cis-Regulatory Sequences and Functions

engrailed contains a TATA-box deficient (TATA-less) promoter. Such TATA-less promoters, including engrailed's, have a conserved sequence motif, A/GGA/TCGTG, termed the downstream promoter element (DPE), located about 30 nucleotides downstream of the RNA start site. 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).

Truncations in the regulatory region of en reduce transcription to levels that depend both upon the tissue and upon the location of the chromosomal break. These mutations affect expression of the linked invected gene, suggesting that en and invected share a complex set of regulatory elements that operate over at least 85 kb (Goldsborough, 1994).

Gap proteins Krüppel and Hunchback to function as transcriptional regulators in cultured cells. Both proteins bind to specific sites in a 100-bp DNA fragment located upstream of the segment polarity gene engrailed, which also contains functional binding sites for a number of homeo box proteins. The Hunchback protein is a strikingly concentration-dependent activator of transcription, capable of functioning both by itself and also synergistically with the pair-rule proteins Fushi tarazu and Paired. In contrast, Krüppel is a transcriptional repressor that can block transcription induced either by Hunchback or by several different homeo box proteins (Zuo, 1991).

Within an engrailed enhancer, adjacent and conserved binding sites for the Fushi tarazu protein and a cofactor are each necessary, and together sufficient, for transcriptional activation. Footprinting shows that the cofactor site can be bound specifically by Ftz-F1, a member of the nuclear receptor superfamily. Ftz-F1 and the Fushi tarazu homeodomain bind the sites with 4- to 8-fold cooperativity, suggesting that direct contact between the two proteins may contribute to target recognition. Even parasegmental reporter expression is dependent on Fushi tarazu and maternal Ftz-F1, suggesting that these two proteins are indeed the factors that act upon the two sites in embryos. The two adjacent binding sites are also required for continued activity of the engrailed enhancer after Fushi tarazu protein is no longer detectable, including the period when both engrailed and the enhancer become dependent upon wingless. A separate negative regulatory element exists that apparently responds to odd-skipped (Florence, 1997).

In Drosophila the Polycomb group genes are required for the long-term maintenance of the repressed state of many developmentally crucial regulatory genes. Their gene products are thought to function in a common multimeric complex that associates with Polycomb group response elements (PREs) in target genes and regulates higher-order chromatin structure. The chromodomain of Polycomb is necessary for protein-protein interactions within a Polycomb-Polyhomeotic complex. Posterior sexcombs protein coimmunoprecipitates Polycomb and Polyhomeotic, indicating that all three are members of a common multimeric protein complex. Immunoprecipitation experiments using in vivo cross-linked chromatin indicate that these three Polycomb group proteins are associated with identical regulatory elements of the selector gene engrailed in tissue culture cells. Polycomb, Polyhomeotic, and Posterior sexcombs are, however, differentially distributed on regulatory sequences of the engrailed-related gene invected. High-resolution mapping shows that Pc binding is maximal in a 1.0-kb element, 400 bp upstream of the inv start of transcription. Pc binding sites in en are found in a fragment that contains repetitive elements. The Pc binding sites and the repetitive elements are separable. In fact, Pc associates with two distinct elements, one covering the first intron and the other 1 kb upstream from the start of transcription. Both these regions have been implicated in regulation of en expression during embryogenesis. The binding site upstream of en overlaps with a number of pairing-sensitive elements which have been suggested to mediate PcG repression. Ph and Psc are present at both Pc binding sites in the en upstream region and first intron. The common Pc-Ph-Psc complex does not appear to funcion at inv: no Psc is associated with inv and Ph is associated with a much more restricted element than Pc (Strutt, 1997).

Polycomb response elements (PREs) in several genes contain conserved sequence motifs. One of these motifs is the binding site for the protein coded for by the recently cloned gene polyhomeotic (pho), the Drosophila homolog of mammalian YY1. The conserved sequence extends beyond the YY1 core consensus sequence suggesting that parts of Pho may impose additional DNA sequence requirements. In this respect and unlike YY1, PHO has an additional 45 amino acids following the fourth zinc finger. It is also possible that Pho may bind to PREs together with another protein in order to fully exploit the conserved sequence. The conserved sequence motif CNGCCATNDNND, includes the YY1 core consensus CCATNWY. Eight consensus sites have been identified in 6 PREs of the bithorax complex (BX-C): bxd, iab-2, Mcp, iab-6, iab-7 and iab-8. The bxd PRE harbors all three characteristics used to define PREs (maintenance of expression of a lacZ reporter assay throughout development; pairing-sensitive repression of a mini-white reporter, and creation of an additional chromosomal binding site of the PcG-repressing complex in a salivary gland assay). The iab-2 PRE contains two homology boxes (a and b) and has been identifed in the maintenance and pairing-sensitive assays. The Mcp and iab-6 PREs have been characterized in the pairing-sensitive assay. The iab-7 PRE contains two homology motifs, a and b. This PRE has been characterized in all three assays. The iab-8 PRE has been identified in the maintenance assay. The conserved sequence motif is found in three PREs from Sexcombs reduced regulatory regions, and has been identified in the pairing-sensitive assay. The sequence motif found in two PREs from the engrailed regulatory region has been characterized in the pairing-sensitive assay. The sequence motif is also found in polyhomeotic, and has been identified in the pairing-sensitive and salivary gland assays (Mihaly, 1998)

Regulatory DNA from engrailed causes silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called 'pairing-sensitive silencing.' The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), the activities of each of these engrailed fragments were tested in a construct designed to detect PRE activity in embryos. Both fragments behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment shows additional silencing capabilities. Using the mini-white reporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggest that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function (Americo, 2002).

An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila engrailed gene

Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. This laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila engrailed gene. One of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. A consensus binding site has been derived for the Sp1/KLF Drosophila family members; this consensus sequence is shown to be present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila (Brown, 2005).

PREs are complex elements -- no single DNA binding site can act as a PRE. Instead, PREs are made up of binding sites for many different proteins. To date most PREs studied at the molecular level have been shown to contain DNA binding sites (usually multiple copies) for the proteins Pleiohomeotic (Pho) and its partially redundant homolog Pleiohomeotic-like (Phol), Zeste, GAGA Factor (GAF)/Pipsqueak (Psq) and Dorsal Switch Protein 1 (Dsp1). Clustered pairs of GAF/Psq, Zeste and Pho/Phol sites can predict the location of many PREs. However it has recently been shown that a combination of Zeste, Dsp1, GAF/Psq and Pho/Phol sites in the same number, orientation and spacing as in the native PRE is insufficient to restore full PRE activity . This suggests that all of the DNA binding sites necessary for PRE activity are still not known (Brown, 2005).

With the aim of identifying the binding sites and factors necessary for PRE function, this lab has been trying to define the components that constitute a minimal 181 bp PRE at ~576 to ~395 upstream of the Drosophila engrailed gene. This element was originally identified in a pairing-sensitive silencing assay, an assay used to detect the function of many PREs. When PREs are included in the vector pCaSpeR, they have an unusual effect on the eye color marker mini-white. Normally, when transgenic flies are made with pCaSpeR, flies homozygous for the transgene have a darker eye color than flies heterozygous for the transgene. However, when a PRE is included in the pCaSpeR vector, the eye color of homozygous flies is often lighter than that of heterozygotes. This phenomenon is dependent on the chromosomes being able to pair. Fragments of DNA that mediate pairing-sensitive silencing are called pairing-sensitive elements (PSEs). Not all PSEs have been shown to act as PREs and vice versa. The engrailed 181 bp element behaves both as a PRE and a PSE (Brown, 2005).

The 181 bp engrailed PRE contains 3 GAF/Psq sites, 2 Pho/Phol sites, 2 potential Zeste sites and 1 Dsp1 site that almost entirely overlaps the Pho/Phol site that has been studied by mutational analysis. In addition to these known sites, a number of other protein-binding sites required for pairing-sensitive silencing have been identified. This study investigated the role of one of these protein-binding sites, Site 2, in PRE function and shows that the Sp1/KLF family of proteins bind to this site. Sp1/KLF binding sites are present in most well characterized PREs (Brown, 2005).

