NORMAL GENOMIC

• Gabler M et al.
• Trans-splicing of the mod(mdg4) complex locus is conserved between the distantly related species Drosophila melanogaster and D. virilis.
• Genetics. 2005; 169: 723-36
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• The modifier of mdg4, mod(mdg4), locus in Drosophila melanogaster represents a new type of complex gene in which functional diversity is resolved by mRNA trans-splicing. A protein family of >30 transcriptional regulators, which are supposed to be involved in higher-order chromatin structure, is encoded by both DNA strands of this locus. Mutations in mod(mdg4) have been identified independently in a number of genetic screens involving position-effect variegation, modulation of chromatin insulators, apoptosis, pathfinding of nerve cells, and chromosome pairing, indicating pleiotropic effects. The unusual gene structure and mRNA trans-splicing are evolutionary conserved in the distantly related species Drosophila virilis. Chimeric mod(mdg4) transcripts encoded from nonhomologous chromosomes containing the splice donor from D. virilis and the acceptor from D. melanogaster are produced in transgenic flies. We demonstrate that a significant amount of protein can be produced from these chimeric mRNAs. The evolutionary and functional conservation of mod(mdg4) and mRNA trans-splicing in both Drosophila species is furthermore demonstrated by the ability of D. virilis mod(mdg4) transgenes to rescue recessive lethality of mod(mdg4) mutant alleles in D. melanogaster.

• Ma J, Hwang KK, Worman HJ, Courvalin JC, Eissenberg JC
• Expression and functional analysis of three isoforms of human heterochromatin-associated protein HP1 in Drosophila.
• Chromosoma. 2001; 109: 536-44
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• Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal protein associated with pericentromeric heterochromatin in Drosophila. HP1-like proteins have also been found associated with heterochromatin in human cells. The goal of this study was to determine whether proteins of the structurally conserved human HP1 family exhibit conserved heterochromatin targeting and silencing properties in Drosophila. We established transgenic lines of Drosophila melanogaster expressing each of the three human HP1 proteins, HP1Hsalpha, HP1HSbeta, and HP1Hsgamma, under the Hsp70 heat shock promoter. We show that all three isoforms of human HP1 are stably expressed in Drosophila and are associated with heterochromatin in Drosophila chromosomes. Like Drosophila HP1, all three human HP1 proteins are delocalized by an HP1-POLYCOMB chimeric protein, implying that both human HP1 and Drosophila HP1 interact in a common protein complex, and that at least some aspects of heterochromatin structure are highly conserved throughout the evolution of eukaryotes. Ectopic expression of two of the three human HP1 family proteins significantly enhances heterochromatic silencing in Drosophila.

• Shareef MM, King C, Damaj M, Badagu R, Huang DW, Kellum R
• Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing.
• Mol Biol Cell. 2001; 12: 1671-85
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• Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

• Birve A et al.
• Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants.
• Development. 2001; 128: 3371-9
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• 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.

• Sedkov Y et al.
• Molecular genetic analysis of the Drosophila trithorax-related gene which encodes a novel SET domain protein.
• Mech Dev. 1999; 82: 171-9
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• The products of the trithorax and Polycomb groups genes maintain the activity and silence, respectively, of many developmental genes including genes of the homeotic complexes. This transcriptional regulation is likely to involve modification of chromatin structure. Here, we report the cloning and characterization of a new gene, trithorax-related (trr), which shares sequence similarities with members of both the trithorax and Polycomb groups. The trr transcript is 9.6 kb in length and is present throughout development. The TRR protein, as predicted from the nucleotide sequence of the open reading frame, is 2431 amino acids in length and contains a PHD finger-like domain and a SET domain, two highly conserved protein motifs found in several trithorax and Polycomb group proteins, and in modifiers of position effect variegation. TRR is most similar in sequence to the human ALR protein, suggesting that trr is a Drosophila homologue of the ALR. TRR is also highly homologous to Drosophila TRITHORAX protein and to its human homologue, ALL-1/HRX. However, preliminary genetic analysis of a trr null allele suggests that TRR protein may not be involved in regulation of homeotic genes (i.e. not a member of the trithorax or Polycomb groups) or in position effect variegation.

