Transcriptional Regulation Table of contents

Structure of promoter

In Drosophila embryos cubitus interruptus activity is both necessary and sufficient to drive expression of HH-responsive genes, including wingless, gooseberry and patched. To demonstrate that ci is required for transduction of the HH signal, expression of wg was examined in ci null embryos when HH is ubiquitously expressed under control of a heat-shock promoter (Hs-hh). In Hs-hh embryos, wg is expressed ectopically in anteriorly expanded stripes. In ci mutants Hs-hh does not induce ectopic expression of wg. Similar results were obtained for gsb. CI is a sequence-specific DNA binding protein that drives transcription from a wingless promoter in transiently transfected cells. CI binds to the same 9 bp consensus sequence -TGGGTGGTC- as mammalian Gli and Gli3. Alteration of a single nucleotide in the core sequence prevents binding. CI activates transcription from a 5-kb fragment of the wg promoter. CI binding sites in the wg promoter are necessary for this transcriptional activation of. CI element maps to a distal 1-kb region of the 5-kb fragment. The wg promoter sequence has 10 possible Gli consensus binding sites, with three pairs of sites in the distal 1.2 kb. When putatitive CI binding sites are mutagenized, mutant fragments show a greater than 90% reduction in CI-dependent transcriptional activation. Mutagenesis of these sites completely eliminates an electrophoretic mobility shift caused by binding of CI to unmutagenized sites (Von Ohlen, 1997a).

A wingless enhancer region has been described whose Cubitus interruptus (Ci) binding sites mediate Ci-dependent transcriptional activation in transiently transfected cells. Hedgehog (Hh) and Patched (Ptc) act through those Ci binding sites to modulate the level of Ci-dependent transcriptional activation in S2 cells. To test for effects of Ptc and Hh, titrations of Ci cDNA in cultured cells were performed on an expression vector regulated by this enhancer region. The titrations were performed either in the presence of Hh cDNA or in the presence of Ptc cDNA. Reporter activity is reduced 3-fold in the presence of co-transfected Ptc. The addition of Hh results in a 1.5-fold increase in reporter activity over that observed for Ci alone. This same wg enhancer region is Hh responsive in vivo and its Ci binding sites are necessary for its activity. This provides strong evidence that Hh affects wg transcription through post-translational activation of Ci (Von Ohlen, 1997b).

Patterning of the Drosophila embryo depends on the accurate expression of wingless (wg), which encodes a secreted signal required for segmentation and many other processes. Early expression of wg is regulated by the nuclear proteins of the gap and pair-rule gene classes but, after gastrulation, wg transcription is also dependent on cell-cell communication. Signaling to the Wg-producing cells is mediated by the secreted protein, Hedgehog (Hh), and by Cubitus interruptus (Ci), a transcriptional effector of the Hh signal transduction pathway. The transmembrane protein Patched (Ptc) acts as a negative regulator of wg expression; ptc- embryos exhibit ectopic wg expression. According to the current models, Ptc is a receptor for Hh. The default activity of Ptc is to inhibit Ci function; when Ptc binds Hh, this inhibition is released and Ci can control wg transcription. An investigation was carried out of the cis-acting sequences that regulate wg during the time that wg expression depends on Hh signaling. A region consisting of 4.5 kb immediately upstream of the wg transcription unit can direct expression of the reporter gene lacZ in domains similar to the normal wg pattern in the embryonic ectoderm. Expression of this reporter construct expands in ptc mutants and responds to hh activity. Within this 4.5 kb, a 150 bp element, highly conserved between D. melanogaster and Drosophila virilis, is required to spatially restrict wg transcription. Activity of this element depends on ptc, but it contains no consensus Ci-binding sites. The 150 bp G box, 91% identical with its counterart from D. virilis, mediates repression of wg in a ptc-dependent manner. The G box is sufficient for conferring wild-type width to reporter stripes, which in turn expand in a ptc mutant background, thus behaving like wg itself. Deletion of element G result in wide stripes in a wild-type embryo, suggesting that this is a binding site for a transcriptional repressor active in cells anterior to the wild-type wg domain. A repressor that binds element G could possibly act in parallel to ptc and hh; in such a case, the repressor's activity would be overcome by an Hh-regulated activator, i.e. Ci. The simpler explanation is that a repressor functions as another endpoint of Hh signaling. The discovery of an element that is likely to bind a transcriptional repressor was unexpected, since the prevailing model suggests that wg expression is principally controlled by Hh signaling acting through the Ci activator. It is shown that wg regulatory DNA can drive lacZ in a proper wg-like pattern without any conserved Ci-binding sites (Lessing, 1998).

Hox genes encode evolutionarily conserved transcription factors that play fundamental roles in the organization of the animal body plan. Molecular studies emphasize that unidentified genes contribute to the control of Hox activity. This study describes a genetic screen designed to identify functions required for the control of the wingless (wg) and empty spiracles (ems) target genes by the Hox Abdominal-A and Abdominal-B proteins. A collection of chromosomal deficiencies were screened for their ability to modify GFP fluorescence patterns driven by Hox response elements (HREs) from wg and ems. Fifteen deficiencies were found that modify the activity of the ems HRE and 18 that modify the activity of the wg HRE. Many deficiencies cause ectopic activity of the HREs, suggesting that spatial restriction of transcriptional activity is an important level in the control of Hox gene function. Further analysis identified eight loci involved in the homeotic regulation of wg or ems. A majority of these modifier genes correspond to previously characterized genes, although not for their roles in the regulation of Hox targets. Five of them encode products acting in or in connection with signal transduction pathways; this suggests an extensive use of signaling in the control of Hox gene function (Marabet, 2002).

