Two distinct domains in Drosophila melanogaster telomeres

Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics.     

Enhancement and Suppression of a Euchromatic Position Effect at NOTCH in Drosophila

The recessive visible rough-eye mutant facet-strawberry, fa^swb, is caused by the deletion of 0.8 kb of base sequences from the 5' end of the Notch locus. Visible deficiencies adjacent to fa^swb suppress this mutant effect of the Notch locus, and in the same region (between salivary bands 3C1 and 3C7), we have demonstrated the presence of at least one partial suppressor and one enhancer of the fa^swb position effect at Notch.—The enhancer seems to be a small inversion approximately equal to the salivary-band doublet 3C2, 3, and the partial suppressor lies between the inversion in 3C2, 3 and the small deletion in fa^swb immediately distal to 3C7. Neither the enhancer, e(fa^swb), nor the partial suppressor, su(fa^swb), can be detected except when linked in cis to fa^swb. The e(fa^swb) and the su(fa^swb), in unison, act antagonistically on the fa^swb position effect.—The fa^swb mutant is interpreted to be a nonvariegating position effect at the Notch locus resulting from a novel euchromatic—euchromatic association of base sequences caused by the small deletion.     

Euchromatic and heterochromatic compositional properties emerging from the analysis of Solanum lycopersicum BAC sequences

The consortium responsible for the sequencing of the tomato (Solanum lycopersicum) genome initially focused on the sequencing of the euchromatic regions using a BAC-by-BAC strategy. We analyzed the compositional features of the whole collection of BAC sequences publically available. This analysis highlights specific peculiarities of heterochromatic and euchromatic BACs, in particular: the whole BAC collection has i) a large variability in repeat and gene content, ii) a positive and significant correlation of LTR retrotransposons of the Gypsy class with the repeat content and iii) the preferential location of the SINEs (short interspersed nuclear elements) in BAC sequences showing a low repeat content. Our results point out a typical design of the tomato chromosomes and pave the way for further investigations on the relationship between DNA primary structure and chromatin organization in Solanaceae genomes.     

Genomic analysis of Drosophila melanogaster telomeres: full-length copies of HeT-A and TART elements at telomeres

The repetitive nature of heterochromatin hampers its analysis in general genome-sequencing projects. Specific studies are needed to extend the sequence into telomeric and centromeric heterochromatin. Drosophila telomeres lack the telomerase-generated repeats that are characteristic of other eukaryotic chromosomes. Instead, they consist of tandem arrays of HeT-A and TART elements. Herein, we present the genomic organization of the telomeres in the isogenic strain (y; cn bw sp) that was used for the Drosophila melanogaster sequencing project. The data indicate that the canonical features of telomere organization are widely conserved in evolution. In addition, we have identified full-length elements, likely competent elements, for HeT-A and TART.     

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.     

Cis-Effects of Heterochromatin on Heterochromatic and Euchromatic Gene Activity in Drosophila Melanogaster

Chromosomal rearrangements that juxtapose heterochromatin and euchromatin can result in mosaic inactivation of heterochromatic and euchromatic genes. This phenomenon, position effect variegation (PEV), suggests that heterochromatic and euchromatic genes differ in their regulatory requirements. This report describes a novel method for mapping regions required for heterochromatic genes, and those that induce PEV of a euchromatic gene. P transposase mutagenesis was used to generate derivatives of a translocation that variegated for the light(+) (lt(+)) gene and carried the euchromatic white(+) (w(+)) gene on a transposon near the heterochromatin-euchromatin junction. Cytogenetic and genetic analyses of the derivatives showed that P mutagenesis resulted in deletions of several megabases of heterochromatin. Genetic and molecular studies showed that the derivatives shared a euchromatic breakpoint but differed in their heterochromatic breakpoint and their effects on seven heterochromatic genes and the w(+) gene. Heterochromatic genes differed in their response to deletions. The lt(+) gene was sensitive to the amount of heterochromatin at the breakpoint but the heterochromatic 40Fa gene was not. The severity of variegated w(+) phenotype did not depend on the amount of heterochromatin in cis, but varied with local heterochromatic environment. These data are relevant for considering mechanisms of PEV of both heterochromatic and euchromatic genes.     

Euchromatic domains in plant chromosomes as revealed by H4 histone acetylation and early DNA replication.