• Ingram R, Charrier B, Scollan C, Meyer P
• Transgenic tobacco plants expressing the Drosophila Polycomb (Pc) chromodomain show developmental alterations: possible role of Pc chromodomain proteins in chromatin-mediated gene regulation in plants.
• Plant Cell. 1999; 11: 1047-60
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• The chromodomain of the Drosophila Polycomb (Pc) protein has been introduced into tobacco nuclei to determine its location in the nucleus and its effect on plant development. Pc is a repressor of homeotic Drosophila genes that shares a well-conserved, although not identical, chromodomain with a structural heterochromatin component, Heterochromatin Protein 1. The chromodomains might therefore play a common role in chromatin repression. An analysis of transgenic plants expressing the Pc chromodomain, which was linked to the green fluorescent protein, suggested that the Pc chromodomain has distinct target regions in the plant genome. Transgenic plants expressing the Pc chromodomain had phenotypic abnormalities in their leaves and flowers, indicating a disruption in development. In axillary shoot buds of plants displaying altered leaf phenotypes, enhanced expression of a homeodomain gene, which is downregulated in wild-type leaves, was found. In Drosophila, Pc has been shown to possess distinct chromosome binding activity and to be involved in the regulation of development-specific genes. Our results support the assumptions that the heterologous chromodomain affects related functions in Drosophila and in plants, and that chromatin modification mechanisms are involved in the regulation of certain plant genes, in a manner similar to chromatin-mediated gene regulation in Drosophila.

• Stankunas K, Berger J, Ruse C, Sinclair DA, Randazzo F, Brock HW
• The enhancer of polycomb gene of Drosophila encodes a chromatin protein conserved in yeast and mammals.
• Development. 1998; 125: 4055-66
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• The Polycomb group of genes in Drosophila are homeotic switch gene regulators that maintain homeotic gene repression through a possible chromatin regulatory mechanism. The Enhancer of Polycomb (E(Pc)) gene of Drosophila is an unusual member of the Polycomb group. Most PcG genes have homeotic phenotypes and are required for repression of homeotic loci, but mutations in E(Pc) exhibit no homeotic transformations and have only a very weak effect on expression of Abd-B. However, mutations in E(Pc) are strong enhancers of mutations in many Polycomb group genes and are also strong suppressors of position-effect variegation, suggesting that E(Pc) may have a wider role in chromatin formation or gene regulation than other Polycomb group genes. E(Pc) was cloned by transposon tagging, and encodes a novel 2023 amino acid protein with regions enriched in glutamine, alanine and asparagine. E(Pc) is expressed ubiquitously in Drosophila embryogenesis. E(Pc) is a chromatin protein, binding to polytene chromosomes at about 100 sites, including the Antennapedia but not the Bithorax complex, 29% of which are shared with Polycomb-binding sites. Surprisingly, E(Pc) was not detected in the heterochromatic chromocenter. This result suggests that E(Pc) has a functional rather than structural role in heterochromatin formation and argues against the heterochromatin model for PcG function. Using homology cloning techniques, we identified a mouse homologue of E(Pc), termed Epc1, a yeast protein that we name EPL1, and as well as additional ESTs from Caenorhabditis elegans, mice and humans. Epc1 shares a long, highly conserved domain in its amino terminus with E(Pc) that is also conserved in yeast, C. elegans and humans. The occurrence of E(Pc) across such divergent species is unusual for both PcG proteins and for suppressors of position-effect variegation, and suggests that E(Pc) has an important role in the regulation of chromatin structure in eukaryotes.

• Laible G et al.
• Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres.
• EMBO J. 1997; 16: 3219-32
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• Gene silencing is required to stably maintain distinct patterns of gene expression during eukaryotic development and has been correlated with the induction of chromatin domains that restrict gene activity. We describe the isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila Polycomb-group (Pc-G) gene Enhancer of zeste [E(z)], a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian homologues of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development. The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing. Together, these data provide a functional link between Pc-G-dependent gene repression and inactive chromatin domains, and indicate that silencing mechanism(s) may be broadly conserved in eukaryotes.

• Sathe SS, Harte PJ
• The extra sex combs protein is highly conserved between Drosophila virilis and Drosophila melanogaster.
• Mech Dev. 1995; 52: 225-32
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• Extra sex combs (esc) is one of the Polycomb Group genes, whose products are required for long term maintenance of the spatially restricted domains of homeotic gene expression initially established by the products of the segmentation genes. We recently showed that the esc protein contains five copies of the WD motif, which in other proteins has been directly implicated in protein-protein interactions. Mutations affecting the WD repeats of the esc protein indicate that they are essential for its function as a repressor of the homeotic genes. We proposed that they may mediate interactions between esc and other Polycomb Group proteins, recruiting them to their target genes, perhaps by additional interactions with transiently expressed repressors such as hunchback. To further investigate the functional importance of the WD motifs and identify other functionally important regions of the esc protein, we have begun to determine its evolutionary conservation by characterizing the esc gene from Drosophila virilis, a distantly related Drosophila species. We show that the esc protein is highly conserved between these species, particularly its WD motifs. Their high degree of conservation, particularly at positions which are not conserved in the WD consensus derived from alignment of all known WD motifs, suggests that each of the WD repeats in the esc protein is functionally specialized and that this specialization has been highly conserved during evolution. Its highly charged N-terminus exhibits the greatest divergence, but even these differences are conservative of its predicted physical properties. These observations suggest that the esc protein is functionally compact, nearly every residue making an important contribution to its function.

