Genetic interactions and dosage effects of Polycomb group genes of Drosophila

The Polycomb (Pc) group of genes are required for maintenance of cell determination in Drosophila melanogaster. At least 11 Pc group genes have been described and there may be up to 40; all are required for normal regulation of homeotic genes, but as a group, their phenotypes are rather diverse. It has been suggested that the products of Pc group genes might be members of a heteromeric complex that acts to regulate the chromatin structure of target loci. We examined the phenotypes of adult flies heterozygous for every pairwise combination of Pc group genes in an attempt to subdivide the Pc group functionally. The results support the idea that Additional sex combs (Asx), Pc, Polycomblike (Pcl), Posterior sex combs (Psc), Sex combs on midleg (Scm), and Sex combs extra (Sce) have similar functions in some imaginal tissues. We show genetic interactions among extra sex combs (esc) and Asx, Enhancer of Pc, Pcl, Enhancer of zeste E(z), and super sex combs and reassess the idea that most Pc group genes function independently of esc. Most duplications of Pc group genes neither exhibit anterior transformations nor suppress the extra sex comb phenotype of Pc group mutations, suggesting that not all Pc group genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of esc mutants. Flies with increasing doses of esc ⁺ exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes. The results are discussed with respect to current models of Pc group function.     

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.     

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.     

Ten different Polycomb group genes are required for spatial control of the abdA and AbdB homeotic products.

Mutations in genes of the Polycomb (Pc) group cause abnormal segmental development due to ectopic expression of the homeotic products of the Antennapedia and bithorax complexes. Here the requirements for Pc group genes in controlling the abdA and AbdB products of the bithorax complex are described. Embryos containing mutations in the genes Polycomb (Pc), extra sex combs (esc), Enhancer of zeste [E(z)], polyhomeotic (ph), Sex comb on midleg (Scm), Polycomb-like (Pcl), Sex comb extra (Sce), Additional sex combs (Asx), Posterior sex combs (Psc) and pleiohomeotic (pho) were examined. In every case, both abdA and AbdB are expressed outside of their normal domains along the anterior-posterior (A-P) axis, consistent with these Pc group products acting in a single pathway or molecular complex. The earliest detectable ectopic expression is highest in the parasegments immediately adjacent to the normal expression boundary. Surprisingly, in the most severe Pc group mutants, the earliest ectopic AbdB is distributed in a pair-rule pattern. At all stages, ectopic abdA in the epidermis is highest along the anterior edges of the parasegments, in a pattern that mimics the normal abdA cell-specific pattern. These examples of highly patterned mis-expression show that Pc group mutations do not cause indiscriminate activation of homeotic products. We suggest that the ectopic expression patterns result from factors that normally activate abdA and AbdB only in certain parasegments, but that in Pc group mutants these factors gain access to regulatory DNA in all parasegments.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Effects of Polycomb Group Mutations on the Expression of Ultrabithorax in the Drosophila Visceral Mesoderm

The Polycomb group (PcG) genes encode repressors of many developmental regulatory genes including homeotic genes and are known to act by modifying chromatin structure through complex formation. We describe how Ultrabithorax (Ubx) expression is affected by the PcG mutants in the visceral mesoderm. Mutant embryos of the genes extra sex combs (esc), Polycomb (Pc), additional sex combs (Asx) and pleiohomeotic (pho) were examined. In each mutation, Ubx was ectopically expressed outside of their normal domains along the anterior-posterior axis in the visceral mesoderm, which is consistent with the effect of PcG proteins repressing the homeotic genes in other tissues. All of these four PcG mutations exhibit complete or partial lack of midgut constriction. However, two thirds of esc mutant embryos did not show Ubx expression in parasegment 7 (PS7). Even in the embryos showing ectopic Ubx expression, the level of Ubx expression in the PcG mutations was weaker than that in normal embryos. We suggest that in PcG mutations the ectopic Ubx expression is caused by lack of PcG repressor proteins, while the weaker or lack of Ubx expression is due to the repression of Ubx by Abd-B protein which is ectopically expressed in PcG mutations as well.     