This study surveyed 60% of the genome and 11 genomic regions were found acting as recessive activators of ems HRE; 4 were found acting as recessive repressors of ems HRE, and 18 were found acting as recessive repressors of wg HRE. So far, the only known gene in addition to AbdB required for ems activation is lines. Df(2R)H3E1, which uncovers lines, has been recovered from the screen for AbdB modifiers. A search for discrete mutations that reproduce the deficiency phenotypes allowed identification of four ems HRE modifier genes: dally, ds, scw, and ttk. Although ttk and scw have already been linked to filzkörper development, none of the four genes had previously been involved in the control of ems expression in posterior spiracles. The screen for AbdA modifiers was restricted to genomic regions leading to ectopic activation of the wg HRE; these response elements relate to functions that repress the enhancer. Accordingly, genomic regions or genes already known to play a role in wg activation, such as abdA, exd, hth, or genes coding for components of the Dpp signaling pathway, were not recovered. Five mutations at specific loci reproduce the phenotypes caused by original deficiencies. Four of these mutations identify tsl, ttk, and genes encoding a putative MPK and a putative CBP as candidate modifiers of wg HRE. None of these genes has so far been involved in the regulation of wg in the visceral mesoderm (Marabet, 2002).

Two modifier genes obtained from the wg screen are presumably involved in the signal transduction cascade. The first, tsl, encodes a ligand for the RTK Torso receptor and the second encodes a putative MKP. Signaling by Ras/MAPK could thus be part of the genetic network that controls wg expression in the midgut, which has been confirmed by showing that wg transcription is impaired by a constitutive active form of Ras. Interestingly, the Ras/MAPK pathway has been implicated in regulation of the Ubx and lab enhancer in the central midgut, and the ETS-domain-containing transcription factor Pointed, which acts as a nuclear effector of the Ras/MAPK pathway, is expressed in the third midgut chamber (Marabet, 2002).

Several modifiers of wg and ems HRE activities identified in this study encode molecules acting in signal transduction cascades. This indicates that signaling processes play important roles in the control of Hox gene function and extends previous observations from a screen for modifiers of a dominant Pb phenotype. Understanding how cell signaling and transcriptional control by Hox protein are mechanistically integrated requires further study (Marabet, 2002).

Hox proteins play fundamental roles in generating pattern diversity during development and evolution, acting in broad domains but controlling localized cell diversification and pattern. Much remains to be learned about how Hox selector proteins generate cell-type diversity. In this study, regulatory specificity was investigated by dissecting the genetic and molecular requirements that allow the Hox protein Abdominal A to activate wingless in only a few cells of its broad expression domain in the Drosophila visceral mesoderm. The Dpp/Tgfß signal controls Abdominal A function, and Hox protein and signal-activated regulators converge on a wingless enhancer. The signal, acting through Mad and Creb, provides spatial information that subdivides the domain of Abdominal A function through direct combinatorial action, conferring specificity and diversity upon Abdominal A activity (Grienenberger, 2003).

AbdA is expressed and is active in the third and fourth compartments of the midgut (PS8-PS12), and yet it activates the wg target gene only in PS8. Dpp secreted from PS7 is shown to provide the spatial information required for PS8-localized wg activation and, acting through a newly identified 546 bp enhancer, AbdA and Mad, a transcriptional effector of the Dpp pathway, directly control wg transcription. The convergence of Hox function and Dpp signaling therefore occurs at the levels of DNA and transcription, and endows AbdA with PS8-specific regulatory properties (Grienenberger, 2003).

To identify the enhancer responsible for wg expression in the VM, subfragments of a 9kb genomic region known to drive wg embryonic expression were analyzed in transgenic lines transformed with lacZ reporter constructs. The smallest fragment that drives accurate expression in the VM is a 546 bp XhoI/ClaI (XC) restriction fragment. Its activity is first detected during germ-band retraction, when wg transcripts are visualized in the VM by in situ hybridization, and only in PS8 VM cells. During subsequent development, XC enhancer activity still mimics wg expression, and is associated with the site of central midgut constriction formation. Thus, from early on to the end of embryogenesis, the XC enhancer exclusively and accurately recapitulates wg spatiotemporal expression in the VM (Grienenberger, 2003).

To address whether AbdA and Dpp signaling could directly regulate wg, the sequence of the XC enhancer was examined for the presence of putative binding sites for AbdA and for Mad/Medea (referred to as DRS, for Dpp response sequence), the canonical transcriptional effectors of the Dpp signaling pathway known to recognize identical target sequences. Since genetic and molecular data led to the proposal that, in Drosophila, the CRE sequences to which Creb proteins bind are required to respond to Dpp in addition to DRSs, potential Creb binding sites were sought. Six TAAT core sequences and four sequences resembling the consensual Hox/Pbx binding sites (TGATNNATG/TG/A) were identified as potentially mediating AbdA function. The Hox/Pbx 3 and 2 sequences strongly match the consensus, with seven or six of the eight consensus nucleotides conserved, respectively. Hox/Pbx sequences 1 and 4 only have five of the eight consensus nucleotides conserved. The XC fragment contains three sequences matching DRSs and two potential CRE sites (Grienenberger, 2003).