Using specific polyclonal antisera raised against acetylated isoforms of histone H4, we have analyzed their distribution in the dioecious plant Silene latifolia (syn. Melandrium album) possessing heteromorphic sex chromosomes. Our previous studies on this species have shown that one of the two X chromosomes in homogametic female cells is heavily methylated and late replicating, as a possible consequence of dosage compensation. Here we report that there are no detectable differences in intensity and distribution of H4 acetylation between these two X chromosomes. In S. latifolia only distal-subtelomeric chromosome regions, on both the sex chromosomes and autosomes, display strong signals of H4 acetylation at N-terminal lysines 5, 8, and 12. These acetylated domains correspond to the very early replicating distal chromosome regions as revealed by 5-bromodeoxyuridine pulses followed by the indirect immunofluorescence microscopy. The distribution of H4 acetylated at lysine 16 was uniform along the chromosomes. The unique distal-subtelomeric H4 acetylation signals were also observed in three other Silene species (S. vulgaris, S. pendula, and S. chalcedonica), but not in two non-related plant species tested (Allium cepa and Nicotiana tobacum). The presented data as well as our recent studies on the structure of S. latifolia chromosome ends indicate that Silene species possess the specific distal-subtelomeric location of euchromatin, gene-rich regions on chromosomes.     

Colonization of heterochromatic genes by transposable elements in Drosophila

As a further step toward understanding transposable element-host genome interactions, we investigated the molecular anatomy of introns from five heterochromatic and 22 euchromatic protein-coding genes of Drosophila melanogaster. A total of 79 kb of intronic sequences from heterochromatic genes and 355 kb of intronic sequences from euchromatic genes have been used in Blast searches against Drosophila transposable elements (TEs). The results show that TE-homologous sequences belonging to 19 different families represent about 50% of intronic DNA from heterochromatic genes. In contrast, only 0.1% of the euchromatic intron DNA exhibits homology to known TEs. Intraspecific and interspecific size polymorphisms of introns were found, which are likely to be associated with changes in TE-related sequences. Together, the enrichment in TEs and the apparent dynamic state of heterochromatic introns suggest that TEs contribute significantly to the evolution of genes located in heterochromatin.     

Pairing-dependent mislocalization of a Drosophila brown gene reporter to a heterochromatic environment.

We describe the precise positioning of a reporter gene within heterochromatin where it may be silenced. A transposition of the 59E-60A region into pericentric heterochromatin ensnares distal 59E-60A via somatic pairing. The frequency with which a brown (bw) reporter gene in 59E is silenced is influenced by chromosomal configurations. Silencing occurs only when the bw+ reporter is unpaired due to heterozygosity with a deficiency, where the frequency of bw+ reporter expression is correlated with the extent of bw gene and flanking sequence present. Surprisingly, the frequency of pairing between the transposition in heterochromatin and distal 59E observed cytologically is indistinguishable from the frequency of pairing of homologous chromosomes at 59E in wild-type larval brains, regardless of configuration. Therefore, bringing a susceptible reporter gene into close proximity with heterochromatin does not necessarily affect its expression, but local pairing changes resulting from altered chromosomal configurations can lead to silencing. We also find that an ensnared distal copy of bw that is interrupted by a heterochromatic insertion enhances silencing. This demonstrates that bw can be simultaneously acted upon by pericentric and distal blocks of heterochromatin.     

Somatic synapsis of eu- and heterochromatic regions of Drosophila melanogaster chromosomes

Quantitative cytogenetical analysis has been used to study the synapsis of D. melanogaster neuroblast mitotic chromosomes from normal females, flies with heterozygous deletions, duplications or inversions in the heterochromatic regions of chromosome 2 and in triploid females. In all these genotypes chromocentric fusion of heterochromatic regions of heterologous chromosomes is observed. Eu- and heterochromatic regions of homologous chromosomes are intimately paired at the same time during the cell cycle. The structural rearrangements lead to reduced frequencies of chromocentric association as well as of homologous synapsis compared with the frequencies in the wild-type. The results obtained are discussed with respect to the general problem of the homologous interaction of chromosomes and the significance of heterochromatin for these processes.     

