Related chromosome binding sites for zeste, suppressors of zeste and Polycomb group proteins in Drosophila and their dependence on Enhancer of zeste function.

Polycomb group genes are necessary for maintaining homeotic genes repressed in appropriate parts of the body plan. Some of these genes, e.g. Psc, Su(z)2 and E(z), are also modifiers of the zeste-white interaction. The products of Psc and Su(z)2 were immunohistochemically detected at 80-90 sites on polytene chromosomes. The chromosomal binding sites of these two proteins were compared with those of zeste protein and two other Polycomb group proteins, Polycomb and polyhomeotic. The five proteins co-localize at a large number of sites, suggesting that they frequently act together on target genes. In larvae carrying a temperature sensitive mutation in another Polycomb group gene, E(z), the Su(z)2 and Psc products become dissociated from chromatin at non-permissive temperatures from most but not all sites, while the binding of the zeste protein is unaffected. The polytene chromosomes in these mutant larvae acquire a decondensed appearance, frequently losing characteristic constrictions. These results suggest that the binding of at least some Polycomb group proteins requires interactions with other members of the group and, although zeste can bind independently, its repressive effect on white involves the presence of at least some of the Polycomb group proteins.     

Genetic interactions of the suppressor 2 of zeste region genes

A wide variety of gain of function mutations have been induced in the Posterior Sex Comb (Psc)--Aristapedioid (Arp)--Suppressor 2 of zeste (Su(z)2) region of the second chromosome of Drosophila. This region contains at least three apparently related genes, two of which we have been studying. Psc1 has previously been used to identify Psc as a Pc group gene; however, it is a complex mutation with both gain and loss of function character. We report here that the Pc group character of Psc is not due to a gain of function and presumably reflects the function of the wild-type gene. We also provide evidence for a maternal function for Psc, as well as the neighboring Su(z)2 gene. Su(z)2 does not appear to be a Pc group gene as it does not act in a synergistic fashion with other Pc group genes in promoting posteriorly directed transformations. However, we have found that mutations in Su(z)2 do interact in a variety of interesting ways with mutations in Pc group genes.     

Comparison of Germline Mosaics of Genes in the Polycomb Group of Drosophila Melanogaster

The genes of the Polycomb group (PcG) repress the genes of the bithorax and Antennapedia complexes, among others. To observe a null phenotype for a PcG gene, one must remove its maternal as well as zygotic contribution to the embryo. Five members of the PcG group are compared here: Enhancer of Polycomb [E(Pc)], Additional sex combs (Asx), Posterior sex combs (Psc), Suppressor of zeste 2 [Su(z)2] and Polycomblike (Pcl). The yeast recombinase (FLP) system was used to induce mitotic recombination in the maternal germline. Mutant embryos were analyzed by staining with antibodies against six target genes of the PcG. The loss of the maternal component leads to enhanced homeotic phenotypes and to unique patterns of misexpression. E(Pc) and Su(z)2 mutations had only subtle effects on the target genes, even when the maternal contributions were removed. Asx and Pcl mutants show derepression of the targets only in specific cell types. Psc shows unusual effects on two of the targets, Ultrabithorax and abdominal-A. These results show that the PcG genes do not act only in a common complex or pathway; they must have some independent functions.     

Rex and a Suppressor of Rex Are Repeated Neomorphic Loci in the Drosophila Melanogaster Ribosomal DNA

We report the molecular characterization of the Posterior sex combs-Suppressor 2 of zeste region of Drosophila melanogaster. The distal breakpoint of the Aristapedioid inversion divides the region into two parts. We have molecularly mapped the lesions associated with several loss of function mutations in the Polycomb group gene Posterior sex combs (Psc) proximal to this breakpoint. In addition, we have found that lesions associated with several loss of function mutations in the Suppressor 2 of zeste [Su(z)2] gene lie distal to this breakpoint. Since the breakpoint does not cause a loss of function in either gene, no essential sequences are shared by these two neighboring genes. There are three dominant gain of function mutations in the region that result in abnormal bristle development. We find that all three juxtapose foreign DNA sequences upstream of the Su(z)2 gene, and that at least two of these mutations (Arp1 and vgD) behave genetically as gain of function mutations in Su(z)2. Northern and in situ hybridization analyses show that the mutations result in increased accumulation of the Su(z)2 mRNA, which we argue is responsible for the bristle loss phenotype.     

