HP1/ORC complex and heterochromatin assembly

We have used the highly conserved heterochromatin component, heterochromatin protein 1 (HP1), as a molecular tag for purifying other protein components of Drosophila heterochromatin. A complex of HP1 associated with the origin recognition complex (ORC) and an HP1/ORC-associated protein (HOAP) was purified from the maternally loaded cytoplasm of early Drosophila embryo. We propose that the DNA-binding activities of ORC and HOAP function to recruit underphosphorylated isoforms of HP1 to sites of heterochromatin nucleation. The roles of highly phosphorylated HP1, other DNA-binding proteins known to interact with HP1, and histone modifying activities in heterochromatin assembly are also addressed.     

Thoracic underreplication in Drosophila species estimates a minimum genome size and the dynamics of added DNA

Many cells in the thorax of Drosophila were found to stall during replication, a phenomenon known as underreplication. Unlike underreplication in nuclei of salivary and follicle cells, this stall occurs with less than one complete round of replication. This stall point allows precise estimations of early-replicating euchromatin and late-replicating heterochromatin regions, providing a powerful tool to investigate the dynamics of structural change across the genome. We measure underreplication in 132 species across the Drosophila genus and leverage these data to propose a model for estimating the rate at which additional DNA is accumulated as heterochromatin and euchromatin and also predict the minimum genome size for Drosophila. According to comparative phylogenetic approaches, the rates of change of heterochromatin differ strikingly between Drosophila subgenera. Although these subgenera differ in karyotype, there were no differences by chromosome number, suggesting other structural changes may influence accumulation of heterochromatin. Measurements were taken for both sexes, allowing the visualization of genome size and heterochromatin changes for the hypothetical path of XY sex chromosome differentiation. Additionally, the model presented here estimates a minimum genome size in Sophophora remarkably close to the smallest insect genome measured to date, in a species over 200 million years diverged from Drosophila.     

Parental Imprinting in Drosophila

Genetic imprinting is a form of epigenetic silencing. But with a twist. The twist is that while imprinting results in the silencing of genes, chromosome regions or entire chromosome sets, this silencing occurs only after transmission of the imprinted region by one sex of parent. Thus genetic imprinting reflects intertwined levels of epigenetic and developmental modulation of gene expression. Imprinting has been well documented and studied in Drosophila, however, these studies have remained largely unknown due to nothing more significant than differences in terminology. Imprinting in Drosophilais invariably associated with heterochromatin or regions with unusual chromatin structure. The imprint appears to spread from imprinted centers that reside within heterochromatin and these are, seemingly, the only regions that are normally imprinted in Drosophila. This is significant as it implies that while imprinting occurs in Drosophila, it is generally without phenotypic consequence. Hence the evolution of imprinting, at least in Drosophila, is unlikely to be driven by the function of specific imprinted genes. Thus, the study of imprinting in Drosophilahas the potential to illuminate the mechanism and biological function of imprinting, and challenge models based solely on imprinting of mammalian genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Age‐associated de‐repression of retrotransposons in the Drosophila fat body,its potential cause and consequence

Eukaryotic genomes contain transposable elements (TE) that can move into new locations upon activation. Since uncontrolled transposition of TEs, including the retrotransposons and DNA transposons, can lead to DNA breaks and genomic instability, multiple mechanisms, including heterochromatin‐mediated repression, have evolved to repress TE activation. Studies in model organisms have shown that TEs become activated upon aging as a result of age‐associated deregulation of heterochromatin. Considering that different organisms or cell types may undergo distinct heterochromatin changes upon aging, it is important to identify pathways that lead to TE activation in specific tissues and cell types. Through deep sequencing of isolated RNAs, we report an increased expression of many retrotransposons in the old Drosophila fat body, an organ equivalent to the mammalian liver and adipose tissue. This de‐repression correlates with an increased number of DNA damage foci and decreased level of Drosophila lamin‐B in the old fat body cells. Depletion of the Drosophila lamin‐B in the young or larval fat body results in a reduction of heterochromatin and a corresponding increase in retrotransposon expression and DNA damage. Further manipulations of lamin‐B and retrotransposon expression suggest a role of the nuclear lamina in maintaining the genome integrity of the Drosophila fat body by repressing retrotransposons.     