Site 2 is required for pairing-sensitive silencing of the 181 bp engrailed PRE. This study asked whether Site 2 is also important for the PRE activity of that fragment. The bxd-Ubx-lacZ reporter construct was used to test the effect of mutations in Site 2 on PRE activity. In the absence of a PRE, the bxd-Ubx-lacZ reporter construct expresses lacZ throughout the embryo, in both the ectoderm and the nervous system, late in development. When the 181 bp PRE from the Drosophila engrailed gene is included in this vector, lacZ expression is restricted to parasegment six and posterior segments. Restricted expression patterns were seen in 75% of the lines, a number consistent with what has been seen by other investigators as PREs are not active in all chromosomal insertion sites (Brown, 2005).

Mutations were introduced into either Site 2 or the two Pho/Phol binding sites in the 181 bp fragment in the context of the bxd-Ubx-lacZ vector. The effect on PRE activity, as assayed by embryonic ß-galactosidase expression patterns, was analyzed. Mutation of either the Pho/Phol sites or Site 2 caused a reduction in the percentage of lines that had PRE activity. Both mutated constructs gave results that were significantly different from the expected frequency of 75% PRE activity for the unmutated construct. There were still a small percentage of lines with restricted expression in the Pho/Phol (20%), and the Site 2 mutant transgene (29%) as well as in the vector only control (8%). This probably reflects the fact that the bxd enhancer and the Ubx promoter are poised to work with flanking genomic PREs and may contain weak PRE activity on their own. When the first 350 bp of the bxd sequence that borders the 181 bp element in these constructs were scanned for the known PRE DNA binding sites, 3 Pho/Phol sites, 3 Dsp1 sites, 1 Zeste site and 2 potential Sp1 sites were found. This may explain why mutation of Pho/Phol or Site 2 did not completely eliminate the PRE activity of the 181 bp engrailed fragment in this vector. Nevertheless, the results suggest that both Pho/Phol and Site 2 binding sites contribute to the activity of the engrailed PRE, and that neither alone is sufficient for PRE activity (Brown, 2005).

In order to identify the protein(s) that interact with Site 2, a yeast one-hybrid screen was carried using multimerized Site 2 as bait. Four cDNA clones were isolated that showed specific binding to Site 2 but not to a mutated Site 2. Each of these four clones encoded a member of the Sp1/KLF family of zinc finger proteins [CG5669, CG12029 (two different length clones) and luna]. The consensus binding sequence for the Sp1/KLF family based on mutational analysis of a KLF family member (KLF4) binding site is (G/A)(G/A)GG(C/T)G(C/T). The Site 2 sequence contains a perfect match to this consensus sequence (Brown, 2005).

The Sp1/KLF family is an important group of proteins that in mammals have been shown to be involved in cell morphogenesis, differentiation and cancer. These proteins share a high degree of homology over 3 Cys2/His2 zinc fingers (>65% sequence identity with each other), located at or close to the C-terminal end of the protein. The N-terminal regions are generally unique (Brown, 2005).

Three members of the Sp1/KLF family were identified in the yeast one-hybrid screen, however the screen was not saturating. When the Drosophila genome sequence was searched for homology to the zinc finger region of these proteins a total of 10 members of this class was identified in Drosophila. The zinc finger regions of these members are highly conserved. The mammalian factors can be sub-divided into three classes (Sp1/class I family, class II family and class III family) based on closer homology between members of the subfamily relative to members outside the subfamily. This homology sometimes extends to functional protein domains that lie outside the DNA binding domain. The Drosophila proteins can also be placed into different groups based on homology of the zinc fingers with the zinc fingers of the human SP1/KLF proteins. CG5669, dSp1 and Btd are more closely related to the Sp1/Class I proteins than to the KLF proteins. In fact, dSp1 has 97% amino acid identity with hSp8 zinc fingers and also has homology to hSp8 in the region N-terminal to the zinc fingers. For CG5669 and Btd it is hard to assign a homolog since they have significant identity with a number of Class I proteins. Sequence identities of CG5669 with the zinc fingers of Class I range from 76% to 87%, for Btd the range is 65%–74%. CG12029, Luna and CG9895 have closer amino acid identity to the Class II human proteins. Luna is most closely related to KLF6 and KLF7 and shows conservation of a putative activation domain at the N-terminus. Cabot (also known as CG4427) and Bteb2 seem to belong to the class III subclass showing 72%–85% identity with human members of this class. Hkb and CG3065 are harder to place. CG3065 has 62%–65% identity to members of the Sp1 family and 62%–64% identity with KLF9 and KLF14 members of class III. Hkb is the most diverged member of this class of proteins. The highest degree of homology for Hkb is 53% identity with hSp5 and it is difficult to say whether it should be placed in this family at all. It is noted that Hkb binds only weakly to sequences that match the Sp1/KLF consensus (Brown, 2005).

Each of the zinc finger regions of these 10 proteins were cloned in frame into an in vitro transcription/translation vector and expressed in vitro. The products were tested for binding to Site 2. Nine of the 10 Drosophila Sp1/KLF family member zinc fingers show binding to Site 2 and only CG3065 shows no binding. Binding to each of these factors is specific based on competition experiments. Binding of Luna, Cabot and Hkb to Site 2 is very weak. This result is not due to the production of inactive protein since, with the exception of Hkb, strong binding of these factors is seen to other binding site sequences (Brown, 2005).

Identifying which Sp1/KLF factor acts through Site 2 is not a easy task. Not only are a number of members of this class genetically uncharacterized there is also the possibility that there may be functional redundancy as is seen with Pho and Phol. The existence of a viable and fertile Bteb2 mutant suggests that functional redundancy will be observed with the Sp1/KLF family in Drosophila. Experiments using family member-specific antibodies in chromatin-immunoprecipitation experiments on PREs will help elucidate which Sp1/KLF family members play a role in PRE function in Drosophila (Brown, 2005).

What role the Sp1/KLF family of proteins play in recruiting the PcG complexes to the PRE remains to be elucidated. In fact, for most of the other proteins required for PRE function, their roles are not yet clear. Both GAF and Psq bind the sequence GAGAG, a sequence shown to be important for PRE function. Psq has been shown to be in a complex with PcG proteins isolated from the Drosophila cell line SL2 and psq mutations enhance the mutant phenotypes of the PcG genes polyhomeotic (Ph) and Polycomb (Pc) in larval and adult tissues. This suggests that Psq may be important for PRE function. GAF has also been reported to co-purify with some PcG proteins and has been shown by chromatin-immunoprecipitation experiments to be present at PREs. GAF is a member of the TrxG of genes but may also play a role in PcG repression. The DNA binding protein Pho has been shown to bind in vitro to a chromatinized PRE template only if GAF is present. GAF and Psq can interact through their BTB protein–protein interaction domains and it has been proposed that they may function together in vivo (Brown, 2005).

Zeste has been shown to be important for both PRE and TRE activity. Zeste is a stoichiometric component of the biochemically purified PcG complex, PRC1 suggesting a role in PcG repression, and it has been report that Zeste is required for the PcG-mediated repression of an Ubx transgene. In contrast, experiments with the iab-7 PRE have shown that Zeste binding sites are important for the ability of this DNA to act as a TRE, not as a PRE (Brown, 2005).

Pho and Phol have recently been shown to be required to recruit an Esc-E(z) complex to a PRE . In vitro, Pho interacts directly with E(z) and Esc whereas Phol interacts with Esc. Recruitment of the Esc-E(z) complex leads to methylation of lysine 27 of histone H3 by the SET domain of E(z). The methylated K27 recruits a Pc-containing complex through interaction of the chromo-domain of Pc with the methylated histone tails. Pho has also been shown to interact with Pc in vitro. Pho/Phol double mutants have a very strong PcG phenotype, much stronger than mutations in the genes encoding the other PRE-binding factors suggesting that Pho/Phol play a central role in PRE function. It has been proposed that Dsp1 facilitates the binding of Pho/Phol to the PRE (Brown, 2005).

The role that the Sp1/KLF family may play remains to be elucidated but it is intriguing to note that mammalian Sp1 has been reported to interact directly with YY1 (the mammalian homolog of Pho). This interaction requires the first one and a half zinc fingers of YY1, a region that is 96% identical between the Drosophila and mammalian proteins. The 158 amino acid C-terminal region of Sp1 (includes the three zinc fingers and one of the activation domains, domain D), can mediate the interaction leading to an increase in the level of correctly initiated transcripts. These data raise the possibility that Pho or Phol may interact with Sp1/KLF proteins at PREs (Brown, 2005).

