Engrailed Locus Research Articles

Identification of evolutionarily conserved downstream core promoter elements required for the transcriptional regulation of Fushi tarazu target genes.

The regulation of transcription initiation is critical for developmental and cellular processes. RNA polymerase II (Pol II) is recruited by the basal transcription machinery to the core promoter where Pol II initiates transcription. The core promoter encompasses the region from -40 to +40 bp relative to the +1 transcription start site (TSS). Core promoters may contain one or more core promoter motifs that confer specific properties to the core promoter, such as the TATA box, initiator (Inr) and motifs that are located downstream of the TSS, namely, motif 10 element (MTE), the downstream core promoter element (DPE) and the Bridge, a bipartite core promoter element. We had previously shown that Caudal, an enhancer-binding homeodomain transcription factor and a key regulator of the Hox gene network, is a DPE-specific activator. Interestingly, pair-rule proteins have been implicated in enhancer-promoter communication at the engrailed locus. Fushi tarazu (Ftz) is an enhancer-binding homeodomain transcription factor encoded by the ftz pair-rule gene. Ftz works in concert with its co-factor, Ftz-F1, to activate transcription. Here, we examined whether Ftz and Ftz-F1 activate transcription with a preference for a specific core promoter motif. Our analysis revealed that similarly to Caudal, Ftz and Ftz-F1 activate the promoter containing a TATA box mutation to significantly higher levels than the promoter containing a DPE mutation, thus demonstrating a preference for the DPE motif. We further discovered that Ftz target genes are enriched for a combination of functional downstream core promoter elements that are conserved among Drosophila species. Thus, the unique combination (Inr, Bridge and DPE) of functional downstream core promoter elements within Ftz target genes highlights the complexity of transcriptional regulation via the core promoter in the transcription of different developmental gene regulatory networks.

The SERTAD protein Taranis plays a role in Polycomb-mediated gene repression.

The Polycomb group (PcG) proteins have been implicated in epigenetic transcriptional repression in development, stem cell maintenance and in cancer. The chromodomain protein Polycomb (Pc) is a key member of the PcG. Pc binds to the histone mark, trimethylated histone 3 lysine 27 (H3K27me3), to initiate transcriptional repression. How PcG proteins are recruited to target loci is not fully understood. Here we show that the Drosophila SERTA domain protein Taranis (Tara) is involved in transcriptional regulation of Pc target genes. Embryos lacking Tara exhibit a partial homeotic transformation of cuticular the segments, a phenotype associated with the loss of Pc function. Moreover, Drosophila embryos homozygous for a tara hypomorphic allele also misexpress engrailed, a Pc-regulated gene, and this phenotype is associated with the loss of Pc binding to the cis response element in the engrailed enhancer. In relation to that, Pc recruitment is reduced on the salivary gland polytene chromosomes and specifically at the engrailed locus. These results suggest that Tara might be required for positioning Pc to a subset of its target genes.

Enhancer-promoter communication at theDrosophila engrailedlocus

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? We have used homing P[en] constructs to study the enhancer-promoter communication at the engrailed locus. Here we show that 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. First, early acting engrailed stripe enhancers exhibit promoter specificity. Second, a proximal promoter-tethering element is required for the action of the imaginal disc enhancer(s). Our data suggest that there are two partially redundant promoter-tethering elements. Third, 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.

Recruitment of Drosophila Polycomb group proteins to chromatin by DSP1

Polycomb and trithorax group (PcG and trxG) proteins maintain silent and active transcriptional states, respectively, throughout development. In Drosophila, PcG and trxG proteins associate with DNA regions named Polycomb and trithorax response elements (PRE and TRE), but the mechanisms of recruitment are unknown. We previously characterized a minimal element from the regulatory region of the Abdominal-B gene, termed Ab-Fab. Ab-Fab contains a PRE and a TRE and is able to maintain repressed or active chromatin states during development. Here we show that the Dorsal switch protein 1 (DSP1), a Drosophila HMGB2 homologue, binds to a sequence present within Ab-Fab and in other characterized PREs. Addition of this motif to an artificial sequence containing Pleiohomeotic and GAGA factor consensus sites is sufficient for PcG protein recruitment in vivo. Mutations that abolish DSP1 binding to Ab-Fab and to a PRE from the engrailed locus lead to loss of PcG protein binding, loss of silencing, and switching of these PREs into constitutive TREs. The binding of DSP1 to PREs is therefore important for the recruitment of PcG proteins.