Interactions of Polyhomeotic with Polycomb Group Genes of Drosophila Melanogaster

The Polycomb (Pc) group genes of Drosophila are negative regulators of homeotic genes, but individual loci have pleiotropic phenotypes. It has been suggested that Pc group genes might form a regulatory hierarchy, or might be members of a multimeric complex that obeys the law of mass action. Recently, it was shown that polyhomeotic (ph) immunoprecipitates in a multimeric complex that includes Pc. Here, we show that duplications of ph suppress homeotic transformations of Pc and Pcl, supporting a mass-action model for Pc group function. We crossed ph alleles to all members of the Polycomb group, and to E(Pc) and Su(z)2 to look for synergistic effects. We observed extragenic noncomplementation between ph(503) and Pc, Psc(1) and Su(z)2(1) in females, and between ph(409) and Sce(1), Scm(D1) and E(z)(1) mutations in males, suggesting that these gene products might interact directly with ph. Males hemizygous for a temperature-sensitive allele, ph(2), are lethal when heterozygous with mutants in Asx, Pc, Pcl, Psc, Sce and Scm, and with E(Pc) and Su(z)2. Mutations in trithorax group genes were not able to suppress the lethality of ph(2)/Y; Psc(1)/+ males. ph(2) was not lethal with extra sex combs, E(z), super sex combs (sxc) or l(4)102EFc heterozygotes, but did cause earlier lethality in embryos homozygous for E(z), sxc and l(4)102EFc. However, ph(503) did not enhance homeotic phenotypes of esc heterozygotes derived from homozygous esc(-) mothers. We examined the embryonic phenotypes of ph(2) embryos that were lethal when heterozygous or homozygous for other mutations. Based on this phenotypic analysis, we suggest that ph may perform different functions in conjunction with differing subsets of Pc group genes.     

Regulation of Polycomb group genes Psc and Su(z)2 in Drosophila melanogaster

Certain Polycomb group (PcG) genes are themselves targets of PcG complexes. Two of these constitute the Drosophila Psc-Su(z)2 locus, a region whose chromatin is enriched for H3K27me3 and contains several putative Polycomb response elements (PREs) that bind PcG proteins. To understand how PcG mechanisms regulate this region, the repressive function of the PcG protein binding sites was analyzed using reporter gene constructs. We find that at least two of these are functional PREs that can silence a reporter gene in a PcG-dependent manner. One of these two can also display anti-silencing activity, dependent on the context. A PcG protein binding site near the Psc promoter behaves not as a silencer but as a down-regulation module that is actually stimulated by the Pc gene product but not by other PcG products. Deletion of one of the PREs increases the expression level of Psc and Su(z)2 by twofold at late embryonic stages. We present evidence suggesting that the Psc-Su(z)2 locus is flanked by insulator elements that may protect neighboring genes from inappropriate silencing. Deletion of one of these regions results in extension of the domain of H3K27me3 into a region containing other genes, whose expression becomes silenced in the early embryo.     

In vivo assay for protein-protein interactions using Drosophila chromosomes

The ability of a chimeric HP1-Polycomb (Pc) protein to bind both to heterochromatin and to euchromatic sites of Pc protein binding was exploited to detect stable protein-protein interactions in vivo. Previously, we showed that endogenous Pc protein was recruited to ectopic heterochromatic binding sites by the chimeric protein. Here, we examine the association of other Pc group (Pc-G) proteins. We show that Posterior sex combs (Psc) protein also is recruited to heterochromatin by the chimeric protein, demonstrating that Psc protein participates in direct protein-protein interaction with Pc protein or Pc-associated protein. In flies carrying temperature-sensitive alleles of Enhancer of zeste[E(z)] the general decondensation of polytene chromosomes that occurs at the restrictive temperature is associated with loss of binding of endogenous Pc and chimeric HP1-Polycomb protein to euchromatin, but binding of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-dependent binding to heterochromatin by endogenous Pc and Psc proteins at the restrictive temperature, suggesting that interaction of these proteins is mediated by E(z) protein. A myc-tagged full-length Suppressor 2 of zeste [Su(z)2] protein interacts poorly or not at all with ectopic Pc-G complexes, but a truncated Su(z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not recruited to the heterochromatin by the chimeric HP1-Polycomb protein, suggesting either that this protein does not interact directly with Pc-G complexes or that such interactions are regulated. Ectopic binding of chimeric chromosomal proteins provides a useful tool for distinguishing specific protein-protein interactions from specific protein-DNA interactions important for complex assembly in vivo.     

Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants

In both Drosophila and vertebrates, spatially restricted expression of HOX genes is controlled by the Polycomb group (PcG) repressors. Here we characterize a novel Drosophila PcG gene, Suppressor of zeste 12 (Su(z)12). Su(z)12 mutants exhibit very strong homeotic transformations and Su(z)12 function is required throughout development to maintain the repressed state of HOX genes. Unlike most other PcG mutations, Su(z)12 mutations are strong suppressors of position-effect variegation (PEV), suggesting that Su(z)12 also functions in heterochromatin-mediated repression. Furthermore, Su(z)12 function is required for germ cell development. The Su(z)12 protein is highly conserved in vertebrates and is related to the Arabidopsis proteins EMF2, FIS2 and VRN2. Notably, EMF2 is a repressor of floral homeotic genes. These results suggest that at least some of the regulatory machinery that controls homeotic gene expression is conserved between animals and plants.     