Enhancer-promoter communication at the Drosophila engrailed locus

Enhancers are often located many tens of kilobases away from the promoter they regulate, sometimes residing closer to the promoter of a neighboring gene. How do they know which gene to activate? This study used homing P[en] constructs to study the enhancer-promoter communication at the engrailed locus. engrailed enhancers can act over large distances, even skipping over other transcription units, choosing the engrailed promoter over those of neighboring genes. This specificity is achieved in at least three ways: (1) early acting engrailed stripe enhancers exhibit promoter specificity; (2) a proximal promoter-tethering element is required for the action of the imaginal disc enhancer(s). The data suggest that there are two partially redundant promoter-tethering elements. (3) The long-distance action of engrailed enhancers requires a combination of the engrailed promoter and sequences within or closely linked to the promoter proximal Polycomb-group response elements. These data show that multiple mechanisms ensure proper enhancer-promoter communication at the Drosophila engrailed locus (Kwon, 2009).

engrailed gene exists in a gene complex with the coregulated invected (inv) gene. en and inv are co-expressed in a complex manner throughout development. Early in development, they are required for segmentation, and are expressed in a series of stripes continually throughout embryogenesis. Although the location of En stripes does not change throughout embryonic development, the enhancers and the trans-acting proteins that regulate their expression do change. For example, separate fragments of regulatory DNA act as enhancers for activation by the pair-rule genes, for activation by Wingless signaling and for regulation by the trithorax and Polycomb group genes. en and inv are also expressed in the hindgut, clypeolabrum, central nervous system (CNS), peripheral nervous system (PNS), fat body and the posterior compartments of the imaginal discs. The regulatory sequences of engrailed are distributed throughout a 70 kb region. Interestingly, the en and inv promoters are separated by ~54 kb, yet they appear to be regulated by the same enhancers, suggesting that en/inv enhancers must be able to act over long distances. What ensures they activate only en/inv and not flanking genes? Homing P-transgenes were used to address this question (Kwon, 2009).

Most P-based constructs insert in the genome in a non-selective manner. However, a few pieces of regulatory DNA have been found to alter the insertional specificity of P-constructs, causing the P-construct to insert near the gene that the regulatory DNA came from. This phenomenon is called P-element homing and was first observed with DNA from the en gene. DNA fragments from the bithorax complex, linotte gene and, most recently, even skipped, have also been shown to mediate homing. For en, P-constructs containing a DNA fragment including the engrailed promoter and 2.4 kb of upstream sequences (P[en-lacZ]) cause homing to the en region of the chromosome (Kassis, 1992). Insertions are not site specific, but occur over a region of ~300 kb, including en and inv and flanking genes. P[en-lacZ] has no enhancer activity on its own, but acts as an enhancer detector; that is, its expression is directed by flanking genomic enhancers. It has recently been shown that P[en-lacZ] can be stimulated by en enhancers even when it is inserted into neighboring genes (DeVido, 2008). Furthermore, this long-distance enhancer activity was dependent upon en DNA fragments that also act as Polycomb-group response elements (PREs). PREs are DNA elements that bind and mediate the action of the Polycomb group of transcriptional repressors. It is not known whether the PRE activity can be separated from the enhancer-detection activity of these DNA fragments (Kwon, 2009).

This study shows that, in addition to the PRE fragments, the en promoter is necessary for long-distance interactions with en/inv enhancers. The data suggest that enhancer-promoter specificity at the en locus is complex, using different mechanisms for different enhancers: (1) the early stripe enhancers, which respond to the pair-rule transcriptional activators, exhibit promoter specificity; (2) a promoter-tethering element is required for interactions with the imaginal disc enhancer(s). Finally, both the promoter and the DNA fragment that includes the promoter-proximal PREs are important for the long-range action of en enhancers (Kwon, 2009).

en and inv exist in a gene complex, encode related proteins with redundant functions and share regulatory DNA. Thus, en enhancers must be able to activate both the en and inv promoters, which are separated by 54 kb. What properties do these two promoters share? First, en and inv are both TATA-less promoters. Both en and inv have the initiator promoter element (Inr) and the downstream promoter element (DPE). The inv promoter has a perfect match to the Inr consensus sequence for Drosophila (at nucleotide 7,363,212), and a near match to the DPE 28 bp after the initiating adenine. The en promoter has a near match to the Inr consensus and a perfect match to the DPE located 30 bp downstream of the third nucleotide of the Inr sequence. Second, both promoters have binding sites for the transcription factor GAGA, which are located just upstream of the transcription start site. GAGA-binding sites greatly increase the activity of the en promoter. Third, both have Polycomb response elements (PREs) located very close to the promoters. Finally, the DNA sequences from 600 bp upstream to 400 bp downstream of the inv promoter were compared with the 588 bp en promoter fragment used in this study and a few stretches of sequence identity were found. The longest was a 14/15 bp match located from -57 to -42 upstream of the en transcription start site and from -40 to -25 bp upstream of the inv transcription start site. The functional significance of this is unknown (Kwon, 2009).

The sequences around the presumed transcription start sites for the different transcripts were examined. Strikingly, aside from sprt (well upstream of inv), none of these genes had sequences that matched the TATA, Inr or DPE consensus sequences. Like en and inv, the sprt gene has Inr and DPE elements. Unlike en and inv, no PREs were found at the sprt gene (as judged by the binding of PcG proteins). It is suggested that sprt is not activated by en enhancers because it lacks the PREs (or associated sequences) that are necessary for the long-distance action of the en enhancers (Kwon, 2009).

The minimal heat shock promoter present in P[enHSP] contains sequences -44 to +204 bp of the HSP70 promoter. It contains the TATA element but does not have any of the GAGA sites that are located further upstream. It has been found that a slightly different version of this promoter (from -73 to +70 bp) would not function in a reporter construct with the en stripe enhancer present in the intron, although it was able to function with enhancers that drive expression in the hindgut, fat body and posterior spiracles. Those data, combined with the current results, clearly show that different en enhancers have different promoter requirements. The ability of different types of core promoters to recognize different enhancers has been reported by many other investigators and may be a common mechanism to achieve enhancer specificity in Drosophila (Kwon, 2009).

At least three distinct processes mediate promoter specificity at en. (1) The early stripe enhancers, those activated by the pair-rule transcription activators, require the en promoter; they are not able to stimulate the heat shock promoter. It is suggested that this could be due to the type of core promoter present at en, or to sequences very near the transcription start site. The en allele enJ86 contains a deletion from -412 to -73 bp upstream of the en transcription start site and shows no disruption of early en expression. Thus, sequences within 73 bp of the transcription start site are sufficient for interaction with early stripe enhancers. Caudal, an early acting developmental transcription factor, was recently found to specifically activate DPE-containing promoters. It would be interesting to test whether pair-rule proteins also exhibit promoter specificity (Kwon, 2009).

(2) It is proposed that there are two promoter-tethering elements that mediate interactions with the imaginal disc enhancers. One of them is located in the 181 bp element, PRE2, and another is located between -273 and -73 bp. en joins a growing list of Drosophila genes that have promoter-tethering elements, including the homeotic genes Scr and Abd-B, as well as the white and string genes. It is likely that many other genes with extensive regulatory regions have promoter-tethering elements (Kwon, 2009).

(3) It has been shown that the 2 kb PRE fragment, from -2.4 to -0.4 kb, is required for distantly located transgenes to interact with the en enhancers (DeVido, 2008). The current study shows that the en promoter is also required for long-range enhancer-promoter interactions. It is suggested that both the promoter and the PRE fragment are necessary to form the correct chromatin structure to allow interactions with distant en enhancers. In conclusion, these data suggest that multiple mechanisms exist to ensure that en enhancers activate the correct promoters (Kwon, 2009).

