Promoter Structure

The transcripts from each of the two Antp promoters accumulate in distinct, but overlapping patterns during embryogenesis. The results demonstrate that the two Antp promoters are differentially regulated in embryos and provide a basis for examining the regulation of the two promoters and characterizing more fully the function of Antp during embryogenesis. In BX-C- embryos both promoters are derepressed in the abdomen. The P1 transcription unit is required for anterior spiracle eversion and dorsal thoracic development, while the P2 unit is required for embryonic viability and leg development. The posterior system regulates P2 but not P1. Krüppel activates P1 but not P2, while Hunchback and Fushi tarazu activate P2 but not P1 (Bermingham, 1990 and references).

Antennapedia P2 contains a TATA-box deficient (TATA-less) promoter. Such promoters have a conserved sequence motif, A/GGA/TCGTG, termed the downstream promoter element (DPE), which is located about 30 nucleotides downstream of the RNA start site of many TATA-less promoters, including Antennapedia P2. DNase I footprinting of the binding of epitope-tagged TFIID to TATA-less promoters reveals that the factor protects a region that extends from the initiation site sequence (about +1) to about 35 nucleotides downstream of the RNA start site. There is no such downstream DNase I protection induced by TFIID in promoters with TATA motifs. This suggests that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters (Burke, 1996).

There are 30 common binding sites for the proteins encoded by the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes within a negatively regulated target, the P2 promoter of the Antennapedia (Antp) gene. By systematically mutagenizing binding sites and observing the resulting P2 expression pattern in embryos, evidence has been found for cell-type-specific interactions that are mediated by these sequences. In certain neuronal cells, UBX and ABD-A proteins appear to repress by competing for common binding sites with another homeodomain protein, which may be ANTP acting to induce P2 transcription in an autoregulatory manner. In sets of cells that contribute to the tracheal system, UBX and ABD-A repress by counteracting the function of a factor acting at independent sites. The latter mechanism of repression requires only that multiple homeodomain binding sequences be present and is not dependent on any particular binding site (Appel, 1993).

Caudal, targeting Antp P2 and Scr promoters, is a DPE-specific transcriptional factor

The regulation of gene transcription is critical for the proper development and growth of an organism. The transcription of protein-coding genes initiates at the RNA polymerase II core promoter, which is a diverse module that can be controlled by many different elements such as the TATA box and downstream core promoter element (DPE). To understand the basis for core promoter diversity, potential biological functions of the DPE were explored. It was found that nearly all of the Drosophila homeotic (Hox) gene promoters, which lack TATA-box elements, contain functionally important DPE motifs that are conserved from Drosophila melanogaster to Drosophila virilis. It was then discovered that Caudal, a sequence-specific transcription factor and key regulator of the Hox gene network, activates transcription with a distinct preference for the DPE relative to the TATA box. The specificity of Caudal activation for the DPE is particularly striking when a BREu core promoter motif is associated with the TATA box. These findings show that Caudal is a DPE-specific activator and exemplify how core promoter diversity can be used to establish complex regulatory networks (Juven-Gershon, 2008).

This study found that the DPE is used extensively in the network of genes that are involved in the development of the early Drosophila embryo. Nearly all of the Drosophila Hox gene promoters, which have been known to be TATA-less, contain functionally essential DPE motifs that are conserved from D. melanogaster to D. virilis. The two Hox genes lacking DPE motifs are also the most recent Hox genes from an evolutionary standpoint. Thus, the DPE is a critical yet previously unrecognized component of the Hox genes (Juven-Gershon, 2008).

The DPE is not only in the Hox genes, but is also present in ftz, gt, h, fkh, cad (zygotic promoter), and en, which are involved in early embryonic development. This finding suggests that the DPE is used broadly throughout the network of genes that mediate the development of the embryo. This hypothesis was tested by analyzing the transcriptional properties of Caudal, a ParaHox protein and sequence-specific DNA-binding factor that regulates ftz, gt, h, and fkh. These studies revealed that Caudal is a DPE-specific activator. The preference of Caudal for activating transcription from DPE- versus TATA-dependent core promoters is seen most distinctly either with the natural ftz enhancer-promoter region or with a core promoter containing a BREu motif associated with the TATA box. The effect of Caudal on transcription of two Hox genes, Antp and Scr, was examined and it was found that Caudal activates the TATA-less, DPE-dependent Antp P2 and Scr promoters. These findings thus provide a direct link between Caudal, a DPE-specific activator, and the DPE-containing Hox genes (Juven-Gershon, 2008).

The discovery that Caudal is a DPE-specific activator provides new insight into the basic mechanisms of transcriptional regulation. Previous enhancer-trapping experiments have shown that there are enhancers that activate DPE-dependent promoters but not TATA-dependent promoters; however, neither the cis-acting elements nor the trans-acting factors that are responsible for the DPE-specific activation had been identified. Therefore, these studies demonstrate the existence of a DPE-specific enhancer-binding factor. Moreover, it is likely that other core-promoter-specific enhancer-binding factors, such as TATA-specific activators, will be discovered (Juven-Gershon, 2008).