To assess the evolutionary conservation of the XC enhancer, an homologous fragment from Drosophila virilis was isolated and analyzed for its in vivo activity by transgenesis in Drosophila melanogaster. The D. virilis fragment drives expression in a pattern very similar to that of the XC enhancer, suggesting that sequences conserved between these two enhancers may be important for wg regulation in the midgut. Sequence comparison, including sequences from D. pseudoobscura, revealed that a majority of the TAAT core motifs, the DRSs and the putative Creb-binding sequences are evolutionarily conserved, whereas sequences that match heterodimeric Hox/Pbx consensus binding sites are not. The existence of two large conserved sequences, Box 1 and 2, is noted. Since Box1 lies in a fragment that does not drive reporter gene expression in transgenic flies, particular attention was paid to Box2 (Grienenberger, 2003).

Hox signaling integration was examined to determine whether signaling pathways contribute towards specifying how AbdA, a widely expressed Hox selector protein, controls the development of distinct pattern elements at different locations. Dpp signal secreted from PS7 provides the positional cue responsible for localized activation of wg by AbdA. Biochemical and reverse genetics experiments have established that AbdA and Mad directly regulate wg transcription through the XC enhancer, which thus serves as an integrator of Hox and Dpp input. AbdA is impotent with respect to this enhancer in the absence of the Dpp signal, though it can function perfectly well on other genes without Dpp. Therefore, functional interactions between selector proteins and signaling pathways confer specificity to signaling pathways, and reciprocally confer functional diversity to selector proteins (Grienenberger, 2003).

This study provides a conceptual framework for understanding the molecular basis of regional Hox protein transcriptional activity. Dpp and Wg signaling subdivide the AbdA Hox domain, allowing activation of pointed (pnt) and opa target genes in the third and fourth midgut chambers, respectively. Based upon the data presented here, it is suspected that the localized activation of pnt and opa by AbdA also relies on direct enhancer integration of Hox and signaling inputs. Accordingly, a Hox/signaling combinatorial code functionally subdivides the domain where a single Hox protein is made, giving rise to discrete patterns of target gene activation. The structures of relevant cis-regulatory regions of AbdA target genes are instrumental for determining which signal is required to allow activation by AbdA. The pnt midgut enhancer would contain AbdA and Wg response elements and would be activated by AbdA specifically in the third midgut chamber through the combinatorial action of AbdA and the Drosophila Tcf/Arm transcriptional effector of Wg signaling. Similarly, the opa midgut enhancer would contain AbdA and Dpp response elements and would be activated only in the fourth gut chamber by AbdA, in this case because of an inhibitory effect of the Dpp-regulated transcription factor on AbdA activity (Grienenberger, 2003).

Further studies are required to understand how Hox selector proteins functionally interact with nuclear effectors of signaling pathways to generate specific transcriptional patterns. In the control of wg by AbdA, several scenarios can be envisioned. In one, the effect of the Dpp transcriptional effector Mad on AbdA activity would be indirect, by antagonizing the function of a repressor that would otherwise act on the XC enhancer to prevent wg expression. The absence of a binding site for this hypothetical repressor in Box2 could explain how Box2 drives AbdA-dependent transcription even without Dpp transcriptional effector binding sites. In a second scenario, Dpp transcriptional effectors would more directly control the activity of AbdA by influencing its DNA binding or transregulatory properties. A direct interaction of HoxC8 and Smad1 has been reported to induce osteoblast differentiation in mammals, suggesting that the coordinate action of AbdA and Dpp signaling might rely on direct AbdA-Mad interaction. In wg regulation, the situation may be different, as additional regulatory inputs are involved. bin and hth are essential, and Wg signaling is required for accurate levels of wg expression. The contribution of Creb might indicate that the Ras/Mapk signaling pathway is involved as well. Ras signaling has been proposed to play a permissive role by acting on CRE sequences of the Ubx and lab enhancers. These observations suggest that AbdA and Hox proteins in general attain specificity and diversity by participating in a variety of protein interactions in enhancer-binding complexes (Grienenberger, 2003).

A 3' cis-regulatory region controls wingless expression in the Drosophila eye and leg primordia

The precise regulation of wingless (wg) expression in the Drosophila eye disc is key to control the anteroposterior and dorsoventral patterning of this disc. This study identifies an eye disc-specific wg cis-regulatory element that functions as a regulatory rheostat. Pannier (Pnr), a transcription factor previously proposed to act as an upstream activator of wg, is sufficient to activate the eye disc enhancer but required for wg expression only in the peripodial epithelium of the disc. It is proposed that this regulation of wg by Pnr appeared associated to the development of the peripodial epithelium in higher dipterans and was added to an existing mechanism regulating the deployment of wingless in the dorsal region of the eye primordium. In addition, this analysis identifies a separate ventral disc enhancer that lies adjacent to the eye-specific one, and thus altogether, they define a 1-kb genomic region where disc-specific enhancers of the wg gene are located (Pereira, 2006).