The mechanism of telomere protection: a comparison between Drosophila and humans

Drosophila telomeres are maintained by transposition of specialized retrotransposons rather than by telomerase activity, and their stability is independent of the sequence of DNA termini. Recent studies have identified several proteins that protect Drosophila telomeres from fusion events. These proteins include the telomere capping factors HP1/ORC-associated protein (HOAP) and heterochromatin protein 1 (HP1), the Rad50 and Mre11 DNA repair proteins that are required for HOAP and HP1 localization at telomeres, and the ATM kinase. Another telomere-protecting factor identified in Drosophila is UbcD1, a polypeptide highly homologous to class I ubiquitin-conjugating E2 enzymes. In addition, it has been shown that HP1 and both components of the Drosophila Ku70/80 heterodimer act as negative regulators of telomere length. Except for HOAP, all these proteins are conserved in humans and are associated with human telomeres. Collectively, these results indicate that Drosophila is an excellent model system for the analysis of the mechanisms of telomere maintenance. In past and current studies, 15 Drosophila genes have been identified that prevent telomeric fusion, and it has been estimated that the Drosophila genome contains at least 40 genes required for telomere protection. We believe that the molecular characterization of these genes will lead to identification of many novel human genes with roles in telomere maintenance.     

Roles of the sister chromatid cohesion apparatus in gene expression,development, and human syndromes

The sister chromatid cohesion apparatus mediates physical pairing of duplicated chromosomes. This pairing is essential for appropriate distribution of chromosomes into the daughter cells upon cell division. Recent evidence shows that the cohesion apparatus, which is a significant structural component of chromosomes during interphase, also affects gene expression and development. The Cornelia de Lange (CdLS) and Roberts/SC phocomelia (RBS/SC) genetic syndromes in humans are caused by mutations affecting components of the cohesion apparatus. Studies in Drosophila suggest that effects on gene expression are most likely responsible for developmental alterations in CdLS. Effects on chromatid cohesion are apparent in RBS/SC syndrome, but data from yeast and Drosophila point to the likelihood that changes in expression of genes located in heterochromatin could contribute to the developmental deficits.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. n. albomicana

Heterochromatin distribution and differentiation in metaphase chromosomes of two morphologically identical Drosophila races, D. nasuta nasuta and D. n. albomicana, have been studied by C- and N-banding methods. — The total heterochromatin values differ only slightly between these races. However, homologous chromosomes of the two Drosophila forms show striking differences in the size of heterochromatin regions and there is an alternating pattern in D. n. nasuta and D. n. albomicana of chromosomes which contain more, or respectively less heterochromatin than their counterparts in the other race. — Three different N-banding patterns could be obtained depending on the conditions of the method employed: One banding pattern occurs which corresponds to the C-banding pattern. Another pattern is the reverse of the C-band pattern; the euchromatic chromosome regions and the centromeres are stained whereas the pericentric heterochromatin regions remain unstained. In the Y chromosomes of both races and in chromosome 4 of D. n. albomicana, however, the heterochromatin is further differentiated. In the third N-banding pattern only the centromeres are deeply stained. Furthermore, between the races, subtle staining differences in the pericentric heterochromatin regions can be observed as verified in F₁ hybrids. On the basis of C- and N-banding results specific aspects of chromosomal differences between D. n. nasuta and D. n. albomicana are discussed.Dedicated to Prof. W. Beermann on the occasion of his 60th birthday     

SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing

This review summarizes genetic, molecular and biochemical studies of the SU(VAR)3-9 protein and the evidence for its key role in heterochromatin formation and heterochromatic gene silencing. The Su(var)3-9 locus was first identified as a dominant modifier of position-effect variegation (PEV) in Drosophila melanogaster. Together with Su(var)2-5 and Su(var)3-7, Su(var)3-9 belongs to the group of haplo-suppressor loci which show a triplo-dependent enhancer effect. All three genes encode heterochromatin-associated proteins. Su(var)3-9 is epistatic to the PEV modifier effects of Su(var)2-5 and Su(var)3-7, and it also dominates the effect of the Y chromosome on PEV. These genetic data support a central role of the SU(VAR)3-9 protein in heterochromatic gene silencing, one that is correlated with its activity as a histone H3-K9 methyltransferase (HMTase). In fact, SU(VAR)3-9 is the main chromocenter-specific HMTase of Drosophila. SU(VAR)3-9 and HP1, the product of Su(var)2-5, are main constituents of heterochromatin protein complexes and the interaction between these two proteins is interdependent. Functional analysis in fission yeast, Drosophila and mammals demonstrate that SU(VAR)3-9-dependent gene silencing processes are conserved in these organisms. This is also demonstrated by the rescue of Drosophila Su(var)3-9 mutant phenotypes with human SUV39H1 transgenes.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. nasuta albomicana

The microchromosomes of the totally cross fertile Drosophila races, D. nasuta nasuta and D. nasuta albomicana have been studied in nietaphase and polytene nuclei. In metaphase the microchromosome of D. n. albomicana is nearly five times longer than the homologous chromosome in D. n. nasuta. As shown by C-banding these length differences are mainly due to a massive addition of heterochromatin to the D. n. albomicana chromosome. In polytene nuclei these striking heterochromatin differences between the microchromosomes of the two Drosophila races cannot be observed. Analysis of the polytene banding pattern shows that the microchromosomes of both races differ by an inversion and by a duplication, present only in D. n, albomicana. The location and orientation of the duplicated regions in D. n. albomicana leads to a specific loop like chromosome configuration. On the basis of these differences within the Drosophila races studied it is assumed that the karyotype of D. n. albomicana is a more recent evolutionary product.     