The doublesex locus of Drosophila melanogaster and its flanking regions: a cytogenetic analysis.

The region of the third chromosome (84D-F) of Drosophila melanogaster that contains the doublesex (dsx) locus has been cytogenetically analyzed. Twenty nine newly induced, and 42 preexisting rearrangements broken in dsx and the regions flanking dsx have been cytologically and genetically characterized. These studies established that the dsx locus is in salivary chromosome band 84E1-2. In addition, these observations provide strong evidence that the dsx locus functions only to regulate sexual differentiation and does not encode a vital function. To obtain new alleles at the dsx locus and to begin to analyze the genes flanking dsx, 59 lethal and visible mutations in a region encompassing dsx were induced. These mutations together with preexisting mutations in the region were deficiency mapped and placed into complementation groups. Among the mutations we isolated, four new mutations affecting sexual differentiation were identified. All proved to be alleles of dsx, suggesting that dsx is the only gene in this region involved in regulating sexual differentiation. All but one of the new dsx alleles have equivalent effects in males and females. The exception, dsxEFH55, strongly affects female sexual differentiation, but only weakly affects male sexual differentiation. The interactions of dsxEFH55 with mutations in other genes affecting sexual differentiation are described. These results are discussed in terms of the recent molecular findings that the dsx locus encodes sex-specific proteins that share in common their amino termini but have different carboxyl termini. The 72 mutations in this region that do not affect sexual differentiation identify 25 complementation groups. A translocation, T(2;3)Es that is associated with a lethal allele in one of these complementation groups is also broken at the engrailed (en) locus on the second chromosome and has a dominant phenotype that may be due to the expression of en in the anterior portion of the abdominal tergites where en is not normally expressed. The essential genes found in the 84D-F region are not evenly distributed throughout this region; most strikingly the 84D1-11 region appears to be devoid of essential genes. It is suggested that the lack of essential genes in this region is due to the region (1) containing genes with nonessential functions and (2) being duplicated, possibly both internally and elsewhere in the genome.

Region-specific recombination and expression are directed by portions of the Drosophila engrailed promoter.

The Drosophila engrailed gene is expressed in the cells of the posterior developmental compartments. To investigate how the engrailed gene is regulated, chimeric genes consisting of fragments of the engrailed promoter and Escherichia coli lacZ were incorporated into the Drosophila germ line by P-element-mediated recombination. Fusion constructs with 7.5 kb of 5'-flanking sequence contain sufficient information to promote expression in most of the embryonic, larval, and imaginal posterior compartments; transformants with smaller fragments of the 5' region do not. Remarkably, of 20 independent transformants with constructs containing more than 1 kb of 5'-flanking DNA, 7 integrated in or around the engrailed locus. These strains inactivate engrailed function to varying degrees, and some express lacZ with a position- and temporal-specific program that is indistinguishable from the normal engrailed gene. Presumably, in these strains, lacZ is expressed in the context of the engrailed promoter.

Allelic interactions at the engrailed locus of Drosophila: engrailed protein expression in imaginal discs

Many embryonic lethal engrailed ( en lethal ) mutations are known to partially complement the cuticular defects of the original engrailed mutation, en 1 . To explore the nature of this complementation, the adult phenotypes of several different en 1 en lethal transheterozygotes were compared with the corresponding patterns of engrailed protein expression in third larval instar imaginal discs (determined by immunofluorescence). Transheterozygotes of en 1 and deletions of the locus ( en Df ) typically show slight complementation in the adult cuticle. The pattern of engrailed protein expression in some en 1 en Df wing discs is indistinguishable from en 1 homozygotes, but in others the pattern is nearly normal. en 1 en Df leg discs appear to express engrailed protein normally. Transheterozygotes of en 1 and EMS-induced, cytological normal en lethal alleles have almost normal adult cuticle phenotypes and also exhibit normal patterns of engrailed protein expression in all of the thoracic imaginal discs. Surprisingly, the intensity of anti- engrailed staining in these discs is elevated relative to that in wild type. en 2 is an unusual lethal allele in that it does not complement either the en 1 adult cuticle phenotype or the protein expression pattern in imaginal discs. Moreover, the cytologically normal en lethal alleles also complement en 2 , at least partially. Both wing and leg imaginal discs from en 2 en lethal transheterozygotes show abnormal patterns of engrailed protein expression. These results are discussed in the context of an autoregulatory model for engrailed regulation.

