesses an insulator activity that can be uncoupled from the core elements that mediate competition. The type 1 even-skipped (eve) promoter contains an optimal TATA element and a GAGA sequence.
The eve promoter insulator activity can be uncoupled from the TATA, Inr, and Dpe core elements. Mutations in a GAGA element,
located between TATA and the transcription start site, impair this insulator activity, so that genes residing
5' from an otherwise normal eve promoter are now activated by a 3' enhancer. Similar results were obtained
in trithorax-like (trl) mutants that diminish the levels of the Trl protein. Mutations in the GAGA element do not
diminish eve promoter function in competition assays. It is suggested that the Trl protein-GAGA element traps distal enhancers
by stabilizing enhancer-promoter interaction (Ohtsuki, 1998).
Promoters that possess enhancer blocking activities should facilitate the orderly trafficking of
cis-regulatory elements. For example, eve stripe enhancers located 3' from the transcription unit should be
unable to interact with neighboring genes located 5' from eve. Similarly, the ftz promoter contains a
GAGA element located 5' to TATA. This configuration of core elements should allow the ftz promoter to be both
transcriptionally active and able to block distal enhancers. Perhaps the ftz promoter helps inhibit
interactions between 3' Antp enhancers and 5' homeotic genes [Dfd (Deformed) and Scr] within the
ANT-C. It is conceivable that many promoters possess an intrinsic enhancer blocking activity. Inspection
of ~250 Drosophila promoter sequences reveals that ~15% contain at least
one optimal GAGA element within 50 bp 5' of the transcription start site. An earlier analysis of one of
these promoters, 1-tubulin, indicates that a GAGA sequence element helps insulate tubulin expression from
position effects. The enhancer blocking activity of the eve promoter appears to be mediated by interactions of Trl with
a GAGA element. Trl has been shown to recruit the NURF protein complex, which facilitates the binding of
upstream activators or core polymerase II components by decondensing chromatin. Trl-GAGA might trap distal enhancers by increasing the
stability of enhancer-promoter interactions through the creation of an open chromatin configuration or by
increasing the occupancy of core Pol II components such as TFIID (Ohtsuki, 1998).
The Drosophila Paired (Prd) transcription factor has homeodomain (HD) and paired domain (PD)
DNA-binding activities required for in vivo function. Correspondingly, Prd activation of late
even-skipped (eve) expression occurs through a conserved target sequence (PTE) with HD and PD
half sites, both of which are required for activation. To investigate the relationship between the HD and
PD, and their roles in conferring specificity to Prd function, altered versions of the Prd
protein and of the PTE target site were investigated using in vivo assays in embryos. It was found that function through
PTE is constrained by the targeting specifications of both the HD and PD as well as the spatial
relationship between these two domains. PTE function is also constrained by the spacing between
the target half sites for the PD and HD, although surprisingly, late eve activation is retained when
PTE is replaced by in vitro optimized binding sites for either the PD alone or for an HD dimer. In
contrast to late eve regulation, other Prd targets tolerate more changes in the Prd protein, suggesting
that their target sequences may be qualitatively different from PTE (Lan, 1998).
Gradients of regulatory factors are essential for establishing
precise patterns of gene expression during development; however,
it is not clear how patterning information in multiple
gradients is integrated to generate complex body plans.Opposing gradients
of two Drosophila transcriptional
repressors, Hunchback (Hb) and Knirps (Kni), position
several segments by differentially repressing two distinct regulatory
regions (enhancers) of the pair-rule gene even-skipped
(eve). Computational and in vivo analyses suggest that enhancer
sensitivity to repression is controlled by the number and affinity
of repressor-binding sites. Because the kni expression domain is
positioned between two gradients of Hb, each enhancer directs
expression of a pair of symmetrical stripes, one on each side of
the kni domain. Thus, only two enhancers are required for the
precise positioning of eight stripe borders (four stripes), or more
than half of the whole eve pattern. These results show that complex
developmental expression patterns can be generated by simple
repressor gradients. They also support the utility of computational
analyses for defining and deciphering regulatory information
contained in genomic DNA (Clyde, 2003).
In Drosophila, the pair-rule gene eve is expressed in a pattern of
seven stripes during the syncytial blastoderm stage of development.
This pattern foreshadows the mature segmented body plan and is
regulated by five enhancers. Three enhancers drive expression of
single stripes (eve 1, eve 2 and eve 5), and the remaining two drive
expression of pairs of stripes (eve 3 - 7 and eve 4 - 6). The best
characterized eve enhancer drives the expression of stripe 2 (eve
2), which is activated in a broad anterior domain by the maternal
morphogens Bicoid and Hb. Borders of the stripe are formed by
repressive interactions involving the gap proteins Giant (Gt) and
Kruppel (Kr), which are expressed in gradients anterior and
posterior to the stripe, respectively. Activation and repression are
mediated by the direct binding of all four proteins to discrete sites in
the enhancer. Thus, this enhancer acts as a transcriptional switch
that senses activator/repressor ratios in individual nuclei (Clyde, 2003).
Considerably less is known about the molecular regulation of the
enhancers that drive two stripes. eve 3 - 7 is activated by ubiquitous
factors including dSTAT92E, and activation of eve 4 - 6 requires
the function of the fish-hook gene, but other activators are
unknown. Genetic studies have shown that the gap genes hb and kni
are required for forming the borders of all four of these stripes. kni is
expressed in a broad posterior domain located between eve stripes 4
and 6. In kni mutants, the two-stripe patterns driven by eve
3 - 7-lacZ and eve 4 - 6-lacZ reporter genes are completely derepressed
in the region between the stripes. By contrast, hb is
expressed in an anterior domain that abuts eve 3 and a broad
posterior stripe that overlaps eve 7. In zygotic hb mutants,
there are marked derepressions of the outer borders of the stripes
driven by both the eve 3 - 7 and eve 4 - 6 reporter genes (Clyde, 2003).
To test whether the eve 3 - 7 and eve 4 - 6 enhancers are
differentially sensitive to Kni- and Hb-mediated repression, the snail(sna) promoter
was used to misexpress these genes along the
ventral surface of the embryo. The ectopic domain
directed by this promoter is uniformly distributed along the
anterior-posterior axis, and forms a ventral to dorsal gradient of
protein diffusion. Since all seven eve stripes are
subject to the same increase in protein concentration, differential
sensitivities among stripes can be assayed directly. Weakly affected
stripes will be repressed only in the ventral-most nuclei, whereas
strongly affected stripes will show repression in more lateral or even
dorsal regions. Ventral expression of either Kni (sna:kni) or Hb (sna:hb) is
sufficient for repression of eve stripes 3, 4, 6 and 7 in ventral regions,
but specific stripes require different quantities of ectopic
protein for repression. One copy of the sna:kni transgene represses
eve stripes 3 and 7, but has little effect on stripes 4
and 6. Two copies repress all four stripes, but stripes 3 and 7
are more strongly repressed than stripes 4 and 6. Misexpression
of Hb shows the opposite effects. One copy of sna:hb
causes a strong repression of stripes 4, 5 and 6, and an anterior
weakening and posterior expansion of stripe 3. The
posterior expansion is probably caused by Hb-mediated repression of kni (Clyde, 2003).