JAK/STAT signaling promotes regional specification by negatively regulating wingless expression in Drosophila

During development, a small number of conserved signaling molecules regulate regional specification, in which uniform populations of cells acquire differences and ultimately give rise to distinct organs. In the Drosophila eye imaginal disc, Wingless (Wg) signaling defines the region that gives rise to head tissue. JAK/STAT signaling was thought to regulate growth of the eye disc but not pattern formation. However, this study shows that the JAK/STAT pathway plays an important role in patterning the eye disc: it promotes formation of the eye field through repression of the wg gene. Overexpression of the JAK/STAT activating ligand Unpaired in the eye leads to loss of wg expression and ectopic morphogenetic furrow initiation from the lateral margins. Conversely, tissue lacking stat92E, which cannot transduce JAK/STAT signals, is transformed from retinal tissue into head cuticle, a phenotype that is also observed with ectopic Wg signaling. Consistent with this, cells lacking stat92E exhibit ectopic wg expression. Conversely, wg is autonomously repressed in cells with hyperactivated Stat92E. Furthermore, the JAK/STAT pathway regulates a small enhancer in the wg 3' cis genomic region. Since this enhancer is devoid of Stat92E-binding elements, it is concluded that Stat92E represses wg through another, as yet unidentified factor that is probably a direct target of Stat92E. Taken together, this study is the first to demonstrate a role for the JAK/STAT pathway in regional specification by acting antagonistically to wg (Ekas, 2006).

Although the majority of functions attributable to STATs involve transcriptional activation, at least one STAT protein, the Dictyostelium Dd-STATa, acts as a functional repressor. Therefore the ability of Stat92E to directly repress wg was tested. A reporter called wg2.11Z, in which ß-galactosidase is driven by a 263 base pair enhancer from the 3' cis wg genomic region, was sufficient to recapitulate wg expression in the dorsal margin of the disc proper and in the dorsal peripodial membrane (Pereira, 2006). This reporter was ectopically expressed in mosaic stat92E clones as well as in stat92E M+ clones in a manner similar to that observed for wgP. Moreover, wg2.11Z was repressed autonomously in hop-expressing clones. These data indicate that Stat92E can regulate dorsal wg expression through the wg2.11Z enhancer. wg2.11Z does not contain any Stat92E binding sites (TTC(N)3GAA), strongly suggesting that Stat92E does not repress dorsal wg directly, but rather regulates another factor which represses wg (Ekas, 2006).

Thus, this paper reports a new role for the Drosophila JAK/STAT pathway. The study demonstrates that JAK/STAT signaling promotes formation of the eye field through repression of wg gene transcription in both the dorsal and ventral halves of the eye disc epithelium. By monitoring Upd expression and activity, it was shown that the JAK/STAT pathway is normally activated early in eye development, during first and second instar. Ectopic activation of this pathway leads to abnormal patterning of the head capsule and a reduction in the inter-eye distance through an increase in dorsal ommatidia. By contrast, loss of activity of this pathway, using strong hypomorphic stat92E mutations, frequently resulted in the development of a rudimentary head. When the head capsule did form, stat92E mutants often had small or ablated adult eyes and excessive head cuticle. wg was ectopically expressed in stat92E clones and hop mutant eye discs, and was repressed by ectopic activation of the JAK/STAT pathway. Reduction in the dose of wg partially rescued stat92E mutants by increasing the rate of eclosion and by mitigating the phenotypes of stat92E mutant animals. Lastly, it was shown that wg regulation by the JAK/STAT pathway is independent of the known wg regulators Eyg, Dac, Hth and Pnr (Ekas, 2006).

These results conflict with those of a previous study, which reported that JAK/STAT signaling does not repress wg in the eye disc. This conclusion was reached on the basis of wild-type Wg protein expression in eye discs that contained ectopic upd-expressing clones (Zeidler, 1999). However, this study found that in the absence of stat92E, wg was ectopically expressed in both dorsal and ventral halves of the eye disc. It is likely that the current examination of the wg gene using the wgP enhancer trap is a more sensitive measure of wg expression than monitoring Wg protein. Zeidler also reported that the JAK/STAT pathway negatively regulates mirr expression. This conclusion was drawn after finding a preponderance of dorsal, mirr-positive ommatidia in adult eyes containing unmarked upd loss-of-function clones (Zeidler, 1999). However, using marked clones, the current study showed that Mirr is expressed normally in eye tissue that is largely homozygous mutant for stat92E. Moreover, stat92E M+ adult eyes are largely composed of Mirr-negative ommatidia, which indicates their ventral origin. Thus, the current data indicate that mirr is not regulated by JAK/STAT pathway activity (Ekas, 2006).

Previous work has shown that the 3' cis region of the wg gene regulates its expression in imaginal discs. Several wg mutations that specifically affect imaginal disc development, as well as discspecific enhancers, map to this region. In this study, it was shown that Stat92E negatively regulates dorsal wg through a small enhancer (wg2.11Z) in the 3' cis genomic region of the wg gene. This enhancer is ectopically expressed in stat92E and hop mutants and is autonomously repressed by ectopic activation of Stat92E. The DNA binding preferences of Stat92E and other STAT proteins have been well characterized. Because there are no Stat92E binding sites in the wg2.11Z enhancer, the interpretation is favoredthat Stat92E does not directly repress dorsal wg but rather acts through another factor. This repressor may be encoded by a direct Stat92E target gene, because wg is autonomously repressed by the JAK/STAT pathway. However, the possibility cannot be ruled out that Stat92E regulates wg through other transcription factors, such as Dorsal or vHNF-4, which have putative sites in wg2.11Z (Pereira, 2006). It is also possible that there are cryptic Stat92E binding sites in this wg enhancer, through which Stat92E may directly repress wg. Additional experiments will be needed to test these possibilities (Ekas, 2006).