These experiments uncovered a novel activity of the BREu core promoter motif. The BREu is a 5' extension of the TATA box that is bound by the TFIIB basal/general transcription factor. Depending on the context, the BREu has been found to have either a positive or a negative effect on transcription. In this study, it was found that the BREu motif has little effect on basal/unactivated transcription, but potently suppresses the ability of Caudal to activate transcription via the TATA box. In contrast, the BREu in its normal upstream location has no effect on Caudal-mediated activation via the DPE. These findings indicate that the BREu can contribute to core-promoter-element-mediated transcriptional regulation. Hence, there is a positive linkage between Caudal and the DPE as well as a negative interaction between Caudal and the BREu-TATA element. The combination of both positive (DPE) and negative (BREu-TATA) interactions yields maximal specificity of Caudal function (Juven-Gershon, 2008).

The new findings lead to the model that transcriptional regulation involves the combined action of sequence motifs in both the core promoter and the enhancer. The ability of Caudal to discriminate among DPE, TATA, and BREu-TATA motifs regulates the flow of information from the enhancer-bound activator to the core promoter -- the site of transcription initiation. In this manner, core promoter elements can be viewed as a component of transcriptional circuits. In these transcriptional circuits, connections between enhancers and core promoters are established and modulated according to the properties of the activators and the sequence motifs in the core promoter. Thus, the discovery that Caudal is a core-promoter-specific activator reveals a new strategy with which complex transcriptional networks can be established. Hence, in a broader sense, these findings show how diversity in core promoter structure can contribute to organismal diversity (Juven-Gershon, 2008).

The core promoter composition establishes a new dimension in developmental gene networks

Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II, which initiates transcription at the core promoter. The dorsal-ventral gene regulatory network (GRN) includes multiple genes that are activated by different nuclear concentrations of the Dorsal transcription factor along the dorsal-ventral axis. Downstream core promoter element (DPE)-containing genes are conserved and highly prevalent among Dorsal target genes. Moreover, the DPE motif is functional in multiple Dorsal target genes, as mutation of the DPE results in the loss of transcriptional activity. Furthermore, analysis of hybrid enhancer-promoter constructs reveals that the core promoter composition plays a pivotal role in the transcriptional output. Importantly, in vivo evidence is provided that expression driven by the homeotic Antennapedia P2 promoter during Drosophila embryogenesis is dependent on the DPE. Taken together, it is proposed that transcriptional regulation results from the interplay between enhancers and core promoter composition, thus establishing a novel dimension in developmental GRNs (Zehavi, 2014).

Stalled Hox promoters as chromosomal boundaries

Many developmental control genes contain stalled RNA Polymerase II (Pol II) in the early Drosophila embryo, including four of the eight Hox genes. Evidence is presented that the stalled Hox promoters possess an intrinsic insulator activity. The enhancer-blocking activities of these promoters are dependent on general transcription factors that inhibit Pol II elongation, including components of the DSIF (Spt4, and Spt5) and NELF complexes. The activities of conventional insulators are also impaired in embryos containing reduced levels of DSIF and NELF. Thus, promoter-proximal stalling factors might help promote insulator-promoter interactions. It is proposed that stalled promoters help organize gene complexes within chromosomal loop domains (Chopra, 2009b).

Hox genes are responsible for the anterior-posterior patterning of most metazoan embryos. They are typically organized in gene complexes containing a series of cis-regulatory DNAs, including enhancers, silencers, and insulator DNAs . In Drosophila, the eight Hox genes are contained within two gene complexes: the Antennapedia complex (ANT-C), which controls the patterning of anterior regions, and the Bithorax complex (BX-C), which controls posterior regions. The proper spatiotemporal transcription of Hox genes is achieved by the coordinated action of linked cis-regulatory DNAs that are organized in a colinear fashion across the ANT-C and BX-C complexes (Chopra, 2009b).

Chromosomal boundary elements, or insulators, are essential for the orderly regulation of Hox gene expression. They are thought to ensure proper cis-regulatory 'trafficking,' whereby the correct enhancers interact with the appropriate target promoters. Insulators might also help control the levels of transcription by attenuating enhancer-promoter interactions. Insulators are sometimes associated with promoter targeting sequences (PTS), which can facilitate enhancer-promoter interactions by modulating the activities of neighboring insulators (Chopra, 2009b).

Recently, long-range cis-regulatory interactions have been mapped in Drosophila Hox complexes using the DamID technique, chromosomal conformation capture (3C) assays, and transgenic approaches. These studies suggest that the Fab7 and Fab8 insulators are associated with the Abd-B promoter under repressed conditions, even though they map >30-50 kb downstream from the promoter. These long-range interactions depend on the CTCF boundary-binding protein, thereby raising the possibility that insulators interact with one another and organize Abd-B cis-regulatory DNAs within chromosomal loop domains. Similarly, the prototypic insulators flanking the heat-shock puff locus, scs and scs', have also been shown to interact with one another. Additional insulator-insulator loops have also been documented. These loops are thought to facilitate the interactions of remote enhancers and silencers with appropriate target promoters. This study presents evidence that Hox promoters with stalled RNA Polymerase II (Pol II) possess an intrinsic insulator activity, which might help foster the formation of insulator-promoter chromosomal loop domains (Chopra, 2009b).