Genetic Analysis of the Additional Sex Combs Locus of Drosophila Melanogaster

Additional sex combs (Asx) is a member of the Polycomb group of genes, which are thought to be required for maintenance of chromatin structure. To better understand the function of Asx, we have isolated nine new alleles, each of which acts like a gain of function mutation. Asx is required for normal determination of segment identity. AsxP1 shows an unusual phenotype in that anterior and posterior homeotic transformations are seen in the same individuals, suggesting that AsxP1 might upset chromatin structure in a way that makes both activation and repression of homeotic genes more difficult. Analysis of embryonic and adult phenotypes of Asx alleles suggests that Asx is required zygotically for determination of segment number and polarity. The expression pattern of even-skipped is altered in Asx mutant embryos, suggesting that Asx is required for normal expression of this gene. We have transposon-tagged the Asx gene, and can thus begin molecular analysis of its function.     

corto genetically interacts with Pc-G and trx-G genes and maintains the anterior boundary of Ultrabithorax expression in Drosophila larvae

In Drosophila melanogaster, segment identity is determined by specific expression of homeotic genes (Hox). The Hox expression pattern is first initiated by gap and pair-rule genes and then maintained by genes of the Polycomb-group (Pc-G) and the trithorax-group (trx-G). The corto gene is a putative regulator of the Hox genes since mutants exhibit homeotic transformations. We show here that, in addition to previously reported genetic interactions with the Pc-G genes Enhancer of zeste, Polycomb and polyhomeotic, mutations in corto enhance the extra-sex-comb phenotype of multi sex combs, Polycomb-like and Sex combs on midleg. corto also genetically interacts with a number of trx-G genes (ash1, kismet, kohtalo, moira, osa, Trithorax-like and Vha55). The interactions with genes of the trx-G lead to phenotypes displayed in the wing, in the postpronotum or in the thoracic mechanosensory bristles. In addition, we analyzed the regulation of the Hox gene Ultrabithorax (Ubx) in corto mutants. Our results provide evidence that corto maintains the anterior border of Ubx expression in third-instar larvae. We suggest that this regulation is accomplished through an interaction with the products of the Pc-G and trx-G genes.     

Maternal expression of genes that regulate the bithorax complex of Drosophila melanogaster

A relatively large number of genes have been described that are required for the normal spatial expression of the genes of the bithorax complex. Most of these regulators appear to act negatively and are required to prevent indiscriminate expression of bithorax complex (BX-C) functions. In this report we examine five negative BX-C regulators to determine whether these are maternally expressed in germ-line derived cells. The genes studied include Additional sex combs (Asx), Polycomblike (Pcl), Sex comb extra (Sce), Sex comb on midleg (Scm), and lethal(4)29 [l(4)29]. The maternal germ-line dependent expression of each of these genes is assessed by comparison of zygotes from mothers whose functional germ cells carry no wild-type alleles to zygotes from mothers whose germ cells contain one wild-type allele. Because mutant alleles of each of the genes studied are recessive lethals, mosaic females with homozygous or hemizygous mutant germ lines were produced by pole cell transplantation. The results demonstrate that all of the negative regulators tested are expressed in the maternal germ line and all play important roles in the regulation of BX-C activities during embryogenesis. The absence of maternally supplied products from all of the genes studied except l(4)29 can be largely or completely compensated for by the activity in the zygote of a paternally contributed wild-type allele. It is argued that, with the exception of l(4)29, the genes studied in this report are qualitatively similar in function to the previously described BX-C regulators Pc, esc, and sxc. The available evidence indicates that genes within this group have functions that are not restricted to the regulation of genes that control segmental identity.     

The Drosophila pleiohomeotic mutation enhances the Polycomblike and Polycomb mutant phenotypes during embryogenesis and in the adult

In Drosophila, the spatially restricted expression of the homeotic genes is controlled by Polycomb group (PcG) repression. PcG proteins appear to form different complexes to repress this gene expression. Although the pleiohomeotic gene (pho) shares mutational phenotypes with other PcG mutations, which demonstrates that PHO binds directly with a Polycomb (Pc)-containing complex, the genetic interactions of pho with other PcG genes have not been examined in detail. Here we investigated whether pho interacts with Polycomblike (Pcl) and Polycomb (Pc) during embryonic and adult development using developmental and genetic approaches. Pcl and Pc strongly enhanced pho phenotypes in the legs and tergite of the adult fly. Embryonic cuticle transformation was also greatly enhanced in Pcl; pho or Pc; pho double mutant embryos. The double mutant phenotypes were more severely affected by the pho maternal effect mutation than in zygotic mutant background, suggesting dosage-dependent processes. Taken together, these results provide genetic evidence of an interaction between PHO with other Polycomb group proteins at the embryonic and adult stages, and of the functioning of PHO as a component of the PcG complex.     

Screen for enhancers of Polycomb and Polycomblike in Drosophila melanogaster

There are 11 Polycomb group genes known in Drosophila. These genes are negative regulators of homeotic gene expression, and may act by modifying chromatin structure. It is not clear how many members of the Polycomb group of genes exist. Many were discovered because of their homeotic phenotypes, or because they enhance homeotic mutations. Systematic screens for enhancers of Polycomb have identified previously known members of the Polycomb group. In an attempt to discover cytological locations of new Polycomb group genes, we crossed deletions uncovering about 20% of the genome to Polycomb-like and Polycomb and scored for enhancement of the extra sex combs phenotype. Haploidy for four regions, 36F7-37A, 43E18; 44B5-9, 70C2-6, and 70C6-15; 70D enhanced the extra sex comb phenotype associated with strong Polycomb group mutations. These regions have homeotic phenotypes either as homozygous embryos or heterozy-gous adults, or both. We also show that spalt enhances Polycomb group mutations. These results are discussed with respect to previous estimates of Polycomb group gene number. © 1994 Wiley-Liss, Inc.     