Two copies of sna:hb cause a stronger repression of stripes 4, 5 and
6, repress stripe 3 completely in ventral-most nuclei, and considerably
affect stripe 7, which seems slightly weaker and expanded
anteriorly, again toward the region normally occupied by kni.
The weaker effect on stripe 7 suggests that higher
concentrations of Hb are required to repress this stripe. This is
consistent with the fact that the posterior hb stripe overlaps stripe 7,
and that additional factors (including Tll) are required for activation
of this stripe8. The strong repressive effect of ectopic Hb on
stripe 5 is unexpected as this stripe seems to be normal in
hb mutants. In addition, computational analysis shows that there
are very few Hb-binding sites in the eve 5 enhancer region.
These results suggest that Hb-mediated repression of this stripe is
indirect (Clyde, 2003).
The above results suggest that the eve 3 - 7 and eve 4 - 6
enhancers respond autonomously to different amounts of the Hb
and Kni repressors. To test this idea further, lacZ reporter genes
driven by the minimal eve 3 - 7 or eve 4 - 6 enhancer were crossed
into embryos carrying the sna:kni or sna:hb misexpression transgene.
Embryos were also stained for endogenous sna
expression, which forms a sharp ventral-lateral border, a landmark
for measuring the extent of repression along the dorsalventral axis.
Ventral repression of the eve 3 - 7-lacZ transgene by Kni (2 sna:kni)
extends at least five nuclei above the sna border, but the eve
4 - 6-lacZ transgene is repressed only within the sna domain. Ventral expression
of Hb (1 sna:hb) causes the opposite
effects: the eve 4 - 6-lacZ transgene is more strongly repressed than
eve 3 - 7-lacZ . These experiments are consistent with
the effects observed for the endogenous eve stripes (Clyde, 2003).
To determine how these enhancers sense differences in repressor
concentration, bioinformatics was used to analyse the distribution
and affinity of Hb- and Kni-binding sites in the eve locus. Position-weighted
matrices (PWMs) for each protein were generated by
compiling and aligning the sequences of all known Hb- and Kni-binding
sites, and a clustering algorithm was used to search the
20-kilobase (kb) region surrounding the eve locus. This analysis
identified only two main clusters for each factor in this region,
which overlap precisely with the positions of the eve 3 - 7 and
4 - 6 enhancers. The composition of sites within these
clusters, however, is very different. The 3 - 7 enhancer contains
considerably more Kni sites with higher PWM scores than does the
4 - 6 enhancer, consistent with its
higher sensitivity to repression by Kni (Clyde, 2003).
For Hb, searching with a low-PWM-cutoff value (.4.0) identified
11 sites in the more sensitive 4 - 6 enhancer and, unexpectedly, 16
sites in the 3 - 7 enhancer. These results are similar to previous
findings; however, 10 of the 11 Hb sites in the 4 - 6 enhancer have
very high PWM scores, as compared with the 3 - 7
enhancer. Also, six of the ten high-scoring sites in the 4 - 6
enhancer are very tightly clustered in a 130-base-pair (bp) interval,
whereas those in the 3 - 7 enhancer are evenly distributed across
the sequence. These results suggest that binding-site affinity and
distribution may be crucial parameters in determining enhancer
sensitivity to Hb-mediated repression (Clyde, 2003).
Next, whether the clustering algorithm could predictably
change enhancer sensitivity was tested using the Kni-binding sites in the
3 - 7 enhancer as a test case. The PWM search identified 12 Kni-binding
sites in the minimal 3 - 7 enhancer; six of these sites were
mutated so that the cluster significance
score of the mutated enhancer (denoted 3 - 7m6K) was intermediate
between those of the wild-type 3 - 7 and 4 - 6 enhancers. Reporter expression
driven by 3 - 7m6K shows a derepression
of the inner borders of stripes 3 and 7, suggesting that Kni-mediated
repression has been compromised by these mutations. The stripe 3
response of the mutated enhancer extends throughout the interstripe
region posterior to eve 3 to the anterior border of, but not
through the region occupied by, eve 4.
Thus, the 3 - 7m6K enhancer is less sensitive to Kni than is the wild-
type 3 - 7 enhancer, but is still more sensitive than the 4 - 6
enhancer. This suggests that the precise positioning of these stripes
is controlled by the strength of Kni site clusters (Clyde, 2003).
Since the normal Hb and Kni gradients set several expression
boundaries in the region between their domains, it is essential
that their relative positions in the embryo are precisely established
and maintained. This could be achieved by mutual repression. To test this,
the effects of ventrally expressed Kni on the expression of hb
messenger RNA was analyzed, and vice versa. Misexpression of Kni causes a
strong reduction in hb mRNA in ventral regions. Similarly,
misexpressed Hb causes a strong repression
of kni (Clyde, 2003).
Loss-of-function experiments lend further weight to the mutual
repression hypothesis. In hb mutants, there is a substantial expansion
of the posterior kni domain. In kni mutants, there is a
slight anterior expansion of the posterior hb domain, but no effect
on the anterior domain. Double mutant embryos that lack kni and
the central gap gene Krüppel (Kr) show, however, a marked expansion
of zygotic hb expression throughout the posterior half of the
embryo. Because misexpression of Kr alone has no
effect on the hb expression pattern, this observation
suggests that Kr and Kni may cooperate in repression of hb.
In conclusion, the principle elements of a
simple repression system have been demonstrated that greatly increases pattern complexity
in the Drosophila embryo. Strong reciprocal repression between kni
and hb positions a symmetrical Kni domain between two opposing
gradients of Hb. This arrangement permits a single
enhancer to make two stripes, one on both sides of the Kni domain.
Two differentially sensitive enhancers effectively double the patterning
information in each gradient, leading to the establishment of
eight expression boundaries. A similar antagonistic relationship
exists between the gap genes gt and Kr,
which are expressed in nonoverlapping
domains, with the central Kr domain positioned
between two gt domains. The eve 2 and eve 5 stripes are formed
on either side of the Kr domain by Kr- and Gt-mediated repression,
but in this case each stripe is regulated by a separate enhancer,
probably because the activators of these stripes are expressed in
localized patterns (Clyde, 2003).
Previous studies have shown that activator gradients are crucial
for differential positioning of target gene expression patterns along
the anterior-posterior and dorsal-ventral axes. This study suggests
that repressor gradients can also specify
several gene expression boundaries by interacting with differentially
sensitive regulatory elements. At the molecular level, repression
mechanisms are flexible: enhancer activation can be prevented by
direct repression or by interfering with the binding or activity of
even a single activator protein. It is proposed that repressor
gradients, owing to this flexibility, are inherently more effective
than activator gradients at providing developmental patterning
information (Clyde, 2003).