Four of the eight Hox genes contained in the ANT-C and BX-C contain stalled Pol II. Interestingly, all four stalled genes map at the boundaries of the two Hox complexes. In contrast, internal Hox genes (pb, Dfd, and Scr within the ANT-C, and abd-A within the BX-C) lack stalled Pol II. This arrangement of stalled Hox genes raises the possibility that stalling contributes to the chromosomal organization of Hox complexes. All four stalled Hox genes (lab, Antp, Ubx, and Abd-B) were tested for enhancer-blocking activity in transgenic embryos, along with the promoter regions of two nonstalled genes (Scr and abd-A). Test promoters were placed 5' of lacZ and inserted between a divergent white reporter gene and 3' iab-5 enhancer (IAB5) (Chopra, 2009b).

IAB5 regulates Abd-B expression in posterior regions of the early embryo, corresponding to the primordia for parasegments 10-14. IAB5 is a robust enhancer, and can activate lacZ and white even when positioned far from the reporter genes. This assay was used to reveal an intrinsic enhancer-blocking activity of the eve promoter region. eve/lacZ fusion genes block the ability of IAB5 to activate a distal CAT reporter gene. However, mutagenized eve promoter sequences lacking a critical proximal GAGA element failed to block IAB5-white interactions. Similarly, the Abd-B proximal promoter (Abd-Bm) and Ubx promoter regions block activation of distal white expression, whereas the abd-A promoter does not interfere with the activation of white expression in the presumptive abdomen by the IAB5 enhancer (Chopra, 2009b).

These results suggest that the stalled Abd-B proximal promoter and Ubx promoters possess an enhancer-blocking activity, whereas abd-A does not. A similar trend was observed for Hox promoter sequences from the ANT-C. The Antp and lab promoters block IAB5-white interactions, whereas the Scr promoter (which lacks stalled Pol II) does not interfere with the activation of white expression in the presumptive abdomen. Stalled genes from the tinman complex (Tin-C), which encode NK homeobox proteins responsible for patterning mesodermal lineages, were also examined. All of the stalled promoters from the Tin-C contain insulator activities. In contrast, nonstalled promoters from lbl and C15 lack such activities when tested in similar transgenic assays. Even the Hsp70 promoter, the classic example of Pol II pausing, displayed insulator activity when tested in similar enhancer-blocking transgenic assays (Chopra, 2009b).

The preceding experiments suggest that stalled Hox gene promoters contain enhancer-blocking activities. However, an alternative possibility is that stalled promoters are 'stronger' than the white promoter, and are able to sequester the shared IAB5 enhancer. To distinguish between competition and insulator activities, the IAB5 enhancer was placed between the divergently transcribed white and lacZ reporter genes. When the white promoter sequence was placed 5' of the lacZ reporter gene, the shared IAB5 enhancer worked equally well to activate both white and lacZ expression. Similar results were obtained when the leftward lacZ reporter gene was placed under the control of either the stalled Abd-B or Ubx promoters. In all of these cases, both white and lacZ are expressed equally well in the presumptive abdomen. These results suggest that stalled promoters do not block enhancer-promoter interactions by a competition mechanism. Rather, they work like insulators and block such interactions only when positioned between the distal enhancer and target promoter (Chopra, 2009b).

To determine whether stalled Pol II is important for the enhancer-blocking activities of Ubx and Abd-B, mutant embryos were examined with reduced levels of critical Pol II elongation factors. Ubx and Abd-B were selected for further studies since optimal expression of both genes depends on the Pol II elongation factors Cdk9 (pTEFb) and Elo-A (Chopra, 2009a). It was reasoned that destabilization of stalled Pol II might reduce the enhancer-blocking activities of the Ubx and Abd-B promoter regions. However, reductions in Cdk9 and Elo-A are expected to stabilize, not destabilize, Pol II stalling since both are positive factors that promote elongation (Saunders, 2006). Indeed, reductions in Cdk9 or Elo-A activity do not alter the enhancer-blocking activities of the Ubx and Abd-B promoters (Chopra, 2009b).

To investigate the link between Pol II stalling and enhancer blocking, two negative elongation factors were examined: NELF (Lee, 2008) and DSIF (Wada, 1998; Yamaguchi, 1998; Kaplan, 2000). The NELF-E protein binds to the short nascent transcripts protruding from the active site of Pol II after transcription initiation and promoter clearance, and thereby inhibits Pol II elongation (Wu, 2005; Lee, 2008). Both NELF and DSIF are thought to help stabilize Pol II at the pause site, typically 20-50 base pairs (bp) downstream from the transcription start site (Saunders, 2006; Gilchrist, 2008; Lee et al. 2008). Since Pol II elongation factors are encoded by essential genes, it is not possible to examine the lacZ/white reporter genes in homozygous mutant embryos. Instead, the transgenes were expressed in embryos derived from heterozygous females, and thereby contain half the normal levels of NELF and DSIF (Spt) subunits. Reductions in Nelf-E, Nelf-A, Spt4, and Spt5 cause clear disruptions in the enhancer-blocking activities of both the Ubx and Abd-B promoters, as seen by the strong activation of the distal white reporter gene. In contrast, white expression is blocked when the same transgenes are expressed in a wild-type background. The simplest interpretation of these results is that reduced levels of the NELF and DSIF inhibitory complexes destabilize stalled Pol II at the pause site. Reduced pausing results in diminished enhancer-blocking activities. There is a similar loss in the enhancer-blocking activities of the eve promoter and Fab7 insulator when the transgenes are expressed in embryos containing reduced levels of the GAGA factor, Trl. It is conceivable that the GAGA factor also contributes to the enhancer-blocking activity of the Ubx promoter since Trl/+ embryos display augmented expression of white (Chopra, 2009b).