Co-regulation of invected and engrailed by a complex array of regulatory sequences in Drosophila

invected (inv) and engrailed (en) form a gene complex that extends about 115kb. These two genes encode highly related homeodomain proteins that are co-regulated in a complex manner throughout development. Dissection of inv/en regulatory DNA shows that most enhancers are spread throughout a 62kb region. Two types of constructs were used to analyze the function of this DNA: P-element based reporter constructs with small pieces of DNA fused to the en promoter driving lacZ expression and large constructs with HA-tagged en and inv inserted in the genome with the phiC31 system. In addition, deletions of inv and en DNA were generated in situ, and their effects on inv/en expression were assayed. The results support and extend knowledge of inv/en regulation. First, inv and en share regulatory DNA, most of which is flanking the en transcription unit. In support of this, a 79-kb HA-en transgene can rescue inv en double mutants to viable, fertile adults. In contrast, an 84-kb HA-inv transgene lacks most of the enhancers for inv/en expression. Second, there are multiple enhancers for inv/en stripes in embryos; some of these may be redundant but others play discrete roles at different stages of embryonic development. Finally, no small reporter construct gave expression in the posterior compartment of imaginal discs, a hallmark of inv/en expression. Robust expression of HA-en in the posterior compartment of imaginal discs is evident from the 79-kb HA-en transgene, while a 45-kb HA-en transgene gives weaker, variable imaginal disc expression. It is suggested that the activity of the imaginal disc enhancer(s) is dependent on the chromatin structure of the inv/en domain (Cheng, 2014).

Transcriptional Regulation

Regulation of engrailed by gap genes

The expression patterns of engrailed and wingless are altered in a single position in crocodile mutant embryos. Within the clypeolabral segment anlage, en expression is absent and the adjacent wg expression domain is significantly expanded and covers the area normally expressing en in addition to wg's normal expresion domain. Wild-type croc activity is therefore required for the control of segment polarity gene expression within a single segment equivalent, i.e. the clypeolabrum, the anterior-most segment equivalent of the head anlage of the embryo (Häcker, 1995).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. A determination was made of whether col mutations affect the expression of wg and En, which mark the anterior and posterior compartments of each head segment, respectively. In col1 hemizygous embryos, both the intercalary stripe of En and the spot of wg expression are missing (Crozatier, 1999).

Regulation of engrailed by pair rule genes

Paired and FTZ activate engrailed, while Naked, Sloppy paired, Runt and Odd paired repress.

How does the pair-rule pattern regulate en expression into a 14 band segmental pattern? A major effect comes from two pair-rule genes, even-skipped and fushi tarazu. Quite early in development at the pre-cellular blastoderm stage, even-skipped, true to its name, is expressed in seven stripes, centered in odd numbered parasegmental primordia. Simultaneously, ftz is expressed in seven stripes, in the even numbered primordia between the even-skipped stripes. In this manner an ordered, symmetrical pattern with a total of 14 stripes is created, subdividing the blastoderm into 14 parasegments, with alternating distributions of FTZ and EVE.

engrailed transcription is inhibited by EVE and activated by FTZ. Therefore, in the absence of any other regulation en would be expressed in each FTZ parasegment. Four other genes are involved however, and most of their influences are negative: Sloppy paired, Runt and Odd-skipped inhibit en transcription, while Paired activates. The overall effect is to repress en in the middle of each FTZ parasegment, and activate en only at the ends. Detailed descriptions of this process are to be found in Baumgartner, 1994, Fujioka, 1995 and Mullen, 1995. Some of the details for Even-skipped, Sloppy paired and Runt are described below:

The early bell-shaped gradient of even-skipped expression is sufficient for generating stable parasegment borders. The anterior portion of each early stripe has morphogenic activity, repressing different target genes at different concentrations. These distinct repression thresholds serve to both limit and subdivide a narrow zone of paired expression. Within this zone, single cell rows express either engrailed, where runt and sloppy-paired are repressed, or wingless, where they are not. While the early eve gradient is sufficient to establish parasegmental borders without refined, more sharply defined late expression, late eve expression has a role in augmenting this boundary to provide for strong, continuous stripes of engrailed expression. The early eve gradient is sufficient, at its posterior edge, for subdividing the ftz domain into engrailed expressing and non-expressing cells (Fujioka, 1995).

The response kinetics of known and putative target genes of Fushi tarazu has been examined in order to distinguish between direct and indirect Ftz targets. This kinetic analysis was achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (less than 10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely to be important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes have been analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy (Nasiadka, 1999).

Besides being autoregulatory, the second best-characterized target of Ftz is the segment polarity gene engrailed. Stripes of en initiate at the anterior edge of each parasegment. Every second stripe overlaps with the anterior portion of each ftz stripe and is lost in ftz mutant embryos. Ubiquitous expression of Ftz causes a broadening of expression of these ftz-dependent en stripes, making them 2-3 cells wide instead of 1-2 cells wide. This expansion occurs in the same cells as endogenous ftz stripe expansion. The kinetics of this response were determined in the same way as for endogenous ftz, using embryos from the same set of collections. The curve generated for en overlaps very closely that of the endogenous ftz response, suggesting that both genes are direct targets of Ftz. If en were regulated indirectly, a delay would be expected reflecting the time required for intermediary gene products to be expressed, to accumulate and to elicit a response (Nasiadka, 1999).

Removal of slp gene causes embryos to exhibit a severe pair-rule/segment polarity phenotype. The en stripes expand anteriorly in slp mutant embryos. slp activity is an absolute requirement for maintenance of wg expression at the same time that wg transcription is dependent on HH. SLP protein is expressed in broad stripes just anterior of the EN-positive cells, overlapping the narrow wg stripes. By virtue of its ability to activate wg and repress en expression, the distribution of SLP defines the wg-competent and en-competent groups. Consistent with this hypothesis, ubiquitous expression of slp throughout the parasegement abolishes en expression and, in ptc mutant embryos, results in a near ubiquitous distribution of WG transcripts. (Cardigan, 1994).

Homeodomain proteins are DNA-binding transcription factors that control major developmental patterning events. Although DNA binding is mediated by the homeodomain, interactions with other transcription factors play an unusually important role in the selection and regulation of target genes. A major question in the field is whether these cofactor interactions select target genes by modulating DNA binding site specificity (selective binding model), transcriptional activity (activity regulation model) or both. A related issue is whether the number of target genes bound and regulated is a small or large percentage of genes in the genome. These issues have been addressed using a chimeric protein that contains the strong activation domain of the viral VP16 protein fused to the Drosophila homeodomain-containing protein Fushi tarazu. Genes previously thought not to be direct targets of Ftz remain unaffected by FtzVP16. Addition of the VP16 activation domain to Ftz does, however, allow it to regulate previously identified target genes at times and in regions that Ftz alone cannot. It also changes Ftz into an activator of two genes that it normally represses. Taken together, the results suggest that Ftz binds and regulates a relatively limited number of target genes, and that cofactors affect target gene specificity primarily by controlling binding site selection (Nasiadka, 2000).

Activity regulation plays an important role in Ftz function, but this role is mainly to refine the temporal and spatial windows of target gene regulation and to modulate levels of expression. This conclusion is supported by the following observations. Five of the genes tested (ftz, odd, slp, en and wg) could be activated ectopically by FtzVP16 in regions and at times that Ftz could not induce a response. This shows that Ftz has the ability to bind to these promoters, but that it must be bound in an inactive state. For Ftz to function in these cells, it probably requires the addition of requisite cofactors, the removal of repressors or both. For the five genes listed above, the VP16 activation domain is able to overcome some of these limitations. The regulation by Ftz of en is a good example of this type of temporal and spatial refinement in activity. Results with FtzVP16 show that Ftz can bind to the en promoter during the time that ftz autoregulation and odd activation are well under way. However, the ability of Ftz to activate en is normally delayed until cellularization is completed (approx. 45 minutes). This delay may be necessary to allow other en regulators to resolve into the complex patterns of expression that are required for en to initiate in 14 narrow stripes. Like most homeodomain proteins, Ftz has the ability to function as both a transcriptional activator and repressor. This dual capacity suggests a requirement for distinct activity-regulating cofactors. However, differential activity can also be achieved, at least in part, by binding to different sites on different genes. For example, the response elements required for repression of the Distalless gene by Ubx and activation by Dfd are different. This also appears to be the case for activation of the dpp gene by Ubx and its repression by Abd-A. The different cofactors that help recruit the three proteins to these sites may also be partly responsible for their differences in transcriptional activity. For example, Exd is thought to generally work as a coactivator, acting in part to alter Hox protein conformation. Other factors bound in the vicinity of these sites are also likely to play a major role in activity regulation (Nasiadka, 2000).