Structure and sequence of the Drosophila zeste gene.

The zeste gene of Drosophila affects the expression of other genes in a manner that depends on the homologous pairing of the chromosomes bearing the target gene. Zeste mediates transvection effects, the ability of one gene to control the expression of its homologous copy on another chromosome. We have determined the structure of the zeste gene and several mutants bearing partial deletions and the sequence of the z+, z1, zop6 and z11G3 alleles. The predicted zeste protein has an unusual structure including runs of Gln, Ala and alternating Gln Ala. Contrary to expectations the z1, zop6 and z11G3 mutations can each be attributed to single amino acid changes. The analysis of the mutants suggests that the zeste gene product is required for normal expression of at least some genes and we argue that za mutants may have residual function.     

Identification of Mutations in Three Genes That Interact with Zeste in the Control of White Gene Expression in Drosophila Melanogaster

Three previously described genes, enhancer of yellow, 1, 2 and 3, are shown to cooperate with the zeste gene in the control of white gene expression. The mutations e(y)1(u1), e(y)3(u1), and to a lesser extent e(y)2(u1), enhance the effect of the zeste null allele z(v77h). Different combinations of e(y)1(u1), e(y)2(u1) and e(y)3(u1) mutations with several other z alleles also enhance the white mutant phenotype, but only to levels characteristic of white alleles containing a deletion of the upstream eye enhancer. Loss of zeste protein binding sites from the white locus does not eliminate the effect of e(y)1(u1) and e(y)3(u1) mutations, suggesting that the products of these genes interact with some other nucleotide sequences. Combinations of either e(y)1(u1) or e(y)2(u1) mutations with e(y)3(u1) are lethal. The products of these three genes may represent, together with zeste, a group of proteins involved in the organization of long-distance interactions between DNA sequences.     

Homeosis and the interaction of zeste and white in Drosophila

Summary Transvection effects in Drosophila melanogaster suggest a form of gene modulation that is responsive to the proximity of homologous genes. These effects have been well characterized at bithorax and decapentaplegic, and in the interaction between the zeste and white genes. The mechanistic basis for transvection is not known. As part of a genetic analysis of transvection, a study is being made of a class of mutation defined as modifiers of the eye color resulting from the interaction of zeste and white. This report details the observations that several of these mutations also have homeotic effects.     

Genetic Analysis of the Enhancer of Zeste Locus and Its Role in Gene Regulation in Drosophila Melanogaster

The Enhancer of zeste [E(z)] locus of Drosophila melanogaster is implicated in multiple examples of gene regulation during development. First identified as dominant gain-of-function modifiers of the zeste1-white (z-w) interaction, mutant E(z) alleles also produce homeotic transformations. Reduction of E(z)+ activity leads to both suppression of the z-w interaction and ectopic expression of segment identity genes of the Antennapedia and bithorax gene complexes. This latter effect defines E(z) as a member of the Polycomb-group of genes. Analysis of E(z)S2, a temperature-sensitive E(z) allele, reveals that both maternally and zygotically produced E(z)+ activity is required to correctly regulate the segment identity genes during embryonic and imaginal development. As has been shown for other Polycomb-group genes, E(z)+ is required not to initiate the pattern of these genes, but rather to maintain their repressed state. We propose that the E(z) loss-of-function eye color and homeotic phenotypes may both be due to gene derepression, and that the E(z)+ product may be a general repressing factor required for both examples of negative gene regulation.     

A Role of Polycomb Group Genes in the Regulation of Gap Gene Expression in Drosophila

Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the hunchback (hb) morphogen. nanos determines the distribution of the maternal Hb protein by regulating its translation. To identify further components of this pathway we isolated suppressors of nanos. In the absence of nanos high levels of Hb protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high Hb levels. The suppressors are alleles of Enhancer of zeste (E(z)) a member of the Polycomb group (Pc-G) of genes. We show that E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal Hb protein gradient. We have identified a small region of the knirps promoter that mediates the regulation by E(z) and hb. Because Pc-G genes are thought to control gene expression by regulating chromatin, we propose that imprinting at the chromatin level underlies the determination of anteroposterior polarity in the early embryo.     

Transvection at the Eyes Absent Gene of Drosophila

The Drosophila eyes absent (eya) gene is required for survival and differentiation of eye progenitor cells. Loss of gene function in the eye results in reduction or absence of the adult compound eye. Certain combinations of eya alleles undergo partial complementation, with dramatic restoration of eye size. This interaction is sensitive to the relative positions of the two alleles in the genome; rearrangements predicted to disrupt pairing of chromosomal homologs in the eya region disrupt complementation. Ten X-ray-induced rearrangements that suppress the interaction obey the same general rules as those tha disrupt transvection at the bithorax complex and the decapentaplegic gene. Moreover, like transvection in those cases, the interaction at eya depends on the presence of normal zeste function. The discovery of transvection at eya suggests that transvection interactions of this type may be more prevalent than generally thought.