In principle, the augmented expression of the white reporter gene might not result from the impaired function of the stalled insulators, but might arise from enhanced activity of the white promoter. To investigate this issue, Pol II chromatin immunoprecipitation (ChIP) assays were performed, coupled with quantitative PCR (qPCR) assays. In DSIF and NELF mutant embryos, there is no increase in Pol II levels at either the white promoter or intronic regions as compared with wild-type embryos. These results suggest that augmented expression of white is due to diminished insulator activities of stalled promoters in embryos containing reduced levels of negative Pol II elongation factors (Chopra, 2009b).

It has been suggested that insulators might work, at least in part, via promoter mimicry. To explore this issue, the impact of reductions in NELF and DSIF on the activities of two known insulators, Fab7 and Fab8, from the BX-C, were examined. Previously published transgenic lines were used that contain Fab7 or Fab8 inserted between the IAB5 and 2XPE (twist) enhancers attached to a leftward lacZ reporter gene and rightward white reporter. In wild-type embryos, the reporter genes are activated only by the proximal enhancer. Thus, white is activated solely in the mesoderm by the 2XPE enhancer, while lacZ is activated in the presumptive abdomen by IAB5. The distal enhancers are blocked by the Fab7 or Fab8 insulators. Consequently, IAB5 fails to activate white and the 2XPE enhancer fails to activate lacZ (Chopra, 2009b).

Very different results are observed when the transgenes are crossed into mutant embryos containing reduced levels of NELF or DSIF (Spt) subunits. There is a loss in the enhancer-blocking activities of the Fab7 and Fab8 insulators and, as a result, white and lacZ display composite patterns of expression in the mesoderm and abdomen since they are now activated by both enhancers. These results suggest that negative Pol II elongation factors are required for the enhancer-blocking activities of the Fab7 and Fab8 insulators (Chopra, 2009b).

It is proposed that insulators interact with stalled promoters to form higher-order chromatin loop domains, similar to those created by insulator-insulator interactions. Perhaps proteins that bind insulators interact with components of the Pol II complex at stalled genes. Indeed, the recent documentation that the BEAF insulator protein binds to many of the same sites as NELF is consistent with a physical link between stalled Pol II and insulators (Jiang, 2009). The resulting chromatin loops can prevent the inappropriate activation of stalled genes by enhancers associated with neighboring loci. As discussed earlier, stalled Hox genes are located at the boundaries of the ANT-C and BX-C. This arrangement might help ensure that cis-regulatory sequences located outside the complexes do not fortuitously interact with genes contained inside the complex and vice versa. The demonstration that stalled Hox promoters possess an intrinsic insulator activity adds to the intricacy of the chromosomal landscapes that control Hox gene expression in both arthropods and vertebrates (Chopra, 2009b).

Stalled Hox promoters may help promote higher-order chromatin organization within the Hox loci (see illustration). These results suggest that the stalled promoters contain intrinsic insulator activity that requires NELF and DSIF proteins, and this may help define higher-order loops within gene complexes such as the Hox complex. The stalled Pol II along with the NELF and DSIF complex may interact with putative insulator sequences, as seen for the Abd-B promoter and the Fab7. These experiments also suggest that that putative insulator sequences also require NELF and DSIF proteins, and this could be due to sharing of these proteins via the formation of higher-order loops. Such loop domains may help in proper regulation of genes and prevent any aberrant activation from neighboring enhancers, thus favoring proper gene regulations at the higher-order level (Chopra, 2009b).

Transcriptional Regulation

To understand the nature of the regulatory signals impinging on the second promoter of the Antennapedia gene (Antp P2), analysis of its expression in mutants and in inhibitory drug-injected embryos has been carried out. The maternally-activated gene oskar is identified as one of two general repressors of P2 which prevent Antp transcription until division cycle 14. Products of the zygotically-active segmentation genes fushi tarazu, hunchback, Krüppel, giant and knirps then act as activators or repressors of Antp P2 in a combinatorial fashion. The timing of these events, and their positive versus negative nature, is critical for generating the expression patterns normal for Antp (Riley, 1991).

Giant regulates the establishment of the expression patterns of Antennapedia and Abdominal-B. In particular, Giant is the factor that controls the anterior limit of early Antennapedia expression (Reinitz, 1990).

A particular deletion mutation in the Antennapedia complex of Drosophila melanogaster, induces both dominant and recessive loss-of-function phenotypes. The deletion is associated with diminished function of proboscipedia (pb), a homeotic gene required for mouthparts formation. This mutation also has associated dominant thoracic defects related to diminished expression of the homeotic Antennapedia gene copy on the homologous chromosome. This is shown to be a consequence of ectopic pb expression in the thorax. Newly juxtaposed Antp sequences provide the pb gene on the deletion bearing chromosome with a second promoter, Antp P1, in addition to its own. Ectopic PB protein expression occurs under Antp P1 control, by alternate splicing, and results in diminished accumulation of ANTP protein in the imaginal disc cells where Antp P1 is normally expressed. The analysis of this mutant chromosome thus demonstrates that PB protein is capable of participating in the negative regulation of a more posteriorly expressed homeotic gene, as well as serving a homeotic "selector" function in the head (Cribbs, 1992).

