The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster

In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, we demonstrate that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, we show that distal heterochromatin (blocks h26-h29) is the only one within the chromocenter to form a big "puff"-like structure. The "puffed" heterochromatin has not only unique morphology but also very special protein composition as well: (i) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (ii) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, we show that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization.     

Interline differences in morphology of the precentromeric region of polytene X-chromosome in Drosophila melanogaster salivary glands]

Morphology of the Drosophila melanogaster polytene X chromosome section 20 in normal flies, in strains carrying inversions that break pericentric heterochromatin at different points, and at the background of the Su(UR)ES mutation has been examined. In all of the strains carrying the Su(UR)ES mutation section 20 displayed a distinct banding pattern till to the section 20F, while in the wild-type strains this region was represented by beta-heterochromatin. The strains carrying different inversions substantially differed in the number and morphology of bands forming section 20. In the Su(UR)ES mutants the most proximal X chromosome euchromatin gene, su(f), is mapped to the boundary between sections 20E and F, while rDNA forming the middle part of the X chromosome mitotic heterochromatin is located in the proximal part of section 20F. All large bands observed in section 20 of the w; Su(UR)ES strain were also present in In(1)sc4; Su(UR)ES, which breaks heterochromatin in the distal part. Hence, the bands of polytene chromosome section 20 are virtually devoid of mitotic heterochromatin.     

An investigation of heterochromatin domains on the fourth chromosome of Drosophila melanogaster

The banded portion of Drosophila melanogaster chromosome 4 exhibits euchromatic and heterochromatic characteristics. Reminiscent of heterochromatin, it contains a high percentage of repetitive elements, does not undergo recombination, and exhibits high levels of HP1 and histone-3 lysine-9 dimethylation. However, in the distal 1.2 Mb, the gene density is typical of euchromatin, and this region is polytene in salivary gland nuclei. Using P-element reporters carrying a copy of hsp70-white, alternative chromatin packaging domains can be distinguished by the eye color phenotype. Mapping studies identified the repetitive element 1360 as a candidate for heterochromatin targeting in the fourth chromosome Hcf region. We report here two new screens using this reporter to look for additional heterochromatin target sites. We confirm that reporter elements within 10 kb of 1360 are usually packaged as heterochromatin; however, heterochromatin packaging occurs in the sv region in the absence of 1360. Analyses of the sequences adjacent to P-element reporters show no simple association between specific repeated elements and transgene expression phenotype on a whole chromosome level. The data require that heterochromatin formation as a whole depends on a more complex pattern of sequence organization rather than the presence of a single sequence element.     

Ectopic HP1 promotes chromosome loops and variegated silencing in Drosophila

A transgene inserted in euchromatin exhibits mosaic expression when targeted by a fusion protein made of the DNA-binding domain of GAL4 and the heterochromatin-associated protein HP1. The silencing responds to the loss of a dose of the dominant modifiers of position-effect variegation Su(var)3-7 and Su(var)2-5, the locus encoding HP1. The genomic environs of the insertion site at 87C1 comprise the dispersed repetitive elements micropia and alphagamma. In the presence of the GAL4-HP1 chimera, the polytene chromosomes of this line form loops between the insertion site of the transgene and six other sections of chromosome 3R, as well as, rarely, with pericentric and telomeric heterochromatin. In contrast to the insertion site of the transgene at 87C, the six loop-forming sites in the euchromatic arm were each previously described as intercalary heterochromatin. Moreover, GAL4-HP1 tethering on one homologue trans-inactivates the reporter on the other. HP1, probably together with other partners, could thus facilitate the coalescence of dispersed middle repetitive sequences, and organize the heterochromatic structure responsible for the variegated silencing of nearby euchromatic genes.     

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.     