In addition to showing that positively acting cofactors are important for Ftz specificity, these data implicate the actions of powerful negative regulators that limit the gene's temporal and spatial domains of activity. The strength and diversity of these negative regulators were emphasized by their ability to suppress the actions of the fused VP16 activation domain despite its previously reported reputation of strength and autonomy. It may be the low DNA binding specificity of the homeodomain that has necessitated this need for diverse mechanisms of repression, since low DNA specificity provides the potential to regulate a large number of inappropriate target genes. Indeed, a rapidly growing number of homeodomain proteins have been shown to be capable of functioning as oncogenes or proto-oncogenes, and oncogenicity can be conferred by fusions to other transcriptional activators. Further studies will be required to identify many of the cofactors and inhibitors that modulate Ftz activity and to determine how they do so (Nasiadka, 2000).

The runt gene is required to generate asymmetries within parasegmental domains. Ectopic runt expression leads to rapid repression of EVE stripes and a somewhat delayed expansion of FTZ stripes. Ectopic runt is a rapid and potent repressor of odd-numbered EN stripes. Two remarkably different segmental phenotypes are generated as a consequence of these effects. The positioning of EN stripes is largely determined by the actions of negative regulators. runt is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of runt and odd by EVE (Manoukian, 1993).

Runt domain family members are defined based on the presence of the 128-amino-acid Runt domain, which is necessary and sufficient for sequence-specific DNA binding. There exists an evolutionarily conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. However, the interaction is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all Runt domain proteins. Drosophila melanogaster Runt and Groucho interact genetically; the in vivo repression of a subset of Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by Runt domain proteins (Aronson, 1997).

Although many of the genes that pattern the segmented body plan of the Drosophila embryo are known, there remains much to learn in terms of how these genes and their products interact with one another. Like many of these gene products, the protein encoded by the pair-rule gene odd-skipped (Odd) is a DNA-binding transcription factor. Genetic experiments have suggested several candidate target genes for Odd, all of which appear to be negatively regulated. Pulses of ectopic Odd expression have been used to test the response of these and other segmentation genes. Three different phenotypes are generated in embryos in which odd is expressed from a heat shock promoter: head defects only, a pair-rule phenotype and a pair-rule phenotype restricted to the dorsal half of the embryo. The head defects only phenotype prevails when Odd is induced between 2:10 and 2:30 hours after egg laying (AEL). The second phenotype is generated when Odd is induced between 2:30 and 2:50 AEL, while the third phenotype prevails when heat shocks are administered between 2:50 and 3:10 AEL. The results are complex, indicating that Odd is capable of repressing some genes wherever and whenever Odd is expressed, while the ability to repress others is temporally or spatially restricted. Previous studies could not establish unambiguously whether Odd acts as a direct or indirect repressor of the en and wg genes. The data presented here show that during gastrulation Odd appears to regulate both genes, not only directly, but indirectly as well. Indirect repression is mediated by selective repression of the en and wg activators: ftz, prd, eve and slp. The result of these interactions in hs-odd embryos is first the loss of all fourteen en and wg stripes due to direct repression and then failure of certain stripes to reinitiate. These results indicate that the activity of Odd is highly dependent on the presence of cofactors and/or overriding inhibitors. Based on these results, and the segmental phenotypes generated by ectopic Odd, a number of new roles for Odd in the patterning of embryonic segments are suggested. These include gap-, pair rule- and segment polarity-type functions (Dréan, 1998).

Krüppel is coexpressed with engrailed in a subset of neurons and glia that include the medial-lateral cluster of en-expressing neurons and the dorsal channel glia cells. In Kr mutants, the medial-lateral cluster is either absent or fails to express en, but the dorsal channel glia cells are not affected. These medial-lateral cluster cells gives rise to serotoninergic neurons, and almost no neurons synthesizing serotonin remain in these mutant embryos. In Kr mutants, the number of gooseberry neural-expressing cells increases from 10% to 50%. Ectopic Kr expression leads to a strong reduction in gsb-n expressing neurons (Romani, 1996).

The odd-paired gene is essential for parasegmental subdivision of the Drosophila embryo. opa is required for the activation of wingless and engrailed in all parasegments. OPA does not act in a spatially restricted manner to establish the position of en and wg expression. Because of its ubiquitous expression, OPA must cooperate with other spatially restricted proteins to achieve proper pair-rule subdivision of the Drosophila embryo (Benedyk, 1994).

The mechanisms of action of cephalic gap genes remain poorly understood. orthodenticle (otd), which establishes a specific region of the anterior head, has been proposed to act in a combinatorial fashion with the cephalic gap genes empty spiracles (ems) and buttonhead (btd) to assign segmental identities in this region. To test this model, a heat-inducible transgene was used to generate pulses of ubiquitous otd expression during embryonic development. Ectopic otd expression causes significant defects in head formation, including the duplication of sensory structures derived from otd-dependent segments. However, these defects do not appear to result from the transformation of head segment identities predicted by the combinatorial model. To determine if the combinatorial model is correct, focus was placed on the dorsomedial papilla (dmp), antennal sense organ (anso), and dorsolateral papilla (dlp). The epidermal portions of these structures serve as markers of the ocular, antennal, and intercalary segments, respectively. According to the combinatorial model, ubiquitous otd expression should cause a transformation of the intercalary segment to a second antennal segment, without affecting the identity of the more anterior ocular segment. This would be indicated by duplication of the anso, loss of the dlp, and no change in the dmp. In a significant fraction of the cuticles that developed after an early pulse of otd expression, the anso is indeed duplicated. Significantly, however, the dlp is generallly not lost in these embryos, indicating that at least part of the intercalary segment is still present. These results do not indicate a transformtion of segmental identity, but rather the specific duplication of otd-dependent sensory structures (Gallitano-Mendel, 1998).

It is likely that misexpression of otd causes intrasegmental transformations rather than intersegmental transformations. The results correlate with specific regulatory effects of otd on the expression of the segment polarity genes engrailed (en) and wingless (wg). In wild-type embryos, en is expressed in each of the head segments. In the anterior head, en expression first appears during germ band extension in the antennal primordium, as a stripe 1-2 cells in width. Expression subsequently appears in the ocular segment (a small spot), the intercalary segment (a small stripe), and eventually in the clypeolabral region. Early induction of otd causes a broadening of the en antennal stripe to a width of as many as eight cells. In wild-type embryos, wg expression first apppears in the forgut primordium at the blastoderm stage. Subsequently, a broad anterior cephalic stripe forms. Following germ-band extension, wg is activated in a discrete stripe or spot in each head segment, anterior and adjacent to the engrailed counterpart. Ubiquitous otd expression causes the reduction or loss of wg in the antennal, intercalary, gnathal, and trunk segments. More anterior wg expression in the forgut, clypeolabral region, and ocular segment is not reduced (Gallitano-Mendel, 1998).

Mutant embryonic cuticles were examined for en or wg: en mutant embryos lack ansos; wg mutant embryos exhibit severe disruptions in head formation. However, unlike in en embryos, the anso is not missing but instead is frequently duplicated. These results indicate that en is required for anso formation. They also suggest that wg plays an inhibitory role in the specification of this sensory strucure. In double en;wg mutant embryos, the anso is absent, indicating that, although the absence of wg permits the formation of multiple ansos, this requires en activity. Although ubiquitous otd represses wg expression in the antennal segment and all segments posterior to it, otd induction has the opposite effect in the ocular segment, positively regulating wg expression. It is concluded that cephalic gap genes define head morphology through the direct modulation of segment polarity gene expression (Gallitano-Mendel, 1998).

In the even-numbered parasegments of the Drosophila embryo, expression of the fushi tarazu (ftz) gene is necessary for transcription of engrailed (en). Yet of those cells expressing ftz+ in a stripe, only the anteriormost come to express en. One explanation is that the level of ftz+ might be graded across the stripe: in order to express en, it would be sufficient for cells to exceed a threshold concentration of Ftz protein. Photographs and microspectrophotometry were used to measure differences in Ftz antigen concentration; no gradient within the Ftz stripe is observed. Rather, the stripe appears to contain cells with similar amounts of antigen plus a few weakly staining cells that are usually at the posterior edge. Further, varying the amount of Ftz protein about fourfold has no effect on en expression. Finally, embryos lacking the even-skipped gene have normal levels of Ftz but do not express en. This result may suggest that wild-type levels of Ftz are necessary but not sufficient to activate en expression. Alternatively, in the absence of Eve, repression by the pair-rule gene odd-skipped (odd) may block the ability of Ftz to activate even-numbered en stripes. This idea is supported by the observation that the Ftz-dependent en stripes are expressed in eve/odd double mutants. These observations appear to rule out the threshold hypothesis (Lawrence, 1998).

Brother and Big brother were isolated as Runt-interacting proteins and are homologous to CBFb, which interacts with the mammalian CBFa Runt-domain proteins. In vitro experiments indicate that Brother family proteins regulate the DNA binding activity of Runt-domain proteins without contacting DNA. In both mouse and human there is genetic evidence that the CBFa and CBFb proteins function together in hematopoiesis and leukemogenesis. Functional interactions between Brother proteins and Runt domain proteins have been demonstrated in Drosophila. A specific point mutation in Runt has been shown to disrupt interaction with Brother proteins but does not affect DNA binding activity. The point mutation was introduced into Runt by a PCR based site-directed mutagenesis. The mutant is dysfunctional in several in vivo assays. The most sensitive targets of Runt are the odd-numbered stripes of engrailed (en) expression. Ectopic expression of the altered Runt[G163R] has no discernible effect on en expression, even at levels that are fivefold greater than required for repression of en by the wild-type Runt protein. To determine whether Runt[G163R] retains any residual activity a heat-shock driven ectopic expression assay was used. The high levels of Runt expression obtained by this method cause alterations in the expression of other pair-rule genes in addition to en. Even under these conditions, the pattern of en expression as well as that of even-skipped and fushi tarazu in embryos expressing Runt[G163R] is indistinguishable from that of wild-type embryos. These results indicate that Runt[G163R] is incapable of regulating expression of several of RuntÂ’s targets in the pathway of segmentation (Li, 1999).

Orphan receptors for whom cognate ligands have not yet been identified form a large subclass within the nuclear receptor superfamily. To address one aspect of how they might regulate transcription, the mode of interaction between the Drosophila orphan receptor FTZ-F1 (NR5A3) and a segmentation gene product Fushi tarazu (FTZ) was investigated. Strong interaction between these two factors was detected by use of the mammalian one- and two-hybrid interaction assays without addition of ligand. This interaction requires the AF-2 core and putative ligand-binding domain of FTZ-F1 and the LXXLL motif of FTZ. The requirement of these elements has been further confirmed by examination of their target gene expression in Drosophila embryos and observation of a cuticle phenotype in transgenic fly lines that express mutated factors (Suzuki, 2001).

Ubiquitous expression of FTZ in early embryos under control of the heat shock promoter broadens even-numbered engrailed (en) stripes, represses alternate wg stripes, and results in a so-called anti-ftz cuticle phenotype, in which roughly reciprocal segments are missing compared with the ftz larval cuticle phenotype. However, ectopic en induction or wg repression was not observed by expressing a construct (mutFTZ) containing substitutions in the tandem leucines, indicating that the LXXLL motif in FTZ is necessary for the ectopic expression of en and the repression of wg. FTZ-dependent en induction and wg repression were also observed by expression of both FTZ-F1 and FTZ under control of the heat shock promoter in the ftz-f1 mutant embryo but not when FTZ-F1DeltaAF2C was used instead of FTZ-F1. An anti-ftz cuticle phenotype was produced by forced expression of wild-type FTZ but not by expression of mutFTZ. In ftz-f1 mutant embryos, anti-ftz phenotypes were obtained when FTZ-F1 and FTZ were coexpressed under heat shock control but not upon replacement of FTZ-F1 with FTZ-F1DeltaAF2C. These observations indicate that interaction through the LXXLL motif in FTZ and AF-2 core in FTZ-F1 is necessary for producing an anti-ftz phenotype and further support the results of the one- and two-hybrid assay (Suzuki, 2001).

Thus, results using embryos strongly suggest that FTZ-F1 activates engrailed in vivo through the AF-2-LXXLL-dependent direct interaction. In early fly embryos, FTZ-F1 seems to function as an activator for engrailed only in regions where FTZ is also present despite the uniform expression of FTZ-F1. Such situations mimic that of the requirement for a ligand by a nuclear receptor in controlling its function and specificity in gene expression. The characteristic cooperation of FTZ-F1 and FTZ provides a novel example of transcriptional regulation by a nuclear receptor, which may be an alternative pathway to the conventional one using lipophilic ligands. From an evolutionary aspect, it has been proposed that the ancestral nuclear receptor had no ligand and the ability to bind a ligand was acquired by a subset of descendent receptors later in evolution. It has also been presumed that FTZ-F1 is one of the most ancient receptors based on its distribution among species. It is believed that transcriptional activation by FTZ-F1 through binding to FTZ might represent a primitive style of regulation by nuclear receptors before the acquisition of ligand-binding ability. The existence is presumbed of yet unidentified corresponding factors for other orphan receptors as well as for ligand-responsive receptors, which may form a new group of nuclear receptor coactivators and play critical roles for development and metabolism (Suzuki, 2001).

During segmentation of the Drosophila embryo, even skipped is required to activate engrailed stripes and to organize odd-numbered parasegments. A 16 kb transgene containing the even skipped coding region can rescue normal engrailed expression, as well as all other aspects of segmentation, in even skipped null mutants. To better understand its mechanism of action, the Even-skipped protein was functionally dissected in the context of this transgene. Even-skipped utilizes two repressor domains to carry out its function. Each of these domains can function autonomously in embryos when fused with the Gal4 DNA-binding domain. A chimeric protein consisting only of the Engrailed repressor domain and the Even-skipped homeodomain, but not the homeodomain alone, is able to restore function, indicating that the repression of target genes is sufficient for even skipped function at the blastoderm stage, while the homeodomain is sufficient to recognize those target genes. When Drosophila Even skipped is replaced by its homologs from other species, including a mouse homolog, these homologs could provide substantial function, indicating that these proteins can recognize similar target sites and also provide repressor activity. Using this rescue system, it has been shown that broad, early even skipped stripes are sufficient for activation of both odd- and even-numbered engrailed stripes. Furthermore, these 'unrefined' stripes organize odd-numbered parasegments in a dose-dependent manner, while the refined, late stripes, which coincide cell-for-cell with parasegment boundaries, are required to ensure the stability of the boundaries (Fujioka, 2002).

Eve has two distinct repressor domains, one dependent on the corepressor Gro and the other Gro-independent. For this study Eve was divided into 4 domains: an N-terminal region; the HD, which includes a conserved flanking region; a repressor domain identified in transient assays in cultured Drosophila cells, and the remaining C terminus, which includes a Gro interaction domain. Paradoxically, a primary function of Eve in segmentation is to allow activation of en stripes in both even- and odd-numbered parasegments. The ability to functionally replace the endogenous eve gene with a transgenic copy was used to evaluate the relative contribution of these and other domains to the function of Eve in this process. Neither repressor domain is sufficient to properly organize the odd-numbered parasegments, although all (or most) en stripes can be restored by either one alone. However, the relative width of the odd-numbered parasegments is reduced, so that they are unstable, and are deleted at later developmental stages. This gives rise to the pair-rule phenotype that earned even skipped its name (the even-numbered abdominal denticle bands are in odd-numbered parasegments) (Fujioka, 2002).

The histone deacetylase Rpd3 affects eve function. In Rpd3 mutant embryos, although the expression pattern of eve is not changed, even-numbered en stripes are very weak or missing owing to a lack of repression of odd. However, odd-numbered en stripes are expressed with only minor alterations. This is in contrast to the relative effects on odd- versus even-numbered en stripes when the eve dose is reduced, or in hypomorphic mutants, suggesting that Rpd3 may affect the repression of odd more than that of slp and prd. The Rpd3 effect similarly contrasts with the effects of removing either of the Eve repressor domains, suggesting that Rpd3 specificity cannot be explained by a selective effect on one of the Eve corepressors. This is true despite the fact that Rpd3 has been shown to mediate Gro repressor activity. Therefore, the apparent specificity of action of Rpd3 during segmentation is not easily explained solely through an effect on Eve activity. Conceivably, Rpd3 might affect the target specificity of the Eve HD, perhaps through selective effects on chromatin structure at different target sites. Another possibility is that it might affect the activities of other pair-rule gene products in addition to Eve. For example, it has been shown that Slp interacts with Gro in vitro. If Rpd3 reduces slp activity, then the effect of Rpd3 on Eve repressor function might be partially antagonized at the odd-numbered parasegment boundaries by its effect on slp (Fujioka, 2002).