The sensitivity of an enhancer is likely to be determined by several
parameters including the number, affinity and arrangement of
repressor-binding sites, but predicting the relative importance of
each of these parameters for a given enhancer is difficult. For the Kni
repressor gradient, the different responses of the 3 - 7 and 4 - 6
enhancers seem to depend on different numbers of binding sites. By
contrast, the different responses of the same enhancers to Hb
repression seem to depend on the affinity and/or arrangement of
sites. Thus, it may be impossible to formulate simple rules that
describe the functional characteristics of most enhancers. However,
future studies that combine computational analyses with experimental
tests will undoubtedly increase the ability to identify and to
characterize the genomic elements that regulate transcription (Clyde, 2003).
The Drosophila Knirps protein is a short-range transcriptional repressor that locally inhibits activators by recruiting the CtBP co-repressor. Knirps also possesses CtBP-independent repression activity. The functional importance of multiple repression activities is not well understood, but the finding that Knirps does not repress some cis-regulatory elements in the absence of CtBP suggested that the co-factor may supply a unique function essential to repress certain types of activators. CtBP-dependent and -independent repression domains of Knirps were assayed in Drosophila embryos; the CtBP-independent activity, when provided at higher than normal levels, can repress an eve regulatory element that normally requires CtBP. Dose response analysis has revealed that the activity of Knirps containing both CtBP-dependent and -independent repression activities is higher than that of the CtBP-independent domain alone. The requirement for CtBP at certain enhancers appears to reflect the need for overall higher levels of repression, rather than a requirement for an activity unique to CtBP. Thus, CtBP contributes quantitatively, rather than qualitatively, to overall repression function. The finding that both repression activities are simultaneously deployed suggests that the multiple repression activities do not function as cryptic 'backup' systems, but that each contributes quantitatively to total repressor output (Struffi, 2004).
The expression of the endogenous eve gene is strongly perturbed by a loss of CtBP, consistent with the important role of this co-repressor in the activity of gap repressors Giant, Krüppel, and Knirps. To study the effectiveness of Knirps repression of individual eve regulatory elements, the expression of eve-lacZ reporter genes was examined. Knirps is required for correct regulation of the eve stripe 3/7 and 4/6 enhancers, as demonstrated by the expression patterns of lacZ reporter genes in kni mutant embryos. The posterior border of eve stripe 3 was not derepressed in a CtBP mutant, consistent with the CtBP-independent activity of Knirps on this enhancer. By contrast, Knirps repression of eve stripe 4/6 is compromised in a CtBP mutant background, indicating that the CtBP-independent repression activity of Knirps is insufficient to regulate this enhancer. Therefore, depending on which part of the eve gene is bound by the Knirps protein, its repression activity is either dependent or independent of the CtBP co-factor (Struffi, 2004).
Previous studies of Krüppel, Giant and Knirps have indicated that CtBP dependence or independence of their repression activities varies according to the specific cis regulatory element involved, suggesting that there are particular enhancer architectures that necessitate CtBP activity. The clearest example of enhancer specific requirements for CtBP is shown in the case of eve enhancers. In nuclei situated between eve stripes 4 and 6, the stripe 4/6 and 3/7 enhancers are both repressed by Knirps in the same
nuclei, yet this repression is independent of CtBP on the 3/7 element and
dependent on CtBP on the 4/6 element. By expressing increasing levels of the CtBP-independent form of Knirps, the requirement for CtBP is obviated. These results suggest that distinct requirements for the CtBP co-factor at different genes or cis regulatory elements can be based on the quantitative levels of repression activity. Indeed, the combination of the CtBP-dependent and CtBP-independent
activities make a particularly powerful repressor, as judged by comparison of repression activities of Knirps 1-429 versus Knirps 1-330 on eve
and other pair-rule genes. These results
suggest that both repression domains can be simultaneously engaged on a given cis regulatory element, rather than a particular repression activity being selectively engaged at particular enhancers. Consistent with this picture, when they are assayed separately as Gal4 fusion proteins in embryos, both CtBP-dependent and CtBP-independent repression domains of Knirps have equal,
modestly effective repression activities. By contrast, a Gal4 protein
containing both domains is much more effective at repressing a strongly
activated promoter (Struffi, 2004).
A model is presented that explains the quantitative contribution of the CtBP co-repressor to Knirps repression activity. At a relatively low level of Knirps protein activity, the eve 3/7 enhancer is repressed, and this level of repression activity is achieved at similar levels of Knirps, regardless of whether or not CtBP contributes to repression. Thus, in the absence of CtBP, the positions at which the stripe 3/7 boundaries form shift very little. The much higher level of repression required by the stripe 4/6 element is achieved
only near the peak of Knirps protein levels. If CtBP is not complexed with
Knirps, the intercept shifts sharply to the right, to a level of Knirps not normally present in the embryo. The sufficient level of repression in the absence of CtBP activity or protein is only achieved under conditions where Knirps is overexpressed (Struffi, 2004).
The threshold model explains how the contributions of separate repression activities act in a quantitative fashion to meet given thresholds, but what is the basis for distinct repression thresholds? There are at least two variables involved in dictating a threshold, namely, regulatory protein levels and the
nature (number, affinity, and placement) of the relevant binding sites within a regulatory element. Varying intranuclear activator levels can influence repression thresholds, as suggested by regulation of the Krüppel gene: Giant requires CtBP for repression of this gene only in nuclei
containing peak levels of the Bicoid activator. Varying
intranuclear repressor levels will dictate how easily those thresholds are met, with or without multiple repression activities. Gap genes, including
knirps, generate protein gradients that have properties of
morphogens, i.e., they trigger differential responses at different threshold levels. The stripe 4/6 and 3/7 modular enhancers of the
even-skipped gene are designed to respond to different levels of
Knirps protein, allowing the embryo to establish multiple stripe boundaries with a single protein gradient. The short-range activity of Knirps allows the two enhancers to act independently, so that activators bound to the stripe 4/6 enhancer activate the gene in nuclei where the levels of Knirps are already sufficiently high to inhibit the stripe 3/7 enhancer (Struffi, 2004).