Specific amino acid residues at the amino end of the Ultrabithorax homeo domain are required to specifically regulate Antennapedia transcription; and in the context of a Deformed protein, these amino-end residues are sufficient to switch from Deformed- to Ultrabithorax-like targeting specificity. Although residues in the amino end of the homeo domain are also important in determining a Deformed-like targeting specificity, other regions of the Deformed homeodomain are also required for full activity (Lin, 1992).

The cut locus codes for a homeodomain protein and controls the identity of a subset of cells in the peripheral nervous system in Drosophila. During a screen to identify cut-interacting genes, it was observed that flies containing a hypomorphic cut mutation and a heterozygous deletion of the Antennapedia complex exhibit a transformation of mouthparts into leg and antennal structures similar to that seen in homozygous proboscipedia (pb) mutants. The same phenotype is produced with all heterozygous pb alleles tested and is fully penetrant in two different cut mutant backgrounds. This phenotype is accompanied by pronounced changes in the expression patterns of both cut and pb in labial discs. These experiments implicate cut in the regulation of expression and/or function of two homeotic genes (Johnston, 1998).

A significant proportion of cut mutant flies that are heterozygous for certain Antennapedia (Antp) alleles have thoracic defects that mimic loss-of-function Antp phenotypes: ectopic expression of Cut in antennal discs results in ectopic Antp expression and a dominant Antp-like phenotype. Evidence for nonautonomous functions of cut can be found in its interaction with Antp, since ectopic Cut expression in a stripe along the anterior-posterior compartment boundary of both portions of the eye-antennal disc results in uniform activation of Antp throughout the disc. The incidence of outgrowths in the dorsal prothoracic region of flies that are mutant for cut and heterozygous for Antp is highest in the presence of the recessive allele Antp11. However, thoracic outgrowths are consistently observed in combination with dominant alleles, such as AntpWu and and AntpR. Many of the dominant Antp alleles are also recessive lethal, so the outgrowths seen in combination with these alleles can still be reconciled with a loss of Antp function. It has been speculated that the outgrowths may be the product of a homeotic change. Although bristles are generally present on all the outgrowths characterized, they are generally malformed and segment or appendage-specific features could not be identified (Johnston, 1998).

The homeotic gene teashirt (tsh) is known to regulate segmental identity of the trunk region in the Drosophila embryo. There is also a requirement for tsh function in the development of adult head structures. Animals homozygous for a viable tsh allele or heterozygous for various embryonic recessive lethal alleles display miniaturized maxillary palps, a phenotype characteristically induced by dominant gain-of-function mutations of the Antennapedia (Antp) homeotic gene. Animals transheterozygous for tsh and Antp mutations display an enhanced antenna-to-leg and a striking reduced-eye phenotype, suggesting aggravated Antp misexpression in eye-antennal discs of these animals. In agreement with this, in the developing eye-antennal discs of the tsh mutant animals, a significant amount of Antp protein is detected overlapping the domains where tsh is normally expressed. teashirt is expressed in the presumptive palpus of the antennal disc and in the primordia of the sensillae, in the rostral membrane. In pharate adults the antennal segments display expression beyond the proximal domain and extending up to the second and third antennal segments Apparently, during later stages of development, tsh expression is not restricted to the proximal domains of the antenna. These results suggest that tsh specifies adult head segments by repressing Antp expression. No obvious antenna-to-leg transformation is seen in any tsh mutants (Bhojwani, 1997).

Transforming growth factor beta at 60A expression had been previously found to be pronounced in mesodermal cells and in cells of the stomadeal and posterior midgut invaginations, foregut and hindgut cells and in the endodermal cells of the anterior and posterior midgut. Chen (1998) sought out dominant enhancer mutations magnifying the effects of a hypomorphic allele of thick veins (tkv), a type I receptor for dpp. Hypomorphic alleles result in partial loss of function of a gene, and dominant enhancers worsten the phenotypic effects of these hypomorphic alleles. Mutations were found in Mad, Medea, punt and thick veins, all of which are known components of the dpp signaling pathway and in Tgfbeta-60A. Phenotypic analysis of Tgfbeta-60A single mutants and tkv 6;Tgfbeta-60A double mutants revealed both dpp-independent and dpp-dependent functions for Tgfbeta-60A. Tgfbeta-60A mutants lack the first constriction of the embryonic midgut and Antennapedia expression in parasegment 6, indicating that 60A is required for the formation of the first constriction, possibly through regulating Antp expression. This function is independent of dpp signaling, since mutations in dpp or its receptors only disrupt the formation of the second but not the first constriction. This also suggests that there is either redundancy or that a different receptor system is responsible for mediating Tgfbeta-60A signaling to pattern the first constriction (Chen, 1998).

Mutations have been isolated in the Drosophila melanogaster gene glass bottom boat (gbb), which encodes a TGF-ß signaling molecule (formerly referred to as 60A) with the highest sequence similarity to members of the bone morphogenetic protein (BMP) subgroup including vertebrate BMPs 5–8. Genetic analysis of both null and hypomorphic gbb alleles indicates that the gene is required in many developmental processes, including embryonic midgut morphogenesis, patterning of the larval cuticle, fat body morphology, and development and patterning of the imaginal discs. In the embryonic midgut, gbb is required for the formation of the anterior constriction and for maintenance of the homeotic gene Antennapedia in the visceral mesoderm. In addition, a requirement has been shown for gbb in the anterior and posterior cells of the underlying endoderm and in the formation and extension of the gastric caecae. gbb is required in all the imaginal discs for proper disc growth and for specification of veins in the wing and of macrochaete in the notum. Significantly, some of these tissues have been shown to also require the Drosophila BMP2/4 homolog decapentaplegic (dpp), while others do not. These results indicate that signaling by both gbb and dpp may contribute to the development of some tissues, while in others, gbb may signal independent of dpp (Wharton, 1999).

Defects in the embryonic midgut are observed in both dpp and gbb mutants, but each BMP appears to play a different role in midgut morphogenesis. gbb is required for the formation of the anterior midgut constriction, while dpp is required for the central constriction. Previous work has indicated that in the developing midgut, the localized visceral mesoderm expression of homeotic genes Antp, Ubx, and abd-A is required for the correct positioning of the anterior, central, and posterior constrictions, respectively. The homeotic genes have been shown to provide regional specification through their regulation of genes encoding secreted factors, such as dpp and wg, which subsequently act on the underlying midgut endoderm. dpp is activated directly by Ubx in a discrete band of cells in PS 7 of the visceral mesoderm from which Dpp is secreted, resulting in the induction of labial expression in underlying endodermal cells. It has been shown that Ubx expression is, in turn, maintained in the visceral mesoderm via a regulatory feedback loop through the action of dpp. In a manner similar to this regulation of Ubx by dpp, the expression of the homeotic gene Antp is regulated by gbb in the visceral mesoderm cells of PS 5 and 6. However, as is true of the regulation of dpp by Ubx, a reciprocal regulation of gbb by Antp is unlikely. The broad expression of gbb throughout the midgut indicates that gbb cannot be regulated exclusively by Antp (Wharton, 1999 and references).

gbb is expressed in both the visceral mesoderm and endoderm, and, as indicated by the regulation of Antp, gbb signaling is required in the visceral mesoderm. gbb signaling is also required in specific regions of the endoderm. The absence of gbb function eliminates the expression of the endodermal marker P-1 from cells in both the anterior and posterior midgut, as well as from cells in the ventriculus, the site from which the gastric caecae bud. The absence of P-1 staining in the primordia of the gastric caecae in gbb mutant embryos is consistent with gastric caecae defects observed in gbb mutant first instar larvae. It appears that although no gastric caecae are evident in stage 17 gbb mutant embryos, gastric caecae do form, albeit abnormally, by the end of the first larval instar. In summary, this analysis indicates, as is true for dpp, that gbb signaling is required in both the visceral mesoderm and endoderm of the Drosophila midgut. At this time, it is unknown which germ layer or layers serve as the source of the gbb signal (Wharton, 1999).

Localized gene expression patterns are critical for establishing body plans in all multicellular animals. In Drosophila, the gap gene hunchback is expressed in a dynamic pattern in anterior regions of the embryo. Hb protein is first detected as a shallow maternal gradient that prevents expression of posterior gap genes in anterior regions. HB mRNA is also expressed zygotically, first as a broad anterior domain controlled by the Bicoid morphogen, and then in a stripe at the position of parasegment 4 (PS4). The PS4-hb stripe changes the profile of the anterior Hb gradient by generating a localized peak of protein that persists until after the broad domain has started to decline. This peak is required specifically for the formation of the mesothoracic (T2) segment. At the molecular level, the PS4-hb stripe is critical for activation of the homeotic gene Antennapedia, but does not affect a gradient of Hb repressive activity formed by the combination of maternal and Bcd-dependent Hb. The repressive gradient is critical for establishing the positions of several target genes, including the gap genes Kruppel, knirps, and giant, and the homeotic gene Ultrabithorax. Different Hb concentrations are sufficient for repression of gt, kni, and Ubx, but a very high level of Hb, or a combinatorial mechanism, is required for repression of Kr. These results suggest that the individual phases of hb transcription, which overlap temporally and spatially, contribute specific patterning functions in early embryogenesis (Wu, 2001).

The expression of the homeotic gene Antennapedia (Antp), which is first activated in late blastoderm embryos as a strong stripe that overlaps PS4, and in the ventral-most cells of PS5, was examined. Loss-of-function Antp mutants show homeotic transformations of T2 and T3 toward a T1 fate, suggesting that Antp is critical for specifying pattern in these segments. The PS4-Antp stripe is abolished in zygotic hb mutants and in ftz mutants, suggesting that these genes are required for its activation. In st2-kni embryos, the PS4-Antp stripe is completely missing, but the ventral expression at PS5 is not affected. When the st2DeltaK-hb-1 transgene is added to these embryos, there is a reactivation of the PS4-Antp stripe, which is then expressed at wild-type levels by the beginning of germ band elongation. The recovery of the Antp stripe suggests that activation of Antp occurs as a direct response to the increase of Hb protein at PS4, since there is no detectable augmentation of the levels of ftz in the rescued embryos (Wu, 2001).