Repression of the Gal4 binding site-containing transgene by the Eve-Gal4 fusion proteins shows a consistently stronger effect on stripe 1 than on stripe 5. Although the stripe 5 element in the reporter is further away from the Gal4 binding sites and is also less well repressed than the stripe 1 element, the apparent specificity of repression is probably not due to a distance effect. This is inferred from the fact that a similar stripe preference is seen when Gal4 binding sites are inserted upstream of the same stripe elements, this time closer to the stripe 5 region. The stronger repression activity on stripe 1 expression may be due to the earlier activity of the stripe 1 enhancer, relative to that of stripe 5. Since these elements are also used to drive the expression of the repressors, the earlier activity of the stripe 1 element causes earlier accumulation of the repressors in the stripe 1 region, which may result in more effective repression. Alternatively, the Eve repressor domains may have some functional specificity that allows them to work more effectively on the stripe 1 enhancer (Fujioka, 2002).

In the complete absence of eve function, en is not expressed in the trunk region, and there is little evidence of segmentation at the end of embryogenesis. In hypomorphic eve mutants, the odd-numbered en stripes are expressed at posteriorly shifted positions, so that the odd-numbered parasegments are too narrow, and are deleted at later embryonic stages. Note: a contribution to the narrowing of odd-numbered parasegments may also come from an anterior shift of even-numbered en stripes. The positions of the odd-numbered parasegment boundaries, which are the anterior edges of odd-numbered en stripes, are foreshadowed by the anterior borders of refined, late eve stripe expression, prompting the suggestion that the late stripes are the more important functional aspects of expression, with the early, broad stripes serving only to help activate the late stripes. However, a previous model of eve function has suggested that the early stripes, acting as morphogenic gradients, independently set the anterior margins of both late eve and odd-numbered en stripes, which coincide because of their similar regulation by repressors (including slp) and the activator prd. These models were tested by removing late eve stripe expression while retaining normal early stripes. In eve null embryos rescued by a transgene deleted for the late expression element, although there is variable partial refinement under the influence of runt, which represses each early stripe from the posterior, the well-refined, late stripes never appear. In these embryos, odd-numbered en stripes form normally. However, they are variably lost during germband extension, coincident with an expansion of slp expression. Nonetheless, without refined, late eve stripes, many embryos are able to survive to fertility. Thus, it appears that the initial expression pattern of en and the overall organization of parasegments are determined primarily by the broad, early stripes. The late, refined stripes are required to maintain the pattern of slp, and to prevent partial repression of en shortly after it is activated. The expansion of slp is probably sufficient to explain the loss of en, since ectopic slp expression causes repression of these en stripes. The hypothesis that early stripes position odd-numbered en stripes in a concentration-dependent manner is also supported by the phenotype of embryos rescued by a transgene missing the stripe 4+6 element: these embryos have severely reduced levels of early stripes 4 and 6, and activate odd-numbered en stripes in those regions at posteriorly shifted positions. A model of these functions of early and late eve expression is presented (Fujioka, 2002).

The prevalence of repression as a mechanism of early developmental regulation among pair-rule and gap genes is striking. In the case of Eve, this activity provides not only for the activation of en with the appropriate spacing between cell rows, but also for the maintenance of en expression in the face of opposing repressive activities. One of these opposing activities is that of slp, which apparently helps to set the anterior boundary of both late Eve and en expression. Thus, spatially localized repressors may have advantages over activators in making and maintaining cell fate decisions, where mutually exclusive patterns of transcription factor expression help to establish and reinforce those decisions. Such mutually exclusive patterns can be directly established and reinforced by repressors acting to repress each others expression in adjacent domains, while activators can do this only indirectly (Fujioka, 2002).

engrailed transcriptional repression by Runt

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. By using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, it is shown that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos: this indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression (Wheeler, 2002).

Raf regulation of engrailed

Raf is an essential downstream effector of activated Ras in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified 'Raf activator.' In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, a genetic screen was conducted to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). A collection of chromosomal deficiencies representing ~70% of the autosomal euchromatic genomic regions was examinied for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, DrafSu2. Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of DrafSu2, causing lethality in DrafSu2 hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. Src64B interacts genetically with Draf and an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, a genetic interaction was discovered between DrafSu2 and Df(3L)vin5 that reveals a novel role for Draf in limb development. Loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs (Li, 2000).

The proper expression of en in the posterior compartment of imaginal discs is essential for maintaining compartmental boundaries and patterning of Drosophila limbs. Despite much insight into the events required for Hh signaling, little is known about the mechanism(s) by which en expression is controlled in the posterior compartment. Two instances have been identified where a further reduction in Draf function, due to the presence of a deficiency, results in defects in posterior pattern elements in the limbs. DrafSu2/Y; Df(3L)vin5/+ male survivors exhibit notching only in the posterior region of the wing, and partial pattern duplications in the posterior compartment. Since no specific role for Draf has been described in the limbs, the requirements for Draf in the imaginal discs were examined. Since clonal analysis with null alleles is uninformative, because Draf mutant clones do not develop, Draf was conditionally provided to the developing animals in a Draf null background (Li, 2000).

As a result of withholding Draf during the second and early third larval instars, animals with anterior pattern element duplications in the posterior compartment were frequently observed. By examining the imaginal discs of these animals, it could be determined that when there are insufficient levels of Draf, en expression is no longer restricted to the normal posterior compartment, which suggests that Draf may act to repress/restrict En expression. Along with ectopic expression of En in the anterior compartment and increased levels of En in the posterior compartment, a new mirror image anterior compartment devoid of en expression was induced. This observation is consistent with the observation that when En is ectopically expressed, ectopic anterior pattern elements are induced. Also, ectopic expression of En in the anterior compartment induces expression of high levels of Hh and Dpp, which are responsible for overgrowth and the duplication of anterior pattern elements. Indeed, when Hh was examined in the partially rescued Draf null males, it was found to be widely ectopically expressed. The posteriorly restricted wing notching observed in DrafSu2/Y; Df(3L)vin5/+ male survivors is also consistent with a requirement for Draf in negatively regulating en, since elevated levels of En expression in the posterior compartment partially inactivate both en and inv, which are necessary for the development and terminal differentiation of posterior fates. Taken together, these observations suggest that the Df(3L)vin5 deficiency contains a gene that participates with Draf in patterning of the limbs (Li, 2000).

Ftz modulates Runt-dependent activation and repression of segment-polarity gene transcription

A crucial step in generating the segmented body plan in Drosophila is establishing stripes of expression of several key segment-polarity genes, one stripe for each parasegment, in the blastoderm stage embryo. It is well established that these patterns are generated in response to regulation by the transcription factors encoded by the pair-rule segmentation genes. However, the full set of positional cues that drive expression in either the odd- or even-numbered parasegments has not been defined for any of the segment-polarity genes. Among the complications for dissecting the pair-rule to segment-polarity transition are the regulatory interactions between the different pair-rule genes. An ectopic expression system that allows for quantitative manipulation of expression levels was used to probe the role of the primary pair-rule transcription factor Runt in segment-polarity gene regulation. These experiments identify sloppy paired 1 (slp1), most appropriately classified as segment polarity genes, as a gene that is activated and repressed by Runt in a simple combinatorial parasegment-dependent manner. The combination of Runt and Odd-paired (Opa) is both necessary and sufficient for slp1 activation in all somatic blastoderm nuclei that do not express the Fushi tarazu (Ftz) transcription factor. By contrast, the specific combination of Runt + Ftz is sufficient for slp1 repression in all blastoderm nuclei. Furthermore Ftz is found to modulate the Runt-dependent regulation of the segment-polarity genes wingless (wg) and engrailed (en). However, in the case of en the combination of Runt + Ftz gives activation. The contrasting responses of different downstream targets to Runt in the presence or absence of Ftz is thus central to the combinatorial logic of the pair-rule to segment-polarity transition. The unique and simple rules for slp1 regulation make this an attractive target for dissecting the molecular mechanisms of Runt-dependent regulation (Swantek, 2004).