• Platero JS, Hartnett T, Eissenberg JC
• Functional analysis of the chromo domain of HP1.
• EMBO J. 1995; 14: 3977-86
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• Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein in Drosophila with dosage-dependent effects on heterochromatin-mediated gene silencing. An evolutionarily conserved amino acid sequence in the N-terminal half of HP1 (the 'chromo domain') shares > 60% sequence identity with a motif found in the Polycomb protein, a silencer of homeotic genes. We report here that point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing. We show that the HP1 chromo domain, like the Polycomb chromo domain, has chromosome binding activity, but to distinct chromosomal sites. We constructed a chimeric HP1-Polycomb protein, consisting of the chromo domain of Polycomb in the context of HP1, and show that it binds to both heterochromatin and Polycomb binding sites in polytene chromosomes. In flies expressing chimeric HP1-Polycomb protein, endogenous HP1 is mislocalized to Polycomb binding sites, and endogenous polycomb is misdirected to the heterochromatic chromocenter, suggesting that both proteins are recruited to their distinct chromosomal binding sites through protein-protein contacts. Chimeric HP1-Polycomb protein expression in transgenic flies promotes heterochromatin-mediated gene silencing, supporting the view that the chromo domain homology reflects a common mechanistic basis for homeotic and heterochromatic silencing.

• Schlossherr J, Eggert H, Paro R, Cremer S, Jack RS
• Gene inactivation in Drosophila mediated by the Polycomb gene product or by position-effect variegation does not involve major changes in the accessibility of the chromatin fibre.
• Mol Gen Genet. 1994; 243: 453-62
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• In Drosophila position effect variegation and Polycomb-dependent regulation of homeotic gene expression are phenomena in which genes are inactivated in a clonally inherited manner. In both processes inactivation involves proteins that interact with the chromosome at or close to the position of inactivated genes. Two models have been proposed to explain this form of genetic silencing. In one, cooperative concatamerisation of a large multisubunit protein complex packages the chromatin fibre into a higher order structure, which is inaccessible for the transcription apparatus. In the second, the chromatin fibre is left unaltered but the region to be silenced is assigned to a compartment within the nucleus to which not all transcription factors have access. To distinguish between these types of model we have used the ligation-mediated PCR procedure to quantitate the accessibility of restriction sites in the chromatin fibre in both the active and inactivated forms. By making use of appropriate mutations and tissues we show that the inactivation of genes by Polycomb or by position effect variegation is not accompanied by a substantial change in the accessibility of the fibre. These results favour models in which the inactivation is achieved by sequestration of the silenced region in a particular nuclear compartment rather than by a chromatin packaging model.

• Tschiersch B, Hofmann A, Krauss V, Dorn R, Korge G, Reuter G
• The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes.
• EMBO J. 1994; 13: 3822-31
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• Modifier mutations of position-effect variegation (PEV) represent a useful tool for a genetic and molecular dissection of genes connected with chromatin regulation in Drosophila. The Su(var)3-9 gene belongs to the group of haplo suppressor loci which manifest a triplo enhancer effect. Mutations show a strong suppressor effect even in the presence of PEV enhancer mutations, indicating a central role of this gene in the regulation of PEV. By molecular analysis, Su(var)3-9 could be correlated with a 2.4 kb transcript which encodes a putative protein of 635 amino acids containing a chromo domain and a region of homology to Enhancer of zeste and trithorax, two antagonistic regulators of the Antennapedia and Bithorax gene complexes, as well as to the human protein ALL-1/Hrx which is implicated in acute leukemias. This region of homology is found in all four proteins at the C-terminus. The homology of Su(var)3-9 to both negative (Polycomb and Enhancer of zeste) and positive (trithorax) regulators of the Antennapedia and Bithorax complexes also suggests similarities in the molecular processes connected with stable transmission of a determined state and the clonal propagation of heterochromatinization.