The preceding experiments suggest that Antp can be efficiently activated with a reduced level of ftz activity. To test this further, the eve stripe 2 enhancer was used to misexpress the pair-rule gene hairy (h), which is a potent repressor of ftz transcription. In wild-type embryos, h is expressed in seven stripes that overlap the anterior portions of the eve stripes. Misexpressing h using the stripe 2 enhancer (st2-h) expands accumulation of H mRNA into the interstripe between h stripes 2 and 3 and causes a deletion of T2. As with the st2-kni deletion, the effect is stronger on the ventral side of the embryo. At the molecular level, st2-h misexpression represses ftz stripe 2 in a dose-dependent manner. High levels completely abolish the stripe, especially in ventral regions, and prevent the activation of en stripe 4. This result is consistent with a direct role for ftz in activation of en. In contrast, the ectopic h does not cause a significant change in activation of PS4-Antp, suggesting that PS4-Antp expression can be activated in the absence of ftz activity. The PS4-hb stripe is also unaffected in these embryos, consistent with the hypothesis that Antp is specifically activated by Hb (Wu, 2001).

pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and has been proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibits several mutant phenotypes such as the transformation of antennae to mesothoracic legs. This study examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, is ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation are also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, is also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes are enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are also PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis (Kim, 2008).

Many PcG genes act as zygotic as well as maternal effect genes during whole Drosophila development, but it is not well known when and how they function. Pho is known to work with its redundant DNA-binding protein, Phol and recruits other PcG complexes by binding its binding sites on PREs. pho functions as a maternal effect gene. Its maternal effect mutant embryos show several segment defects and weak homeotic transformation. When pho functions as a zygotic gene, its zygotic mutant adults show homeotic transformation of antennae and legs. In accord to these results, pho functions in identification of ventral appendage were investigated (Kim, 2008).

Mutations in a few PcG genes result in the transformation of antennae to legs. Mutation in esc induces the ectopic expression of Antp and Ubx in the antennal disc, thus transforming antennae to legs. This indicates that esc represses Antp and Ubx expression in the antennal disc during antennal development. Therefore, the possibility was investigated that pho mutation, like esc mutation, would affect the expression of the selector genes that determine the identity of antenna or leg. In the wild type antennal disc, Antp is not expressed, but hth is expressed in almost all cells except for the presumptive arista, allowing for the development of antenna. However, in the leg disc, Antp is expressed and restricts hth expression to the proximal cells, which permits leg development (Kim, 2008).

Antp is ectopically expressed in the antennal disc of the pho mutant, and its expression subsequently but partially represses hth expression in the presumptive a2 or a3. Moreover, in the pho mutant, dac, which is expressed in the presumptive a3 of wild type antennal discs, is overexpressed in the presumptive a2 or a3 where hth expression is reduced. Ectopic expression of Antp in the presumptive a2 represses hth expression, which subsequently results in the transformation from antenna to leg. Ectopic Antp expression in the presumptive a1 permits expression of hth. In addition, when dac is ectopically expressed in a3 using the UAS/GAL4 system, leg-like bristles are newly formed in a3, indicating transformation of a3 to femur. However, the antennal disc of pho mutant shows that hth expression does not completely disappear in all regions of the presumptive a2 and a3 where Antp is ectopically expressed. These indicate that a pho single mutation partially affects expression of Antp, which leads to the incomplete repression of hth. Moreover, as the increased dosage of PcG mutants causes stronger mutant phenotypes than each single mutant, double mutation of pho and Psc strongly affects the expression of Antp, which leads to the complete repression of hth. Therefore, these results indicate that a pho mutation results in the ectopic expression of Antp, which directly represses hth expression in antennal disc and indirectly regulates dac expression through hth expression, which consequently transforms antennae to legs (Kim, 2008).

In the wing imaginal disc, Polycomb (Pc) and Suppressor of zeste (Su(z)) regulate the expression of teashirt (tsh), which specifies the proximal domain with hth. The polyhomeotic (ph) gene regulates the expression of en and the hedgehog (hh) signaling pathway in the wing imaginal disc. Pc also regulates eye specification genes such as tsh and eyeless (ey). PcG genes have recently been found to regulate organ specification genes in addition to homeotic genes, segmentation genes and cell cycle genes (Kim, 2008).

Therefore, it was proposed that pho might regulate the expression of organ specification genes for several reasons. First, Dll is ectopically expressed in the proximal region of the posterior compartment in the antennal disc of the pho mutant. Additionally, Dll is ectopically expressed in the more proximal region of the leg disc in the pho mutant, while dac is ectopically expressed in both the proximal and distal regions. These ectopic expressions do not antagonize each other in their normal region of expression, and result in duplication of distal tibia. Finally, en expression extends to the anterior compartment of both the antennal and leg discs of the pho mutant (Kim, 2008).

