Interaction of heterochromatin protein 2 with HP1 defines a novel HP1-binding domain

Heterochromatin Protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster localized principally in the pericentric heterochromatin, telomeres, and fourth chromosome, all regions associated with HP1. Mutations in HP2 can suppress position effect variegation, indicating a role in gene silencing and heterochromatin formation [Shaffer, C. D. et al. (2002) Proc. Natl. Acad. Sci.U.S.A. 99, 14332-14337]. In vitro coimmunoprecipitation experiments with various peptides from HP2 have identified a single HP1-binding domain. Conserved domains in HP2, including those within the HP1-binding region, have been identified by recovering and sequencing Su(var)2-HP2 from D. willistoni and D. virilis, as well as examining available sequence data from D. pseudoobscura. A PxVxL motif, shown to be an HP1-binding domain in many HP1-interacting proteins, is observed but is not well-conserved in location and sequence and does not mediate HP2 binding to HP1. The sole HP1-binding domain is composed of two conserved regions of 12 and 16 amino acids separated by 19 amino acids. Site-directed mutagenesis within the two conserved regions has shown that the 16 amino acid domain is critical for HP1 binding. This constitutes a novel domain for HP1 interaction, providing a critical link for heterochromatin formation in Drosophila.     

The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins

Drosophila heterochromatin-associated protein 1 (HP1) is an abundant component of heterochromatin, a highly condensed compartment of the nucleus that comprises a major fraction of complex genomes. Some organisms have been shown to harbor multiple HP1-like proteins, each exhibiting spatially distinct localization patterns within interphase nuclei. We have characterized the subnuclear localization patterns of two newly discovered Drosophila HP1-like proteins (HP1b and HP1c), comparing them with that of the originally described fly HP1 protein (here designated HP1a). While HP1a targets heterochromatin, HP1b localizes to both heterochromatin and euchromatin and HP1c is restricted exclusively to euchromatin. All HP1-like proteins contain an amino-terminal chromo domain, a connecting hinge, and a carboxyl-terminal chromo shadow domain. We expressed truncated and chimeric HP1 proteins in vivo to determine which of these segments might be responsible for heterochromatin-specific and euchromatin-specific localization. Both the HP1a hinge and chromo shadow domain independently target heterochromatin, while the HP1c chromo shadow domain is implicated solely in euchromatin localization. Comparative sequence analyses of HP1 homologs reveal a conserved sequence block within the hinge that contains an invariant sequence (KRK) and a nuclear localization motif. This block is not conserved in the HP1c hinge, possibly accounting for its failure to function as an independent targeting segment. We conclude that sequence variations within the hinge and shadow account for HP1 targeting distinctions. We propose that these targeting features allow different HP1 complexes to be distinctly sequestered in organisms that harbor multiple HP1-like proteins.     

Novel Drosophila heterochromatin protein 1 (HP1)/origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations

Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin.     

Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila.

The Su(var)205 gene of Drosophila melanogaster encodes heterochromatin protein 1 (HP1), a protein located preferentially within beta-heterochromatin. Mutation of this gene has been associated with dominant suppression of position-effect variegation. We have cloned and sequenced the gene encoding HP1 from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with Drosophila melanogaster HP1 shows two regions of strong homology, one near the N-terminus (57/61 amino acids identical) and the other near the C-terminus (62/68 amino acids identical) of the protein. Little homology is seen in the 5' and 3' untranslated portions of the gene, as well as in the intronic sequences, although intron/exon boundaries are generally conserved. A comparison of the deduced amino acid sequences of HP1-like proteins from other species shows that the cores of the N-terminal and C-terminal domains have been conserved from insects to mammals. The high degree of conservation suggests that these N- and C-terminal domains could interact with other macromolecules in the formation of the condensed structure of heterochromatin.     

Functional dissection of the ETS transcription factor MEF

We previously indicated that myeloid elf-1-like factor (MEF) but not elf-1, specifically activated lysozyme gene expression in epithelial cells. MEF is highly homologous at the nucleotide and amino acid level, with elf-1 especially in the ETS domain. Here, we report the functional analysis of the nuclear localization and transactivation properties of MEF. To investigate the intracellular localization of MEF, we transiently transfected MEF-green fluorescence protein (GFP) fusion protein expression vector into HeLa cells. A region spanning residues 177-291 is required for nuclear localization. We produced deletion mutants of MEF to determine the transactivation domain. The data showed that the N-terminal region, encompassing amino acids 1-52 is a potent transactivation domain. The C-terminal region spanning residues 477-663 can also mediate transactivation but not as strongly as the N-terminal region. The activity of the amino acid residues 1-52 was confirmed by experiments with fused constructs of MEF to the DNA binding-domain of the yeast GAL4 protein. These results, which determined the localization of the functional domains of MEF, will provide us with new clues to its transactivation mechanisms to regulate lysozyme gene expression in epithelial cells.     

The HP1 chromo shadow domain binds a consensus peptide pentamer

Heterochromatin-associated protein 1 (HP1) is thought to affect chromatin structure through interactions with other proteins in heterochromatin. Chromo domains located near the amino (amino chromo) and carboxy (chromo shadow) termini of HP1 may mediate such interactions, as suggested by domain swapping, in vitro binding and 3D structural studies . Several HP1-associated proteins have been reported, providing candidates that might specifically complex with the chromo domains of HP1. However, such association studies provide little mechanistic insight and explore only a limited set of potential interactions in a largely non-competitive setting. To determine how chromo domains can selectively interact with other proteins, we probed random peptide phage display libraries using chromo domains from HP1. Our results demonstrate that a consensus pentapeptide is suffident for specific interaction with the HP1 chromo shadow domain. The pentapeptide is found in the amino acid sequence of reported HP1-associated proteins, including the shadow domain itself. Peptides that bind the shadow domain also disrupt shadow domain dimers. Our results suggest that HP1 dimerization, which is thought to mediate heterochromatin compaction and cohesion, occurs via pentapeptide binding. In general, chromo domains may function by avidly binding short peptides at the surface of chromatin-associated proteins.     

Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila

Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1), which is an essential, nonhistone chromosomal protein. Here we investigate the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, we find that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of "junk DNA" in eukaryotic genomes.     

Functional dissection of the Drosophila modifier of variegation Su(var)3-7

An increase in the dose of the heterochromatin-associated Su(var)3-7 protein of Drosophila augments the genomic silencing of position-effect variegation. We have expressed a number of fragments of the protein in flies to assign functions to the different domains. Specific binding to pericentric heterochromatin depends on the C-terminal half of the protein. The N terminus, containing six of the seven widely spaced zinc fingers, is required for binding to bands on euchromatic arms, with no preference for pericentric heterochromatin. In contrast to the enhancing properties of the full-length protein, the N terminus half has no effect on heterochromatin-dependent position-effect variegation. In contrast, the C terminus moiety suppresses variegation. This dominant negative effect on variegation could result from association of the fragment with the wild type endogenous protein. Indeed, we have found and mapped a domain of self-association in this C-terminal half. Furthermore, a small fragment of the C-terminal region actually depletes pericentric heterochromatin from endogenous Su(var)3-7 and has a very strong suppressor effect. This depletion is not followed by a depletion of HP1, a companion of Su(var)3-7. This indicates that Su(var)3-7 does not recruit HP1 to heterochromatin. We propose in conclusion that the association of Su(var)3-7 to heterochromatin depends on protein-protein interaction mediated by the C-terminal half of the sequence, while the silencing function requires also the N-terminal half containing the zinc fingers.     

The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19- amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.     