The role of Runt as a primary pair-rule gene complicates interpreting the alterations in segment-polarity gene expression that are observed in run mutants. Recent experiments utilizing a GAL4-based NGT-expression system [the transgene construct used to express GAL4 maternally contains the nanos promoter and the 3' untranslated region of an alpha-tubulin mRNA and is thus referred to as an NGT transgene (nanos-GAL4-tubulin)] to manipulate expression in the blastoderm embryo have demonstrated that low levels of Runt repress en in odd-numbered parasegments without altering expression of the pair-rule genes eve and ftz. This observation suggested that this approach might provide a useful tool for defining the role of Runt in regulating other segment-polarity genes. A systematic survey was undertaken of the response of other segmentation genes to increasing levels of NGT-driven Runt expression. These experiments revealed significant differences in sensitivity as well as interesting differences in the nature of the response of different genes to ectopic Runt. The odd-numbered en stripes are repressed at both intermediate and high levels of ectopic runt. After en, the second most sensitive target is slp1. This gene shows a partially penetrant and subtle defect in the spacing of the segmentally repeated stripes in embryos with low levels of NGT-driven Runt. A more pronounced alteration is obtained in embryos with intermediate levels of Runt. In these embryos the slp1 pattern is converted from a segment-polarity-like, 14-stripe pattern to a pair-rule-like, seven-stripe pattern. At this level, expression of other segmentation genes is normal although there are subtle changes in the spacing of the wg stripes and a partial loss of the odd-numbered hh stripes. All three of these genes show clearer alterations at higher levels of NGT-driven Runt, with wg responding in a manner similar to slp1 and hh responding in a manner similar to en. High Runt levels also produce spacing defects in the expression of odd and gsb, as well as a more subtle effect on prd. Several of the changes observed at high levels of ectopic Runt are likely to be indirect and due to alterations in the expression of eve, ftz and hairy. The response of slp1 to ectopic Runt is notable both because of its sensitivity and apparent simplicity, thus suggesting that Runt plays a pivotal role in regulating slp1 transcription (Swantek, 2004).

The differential combinatorial effects of Runt and Ftz on segment-polarity gene regulation emerged as a result of analyzing the sensitive and relatively simple response of slp1 to ectopic Runt. The slp1 transcription unit is one of two redundant genes that comprise the slp locus. This locus was initially characterized as having a pair-rule function in the segmentation gene hierarchy based on a weak pair-rule phenotype associated with loss of slp1 function. The slp1 and slp2 genes are expressed in similar patterns during early embryogenesis. Embryos deficient for both slp1 and slp2 have an unsegmented lawn cuticle phenotype similar to that produced by wg mutations. This raises the question of whether it is most appropriate to consider slp as a pair-rule or segment-polarity locus. In the most straightforward interpretation of the segmentation hierarchy, the role of the pair-rule genes is to establish the initial metameric expression patterns of the segment-polarity genes. The initial expression of the key segment polarity genes en and wg is normal in gastrula stage embryos that are deleted for both slp1 and slp2. The expression of wg begins to become abnormal and is lost during early germband extension. These observations are consistent with the proposal that slp expression identifies cells that are competent to maintain wg expression subsequent to the blastoderm stage. Based on these observations, it is concluded that slp1 and slp2 are most appropriately classified as segment polarity genes, not pair-rule genes (Swantek, 2004).

The results point an important component of context-dependent regulation by Runt. The specific combination of Runt + Ftz, which represses slp1, does not always give repression, since these same two factors work together to activate en in some of these same cells at the same stage of development. Thus, cellular context alone cannot fully account for the regulatory differences and there must be a target-gene specific component of context-dependent regulation. A similar gene-specific example of context-dependent regulation has recently been described for the Runx protein Lozenge. In this case, the presence of binding sites for the Cut homeodomain protein helps to stabilize a complex that leads to repression of deadpan transcription in the same cells in which Lozenge is responsible for activation of Drosophila Pax2. In a strict parallel of this model, it would be speculated that the slp1 regulatory region contains binding sites for some factor that helps to stabilize a repressor complex that includes the Runt and Ftz proteins. In a reciprocal, and not mutually exclusive model, perhaps there are binding sites for a factor in the en regulatory region that helps to stabilize a Runt- and Ftz-dependent transcriptional activation complex. Further studies on the en and slp1 cis-regulatory regions are needed in order to address these questions at the molecular level. This future work is crucial for understanding the context-dependent activity of Runt and thus the molecular logic of the control system that underlies the pair-rule to segment-polarity transition in Drosophila segmentation (Swantek, 2004).

Dorsoventral patterning of the Drosophila hindgut is determined by interaction of genes under the control of two independent gene regulatory systems, the dorsal and terminal systems

Dorsoventral (DV) patterning in the trunk region of Drosophila embryo is established through intricate molecular interactions that regulate Dpp/Scw signaling during the early blastoderm stages. The hindgut of Drosophila, which derives from posterior region of the cellular blastoderm, also shows dorsoventral patterning, being subdivided into distinct dorsal and ventral domains. engrailed (en) is expressed in the dorsal domain, which determines dorsal fate of the hindgut. This study shows that a repressor Brk restricts en expression to the dorsal domain of the hindgut. Expression domain of brk during early blastodermal stages is defined through antagonistic interaction with dpp, and expression domains of dpp and brk in the early blastoderm include prospective hindgut domain. After stage 9, dpp expression in the dorsal domain of the hindgut primordium disappears, but, the brk expression in the ventral domain continues. It was found that Dorsocross (Doc), which is a target gene of Dpp, is responsible for restricting brk expression to the ventral domain of the hindgut. On the other hand, activation of en is under the control of brachyenteron (byn) that is regulated independently of dpp, brk, and Doc. The cooperative interaction of common DV positional cues with byn during hindgut development represents another aspect of mechanisms of DV patterning in the Drosophila embryo (Hamaguchi, 2012).

A model is presented of the genetic pathway leading to the DV subdivision of the Drosophila hindgut. The dorsal fate of the hindgut is finally determined by a selector gene en that is expressed in the dorsal domain of the hindgut. The present study revealed that DV patterning of the hindgut is based on antagonistic interaction of Dpp and Brk in the early cellular blastoderm. Dpp expression disappears in the hindgut primordium after stage 9, but, Dpp target gene Doc takes over the repressive effect on Brk, and restricts Brk expression to the ventral domain. In other words, primary role of Dpp in the hindgut development is to activate Doc genes in the dorsal domain for repression of Brk. Eventually, Brk represses the selector gene en in the ventral domain, restricting it to the dorsal domain. This is the outline of gene regulatory pathway of DV patterning of the hindgut. It should be noted that Doc represses brk in the dorsal domain, while Brk does not regulate Doc expression in the hindgut in normal development, since brk mutation does not affect Doc expression in the hindgut. Dpp and Doc do not determine the dorsal fate directly, but, act indirectly by repressing brk in the dorsal domain. In fact, brk; DocA (null) double-mutant embryos, as well as brk; dpp double mutant embryos, expressed en in both dorsal and ventral domains of the hindgut. This regulatory interrelation between brk and Dpp/Doc is partially reminiscent of that in the wing discs, in which primary role of Dpp is repression of brk, and the latter is responsible for defining antero-posterior pattern of gene expression in the wing discs. Repression of brk by Dpp signals has been reported to depend on the zinc finger protein Schnurri (Shn) in some tissues. However, brk expression in the hindgut did not expand dorsally in the shn mutant embryo, and also, en expression was observed in the hindgut in shn mutant embryo. Thus, shn may not be essential for regulation of brk in the hindgut (Hamaguchi, 2012).

However, activation of en in the hindgut, is under the control of byn, the process of which is independent of DV patterning. The expression domain of byn is included in the region where intricate interaction of dpp, brk, and Doc proceeds. The dpp, brk, and Doc genes are all under the control of the dorsal system, while byn is activated under the control of the terminal system that provides AP positional cues in terminal regions of the early blastoderm. In other words, AP positional cues activate en, while DV positional cues repress en. Thus, cooperative interaction of the two independent gene regulatory systems establishes the expression pattern of en in the hindgut (Hamaguchi, 2012).

Regulation of engrailed by segment polarity genes

Continued: see engrailed Transcription regulation part 2/3 | part 3/3

engrailed: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Developmental Biology | Effects of mutation | References

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