Drosophila Telomere Transposons: Genetically Active Elements in Heterochromatin

In Drosophilatwo non-LTR retrotransposons, HeT-Aand TART, offer a novel experimental system for the study of heterochromatin. These elements, found only in heterochromatin, form Drosophilatelomeres by repeated transposition onto chromosome ends. Their transposition yields arrays of repeats larger and more irregular than the repeats produced by telomeras; nevertheless, the transpositions are, in principle, equivalent to the telomere-building action of telomerase. The identification of the HeT-Apromoter has given the first view of the molecular structure of a promoter active in heterochromatin. These telomere-specific elements are unusual in having a large amount of non-coding sequence. Like many other heterochromatic sequences, the HeT-Anon-coding sequence has a repetitive organization strongly conserved within the species, although the sequence itself can undergo significant change between species (atypical example of concerted evolution). Such heterochromatic sequences could be important for the cell, perhaps as docking stations for essential proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants

Background: The accurate duplication and packaging of the genome is an absolute prerequisite to the segregation of chromosomes in mitosis. To understand the process of cell-cycle chromosome dynamics further, we have performed the first detailed characterization of a mutation affecting mitotic chromosome condensation in a metazoan. Our combined genetic and cytological approaches in Drosophila complement and extend existing work employing yeast genetics and Xenopus in vitro extract systems to characterize higher-order chromosome structure and function.Results: Two alleles of the ORC2 gene were found to cause death late in larval development, with defects in cell-cycle progression (delays in S-phase entry and metaphase exit) and chromosome condensation in mitosis. During S-phase progression in wild-type cells, euchromatin replicates early and heterochromatin replicates late. Both alleles disrupted the normal pattern of chromosomal replication, with some euchromatic regions replicating even later than heterochromatin. Mitotic chromosomes were irregularly condensed, with the abnormally late replicating regions of euchromatin exhibiting the greatest problems in mitotic condensation.Conclusions: The results not only reveal novel functions for ORC2 in chromosome architecture in metazoans, they also suggest that the correct timing of DNA replication may be essential for the assembly of chromatin that is fully competent to undergo mitotic condensation.     

Conserved domains control heterochromatin localization and silencing properties of SU(VAR)3–7

The Drosophila protein SU(VAR)3–7 is essential for fly viability, chromosome structure, and heterochromatin formation. We report that searches in silico and in vitro for homologues of SU(VAR)3–7 were successful within, but not outside, the Drosophila genus. Protein sequence homology between the distant sibling species Drosophila melanogaster and Drosophila virilis is low, except for the general organization of the protein and three conserved motives: seven widely spaced zinc fingers in the N-terminal half and the BESS and BoxA motives in the C-terminal half of the protein. We have undertaken a fine functional dissection of SU(VAR)3–7 in vivo using transgenes encoding truncations of the protein. BESS mediates interaction of SU(VAR)3–7 with itself, and BoxA is required for specific heterochromatin association. Both are necessary for the silencing properties of SU(VAR)3–7. The seven zinc fingers, widely spaced over the N-terminal half of SU(VAR)3–7, are required for binding to polytene chromosomes. One finger is necessary and sufficient to determine the appropriate chromatin association of the C-terminal half of the protein. Conferring a function to each of the conserved motives allows us to better understand the mode of action of SU(VAR)3–7 in triggering heterochromatin formation and subsequent genomic silencing.     

The vectoring of cactophilic yeasts by Drosophila

Summary At two locations in the Sonoran Desert, yeasts were sampled from species of Drosophila, the flies' cactus hosts, and other neighboring sources of cactophilic yeasts to determine the relation between the yeasts vectored by the fly and the yeasts found in their breeding sites. D. mojavensis, D. nigrospiracula, and D. mettleri vectored yeast assemblages significantly more similar to the yeast species found on the rot from which the flies were collected than to the yeasts found on other rots from the flies host cactus or other rotting cactus at the same site. Rots with Drosophila had fewer yeast species than those without flies, suggesting that flies were associated with younger rots. Rots with flies and the Drosophila also had more yeast species with the capability to produce ethyl acetate than rots without flies. The results support the contention that cactophilic Drosophila feed on a subset of the yeasts available in an area, and may act to maintain differences among the yeast communities found on different species of cactus.     