Healing of Euchromatic Chromosome Breaks by Efficient de novo Telomere Addition in Drosophila melanogaster

Many arthropod species are infected with maternally inherited endosymbionts that induce a shift in the sex ratio of their hosts by feminizing or killing males (cytoplasmic sex-ratio distorters, or SRDs). These endosymbionts can have profound impacts on evolutionary processes of their hosts. Here, I derive analytical expressions for the coalescent effective size N_e of populations that are infected with SRDs. Irrespective of the type of SRD, N_e for mitochondrial genes is given by the number of infected females. For nuclear genes, the effective population size generally decreases with increasing prevalence of the SRD and can be considerably lower than the actual size of the population. For example, with male-killing bacteria that have near perfect maternal transmission, N_e is reduced by a factor that is given to a good approximation by the proportion of uninfected individuals in the population. The formulae derived here also yield the effective size of populations infected with mutualistic endosymbionts or maternally inherited bacteria that induce cytoplasmic incompatibility, although in these cases, the reduction in N_e is expected to be less severe than for cytoplasmic SRDs.SIMPLE null models are essential in science. In population genetics, this role is filled by the Wright–Fisher model and its retrospective counterpart, the Kingman coalescent. Both of these models have proven to be immensely useful in spite of the fact that natural populations usually violate the assumptions made in these models. The reason for this is that often, the Wright–Fisher model can be rescaled so that it behaves in many important respects like a more complex population model. This rescaling is achieved through the concept of the effective population size, N_e. Roughly speaking, a complex population model is said to have a certain N_e if the haploid Wright–Fisher model with population size N_e experiences the same amount of random genetic drift as the complex model. Reflecting the different ways in which drift can be measured, N_e can be defined in different ways, e.g., as the inbreeding, the variance, or the coalescent effective population size. Different definitions often produce the same value for N_e, but may also yield drastically different numbers (Kimura and Crow 1963).The coalescent effective population size is defined through the factor by which time needs to be rescaled in a complex population model to produce the standard coalescent with time scale given by the population size N (Nordborg and Krone 2002). It has been argued that this is the most useful definition for N_e because “the coalescent essentially embodies all of the information that can be found in sampled genetic data” (Sjödin et al. 2005). More recently, Wakeley and Sargsyan (2009) have proposed two extensions of the coalescent effective population size in which they advocate including a mutation parameter in the definition and also allowing for a nonlinear relationship between N_e and N.One frequently encountered feature in natural populations that complicates population genetics is infection with maternally inherited endosymbionts. In particular, many arthropod species harbor a great number of phylogenetically diverse microorganisms that influence their hosts'' biology in different ways (Bourtzis and Miller 2003; Bourtzis and Miller 2006; Bourtzis and Miller 2009). Because of their maternal transmission, many of these microorganisms—for example, the bacteria Wolbachia pipientis and Cardinium hertigii—have evolved intricate manipulations of their hosts'' reproductive system that allows them to spread in a host population through exploitation of male hosts (reproductive parasitism, reviewed in Engelstädter and Hurst 2009a). Most manipulations involve a shift in the sex ratio of their hosts (both primary and at the population level), and the inducing endosymbionts are consequently referred to as cytoplasmic sex-ratio distorters (SRDs). In some species, genetic males develop into females if they are infected (“feminization”: Martin et al. 1973; Rigaud 1997; Bouchon et al. 1998; Hiroki et al. 2002). More commonly, infected males are killed by the endosymbionts early in their development (male killing: reviewed in Hurst et al. 2003). The adaptive advantage of this strategy is seen in an early fitness boost in the surviving females in a brood, for example, through reduced sibling competition or cannibalism of the dead brothers (Hurst 1991; Hurst and Majerus 1993; Jaenike et al. 2003). Some examples for species infected with male-killing or feminizing SRDs are given in Huigens and Stouthamer 2003).

TABLE 1

Empirical examples for cytoplasmic SRDs with parameter estimates

t-loops at trypanosome telomeres

Mammalian telomeres form large duplex loops (t-loops) that may sequester chromosome ends by invasion of the 3' TTAGGG overhang into the duplex TTAGGG repeat array. Here we document t-loops in Trypanosoma brucei, a kinetoplastid protozoan with abundant telomeres due to the presence of many minichromosomes. These telomeres contained 10-20 kb duplex TTAGGG repeats and a 3' TTAGGG overhang. Electron microscopy of psoralen/UV cross-linked DNA revealed t-loops in enriched telomeric restriction fragments and at the ends of isolated minichromosomes. In mammals, t-loops are large (up to 25 kb), often comprising most of the telomere. Despite similar telomere lengths, trypanosome t-loops were much smaller (approximately 1 kb), indicating that t-loop sizes are regulated. Coating of non-cross-linked minichromosomes with Escherichia coli single-strand binding protein (SSB) often revealed 3' overhangs at both telomeres and several cross-linked minichromosomes had t-loops at both ends. These results suggest that t-loops and their prerequisite 3' tails can be formed on the products of both leading and lagging strand synthesis. We conclude that t-loops are a conserved feature of eukaryotic telomeres.