According to these reasons the following is proposed; first, pho regulates the expression of Antp in the antennal disc, which in turn might activate Dll. It has been shown that Dll is activated in AntpNS discs, which is similar in younger and older pho discs. Second, pho regulates the expression of en, which affects the expression of Dll. As a gene determining the A/P axis during antenna and leg development, en affects expression of wg and dpp, which determine the D/V axis via Hh signaling. Wg and Dpp act as morphogens, restricting the expression domain of hth, dac and Dll. This study has demonstrated that en is misexpressed in the anterior compartment in the antennal and leg discs of the pho mutant, which leads to misexpression of wg in the anterior-dorsal compartment. Although it has been shown that in the pho zygotic mutant embryos en is hardly derepressed, the current study showed that it is depressed in the pho zygotic mutant adults, suggesting that pho is involved in regulation of en expression and indirect regulation of Dll expression. Finally, pho might directly regulate expression of Dll, because recent studies using X-ChIP analysis have shown that PcG proteins bind PREs of appendage genes including Dll and hth. Hence, pho may directly or indirectly maintain the expression of Antp and en and regulates P/D patterning genes during ventral appendage formation (Kim, 2008).

Pho and Phol are the only PcG proteins that have a zinc finger domain. A mutation in pho results in weaker phenotypes than other PcG mutations despite the functioning of Pho as a DNA-binding protein. Therefore, Pho may interact with other corepressors and repress the homeotic selector genes. In fact, Pho binds to PRE, which is facilitated by GAGA. PRE-bound Pho and Phol directly recruit PRC2, which leads to the anchoring of PRC1. Pho interacts with PRC1 as well as with the BRM complex. Pho has recently been used to construct a novel complex, called the Pho-repressive complex (PhoRC), which has selective methyl-lysine-binding activity. It is currently known that pho interacts with two other PcG genes, Pc and Pcl, in vivo (Kim, 2008 and references therein).

Pho binds to approximately 100 sites on the polytene chromosome and colocalizes with PSC in about 65% of these binding sites. PSC is a component of PRC1 and inhibits chromatin remodeling. In the third instar larvae, PSC is found in the nuclei in all regions of all imaginal discs. Therefore, it is possible that pho and Psc interact with each other during the adult structure formation from the imaginal discs. pho and Psc interact in ventral appendage formation. While the Psc heterozygote was normal, it enhanced the adult mutant phenotypes exhibited by the pho homozygous mutant. Antp is more widely expressed in the antennal disc of the double mutant of pho and Psc than in that of the pho single mutant, while Psc mutant clones induced by FRT/FLP system showed normal expression of Antp, which indicated that Psc does not directly act by itself in regulating expression of Antp, but it certainly interacts with pho (Kim, 2008 and references therein).

hth is expressed in the distal region regardless of Antp expression so that dac was expressed not only in presumptive a3 but also in other segments, which results in the formation of a new P/D axis. According to recent study showing that hth may have a PRE, these results suggest that pho and Psc might interact to maintain hth expression during antennal development. Moreover, Dll expression in the antennal disc might be repressed by an unknown factor that was affected by the double mutation of pho and Psc, suggesting that the factor might be regulated by pho interaction with Psc during antennal development. In addition, legs of the double mutant had fused segments and weakly jointed tarsi, which may be because extension of Hh signal lead to the abnormal expression of the P/D patterning genes. In sum, pho functions as a regulator of selector genes for the identification of ventral appendages and axis formation by interaction with Psc during postembryogenesis (Kim, 2008).

In addition, Pho interacts with Phol in ventral appendage formation. Adults of double mutants showed more severe defects in appendage formation than those of single mutant. The stronger ectopic expression of Antp in the antennal disc of phol; pho double mutant seems to be one of reasons for severe defects. While Antp is not expressed in phol mutant clones of the wild type antennal discs, it is more strongly ectopically expressed in phol mutant clones of the pho mutant antennal discs than in their surrounding phol/+; pho/pho cells, indicating that Phol may not regulate the expression of Antp alone, but it may do that by interaction with Pho, suggesting that this may lead to recruit PRC1 including PSC to PRE sites of Antp and other appendage genes (Kim, 2008).

Posttranscriptional regulation

Ultrabithorax and Antennapedia 5' untranslated regions promote developmentally regulated internal translation initiation. In principle, mRNAs that contain unusually long leader sequences with multiple upstream reading frames (URFs) are good candidates for initiating transcription via a cap-independent internal ribosome binding mechanism. The 5' untranslated regions (UTRs) of the Drosophila Ubx and Antp genes were tested for their ability to promote cap-independent translation initiation. The Ubx and the Antp 5' UTR were inserted between the CAT and lacZ coding sequences in a dicistronic gene and tested for internal ribosome entry site (IRES) activity in transgenic Drosophila. Predicted full-length dicistronic mRNAs were present. High CAT activity is expressed from the first cistron from all of the dicistronic constructs introduced into the fly genome. The dicistronic transgenic strains bearing the Ubx and Antp IRES elements express significant levels of beta-galactosidase (betaGAL) from the second cistron whereas little or no betaGAL is expressed in the controls lacking the IRESs. In situ analysis of betaGAL expression in the transgenic strains indicates that expression of the second cistron is spatially and temporally regulated. Although the developmental patterns of expression directed by the Antp and Ubx IRESs overlap, they exhibit several differences indicating that these IRESs are not functionally equivalent (Ye, 1997).

Regulation by Polycomb and trithorax groups

Continued: Antennapedia Transcriptional regulation part 2/2

Antennapedia: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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