Binding site affinity and number have been clearly established to influence threshold responses in the case of transcriptional activators, such as Bicoid and Dorsal. A similar effect is likely to be true for repressors. Sequence analysis of the eve gene indicates that there are more high-affinity Knirps binding sites within the eve stripe 3/7 element than in the 4/6 enhancer, consistent with relative sensitivities of these elements that were determined experimentally. Removal of some of the Knirps binding sites in the eve stripe 3/7 enhancer reduces the sensitivity of this element to the Knirps gradient. However, the number of predicted high-affinity binding sites alone is not sufficient information to predict relative sensitivity to Knirps. If it were, one would expect the eve stripe 2 enhancer, with three predicted Knirps sites, to be more sensitive to Knirps than eve stripe 4/6, with only a single site, yet the reverse is true. This lack of
correlation might be partly attributable to errors in the prediction of binding sites; however, additional factors, such as affinity of binding sites and relative placement with respect to other proteins, are likely to make the decisive difference in determining enhancer sensitivity to Knirps. In the case
of the Giant repressor, small shifts in the placement of the binding site
allows detection of less than two-fold differences in repressor
concentrations, a 'gene tuning' mechanism that seems to have been invoked
during internal evolution of the eve stripe 2 enhancer. The
stoichiometry of activators to repressors has also been suggested to be a
crucial factor in determining repression levels, and direct tests indicate
that Giant and Knirps respond sensitively to differences in activator binding
site number and affinity on defined regulatory elements (Struffi, 2004).
eve stripe 1 lies just posterior to the weak anterior domain of knirps expression, suggesting a possible role for Knirps in regulating that element, but it is not clear whether the relative sensitivity of other eve stripe enhancers normally active outside of the main posterior domain of Knirps expression is of physiological significance. The eve stripe 2 pattern lies outside of the normal area of Knirps expression, and is only repressed at the highest levels of Knirps, suggesting that repression might be through cryptic Knirps sites in the element. The
robust activity of the eve stripe 5 enhancer even under conditions of high levels of Knirps misexpression emphasizes that this regulatory element has been designed to function in nuclei containing peak levels of Knirps protein. Similarly, runt stripe 5 also resists peak levels of ectopic Knirps. Both of these
regulatory elements have few or no predicted Knirps-binding sites. These
elements would provide a useful platform to test the number and placement of
novel Knirps binding sites required to bring the element under the control of this repressor (Struffi, 2004).
The effects of Knirps misexpression on other endogenous pair rule genes
reinforce the lessons learned from eve, regarding the relative
potency of the Knirps repression domains and the sensitivity of different
enhancers. Both the CtBP-independent region of Knirps as well as the intact
protein are capable of repressing the hunchback parasegment 4
stripe, a highly sensitive target of Knirps.
However, hairy, runt and ftz, which have been previously
noted to have a higher threshold to Knirps repression, are noticeably less
affected by Knirps 1-330 compared with Knirps 1-429. Thus, it is likely
that CtBP activity contributes quantitatively to repression of other Knirps target genes in addition to eve (Struffi, 2004).
Repression of central runt stripes is consistent with previous
findings of direct repression by Knirps and the greater sensitivity of stripes 2-4 relative to stripe 1. A greater effect of ectopic expression
of Knirps is observed on hairy than noted in previous experiments, probably on account of higher levels of expression. Knirps expressed under the control of an eve stripe 2 enhancer was previously found to have little effect on anterior hairy expression, except for a delay in stripe 3/4 separation. Heat shock expression of full-length Knirps 1-429, by contrast, results in strong repression of hairy stripes 3, 4 and 7. The hairy stripe 3, 4 and 7 enhancers are predicted to contain Knirps-binding sites, in contrast to the unrepressed stripe 1 and 5 enhancers. The weaker Knirps 1-330 protein had an effect similar to that of full-length Knirps expressed from an eve stripe 2 expression construct, i.e., a delay of stripe 3/4 separation. Interestingly, knirps is important for activation of hairy stripe 6, and the protein can bind to the stripe 6 enhancer directly in vitro. No evidence of activation is seen upon overexpression, however, suggesting that such activation might be indirect (Struffi, 2004).
The derepression of ftz observed between stripes 2-4 and 6-7 is likely due to indirect effects of repression of hairy and
eve expression; both of these genes are thought to repress
ftz directly. By contrast, previous work involving lower levels
of anteriorly expressed Knirps observed only weakened ftz stripes 2
and 3, rather than stripe fusion. This lower level of Knirps had a much less
profound effect on upstream regulators hairy and eve,
suggesting that Knirps might be a direct gap gene input to this pair-rule
gene (Struffi, 2004).
This study suggests that the multiple repression activities of Knirps can be simultaneously mobilized to provide quantitatively correct levels of repression activity, and that the design of cis regulatory elements can elicit CtBP dependence. CtBP-independent activity can in some cases be directly attributed to direct competition with activator for DNA binding; however, the CtBP-independent activity of Knirps can repress activators on elements where sites are not overlapping, and overexpression of the DNA-binding domain of Knirps (Knirps1-105) is insufficient to mediate repression of endogenous eve enhancers. Cell culture and transgenic embryo assays indicate that both CtBP-dependent and independent repression activities of Knirps have very similar characteristics with respect to activator specificity, distance dependence and overall potency, thus the targets and molecular mechanisms might well be similar in each case. Key to a deeper understanding of the molecular circuitry controlled by short-range repressors such as Knirps will be biochemical knowledge of the mechanisms of repression employed on these developmentally
regulated enhancers (Struffi, 2004).
The Drosophila pair-rule gene even skipped is required for embryonic segmentation and later in specific cell lineages in both the nervous system and the mesoderm. eve mesoderm-specific mutants have been generated by combining an eve null mutant with a rescuing transgene that includes the entire locus, but with the mesodermal enhancer removed. This allowed analysis in detail of the defects that result from a precisely targeted elimination of mesodermal eve expression in the context of an otherwise normal embryo. Absence of mesodermal eve causes a highly selective loss of the entire eve-expressing lineage in this germ layer, including those progeny that do not continue to express eve, suggesting that mesodermal eve precursor specification is not implemented. Despite the resulting absence of a subset of muscles and pericardial cells, mesoderm-specific eve mutants survive to fertile adulthood, providing an opportunity to examine the effects of these developmental abnormalities on adult fitness and heart function. In these mutants, flying ability, myocardial performance under normal and stressed conditions, and lifespan are all severely reduced. These data imply a nonautonomous role of the affected pericardial cells and body wall muscles in developing and/or maintaining cardiac performance and possibly other functions contributing to normal lifespan. Given the similarities of molecular-genetic control between Drosophila and vertebrates, these findings suggest that peri/epicardial influences may well be important for proper myocardial function (Fujioka, 2005).
Removing the mesodermal enhancer eme in the context of a rescue transgene removes all detectable mesodermal eve expression without affecting any other aspect of expression. When combined with a null mutation at the endogenous locus, this results in an eve 'mesoderm negative' (eve meso–) mutant. A separate transgene containing eme upstream of a reporter gene (lacZ) can be used to faithfully mark the cells that would normally express eve. In the absence of mesodermal eve, the body wall muscles no longer show reporter gene expression. In cells associated with the heart, however, the stable reporter protein persists until the formation of a seemingly normal heart tube. Except for the lack of eve expression, other heart cell types, particularly the myocardial cells, appear to be present in their normal numbers and positions. In the absence of eve, the cells in which the reporter persists appear to be naïve and able to adopt other (cardiac) fates, consistent with eve acting as an essential factor for specification of their lineage. Interestingly, the vertebrate eve homolog, Evx2, is also expressed in the heart, in an epicardial cell line, and in cells from primary epicardial explants. Thus, eve/Evx may play a role in heart development in both insects and mammals (Fujioka, 2005).