This study also demonstrated that Stat92E represses ventral wg in the eye disc epithelium. This is presumably independent of the wg2.11Z enhancer, which recapitulates wg expression in the dorsal but not the ventral eye disc (Pereira, 2006). Moreover, Stat92E negatively regulates pnr in peripodial cells. In the absence of JAK/STAT signaling, pnr is dramatically expanded into the posterior peripodial membrane. However, it is stressed that because pnr is an intracellular protein, the ectopic pnr in the peripodial membrane cannot account for the ectopic wg observed in the disc proper of stat92E mutants. Currently, it is not know whether Stat92E regulates wg in the ventral eye disc epithelium and the peripodial membrane in the same manner as in the dorsal eye. All three wg expression domains may be regulated by the same as yet unidentified factor. Alternatively, Stat92E may regulate wg expression domains through different mechanisms. For example, dorsal wg may be regulated indirectly, whereas ventral and peripodial wg may be regulated directly by Stat92E. The wg gene 3' cis genomic region contains one putative Stat92E binding site, which resides downstream of the wg2.11Z enhancer. Therefore, it is possible that Stat92E regulates ventral and peripodial wg through this site. Future work will be needed to address these issues (Ekas, 2006).

Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgut morphogenesis

The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. This study reports that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. It was demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells, known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes were dissected. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. It is concluded that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut (Schaub, 2013).

The analysis of org-1 expression and function during visceral mesoderm development defined this gene as a new and essential lineage specific regulator of circular visceral muscle founder cell identities and midgut patterning in Drosophila. The data add new insights into the developmental regulatory mechanisms responsible for the diversification of the circular visceral muscle founder cell lineage and midgut morphogenesis (Schaub, 2013).

The initial expression of org-1 occurs in the segmented trunk visceral mesoderm (TVM), where it is coexpressed with tin, bap, bin and Alk. It has been documented that the induction of tin and bap in the dorsal mesoderm involves the combined binding of Smad proteins (Medea and Mad) and Tin to Dpp-responsive enhancers of the tin and bap genes, whereas the segmental repression of bap is mediated by binding of the sloppy paired (slp) gene product. Genetic analysis of org-1 has shown that org-1 is activated downstream of tin but independently of bap and bin, and that dpp provides the key signals for its induction. This suggests a regulatory mechanism analogous to that of bap, in which the combined binding of Smads and Tin activates a Dpp-responsive org-1 enhancer, whereas Wg activated Slp is required for its mutual segmental repression (Schaub, 2013).

The similarities in the early expression patterns of bap, bin, Alk and org-1 in the trunk visceral mesoderm primordia raise the question of the contribution of org-1 to the early development of the TVM as such. Whereas bap and bin are crucially required for the specification of the trunk visceral mesoderm and visceral musculature, loss of org-1 function, like the loss of Alk, has no obvious impact on the specification of the early TVM. Therefore, it is notable that during the subdivision of the visceral mesoderm primordia into founder and fusion-competent myoblasts (cFCs and FCMs), org-1 expression is extinguished in the FCMs and only sustained in the cFC lineage of the circular visceral musculature. This lineage-specific restriction and maintenance of org-1 expression crucially depends on Jeb mediated Alk/Ras/MAPK signaling and points toward a possible cFC lineage specific function of org-1. The genetic analysis demonstrates that org-1 is not required for cFC specification, but plays a decisive role in the induction of the visceral mesoderm specific expression of patterning genes in the founder cells of the circular musculature. Thus, org-1 is critical for the processes of cell fate diversification that provide individual fields of cells along the anteroposterior axis of the visceral mesoderm with their specific identities (Schaub, 2013).

Proper anteroposterior patterning of the trunk visceral mesoderm and the formation of localized organizer fields are prerequisites for eliciting the morphogenetic events that shape the midgut. The formation of these organizer fields depends on the appropriate spatial expression domains of the homeotic selectors Scr, Antp, Ubx and abd-A, the secreted factors dpp and wg, as well as the zinc finger proteins opa and tsh, which are required for the formation of the midgut constrictions as well as the gastric caeca. The regulatory mechanisms responsible for the establishment of the spatial, temporal and tissue-specific expression patterns of these genes in the TVM are only partially understood. Genetic and molecular analyses with the FoxF gene bin, which is expressed in all trunk visceral mesoderm precursors and their descendents, have demonstrated that bin is a direct upstream regulator of dpp in PS7 and is also required for the expression of wg in PS8 of the TVM. Thus, Bin serves as an essential TVM-specific competence factor in conjunction with the dpp/wg signaling feedback loop. The current findings have defined Org-1 as an additional tissue-specific regulator with an even broader range of downstream patterning genes in the TVM, but with a narrower spatial range of action. org-1 acts specifically within the visceral muscle founder cell lineage as a positive regulator upstream of opa, tsh, Ubx, dpp as well as wg (Schaub, 2013).

This combination of genetic data and functional enhancer analyses provides convincing evidence that both dpp and wg are direct transcriptional targets of Org-1 in the cFCs. Prior dissections of the dpp visceral mesoderm (VM) enhancer had shown that it is also regulated by the direct binding of Ubx, Exd, dTCF (a Wg effector) and Bin, and that minimal synthetic variants that contain only the binding motifs for Ubx, Exd, Bin, and dTCF within conserved sequence contexts (which happen to include the Org-1 motif) are active as VM enhancers. Likewise, the wgXC enhancer fragment integrates Org-1 with the direct regulatory inputs of Abd-A as well as CREB and Smad (Mad/Medea) proteins mediating Dpp signaling (Schaub, 2013).