N-banding in polytene chromosomes of Chironomus and Drosophila

The N-banding patterns of the polytene chromosomes of Drosophila melanogaster, Chironomus melanotus, Ch. th. thummi and Ch. th. thummi x Ch. th. piger were studied. In Chironomus the polytene N-banding patterns correspond to the polytene puffing patterns. This is revealed by comparison of the puffing and N-banding patterns of identical chromosomes. Size and staining intensity of the N-bands reflect the size of the puffs as shown by puff induction. There is no evidence that the N-bands are also located in Chironomus heterochromatin or are restricted to the nucleolar organizer regions. In Drosophila the -heterochromatin is strongly N-positive, whereas the -heterochromatin, as well as the Chironomus heterochromatin is not N-banded. Contrary to Chironomus, the puffs in Drosophila polytene chromosomes do not give rise selectively to well stained N-bands. — The N-banding method is interpreted to stain specifically non-histone protein which is (1) accumulated in genetically active chromosome regions and (2) present in a specific type of heterochromatin (-heterochromatin of Drosophila).     

Chromosomal distribution of heterochromatin protein 1 (HP1) in Drosophila: a cytological map of euchromatic HP1 binding sites

The Heterochromatin Protein 1 (HP1) is a conserved protein which is best known for its strong association with the heterochromatin of Drosophila melanogaster. We previously demonstrated that another important property of HP1 is its localization to the telomeres of Drosophila, a feature that reflects its critical function as a telomere capping protein. Here we report our analysis of the euchromatic sites to which HP1 localizes. Using an anti-HP1 antibody, we compared immunostaining patterns on polytene chromosomes of the Ore-R wild type laboratory strain and four different natural populations. HP1 was found to accumulate at specific euchromatic sites, with a subset of the sites conserved among strains. These sites do not appear to be defined by an enrichment of known repetitive DNAs. Comparisons of HP1 patterns among several Drosophila species revealed that association with specific euchromatic regions, heterochromatin and telomeres is a conserved characteristic of HP1. Based on these results, we argue that HP1 serves a broader function than typically postulated. In addition to its role in heterochromatin assembly and telomere stability, we propose that HP1 plays an important role in regulating the expression of many different euchromatic regions.     

Comparisons of yeast florae from natural substrates and larval guts of southwestern Drosophila

Summary The yeast florae in the natural substrates of four desert and three non-desert Drosophila species were compared both qualitatively and quantatively to the yeast present in the guts of Drosophila larvae living in those substrates. The desert species breed in rotting cacti and the other Drosophila were found breeding in necrotic oranges. Larvae of one cactophilic species, D. mojavensis, and larvae of all of the species utilizing oranges (D. melanogaster, D. pseudoobscura, and D. arizonensis) were found to contain non-random samples of the yeasts available in their respective substrates. Larval preference behavior is most likely responsible for these differences. The other cactophilic Drosophila (D. nigrospiracula, D. mettleri, and D. pachea) did not exhibit significant differences when the yeast florae of their larvae and substrates were compared. Selective feeding by larvae appears to be related to the degree of polyphagy in that only larvae of polyphagous species are selective. Trade-off between generalism and specialism at two biological levels is discussed.     

The effect of JIL-1 on position-effect variegation is proportional to the total amount of heterochromatin in the genome

In this study we have taken advantage of recent whole genome sequencing studies that have determined the DNA content in the heterochromatic regions of each Drosophila chromosome to directly correlate the effect on position-effect variegation of a pericentric insertion reporter line, 118E-10 with the total amount of heterochromatic DNA. Heterochromatic DNA levels were manipulated by adding or subtracting a Y chromosome as well as by the difference in the amount of pericentric heterochromatin between the X and Y chromosome. The results showed a direct, linear relationship between the amount of heterochromatic DNA in the genome and the expression of the w marker gene in the 118E-10 pericentric reporter line and that increasing amounts of heterochromatic DNA resulted in increasing amounts of pigment/gene activity. In Drosophila heterochromatic spreading and gene silencing is counteracted by H3S10 phosphorylation by the JIL-1 kinase, and we further demonstrate that the haplo-enhancer effect of JIL-1 is proportional to the amount of total heterochomatin, suggesting that JIL-1's activity is dynamically modulated to achieve a more or less constant balance depending on the levels of heterochromatic factors present.     

Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.