Mesodermal eve expression normally occurs in founder cells that give rise to a subset of pericardial cells and to 2 muscles per hemisegment, DO2 and DA1. A cell within 1 eve-expressing cluster (cluster 2) initiates expression of Krüppel (Kr). This Kr- and eve-expressing cell (progenitor 2) divides to yield two founder cells that express runt, one of which is the founder of DO2 and continues to express runt, and the other of which is the EPC founder and turns off runt. The DO2 founder turns off eve shortly after runt is activated, whereas the EPC founder and the resulting EPCs continue to express eve. A cell within a second eve-expressing cluster (cluster 15) activates Kr, then divides to yield the DA1 founder and a second cell that is fated to die. The DA1 muscle maintains eve expression (Fujioka, 2005).
Previous studies have suggested that eve function is required for normal EPC differentiation and for the normal pattern of expression of ladybird. As a way to define the role of eve, both runt and Kr expression were examined in eve meso– embryos. In the absence of eve, both runt and Kr expression are either completely absent from or dramatically reduced in these muscle founder lineages. This strongly suggests that eve is required for these cells to adopt their normal fates. Thus, eve has either a direct or an indirect role (repression of a repressor) in activating Kr and runt (Fujioka, 2005).
To determine the extent to which eve function is required for normal muscle formation, the musculature was examined in eve meso– third instar larvae. The normal arrangement of dorsal muscles within each segment is clearly altered. Both DA1 and DO2 are severely defective or aberrant, and other muscles in the vicinity exhibit alterations in their placement and size. A simple interpretation of the effects seen in a majority of segments is that DA1 is missing, and a single, large muscle occupies the normal positions of DO1 and DO2. The muscle occupying the normal position of DA2 is also enlarged. These enlarged muscles suggest that myoblasts that normally fuse with the DA1 and DO2 founders may instead fuse with other muscles nearby, or, alternatively, that in the absence of DA1 and DO2, attachment sites for adjacent muscles expand (Fujioka, 2005).
The DA1 and DO1 muscles are innervated in wild-type embryos by motorneurons that express eve. Transgenes that express green fluorescent protein (GFP) in these neurons were used to label their axons in the muscle field. In eve meso– embryos, one or both muscles in the normal positions of DA1/2 and DO1/2 are innervated by these eve-expressing neurons. It was also found that in eve meso– third instar larvae, both DA1/2 and DO1/2 are innervated (Fujioka, 2005).
The changes in gene expression observed in eve meso– embryos suggest that eve acts, directly or indirectly, as an activator of Kr and runt. Previous analyses of eve function suggested that it acts as a repressor of transcription. If this is true in the mesoderm, then at least one intermediary gene that is repressed by eve normally represses runt and Kr. To study the domain requirements of eve, transgenes were expressed in the eve meso– background that contained the eve mesodermal enhancer driving expression of modified Eve proteins. With the wild-type Eve coding region, the mesodermal defect is completely rescued. In contrast, when the Eve homeodomain (HD)-containing region alone is so expressed, a very limited degree of rescue is observed. Importantly, when the heterologous Engrailed repressor domain is added to the HD construct, full rescuing ability is restored, implying that eve acts exclusively as a repressor in the mesoderm (Fujioka, 2005).
The ability of Eve to act as a direct repressor in the mesoderm was examined by targeting it to a reporter transgene using the Gal4 DNA binding domain. When a Gal4-Eve fusion protein containing both of the repressor domains of Eve is combined with an eve lineage-specific Gal4-UAS-containing reporter, the reporter is strongly repressed (Fujioka, 2005).
eve meso– embryos develop into viable adults, providing an opportunity to examine the role of PC cells in larval and adult heart function. The absence of mesodermal eve does not noticeably affect the assembly of the myocardial cells at the dorsal midline, which will give rise to the contractile part of the heart tube. To examine the contribution of EPCs to larval heart development, eve meso– larvae were dissected and PC cells were counted. Wild-type and wild-type eve-rescued larvae show 7 to 8 PC cells per segement, and eve meso– heterozygotes show 6. In contrast, two independent eve meso– lines displayed a marked reduction, with an average of only 3 to 4 PC cells per segment. Thus, eve is required to produce the normal complement of larval PC cells (Fujioka, 2005).
Because eve meso– animals are missing half or more of their PC cells, the effect on heart function was examined in pupae and adults. Neither wild-type eve-rescued pupae nor those heterozygous for eve and carrying one copy of a meso– rescue transgene exhibited heart rates that differed significantly from controls. However, all 3 meso– lines, which carry independent transgene insertions, exhibited a significant reduction (30% to 50%) in heart rate (Fujioka, 2005).
Adults of the same genotypes were examined to assess whether defective functions persist through partial remodeling of the heart during metamorphosis. The wild-type heart rate is 2.9 beats/second (Hz) in 1-week-old adults and 2.6 Hz in 3-week-old adults. Compared with wild type, both the eve null background rescued by a wild-type rescue transgene and the eve meso– heterozygotes (J49/CyO) exhibit a reduced heart rate at both ages, probably attributable to a genetic background effect inherent to these eve rescue lines that serve as controls. eve meso– flies, however, develop a dramatically lower heart rate with age, and one of the meso– lines also shows a severely reduced heart rate at an early age (Fujioka, 2005).
Heart function can also be assayed by quantifying stress tolerance, using an external current to briefly pace the heart to about twice the normal rate, then charting the percentage that undergo either fibrillation or cardiac arrest (termed heart failure). In wild-type flies, the ability of the heart to withstand such stress is highly age-dependent, with stress-induced failure rates increasing dramatically (2- to 3-fold) between 1 and 5 weeks of age.Because eve meso– flies seldom reach 5 weeks of age, flies at one and three weeks of age were examined. Neither heterozygous eve meso– nor eve rescued flies differed from wild type. In contrast, one eve meso– line showed a significantly increased failure rate at 1 week of age, whereas the other showed a disastrously high failure rate at 3 weeks of age. Although dorsal somatic muscle defects might also conceivably affect heart function, these results suggest that the reduction in PC cell number causes a slowed heartbeat and reduces cardiac stress resistance (Fujioka, 2005).
To further assess the importance of fully functional heart and muscle activity and potentially of other results of mesodermal eve expression, the life spans of eve meso– flies was examined. Such flies display a significantly reduced mean and maximal lifespan, suggesting that the presence of mesodermal Eve is required not only for normal activity levels but also for a normal lifespan (Fujioka, 2005).