Org-1 is the first transcription factor known to be required for Ubx expression in PS7 of the visceral musculature. Extensive work on an Ubx visceral mesoderm CRM (UbxRP) indicated that dpp and wg regulate Ubx through indirect autoregulation. Of note, in bin embryos, which also lack visceral mesodermal dpp and wg expression, Ubx is still expressed. Genetic data show that the UbxRP element, while requiring org-1, is not directly regulated by Org-1, since mutation of its four predicted T-Box binding sites did not have any effects. Taking into account that no UbxRP reporter activity was detected in the cFCs at pre-fusion stages, it is suggested that UbxRP represents a late enhancer element and responds to dpp and wg only after they are activated by Org-1 in the founder cells. To clarify whether the regulation of Ubx by Org-1 is direct or indirect, the identification and dissection of a founder cell specific CRM will be required (Schaub, 2013).

tsh and opa were described as homeotic target genes of Antp in PS4-6 (tsh) and PS4-5 (opa) as well as of abd-A in PS8 (tsh) and PS9-12 (opa) of the visceral musculature. The current data show that tsh and opa expression is already activated in the respective cFCs of the visceral parasegments where it requires org-1. The later activation of tsh in PS8 during muscle fusion follows the org-1 dependent founder cell specific initiation of wg in PS8, which acts upstream of tsh. Thus it was conceivable that the regulation of tsh by org-1 is indirect. However, ectopic activation of wg in an org-1 loss of function background is not able to rescue tsh expression and Antp and abd-A expression is not altered upon loss of org-1. These observations suggest that Org-1 acts directly on tsh and opa, e.g., via functional cooperation with Antp and Abd-A, respectively, during the early activation of tsh and opa in the founder cells (Schaub, 2013).

It was reported that the absence of Jeb/Alk signaling causes loss of dpp expression in the founder cells in PS7 of the visceral mesoderm. In light of the current findings that org-1 loss-of-function produces a similar phenotype, and of the previous demonstration that org-1 expression is downstream of Jeb/Alk, this observation could simply be explained by the action of a linear regulatory cascade from Jeb/Alk via org-1 towards dpp. Alternatively, Jeb/Alk may provide additional inputs towards dpp (and other patterning genes) in parallel to org-1, which could explain the slightly stronger phenotype of Alk as compared to org-1 mutations with respect to dpp. A possible candidate for an additional effector of Jeb/Alk signals in this pathway is extradenticle (exd), which is known to be required for normal dpp expression in PS7 of the visceral mesoderm, presumably through direct binding of Exd in a complex with Hox proteins and Homothorax (Hth) to a PS7-specific enhancer element (a derivative of which was used in this study). Like org-1, exd is also needed for the expression of tsh and wg in the visceral mesoderm (Additionally, it represses dpp in PS4-6 through sequences not contained in the minimal PS7 enhancer). It is thought that Exd complexed with Hox proteins and Hth increases the binding preference of these Hox complexes for specific binding sites within visceral mesodermal enhancers of their target genes (Schaub, 2013).

Since exd is expressed in both founder and fusion-competent cells in the visceral mesoderm, it is unlikely that it fulfills its roles in the regulation of dpp, wg, and tsh in the founder cells as a downstream gene of org-1. However, it is known that Exd requires nucleocytoplasmic translocation for it to be functiona and, interestingly, it has been shown that Jeb/Alk signals trigger nuclear localization of Exd specifically in the cFCs of the visceral mesoderm. Because nuclear Exd appears to be hyperphosphorylated as compared to cytoplasmic Exd, nuclear translocation of Exd may be triggered by Alk-mediated phosphorylation. Alternatively, Jeb/Alk signals may induce the expression of hth in the cFCs and Hth could then translocate Exd to the nuclei, as has been shown in other contexts. This would be compatible with the observation that Hth is upregulated in the founder cells in an org-1-independent manner (Schaub, 2013).

The combined data show that Jeb/Alk signals exert at least two parallel inputs towards patterning genes in the cFCs, which are the induction of org-1 and the nuclear translocation of Exd. Taken altogether, a model is suggested in which combinatorial binding of Org-1, nuclear Exd/Hth and the homeotic selector proteins to the corresponding visceral mesoderm specific CRMs is required for the initiation of lineage specific expression of opa, tsh, dpp, Ubx and wg in the founder cells of the respective parasegments. As shown in the examples of dpp (PS7) and wg (PS8), accessory Bin is required for the activation as a general visceral mesodermal competence factor, whereas Dpp and Wg effectors mediate autoregulatory stabilization of their expression (Schaub, 2013).

Extensive work has shown that during somatic muscle development individual founder myoblasts acquire distinct identities, which are adopted by the newly incorporated nuclei upon myoblast fusion, thus leading to the morphological and physiological diversification of the differentiating muscles. It is proposed that the same principle is active during visceral muscle development. In this view, Org-1 acts as a muscle identity factor in both the somatic and visceral mesoderm. In the visceral mesoderm, Org-1 helps diversifying founder cell identities and, after myoblast fusion, their differential identities are transmitted to the respective differentiating circular gut muscles. The activation of downstream targets of this identity factor in the developing muscles leads to the observed morphogenetic differentiation events of the midgut and the establishment of the signaling center in PS7/8 that is also required for Dpp and Wg mediated induction of labial in the endodermal germ layer. As is the case for identity factors in the somatic muscle founders, Org-1 in the visceral mesoderm acts in concert with other, spatially restricted activities such as Hox factors and signaling effectors to achieve region-specific outputs. The main difference is that, in the trunk visceral mesoderm, Org-1 is present in all founder cells whereas in the somatic mesoderm this identity factor (like others) is expressed in a particular subset of founder myoblasts. Thus, in contrast to the somatic mesoderm, the spatial expression of Org-1 does not contribute to its function in visceral muscle diversification and instead, it solely relies on spatially-restricted co-regulators during this process (Schaub, 2013).