The transcription unit of the Drosophila engrailed locus: an unusually small portion of a 70,000 bp gene.

Transcripts from the engrailed gene of Drosophila melanogaster have been characterized by Northern, S1 nuclease sensitivity, and primer extension analyses. The engrailed gene encodes three poly(A)+ transcripts (3.6 kb, 2.7 kb, and 1.4 kb) that derive from a 3.9-kb portion of the genome. No other transcribed regions were found up to 16 kb downstream and 48 kb upstream of the engrailed transcription unit, the portion of the genome to which engrailed mutations have been mapped. The structures of the engrailed transcripts are unaffected by lethal engrailed mutations that break the locus at points in the transcriptionally silent regions. Transcripts expressed by 145 kb of DNA that surround the engrailed locus were also identified by Northern analysis. In contrast to the large portion of the engrailed gene that is transcriptionally inactive, most of the surrounding regions are 10-fold more densely populated with transcripts. We presume that the unusually large silent region at the periphery of the engrailed transcription unit signify the presence of special mechanisms that regulate its expression.

Electron microscopic heteroduplex mapping identifies regions of the engrailed locus that are conserved between Drosophila melanogaster and Drosophila virilis.

Physical localization of mutations in the engrailed (en) gene suggested that at least 70 kilobases (kb) of genomic sequences contribute to the normal function of this gene. Molecular characterization has suggested that en function is encoded in a small, 4.5-kb primary transcript. To identify functional regions within the 70 kb of the en locus of D. melanogaster, we identified sequences conserved in the D. virilis genome (estimated divergence time, 60 million years). Based on homology to D. melanogaster, we isolated en DNA from a D. virilis genomic library. Electron microscopic heteroduplex analysis indicated that in 70 kb there is 20 kb of conserved DNA in 33 different regions dispersed throughout the en locus, including two which encode parts of the major embryonic transcript. The conserved regions are in the same linear order and are spaced by similar lengths of nonconserved sequences in the D. virilis and D. melanogaster DNAs. What functional constraints have enforced conservation of sequences throughout the entire 70 kb and protected the region from divergence of size and arrangement? Our working hypothesis is that sequences necessary for the complex spatial and temporal pattern of en expression are dispersed throughout the 70-kb en locus and that selection for proper regulation restricts evolutionary divergence.

The engrailed locus of drosophila: In situ localization of transcripts reveals compartment-specific expression

The engrailed locus plays a unique and critical role in organizing the segmented body plan of Drosophila. Embryos lacking engrailed function die with fused, abnormal segments. Adult mosaics with patches of engrailed cells similarly suffer defects in all of their segments, but only with mutant cells that are in the posterior developmental compartment of each segment. The non-uniform requirement for engrailed function reflects the position-dependent expression of the engrailed locus and we demonstrate it here unambiguously by directly visualizing engrailed transcripts in frozen sections of embryos and larvae and in whole imaginal discs. These results demonstrate that developmental compartments subdivide the embryonic insect segments. In these and in the compartments of the later developmental stages the engrailed locus is expressed in the posterior but not the anterior compartments. With its role in controlling the developmental pathway of the posterior compartment cells, the engrailed locus may be an example of a binary developmental switch.

Isolation of a homoeo box-containing gene from the engrailed region of Drosophila and the spatial distribution of its transcripts

The engrailed locus of Drosophila melanogaster has the characteristics of both a homoeotic gene and a segmentation gene: like a homoeotic gene, it specifies the development of specific compartments of the Drosophila embryo (the posterior compartments of each segment), and, like mutations of segmentation genes, lethal alleles of engrailed affect also the pattern of segmentation of the embryo. Here we report that like many of the homoeotic genes of the bithorax and Antennapedia complexes, engrailed has a 'homoeo box' sequence: also, like the segmentation gene fushi tarazu, the engrailed gene displays a periodic pattern of expression in Drosophila embryos.