Thus eve is required not only in the eve-expressing lineages in which it is maintained during terminal differentiation (the eve-expressing pericardial cells and the DA1 muscle), but also in the lineage in which it is expressed in the progenitor but turned off in the muscle founder cell and the resulting DO2 muscle. Thus, both DA1 and DO2 require mesodermal eve to be specified, and without this specification, the pattern and size control of some remaining muscles are compromised. Importantly for heart function, in the absence of mesodermal eve expression, the majority of the large larval PC cells are missing (Fujioka, 2005).
It is intriguing that even though ectopic Eve expression can interfere with the DO2 fate, and eve is normally turned off as runt is activated in the lineage, eve function is nonetheless required for DO2 formation, apparently because of a requirement in the progenitor before the lineage divisions (Fujioka, 2005).
When normal eve expression in the mesoderm is replaced by expression of the eve HD (with repressor domains deleted), a similar but less severe muscle deficiency is observed compared with the complete absence of mesodermal eve. In particular, a muscle in the DO2 position is usually formed, whereas DA1 is still absent. Additionally, there is occasionally an extra muscle ventral to DO2, as if the DO2 founder was duplicated. Importantly, however, when the heterologous Engrailed repressor domain is added to the HD construct, full rescuing ability is restored. This suggests that Eve functions in the mesoderm primarily or exclusively as a repressor, and in turn that eve acts indirectly to activate Kr and runt in the mesoderm. Good candidates for intermediary repressors are ladybird and the muscle identity gene msh (Fujioka, 2005).
A reduced number of larval PC cells (and dorsal somatic muscles) caused by a lack of mesodermal eve expression results in severely compromised heart function and is likely to contribute to a shortened lifespan. A less drastic effect on cardiac performance and lifespan is observed when manipulating insulin signaling exclusively in the heart. The functional role of pericardial cells in insect hearts is not well understood, but they may contribute to heart function by secreting hormones or by gathering such peptides from circulating hemolymph and 'presenting' them to the myocardium. It has been suggested that pericardial cells may function as nephrocytes, and at this point it cannot be ruled out that a potential accumulation of toxic agents, as a consequence of fewer pericardial cells, contributes to the observed phenotypes (Fujioka, 2005).
As in insects, the developmental and functional interactions between the vertebrate epicardium and the myocardium are not well understood. Recent studies have suggested that the loss of epicardial function results in impaired growth of the myocardium at mid-gestation. The epicardium is thought to be a source of signals and secreted factors that affect myocardial proliferation and differentiation, as well as influencing formation of the conduction system. Even though it cannot yet be decided whether the mammalian epicardium has a developmental program in common with a fly’s pericardial cells, they both depend on GATA factors for formation. In addition, the Evx2 homolog of eve is indeed expressed in the mammalian heart, including in epicardial tissue. These findings are consistent with pericardial cells in Drosophila functioning as a source of signals that affect the myocardium. Possibly because the myocardium, which is maintained by proliferation in vertebrates, does not proliferate in flies after it is developmentally specified, pericardial deficiency does not appear to result in morphological heart defects. Rather, defects manifest themselves as functional deficits. This provides an opportunity to study the influence of these heart-associated cell types on cardiac physiology in the absence of myocardial defects. Epicardial lineages in vertebrates may contribute analogously to normal cardiac physiology and performance (Fujioka, 2005).
Although epigenetic maintenance of either the active or repressed transcriptional state often involves overlapping regulatory elements, the underlying basis of this is not known. Epigenetic and pairing-sensitive silencing are related properties of Polycomb-group proteins, whereas their activities are generally opposed by the trithorax group. Both groups modify chromatin structure, but how their opposing activities are targeted to allow differential maintenance remains a mystery. This study identified a strong pairing-sensitive silencing (PSS) element at the 3' border of the Drosophila even skipped (eve) locus. This element can maintain repression during embryonic as well as adult eye development. Transgenic dissection revealed that silencing activity depends on a binding site for the Polycomb-group protein Pleiohomeotic (Pho) and on pho gene function. Binding sites for the trithorax-group protein GAGA factor also contribute, whereas sites for the known Polycomb response element binding factors Zeste and Dsp1 are dispensible. Normally, eve expression in the nervous system is maintained throughout larval stages. An enhancer that functions fully in embryos does not maintain expression, but the adjacent PSS element confers maintenance. This positive activity also depends on pho gene activity and on Pho binding. Thus, a DNA-binding complex requiring Pho is differentially regulated to facilitate epigenetic transcriptional memory of both the active and the repressed state (Fujioka, 2008).
This study dissected a strong pairing sensitive silencing element
from the 3' boundary of the eve locus. It was found that silencing
activity depends on a single Pho-binding site, whereas sites for a number of
other proteins found in such elements are less important. The element is
genetically responsive to PcG-group activity, as it depends on pho
gene function. This eve 3' PRE has bona fide PRE activity,
which can maintain a silenced state established in embryos (Fujioka, 2008).
Previous studies have suggested that PRE-containing P-element-based
transgenes have a tendency to insert near endogenous PREs, and that this can
bias reporter gene expression. This study applied both P-element analysis and
the {Phi}C31 recombinase-mediated cassette exchange (RMCE) system to compare the effects of mutating binding sites. The data
reveal that there is also variation in PRE effects using RMCE into different
target sites. Therefore, it would seem important to test several target sites
when using RMCE, to ensure that results are not specific to one chromosomal
location. Furthermore, where sensitivity to position effects is high, such as
with GAGA factor (GAF) site-mutated PRE, it remains valuable to use the standard
methodology to probe a variety of insertion sites (Fujioka, 2008).
Surprisingly, it ws found that the eve PRE is also required for
positive maintenance of expression in the larval CNS, and that this activity
requires both the Pho-binding site and pho gene function. Together,
these data strongly suggest that Pho is directly involved in positive
maintenance of gene activity. This is surprising because Pho has heretofore
been associated only with direct repression of target genes, by recruiting the
PRC2 complex and other PcG proteins. However, recent studies have blurred the
distinction between PcG genes and trxG genes, as some members of each class
appear to have dual functions. Furthermore, PREs usually reside in close proximity to
TREs, and an element from the promoter region of engrailed that mediates PSS and can act as a PRE was recently shown to have an activating role in its natural context (Devido, 2008). Recent studies of the Ubx locus have indicated that PcG proteins are present at PREs in both the off and the on state, and that binding of Ash1 prevents
silencing by the PRC complex in cells where Ubx is expressed,
suggesting that silencing is actively prevented. A
similar situation may pertain to Pho function in the eve locus (Fujioka, 2008).
Because trxG proteins are known to be involved in positive regulation by
other maintenance elements, it was of interest to see in whether they are involved in
pho-dependent positive maintenance by the eve PRE. It was
also of interest to see in whether other PcG proteins are involved. Because the
positive maintenance assay requires survival to the third larval instar, so far it has not been possible to test only weak alleles of trx, Trl and
E(z), none of which showed discernable effects in the assays that were used. At
this point, it cannot definitively be said whether other trxG or PcG proteins are
involved in the positive maintenance function of the eve PRE.