Electron microscope mapping of the pericentric and intercalary heterochromatic regions of the polytene chromosomes of the mutant Suppressor of underreplication in Drosophila melanogaster

Breaks and ectopic contacts in the heterochromatic regions of Drosophila melanogaster polytene chromosomes are the manifestations of the cytological effects of DNA underreplication. Their appearance makes these regions difficult to map. The Su(UR)ES gene, which controls the phenomenon, has been described recently. Mutation of this locus gives rise to new blocks of material in the pericentric heterochromatic regions and causes the disappearance of breaks and ectopic contacts in the intercalary heterochromatic regions, thereby making the banding pattern distinct and providing better opportunities for mapping of the heterochromatic regions in polytene chromosomes. Here, we present the results of an electron microscope study of the heterochromatic regions. In the wild-type salivary glands, the pericentric regions correspond to the beta-heterochromatin and do not show the banding pattern. The most conspicuous cytological effect of the Su(UR)ES mutation is the formation of a large banded chromosome fragment comprising at least 25 bands at the site where the 3L and 3R proximal arms connect. In the other pericentric regions, 20CF, 40BF and 41BC, 15, 12 and 9 new bands were revealed, respectively. A large block of densely packed material appears in the most proximal part of the fourth chromosome. An electron microscope analysis of 26 polytene chromosome regions showing the characteristic features of intercalary heterochromatin was also performed. Suppression of DNA underreplication in the mutant transforms the bands with weak spots into large single bands.     

HP2-like protein: a new piece of the facultative heterochromatin puzzle

In Drosophila melanogaster, the two chromosomal proteins HP1 and HP2 colocalize on heterochromatic and euchromatic sites in polytene chromosomes. Mutations in the HP2 gene act as dominant suppressors of position effect variegation, demonstrating a role for HP2 in the formation or maintenance of heterochromatin. In this paper, we investigated whether a putative homolog of the D. melanogaster HP2 is involved in the facultative heterochromatinization process in mealybugs. Using an antibody raised against the Drosophila HP2, we identified in the mealybug Planococcus citri a cross-reactive epitope, which we refer to as HP2-like. We investigated the HP2-like pattern during the male embryo development where the entire paternal haploid chromosome set becomes heterochromatic. The HP2 antibody heavily decorates the chromocenters, where it localizes with HP1, and marks the chromatin before it acquires the full cytological characteristics of the male-specific heterochromatin. In euchromatic chromosomes, HP2-like is mainly concentrated at telomeric sites. The interplay between HP2-like and HP1-like was studied by dsRNA interference experiments. Extinguishing HP1-like expression by RNAi does not prevent the association of HP2-like with facultative heterochromatin, implying that HP2-like binds to chromatin in a HP1-independent manner. Our results confirm and extend the structural and functional conservation of proteins involved in heterochromatin assembly. Silvia Volpi and Silvia Bongiorni contributed equally to the work.     

A combined approach to mapping the proximal border of the right arm of polytene chromosome 2 in Drosophila melanogaster otu 11

A combined approach based on cytological observations in situ hybridization, and qualitative Southern-blot analyses were used to localize the proximal border of the right arm of polytene chromosome 2 in Drosophila melanogaster otu 11 strain. A genetically functional chromosome 2 is bounded by "deletions" C', C, D, B, A and ms2-10. Using in situ hybridization in conjunction with comparative quantitative Southern-blot hybridization to deletions in centromeric heterochromatin, DNA of specific centromeric clone lambda20p1.4 was localized with respect to "deletions" and on otu 11 polytene chromosomes. Comparison of hybridization sites of lambda20p1.4 on polytene chromosomes, and its amount in mutant lines of D. melanogaster carrying known "deletions" in the centromeric heterochromatin enabled us to localize the proximal border of the right arm of chromosome 2 in D. melanogaster otu 11 strain between the 39/40 region and hybridization site of the k20p1.4 DNA fragment.     

Effects of tethering HP1 to euchromatic regions of the Drosophila genome

Heterochromatin protein 1 (HP1) is a conserved non-histone chromosomal protein enriched in heterochromatin. On Drosophila polytene chromosomes, HP1 localizes to centric and telomeric regions, along the fourth chromosome, and to specific sites within euchromatin. HP1 associates with centric regions through an interaction with methylated lysine nine of histone H3, a modification generated by the histone methyltransferase SU(VAR)3-9. This association correlates with a closed chromatin configuration and silencing of euchromatic genes positioned near heterochromatin. To determine whether HP1 is sufficient to nucleate the formation of silent chromatin at non-centric locations, HP1 was tethered to sites within euchromatic regions of Drosophila chromosomes. At 25 out of 26 sites tested, tethered HP1 caused silencing of a nearby reporter gene. The site that did not support silencing was upstream of an active gene, suggesting that the local chromatin environment did not support the formation of silent chromatin. Silencing correlated with the formation of ectopic fibers between the site of tethered HP1 and other chromosomal sites, some containing HP1. The ability of HP1 to bring distant chromosomal sites into proximity with each other suggests a mechanism for chromatin packaging. Silencing was not dependent on SU(VAR)3-9 dosage, suggesting a bypass of the requirement for histone methylation.     