Posterior-to-anterior transformation in engrailed wing imaginal disks of Drosophila.

Segments in the Drosophila adult are divided into clonally distinct anterior and posterior compartments. Mutations at the engrailed locus can affect the pattern of cuticular structures in the posterior compartments of segments, but have no obvious effect on anterior structures; for example, bristles that are normally seen only on the anterior wing margin in wild-type flies can be found on the posterior margin of engrailed wings. These and clonal analysis data led to the hypothesis that engrailed causes a transformation of posterior to anterior identity in the wing cells. Despite some striking examples of this transformation, a common engrailed phenotype is the disruption or elimination of posterior pattern elements, without a clear replacement by anterior structures; this, together with indications that localized cell death can mimic some of the observed posterior-to-anterior transformations, has led some investigators to question the original engrailed hypothesis. Recently, monoclonal antibodies displaying region-specific binding patterns on the wing imaginal disk have been described, and one of these antibodies in particular provides a novel probe for the engrailed phenotype in the larval precursors of the adult wing. Here I compare the antibody binding patterns on engrailed and wild-type wing disks. The results strongly support the notion that engrailed mutations cause a posterior-to-anterior transformation in these cells.

Effects of engrailed in the genital disc of Drosophila melanogaster

engrailed has been postulated to be the “selector gene” involved in the establishment of the anterior-posterior compartment border in several imaginal discs and in at least the first two abdominal segments of Drosophila melanogaster. Our study of the effects of different mutant engrailed genotypes on genital disc development provided the following major results: All three terminal primordia (female and male genitalia, and analia) were affected. Different heteroallelic combinations showed different expressivities, and the three terminal primordia were differently affected by the same mutant genotype. The engrailed genotypes deleted specific elements of the adult terminalia without causing associated pattern duplications. The reduced morphology of the male engrailed genital disc was analogous to the pattern deletions observed in the adult terminalia. That the engrailed phenotype is stable was demonstrated by culturing in vivo intact and fragmented engrailed genital discs. Cell death was found in a significant number of mature male en 2 en 3 genital discs. The results are discussed in terms of the segmental organization of the genital disc and in terms of the “selector gene” function postulated for the engrailed locus. The interpretation that each terminal primordium has an anterior and a posterior compartment is presented and it is assumed that in the genital disc engrailed transforms posterior cells into anterior cells that do not develop, thereby causing the deficiency pattern of the engrailed phenotype.

Further studies of the engrailed phenotype in Drosophila.

Although most mutations at the engrailed locus of Drosophila cause embryonic death when homozygous, they are viable in clones of cells. We describe the phenotype of such clones in the eye-antenna, proboscis, humerus, wing, legs, and terminalia. When in anterior compartments the clones are normal, but in most posterior compartments they are abnormal and fail to respect the anteroposterior compartment boundary. We find that the yield of engrailed-lethal clones in posterior compartments is often significantly lower than expected, indicating that these clones are lost during development. Mutant clones are abnormal in the analia and rare in the humerus, suggesting that both structures are of posterior provenance. These results support the hypothesis that the engrailed+ gene is required exclusively in cells of posterior compartments to specify their characteristic cell affinities and pattern.

Compartments in the abdomen of Drosophila and the role of the engrailed locus

A clonal analysis has shown that the dorsal surface of the first abdominal segment of Drosophila melanogaster is subdivided into anterior and posterior compartments. Cells of the posterior compartment grow up to but not beyond the anterior-posterior compartment border within the first abdominal segment and the intersegmental border that defines the boundary between the first and second abdominal segments. Growing within these boundaries, a narrow band of tissue clonally isolated from the adjoining tissue is formed. When these posterior cells are deficient for the engrailed locus, however, neither the compartment nor the segment border is maintained. The implications, that compartmentalization is essential for segmentation, and that all insect segments are subdivided by anterior and posterior compartments, are discussed.

Engrailed: a gene controlling compartment and segment formation in Drosophila.

A total of 58 mutations at the engrailed locus were isolated. Analysis suggests that this genetic locus is necessary for survival but required only in the cells of the posterior compartments. Inactivation of the engrailed locus renders the animal incapable of maintaining the separation between the groups of cells that constitute either the compartments that subdivide each segment or the individual segments themselves.