The pool of trunk visceral mesodermal fusion-competent cells contributes to the formation of both circular and longitudinal midgut muscles, depending on whether they fuse with resident founder cells of the trunk visceral mesoderm or with founders that migrated in from the caudal visceral mesoderm. The restricted expression of the identity factor Org-1 in the founder myoblasts in the trunk visceral mesoderm and its exclusion from the FCMs represents an elegant mechanism to ensure that the respective patterning events only occur in the developing circular musculature but not in the longitudinal muscle fibers, which extend as multinucleate syncytia throughout the length of the midgut (Schaub, 2013).

Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

In the era of functional genomics, the role of transcription factor (TF)-DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. This study investigated the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients. It is noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to an initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp, wingless and stripe, by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, it was found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, patterning defects were observed in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. It is proposed that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes (Ramos, 2013).

This study present in vivo evidence corroborating previous findings that multiple tissue-specific enhancers require low-affinity Ci binding sites for optimal activation by Hh/Ci. Most of the Hh target enhancers identified up to this point in Drosophila and mouse are regulated by degenerate Ci/Gli binding sites of low predicted affinity. The prevalence of these non-consensus sites in Hh target enhancers across species demonstrates their importance in regulating the Hh response. The transcriptional relevance of low-affinity TF binding is not limited to Hh/Ci regulated enhancers. For instance, two phylogenetically conserved low-affinity binding sites in the mouse Pax6 lens enhancer have been shown to be critical to promote gene expression at the right stage of development (Ramos, 2013).

A mechanistic explanation is provided as to why these Hh/Ci-regulated elements require low-affinity sites to activate transcription in cells with moderate signalling levels. Clusters of high-affinity sites mediate a restricted response in cells with high levels of Hh signalling, most likely as a result of cooperative interactions among Ci-Rep molecules in highly occupied Ci binding sites, whereas clusters of low-affinity sites mediate a broader response by having lower occupancy by Ci. Using synthetic enhancer reporters with high- or low-affinity Ci binding sites, this effect was confirmed in the wing, but not in embryos. This tissue-specific discrepancy may imply a context-dependent function for some non-consensus Ci binding sites. As in the Pax6 lens enhancer (Rowan, 2010), it is possible that some low-affinity binding sites are required specifically during earlier stages of development to interpret overall lower levels of Hh signalling (Ramos, 2013).

Finally, clues are provided as to additional regulatory inputs into dppD by showing a requirement for conserved consensus homeodomain (HD) binding sites. Cooperation between Glis and HD proteins has been recently shown in the mouse neural tube. In this case, HD proteins are critical to repress Hh-regulated neural tube enhancers, whereas in dppD they are critical to activate gene expression (Ramos, 2013).

The limited number of known, experimentally confirmed, direct Hh/Gli target enhancers may reflect the widespread, practical tendency to search for consensus or near-consensus motifs, and to focus on the highest peaks of TF-DNA binding, when hunting for cis-regulatory sequences. From a biochemical standpoint—for example, when mining ChIP-seq data—low-affinity DNA-binding interactions are troublesome because they are much more common, by definition, than the top 1% of peaks. It is important to note that iy is not always useful to strictly equate ChIP peak height with TF binding affinity, nor to equate in vitro binding or in silico 'motif quality' with in vivo TF occupancy, though these properties may often be roughly correlated. Separating the weak but functional binding events from weak and non-functional binding events is extremely challenging, and some have proposed that low-affinity genome-binding interactions can be categorically ignored. This certainly simplifies the problem from a computational perspective, but the findings discussed here and elsewhere suggest a risk of discarding functional sequences. Similar challenges confront in silico genomic screens to identify clusters of predicted TF binding sites: these necessarily filter out binding events of low predicted affinity, because there are many more predicted low-affinity binding motifs than consensus high-affinity motifs in any given sequence. Binding site predictions have been supported by taking evolutionary sequence conservation into account, but this risks filtering out true positives: as shown in Ci motif alignments, lower-affinity binding sites seem to be less constrained with respect to sequence variation, even in cases when the presence of the site itself is highly conserved. This is presumably because, for each non-consensus binding motif, there are multiple alternative sequences with similar affinity and thus equivalent functionality. Importantly, this type of degenerate motif conservation is easily missed: for example, some of the well-conserved Ci motifs described in this study are not properly aligned in the UCSC Genome Browser, because they do not constitute contiguous blocks of perfect sequence identity. To avoid these pitfalls, it is important to use phylofootprinting approaches that account for these alignment flaws. In contrast to most of the low-affinity binding sites discussed in this study, optimal-affinity Ci motifs in the ptc enhancer have been preserved throughout the evolution of the genus Drosophila, and perhaps much farther: GACCACCCA motifs occur in promoter-proximal regions of multiple vertebrate orthologues of ptc (Ramos, 2013).

Evolutionary enhancer sequence alignments, along with limited experimental data, also suggest that, although many predicted low-affinity sites are poorly conserved, overall TF occupancy on an enhancer may be maintained despite significant sequence turnover. This may occur either through the rapid gain and loss of individual sites, or through the maintenance of relatively weak binding affinity at a site that is unstable at the level of DNA sequence. While this last idea requires further direct testing, it is consistent with the fact that Gli sites of moderate predicted affinity have many sequence variants of similar quality, whereas the highest-affinity motifs have far fewer alternatives of similar quality. In other words, there are many more ways to be a weak binding site than a strong site. For example, among all possible 9-mer sequences, there are 654 motifs with Ci matrix similarity scores between 70 and 75 (inclusive), but only 12 motifs with scores between 90 and 95, and one motif with a score above 95. Therefore, weaker binding sites, and the enhancers containing them, have a far greater volume of sequence space in which to roam without strongly impacting transcriptional output. A thermodynamics-based simulation of enhancer evolution has shown that there is a greater number of fit solutions using weak TF sites than using high-affinity sites for a given gene expression problem (Ramos, 2013).

Equally consistent with the view of TF binding site evolution is the fact that it is much easier (that is, more likely) to create a low-affinity, non-consensus binding motif with a single mutation than a high-affinity consensus motif. An enhancer-sized DNA sequence can acquire a weak Gli motif with single-nucleotide substitutions at any of a large number of positions, as demonstrated by simulations. These arguments may help to explain why sequence conservation is not a foolproof test of the functional relevance of non-consensus TF binding sites (Ramos, 2013).

While there is no simple answer to the technical challenges facing those who hunt enhancers, the findings described in this report lead to a conclusion that low-affinity TF-DNA interactions, mediated by non-consensus and often poorly conserved sequence motifs, play important and widespread roles in developmental patterning and cis-regulatory evolution, and therefore cannot be safely ignored (Ramos, 2013).

Dependence on signaling from cells of the adjacent anterior compartment

Hedgehog is produced by engrailed expressing cells in anterior parasegmental compartments. Genetic analysis has identified wg transcription in posterior compartments as one of the targets of HH activity. It has been suggested that the spatial control of wg expression depends on the limited range of the HH signal and the differential competence of responding cells. Ubiquitous expression of the hh gene causes the ectopic activation of wg in only a subset of the cells of each parasegment. Competence of cells to express wg is independent of their ability to receive the HH signal (Ingham, 1993).

Localized or ubiquitous expression of the N-terminal domain of HH, a biologically active form of the protein that lacks the normal lipophilic modification, causes an expansion of wingless expression, ventral cuticle defects including a rectangular rather than trapezoidal shape for the denticle belts and loss of denticle diversity, dorsal cutical defects and embryo lethality. This suggests a role for HH autoprocessing (lipid modification) in spatial regulation of hedgehog signaling (Porter, 1996).

Cyclic AMP (cAMP)-dependent Protein kinase A (PKA) is essential during limb development to prevent inappropriate decapentaplegic and wingless expression. A constitutively active form of PKA can prevent inappropriate dpp and wg expression, but does not interfere with their normal induction by hh. It seems that the basal activity of PKA imposes a block on the transcription of dpp and wg and that hh exerts its organizing influence by alleviating this block (Jiang, 1995).

Considered now to be the HH receptor, Patched acts negatively, both in early segment development and in imaginal discs. PTC represses wingless in posterior parasegment domains and acts to repress dpp in the anterior compartment of wing imaginal discs (Schuske, 1994). Unrestricted expression of ptc from a heat-shock promoter has no adverse effect on development of Drosophila embryos. The heat-shock construct can also rescue ptc mutants, restoring wg expression to its normal narrow stripe. This implies that despite its localized requirement, the restricted expression of ptc does not itself allocate positional information (Sampedro, 1991). Thus the role of ptc in positional signaling is permissive rather than instructive, its activity being required to suppress wg transcription in cells predisposed to express wg. According to this view, expression of wg is normally maintained only in those cells receiving an extrinsic signal, encoded by hedgehog, that antagonizes the repressive activity of ptc (Ingham, 1991).

Transient overexpression of ptc in all cells has little or no effect on the segmental pattern. Repeated pulses of ptc production drastically alter the segment pattern to mimic embryos lacking wg. Repeated overexpression results in repression of wg and gooseberry transcription in the germband ectoderm but not in the head. (gooseberry is a wg class segment polarity gene). Expression is unaffected for two other segment polarity genes: engrailed and cubitus interruptus. Thus excess ptc is capable of overcoming the neutralizing signal presumably carried by hedgehog (Schuske, 1994).

Cubitus interruptus is required for wg induction. CI is downstream of ptc and zeste-white 3 which both act negatively on the induction of wg by CI. Fused is required for CI induction of wg (Motzny, 1995).

Misexpression of CI in the Engrailed domain (by placing CI under the control of an en promoter) activates wingless transcription. The expression domains of wingless in such embryos are significantly broader than in wild-type cells. Placing ci under the control of the hairy promoter (hairy is expressed only in alternating parasegments), and testing such a construct in hh mutant embryos, results in a pair-rule phenotype, where every other segment shows naked cuticle. In these embryos, wg expression is present in alternate parasegment. This provides conclusive evidence the CI regulates wingless (Alexandre, 1996).

Transcriptional Regulation Table of contents

wingless continued: Biological Overview | Evolutionary Homologs |Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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