SU(VAR)3-7, a Drosophila heterochromatin-associated protein and companion of HP1 in the genomic silencing of position-effect variegation.

An increase in the dose of the Su(var)3-7 locus of Drosophila melanogaster enhances the genomic silencing of position-effect variegation caused by centromeric heterochromatin. Here we show that the product of Su(var)3-7 is a nuclear protein which associates with pericentromeric heterochromatin at interphase, whether on diploid chromosomes from embryonic nuclei or on polytene chromosomes from larval salivary glands. The protein also associates with the partially heterochromatic chromosome 4. As these phenotypes and localizations resemble those described by others for the Su(var)2-5 locus and its heterochromatin-associated protein HP1, the presumed co-operation of the two proteins was tested further. The effect of the dose of Su(var)3-7 on silencing of a number of variegating rearrangements and insertions is strikingly similar to the effect of the dose of Su(var)2-5 reported by others. In addition, the two loci interact genetically, and the two proteins co-immunoprecipitate from nuclear extracts. The results suggest that SU(VAR)3-7 and HP1 co-operate in building the genomic silencing associated with heterochromatin.     

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.     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

The large isoform of Drosophila melanogaster heterochromatin protein 2 plays a critical role in gene silencing and chromosome structure

Drosophila melanogaster heterochromatin protein 2 (HP2) interacts with heterochromatin protein 1 (HP1). In polytene chromosomes, HP2 and HP1 colocalize at the chromocenter, telomeres, and the small fourth chromosome. We show here that HP2 is present in the arms as well as the centromeric regions of mitotic chromosomes. We also demonstrate that Su(var)2-HP2 exhibits a dosage-dependent modification of variegation of a yellow reporter transgene, indicating a structural role in heterochromatin formation. We have isolated and characterized 14 new mutations in the Su(var)2-HP2 gene. Using wm4h, many (but not all) mutant alleles show dominant Su(var) activity. Su(var)2-HP2 mutant larvae show a wide variety of mitotic abnormalities, but not the telomere fusion seen in larvae deficient for HP1. The Su(var)2-HP2 gene codes for two isoforms: HP2-L (approximately 365 kDa) and HP2-S (approximately 175 kDa), lacking exons 5 and 6. In general, mutations that affect only the larger isoform result in more pronounced defects than do mutations common to both isoforms. This suggests that an imbalance between large and small isoforms is particularly deleterious. These results indicate a role for HP2 in the structural organization of chromosomes and in heterochromatin-induced gene silencing and show that the larger isoform plays a critical role in these processes.     

Drosophila: the genetics of two major larval proteins.

A series of irradiation-induced deficiencies covering 62 polytene chromosome bands in chromosome arm 3L of Drosophila melanogaster includes the loci of two abundant developmentally regulated larval proteins. The structural gene for larval serum protein 2 (LSP 2) lies at 68E3 or 4, and that for salivary glue secretion protein 3 between 68A8 and 68C11, coincident with a major intermoult puff active in the salivary gland at the time of glue synthesis. The structural genes for esterase 6 and four visible recessive loci lie within the same region.     

Heterochromatin protein 1 binds transgene arrays

Heterochromatin protein 1 (HP1) of Drosophila and its homologs in vertebrates are key components of constitutive heterochromatin. Here we provide cytological evidence for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation. Received: 26 June 1998; in revised form: 6 July 1998 / Accepted: 12 July 1998     

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.     

DDP1, a single-stranded nucleic acid-binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy.

The centromeric dodeca-satellite of Drosophila forms altered DNA structures in vitro in which its purine-rich strand (G-strand) forms stable fold-back structures, while the complementary C-strand remains unstructured. In this paper, the purification and characterization of DDP1, a single-stranded DNA-binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca-satellite C-strand, is presented. In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca-satellite sequences. DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts. During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes. In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co-localizing with the heterochromatin protein HP1. DDP1 is a multi-KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy. Expression of DDP1 complements a Deltascp160 deletion in yeast. These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin.