The role of Polycomb-group response elements in regulation of engrailed transcription in Drosophila

Polycomb group proteins are required for long-term repression of many genes in Drosophila and all metazoans. In Drosophila, DNA fragments called Polycomb-group response elements (PREs) have been identified that mediate the action of Polycomb-group proteins. Previous studies have shown that a 2 kb fragment located from -2.4 kb to -395 bp upstream of the Drosophila engrailed promoter contains a multipartite PRE that can mediate mini-white silencing and act as a PRE in an Ubx-reporter construct. Here, we study the role of this 2 kb fragment in the regulation of the engrailed gene itself. Our results show that within this 2 kb fragment, there are two subfragments that can act as PREs in embryos. In addition to their role in gene silencing, these two adjacent PRE fragments can facilitate the activation of the engrailed promoter by distant enhancers. The repressive action of the engrailed PRE can also act over a distance. A 181 bp subfragment can act as a PRE and also mediate positive effects in an enhancer-detector construct. Finally, a deletion of 530 bp of the 2 kb PRE fragment within the endogenous engrailed gene causes a loss-of-function phenotype, showing the importance of the positive regulatory effects of this PRE-containing fragment. Our data are consistent with the model that engrailed PREs bring chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments.     

A complex array of DNA-binding proteins required for pairing-sensitive silencing by a polycomb group response element from the Drosophila engrailed gene

Regulatory DNA from the Drosophila gene 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), we tested the activities of each of these engrailed fragments in a construct designed to detect PRE activity in embryos. Both fragments were found to behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment showed 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 suggested 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.     

Unusual Properties of Regulatory DNA from the Drosophila Engrailed Gene: Three ``pairing-Sensitive'''' Sites within a 1.6-Kb Region

We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called ``pairing-sensitive sites' (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter.     

Engrailed and polyhomeotic maintain posterior cell identity through cubitus-interruptus regulation

In Drosophila, the subdivision into compartments requires the expression of engrailed (en) and hedgehog (hh) in the posterior cells and of cubitus-interruptus (ci) in the anterior cells. Whereas posterior cells express hh, only anterior cells are competent to respond to the hh signal, because of the presence of ci expression in these cells. We show here that engrailed and polyhomeotic (ph), a member of the Polycomb Group (PcG) genes, act concomitantly to maintain the repression of ci in posterior compartments during development. Using chromatin immunoprecipitation (ChIP), we identified a 1 kb genomic fragment located 4 kb upstream of the ci coding region that is responsible for the regulation of ci. This genomic fragment is bound in vivo by both Polyhomeotic and Engrailed. In particular, we show that Engrailed is responsible for the establishment of ci repression early during embryonic development and is also required, along with Polyhomeotic, to maintain the repression of ci throughout development.     

The evolution of hexapod engrailed-family genes: evidence for conservation and concerted evolution

Phylogenetic analyses imply that multiple engrailed-family gene duplications occurred during hexapod evolution, a view supported by previous reports of only a single engrailed-family gene in members of the grasshopper genus Schistocerca and in the beetle Tribolium castaneum. Here, we report the cloning of a second engrailed-family gene from Schistocerca gregaria and present evidence for two engrailed-family genes from four additional hexapod species. We also report the existence of a second engrailed-family gene in the Tribolium genome. We suggest that the engrailed and invected genes of Drosophila melanogaster have existed as a conserved gene cassette throughout holometabolous insect evolution. In total 11 phylogenetically diverse hexapod orders are now known to contain species that possess two engrailed-family paralogues, with in each case only one paralogue encoding the RS-motif, a characteristic feature of holometabolous insect invected proteins. We propose that the homeoboxes of hexapod engrailed-family paralogues are evolving in a concerted fashion, resulting in gene trees that overestimate the frequency of gene duplication. We present new phylogenetic analyses using non-homeodomain amino acid sequence that support this view. The S. gregaria engrailed-family paralogues provide strong evidence that concerted evolution might in part be explained by recurrent gene conversion. Finally, we hypothesize that the RS-motif is part of a serine-rich domain targeted for phosphorylation.     

A cloned fragment of HeLa DNA containing consensus sequences of satellite II and III DNA hybridizes with the Drosophila P-element and with the 1.8 kb family of human KpnI fragments

We have cloned a repetitive EcoRI fragment from the human genome which displays weak homologies with the Drosophila melanogaster transposable P-element. This cloned DNA appeared not to be a mobile element but, instead, a divergent member of human satellite II or III DNAs. We present here the first complete nucleotide sequence of a 1.797 kilobase pair (kb) satellite-like DNA. Moreover, this EcoRI satellite monomer contains a unique sequence of 49 basepairs (bp) that is devoid of the satellite consensus repeat 5'TTCCA3'. Southern hybridization analysis revealed that the cloned insert is closely related to a highly repetitive 1.8 kb KpnI family of tandemly organized satellite DNAs. Thus, the relationships among these satellite DNA families appear to be complex and may be a factor in their copy number, position and spatial organization.     

The initiator tRNA genes of Drosophila melanogaster: evidence for a tRNA pseudogene

We have isolated four segments of Drosophila melanogaster DNA that hybridize to homologous initiator tRNAMet. Three of the cloned fragments contain initiator tRNA genes, each of which can be transcribed in vitro. The fourth clone, pPW568, contains an initiator tRNA pseudogene which is not transcribed in vitro by RNA polymerase III. The pseudogene is contained in a 1.15 kb DNA fragment. This fragment has the characteristics of dispersed repetitive DNA and hybridizes in situ to at least 30 sites in the Drosophila genome. The arrangement of the initiator tRNA genes we have isolated, is different to that of other Drosophila tRNA gene families. The initiator tRNA genes are not clustered nor intermingled with other tRNA genes. They occur as single copies within an approximately 415-bp repeat segment, which is separated from other initiator tRNA genes by a mean distance of 17 kb. In situ hybridization to polytene chromosomes localizes these genes to the 61D region of the Drosophila genome. Hybridization analysis of genomic DNA indicates the presence of 8-9 non-allelic initiator tRNA genes in Drosophila melanogaster.     

Cloning of segment polarity gene homologues from the unsegmented brachiopod Terebratulina retusa (Linnaeus).

We have used the polymerase chain reaction (PCR) to amplify, clone and sequence homologues of the Drosophila segment polarity genes engrailed (en), cubitus interruptus Dominant (ciD) and wingless (wg) from the genome of the brachiopod, Terebratulina retusa (Linnaeus). The deduced translation products of brachiopod en and ciD share high levels of sequence identity with their Drosophila homologues. The brachiopod wg-related clone is divergent from Drosophila wg, although clearly a member of the wg/Wnt gene family. These results indicate that structural diversity of Drosophila segment polarity genes has been evolutionarily conserved in a divergent, ancient and unsegmented animal phylum.     

A dense genetic map of the silkworm,Bombyx mori,covering all chromosomes based on 1018 molecular markers.

A dense linkage map was constructed for the silkworm, Bombyx mori, containing 1018 genetic markers on all 27 autosomes and the Z chromosome. Most of the markers, covering approximately 2000 cM, were randomly amplified polymorphic DNAs amplified with primer-pairs in combinations of 140 commercially available decanucleotides. In addition, eight known genes and five visible mutations were mapped. Bombyx homologues of engrailed and invected genes were found to be closely linked, as in Drosophila melanogaster. The average interval between markers was approximately 2 cM, equal to approximately 500 kb. The correspondence of seven linkage groups to counterparts of the conventional linkage map was determined. This map is the first linkage map in insects having a large number of chromosomes (n = 28) that covers all chromosomes without any gaps.     

Functional Anatomy of Polycomb and Trithorax Chromatin Landscapes in Drosophila Embryos

Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N–bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.     

Isolation and chromosomal localization of the human En-2 gene

By low stringency hybridization we have isolated from a human cosmid genomic library sequences homologous with a probe from the Drosophila engrailed gene. Partial nucleotide sequence analysis shows a consensus splice acceptor site followed by an open reading frame (ORF) that can encode 104 amino acids; the first 94 amino acids have 71% identity with the Drosophila engrailed protein. The shared region contains a homeo domain and is within the region of engrailed shared with the Drosophila invected gene and the mouse En-1 and En-2 genes. At the amino acid level, the human sequence is 85% identical with the mouse En-1 gene and 100% identical with the mouse En-2 gene. Hybridization against a panel of human-hamster somatic cell hybrids maps this human En-2 gene to chromosome 7, and regional mapping by in situ hybridization to human chromosomes localizes it to region 7q36 at the end of the long arm.     

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.