However, the observation that a consensus Grh binding site is present in the
more active half of PRE300 suggests the involvement of Grh. Indeed, Grh has
been shown to interact genetically with Pho, and to facilitate cooperative
interaction with Pho in vitro (Fujioka, 2008).
Consistent with the broad overexpression of eve seen in the CNS of
ph mutants, the eve PRE may silence expression in many cells
by forming a silencing complex. In wild-type embryos, in the subset of CNS
cells where eve is expressed, the same Pho-dependent DNA binding
platform may recruit a distinct complex that maintains the active state.
Consistent with this model, it was found that expression driven by the
eve RP2+a/pCC enhancer fades prematurely in late stage embryos in ph mutants, at the same time that endogenous eve is broadly overexpressed. It will be interesting to determine the composition of Pho-dependent complexes in cells where eve is on, and in those where eve is off (Fujioka, 2008).
How can a region 9 kb away from the basal promoter affect the state of gene
expression? There are accumulating data suggesting that locus-wide regulation
occurs through direct interactions of the promoter with enhancers and locus
control regions. For example, a recent study showed that silencing by the
bxd PRE directly affects the activity of the transcriptional
machinery at the promoter. In the eve locus, there are PSEs both at the
3' end of the locus and at the promoter. Both contain clusters of
binding sites typical of a PRE/TRE. It has been suggested that PRE-containing
transgenes have a tendency to insert near endogenous PREs, which
might be expected if they mediate long-range interactions. Putting these ideas
together, the eve 3' PRE may physically interact with the
promoter region in a Pho-dependent manner. This may serve to keep eve
on in some cells and to keep it off in others, depending on whether activating
or repressive complexes mediate the association (Fujioka, 2008).
Insulator sequences help to organize the genome into discrete functional regions by preventing inappropriate cross-regulation. This is thought to be mediated in part through associations with other insulators located elsewhere in the genome. Enhancers that normally drive Drosophila even skipped (eve) expression are located closer to the Ter94 transcription start site than to that of eve. It was discovered that the region between these genes has enhancer-blocking activity, and that this insulator region also mediates homing of P-element transgenes to the eve-TER94 genomic neighborhood. Localization of these activities to within 0.6 kb failed to separate them. Importantly, homed transgenic promoters respond to endogenous eve enhancers from great distances, and this long-range communication depends on the homing/insulator region, which has been called Homie. The eve promoter contributes to long-distance communication. However, even the basal hsp70 promoter can communicate with eve enhancers across distances of several megabases, when the communication is mediated by Homie. These studies show that, while Homie blocks enhancer-promoter communication at short range, it facilitates long-range communication between distant genomic regions, possibly by organizing a large chromosomal loop between endogenous and transgenic Homies (Fujioka, 2009).
Some of the eve enhancers are close to the TER94
promoter, yet they do not activate TER94. Although TER94 is
expressed nearly ubiquitously in embryos, it is expressed only at a low level
in the mesoderm and anal plate, where eve expression is high in a
subset of cells, making it unlikely that eve enhancers acting on
TER94 would be masked by this expression. Therefore, something
isolates TER94 from eve enhancers (and probably vice versa).
Indeed, the region between the 3'-most eve regulatory element,
a PRE, and the TER94 transcription start site has the properties of
an enhancer-blocking insulator. It exhibits directional
enhancer blocking in transgenes carrying eve enhancers in combination
with either the eve promoter region or heterologous promoters, as
well as between heterologous enhancers and promoters (Fujioka, 2009).
This insulator region was dissected in the context of transgenes carrying
two different enhancers between divergently transcribed reporter genes. Some deletion mutants were still able to block the AR enhancer from activating the
mini-white reporter, while allowing the eve
mesodermal enhancer to activate the eve-promoter-lacZ
reporter across the mutant insulator. This might result from a relatively weak interaction between the eve AR enhancer and the heterologous mini-white promoter, which suggests a degree of specificity of eve enhancers for their cognate promoter. This mechanism also contributes to long-range E-P communication mediated by the insulator. Furthermore, the
recently discovered presence of an insulator at the 3' end of
mini-white might contribute to stronger enhancer blocking in this
direction (Fujioka, 2009).
First enhancer-blocking activity was narrowed down to an 800 bp sequence
that spans the 5' end of TER94. Further dissection showed that the start site of TER94 is not required. This makes it unlikely
that transcriptional interference makes a
strong contribution to the results, although it could be significant in some
cases, such as for δF, which retains the TER94 start site.
Notably, region F, extending from ~150 to 45 bp upstream of this start
site, seems particularly important for enhancer blocking. A similar situation
pertains to the well-studied insulators scs and scs'. Perhaps
some promoter regions induce a chromatin configuration that blocks the
progression of activating complexes or chromatin modifications, through which
enhancers communicate with target promoters (Fujioka, 2009).
The region between eve and TER94 also induces transgene homing. About 7% of transgenes carrying this region (27 out of 380 lines tested) inserted within 180 kb of eve. Among 27 homed lines, eight inserted within 1.5 kb of the endogenous insulator, suggesting that homing involves direct tethering, possibly through a homophilic protein complex formed on the element in the germline, where transgenic insertion occurs. The responsible element has been called Homie, for homing insulator at eve (Fujioka, 2009).
Although it is more difficult to dissect the region required for homing
than it is to dissect the region required for enhancer blocking (due to the
number of transgenic insertions required to validate a negative result), there
is a clear correlation between these activities. Of the 210 transgenes tested
for homing that carry all or part of the 800 bp R100 insulator, nine of them (4.3%) were homed, even though the `homed' region is less than 0.4% of the genome. Protein-protein interactions among insulators, when they occur in the
germline, might lead to transgene homing (Fujioka, 2009).
In previous studies of the eve 3' region, hundreds of lines were produced that carried the eve PRE, yet homing was not observed. Therefore, the eve PRE is not sufficient for homing.
Furthermore, as the minimal homing element does not contain the PRE, this PRE
is not required for either homing activity or long-range E-P communication.
However, the engrailed homing region has PRE activity,
indicating that some PREs may engage in homotypic interactions that facilitate
homing. Consistent with this, long-range interactions among PREs were seen in
the BX-C. Furthermore, the engrailed PRE may also facilitate
long-distance E-P communication (Fujioka, 2009).
The eve-promoter-lacZ reporter in a homed transgene is
usually expressed in a full eve pattern, showing communication with
all of the endogenous eve enhancers from as far away as 180 kb, and
across a number of other genes. Beyond
the homing target region, there is a tendency for Homie-carrying transgenes to
insert on chromosome 2R, particularly centromere proximal from eve. These insertions have not been referred to as 'homed',
mainly to distinguish them from transgenes that pick up a full eve
pattern of expression. However, they usually (9 out of 12) pick up a partial
eve pattern. Intriguingly, Homie-carrying transgenes inserted as far
as 3300 kb away, are capable of interacting with the
endogenous eve AR and mesodermal enhancers. Previous
indications of long-range E-P interactions mediated by transgenic insulators
have come from the genetic and phenotypic analysis of transvection and
related regulatory interactions (Fujioka, 2009).