Heterochromatin protein HP1Hsβ(p25β) and its localization with centromeres in mitosis

Some autoimmune sera containing anticentromere autoantibodies also recognize a doublet of Mr 23 000 (p23) and 25 000 (p25) in addition to CENP (centromere protein)-A (Mr 19 000), -B (Mr 80 000), and -C (Mr 140 000). A p25 antigen (HP1^Hsα) has been shown to be a human homolog of Drosophila HP1 (heterochromatin protein 1). We have isolated a cDNA clone encoding another form of p25 (HP1^Hsβor p25β) from a λZap HepG2 library using human autoimmune serum. The deduced amino acid sequence of the clone contained a conserved chromodomain (chromatin modifier domain) in the N-terminal region and a heterochromatin binding domain in the C-terminal region. In immunofluorescence experiments, only affinity purified antibodies reactive with the C-terminal (amino acids 70–185) domain showed nucleoplasmic and heterochromatin staining, whereas N-terminal (amino acids 1–115) specific antibodies were nonreactive. In metaphase chromosome spreads, the C-terminal domain antibody was also localized to the centromeric regions of chromosomes. Association with centromeres was most prominent at anaphase and changed to a more generalized association with whole chromosomes in telophase. The cooccurrence of autoantibodies to centromere proteins and HP1 in certain autoimmune diseases might be a reflection of coordinated immune responses to these closely associated sets of proteins. Received: 8 August 1996; in revised form: 4 December 1996 / Accepted: 17 December 1996     

Expression and functional analysis of three isoforms of human heterochromatin-associated protein HP1 in Drosophila

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.     

Functional analysis of the chromo domain of HP1.

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.     

Drosophila DDP1, a multi-KH-domain protein, contributes to centromeric silencing and chromosome segregation

BACKGROUND: The Drosophila melanogaster DDP1 protein is a highly evolutionarily conserved protein that is characterised by the presence of 15 tandemly organized KH domains, known for mediating high-affinity binding to single-stranded nucleic acids (RNA and ssDNA). Consistent with its molecular organization, DDP1 binds single-stranded nucleic acids with high affinity, in vitro. It was shown earlier that, in polytene chromosomes, DDP1 is found in association with chromocenter heterochromatin, suggesting a contribution to heterochromatin formation and/or maintenance. RESULTS: In this paper, the actual contribution of DDP1 to the structural and functional properties of heterochromatin was determined through the analysis of the phenotypes associated with the hypomorphic ddp1(15.1) mutation that was generated through the mobilization of a P element inserted in the second intron of ddp1. ddp1(15.1) behaves as a dominant suppressor of PEV in the variegated rearrangement In(1)w(m4) as well as in several transgenic lines showing variegated expression of a hsp70-white(+) transgene. In polytene chromosomes from homozygous ddp1(15.1) larvae, histone H3-K9 methylation and HP1 deposition at chromocentre heterochromatin are strongly reduced. Our results also show that, when the maternal contribution of DDP1 is reduced, chromosome condensation and segregation are compromised. Moreover, in a ddp1(15.1) mutant background, transmission of the nonessential Dp1187 minichromosome is reduced. CONCLUSIONS: We conclude that DDP1 contributes to the structural and functional properties of heterochromatin. These results are discussed in the context of current models for the formation and maintenance of heterochromatin; in these models, HP1 deposition depends on H3-K9 methylation that, in turn, requires the contribution of the RNAi pathway.     

Bipartite Nuclear Localization Signals in the C Terminus of Human Topoisomerase IIα

DNA topoisomerase IIα is the intracellular target for several important chemotherapeutic agents, and drug-resistant human tumor cell lines have been described in which deletions in the C-proximal region of this enzyme are associated with its cytoplasmic localization. We have identified multiple potential bipartite nuclear localization signal (NLS) sequences in this region using a modified definition of the motif, and in the present study, we have expressed five of these as fusion proteins with β-galactosidase. Only one sequence (spanning amino acids 1454 to 1497) was sufficient to cause strong nuclear localization. Subsequent mutation analyses indicated that this NLS sequence was bipartite and that both domains contain more than two basic amino acids. Substitution of the lysine residue at position 1492 in the second basic domain with glutamine resulted in a fusion protein that localized inefficiently to the nucleus, indicating that all three basic residues in this domain are necessary. Our results confirm that a broader definition is required to detect all potential bipartite NLS motifs in a polypeptide sequence, although functional tests are still essential for identification of those sequences actually capable of directing nuclear localization.     

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.