Polyhomeotic: A gene of Drosophila melanogaster required for correct expression of segmental identity

Summary A new locus in Drosophila melanogaster that is required for the correct expression of segmental identity has been discovered. The new locus, termed polyhomeotic (ph), is X-linked and maps cytologically to bands 2D2-3. Homozygous ph flies have homeotic transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes (ANT-C, BX-C), and in addition show loss of the humerus. ph interacts with three other similar mutations: Polycomb (Pc), Polycomblike (Pcl), and extra sex comb (esc), and acts as a dominant enhancer of Pc. The expression of ph depends on the ANT-C and BX-C dosage. ph has no embryonic phenotype, but temperature shift studies on ph ² show that the ph ⁺ product is required during embryogenesis and larval development. We propose that ph mutants in some way disrupt the normal expression of the ANT-C and BX-C, and, therefore, that ph ⁺ is needed for maintenance of segmental identity.     

Interactions of thePolycomb group of genes with homeotic loci ofDrosophila

Summary Members of thePolycomb (Pc) group of genes are required for the correct determination of segment identity, and are thought to be negative regulators of thebithorax andAntennapedia complexes. This hypothesis has been tested molecularly for only some members of thePc group. Here, we examine the distribution ofUltrabithorax (Ubx),Antennapedia (Antp), andSex combs reduced (Scr) proteins in the epidermis, central nervous system, and midgut of embryos homozygous for mutations in tenPc group genes. We show that zygotic loss of mostPc group genes causes ectopic expression ofUbx andAntp, but that there are differences in time and tissue-specificity. FivePc group mutations lack midgut constrictions and also exhibit ectopic or suppressedUbx expression and suppression ofAntp expression. Distribution ofAntp is upset earlier than distribution ofUbx in the central nervous system of everyPc group mutant affecting both genes. Loss of the zygotic products ofPolycomb, extra sex combs, andAdditional sex combs cause ectopic expression ofScr in epidermis, and allPc group genes exceptPsc have suppressedScr expression in the nervous system. These results are discussed with respect to the function of thePc group.     

The Drosophila Polycomb group gene Sex combs extra encodes the ortholog of mammalian Ring1 proteins

In Drosophila, the Polycomb group (PcG) of genes is required for the maintenance of homeotic gene repression during development. Here, we have characterized the Drosophila ortholog of the products of the mammalian Ring1/Ring1A and Rnf2/Ring1B genes. We show that Drosophila Ring corresponds to the Sex combs extra (Sce), a previously described PcG gene. We find that Ring/Sce is expressed and required throughout development and that the extreme Pc embryonic phenotype due to the lack of maternal and zygotic Sce can be rescued by ectopic expression of Ring/Sce. This phenotypic rescue is also obtained by ectopic expression of the murine Ring1/Ring1A, suggesting a functional conservation of the proteins during evolution. In addition, we find that Ring/Sce binds to about 100 sites on polytene chromosomes, 70% of which overlap those of other PcG products such as Polycomb, Posterior sex combs and Polyhomeotic, and 30% of which are unique. We also show that Ring/Sce interacts directly with PcG proteins, as it occurs in mammals.     

DSP1, an HMG-like protein, is involved in the regulation of homeotic genes

The Drosophila dsp1 gene, which encodes an HMG-like protein, was originally identified in a screen for corepressors of Dorsal. Here we report that loss of dsp1 function causes homeotic transformations resembling those associated with loss of function in the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Abdominal-B. The expression pattern of Scr is altered in dsp1 mutant imaginal discs, indicating that dsp1 is required for normal expression of this gene. Genetic interaction studies reveal that a null allele of dsp1 enhances trithorax-group gene (trx-G) mutations and partially suppresses Polycomb-group gene (Pc-G) mutations. On the contrary, overexpression of dsp1 induces an enhancement of the transformation of wings into halteres and of the extra sex comb phenotype of Pc. In addition, dsp1 male mutants exhibit a mild transformation of A4 into A5. Comparison of the chromatin structure at the Mcp locus in wild-type and dsp1 mutant embryos reveals that the 300-bp DNase I hypersensitive region is absent in a dsp1 mutant context. We propose that DSP1 protein is a chromatin remodeling factor, acting as a trx-G or a Pc-G protein depending on the considered function.