The requirement for Homie in long-range E-P
communication was directly tested using PhiC31-RMCE to compare transgenes with and without this region at the same chromosomal insertion site. Removal of Homie resulted in complete loss of the eve pattern. The same results were obtained at two different landing sites, at opposite ends of the homing region.
Communication of distant 'shadow' enhancers with promoters across several
intervening genes has recently been proposed, based upon bioinformatics-based
identification of functionally conserved enhancer regions with no other
apparent target promoters. The results suggest that for such distant enhancers to
communicate effectively, they may need promoter-targeting and/or
promoter-tethering sequences, and that some of these sequences might also act as
insulators, generating a chromosomal architecture that facilitates functionally important interactions while preventing deleterious ones (Fujioka, 2009).
How does Homie mediate such long-range E-P communication? Both preferential
insertion and the ability to pick up a partial eve pattern from long
range could be explained by a homologous tethering mechanism, if it is
assumed that this region of 2R is in relative proximity to the eve
locus within a chromosome territory,
both in the germline and in the developing AR and mesoderm. Homologous
tethering might stabilize a functional E-P interaction, which in turn might
facilitate transcription initiation through a combination of mechanisms,
including targeting to regions of active transcription within the nucleus (Fujioka, 2009).
{Phi}C31 recombinase-mediated cassette exchange was used to test the role of promoter specificity in long-range communication. Exchanging a basal hsp70 promoter for the eve promoter caused a complete loss of communication with some endogenous eve enhancers but not others. The communication that remained was with the AR and mesodermal enhancers, the same ones that often communicate with either the eve or hsp70 promoters in transgenes
inserted up to 3300 kb away. The ability of these enhancers to communicate at a much longer range than others might indicate relatively stable E-P interactions that can survive entropic forces tending to randomize their positions in the nucleus. Alternatively, the interactions of these enhancers might be specifically facilitated by Homie (Fujioka, 2009).
Another indication of the effects of promoter specificity in long-range E-P
communication is that when the eve promoter was replaced by that of
hsp70, β-gal reporter expression in the CNS changed from an
eve-like pattern to one similar to that of TER94. Although it is
possible that this TER94-like expression is driven by enhancers
located near the insertion site, it is clear that which enhancers are targeted
by the transgenic promoter depends in part on promoter specificity. Similar
influences have recently been found on E-P communication at the
engrailed locus (Fujioka, 2009).
How can Homie act as an insulator and also mediate long-range
communication? The key may lie in the details of the resulting chromosomal
architecture. Precedence for this idea comes from the phenomenon of insulator
bypass, in which the enhancer-blocking activity of a single insulator can be
negated by placing a second insulator between the enhancer and promoter. This
phenomenon is consistent with data from those homed insertions that lie just
downstream of endogenous Homie. In these cases, both the transgenic and
endogenous Homies are interposed between the lacZ reporter and the
endogenous enhancers that drive its expression. The data also show that the
apparent bypass of endogenous Homie does not require that transgenic Homie
lies between the interacting enhancer and promoter. In one case, the
transgenic promoter lies between the two Homies, with the interacting
enhancers on the outside. It is proposed that Homie has directionality, so that
the two copies of Homie line up in parallel with each other within a wall-like
structure. In the cases where both Homies are between the interacting enhancer
and promoter, the Homies are inverted in orientation, whereas in the other
case they are in the same orientation. In both cases, their lining up in
parallel would tend to place the interacting enhancer and promoter on the same
side of this wall-like structure, facilitating their communication. By
contrast, a single copy of Homie would tend to block communication between
sequences on either side, by placing them on opposite sides of the structure.
Similar effects of insulator directionality have been seen for the
Fab-8 and Mcp insulators (Fujioka, 2009).
In most homed lines, mini-white expression is not seen in an
eve pattern. This might be due to the mini-white promoter
being relatively weak and/or less compatible with eve enhancers than
is the eve promoter, or even the hsp70 promoter, which also
often picked up AR or mesodermal enhancer activity from great distances
(facilitated by Homie). Intriguingly, however, although in most of the
transgenes carrying Homie its 5' end was oriented toward the
lacZ reporter, in one line (inserted at +46 kb), this orientation was
reversed, and in that line mini-white was expressed in the
eve pattern. Thus, it is possible that Homie directionality, through
the mechanism described above for insulator bypass, might play a role in
determining whether or not a weak E-P interaction is facilitated (Fujioka, 2009).
There are two likely possibilities for how Homie functions in the
regulation of eve and TER94. The first is that it simply
prevents eve enhancers from activating TER94, and also
prevents eve from being expressed broadly in the CNS like
TER94, which would probably cause mis-specification of neurons.
Another, not mutually exclusive, possibility is that Homie works in
conjunction with the nearby PRE to orchestrate functionally appropriate
chromosomal architectures during development. Known insulators in the BX-C are
each situated near a PRE, and these PRE-insulator regions interact with promoters in several contexts. The data suggest a similar interaction with the
eve promoter region, based on the fact that three of the homed lines
are inserted within the eve promoter region. Such an interaction
might help enhancers from the 3' end of the eve locus
communicate with the eve promoter, while also preventing
inappropriate interaction with TER94 enhancers. One motivation for
such a model is that in mutants for the PcG gene polyhomeotic, eve is
ectopically expressed throughout the CNS, which
is reminiscent of normal TER94 expression. Thus a loss of PcG
repression, acting through the PRE, might disrupt the normal insulator
function that prevents inappropriate activation of eve. This suggests
that the functions of the PRE and Homie are coordinated during development,
allowing the PRE to maintain either an activated or repressed state of
eve in different cells, while maintaining the functional isolation of
eve from TER94 (Fujioka, 2009).
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).
Immunostaining experimental data was examined for the formation of stripe 2 of even-skipped (eve) transcripts on D. melanogaster embryos. An estimate of the factor converting immunofluorescence intensity units into molecular numbers is given. The analysis of the eve dynamics at the region of stripe 2 suggests that the promoter site of the gene has two distinct regimes: an earlier phase when it is predominantly activated until a critical time when it becomes mainly repressed. That suggests proposing a stochastic binary model for gene transcription on D. melanogaster embryos. The model has two random variables: the transcripts number and the state of the source of mRNAs given as active or repressed. It was possible to reproduce available experimental data for the average number of transcripts. An analysis of the random fluctuations on the number of eve mRNAs and their consequences on the spatial precision of stripe 2 is presented. The position of the anterior or posterior borders was shown to fluctuate around their average position by approximately 1% of the embryo length, which is similar to what is found experimentally. The fitting of data by such a simple model suggests that it can be useful to understand the functions of randomness during developmental processes (Prata, 2016).
Continued: even-skipped Transcriptional regulation part 3/3 | back to
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