Drosophila telomeres: two transposable elements with important roles in chromosomes

Telomeres in Drosophila melanogaster are composed of multiple copies of two retrotransposable elements, HeT-A and TART instead of the short DNA repeats generated by telomerase in most organisms. Transpositions of HeT-A and yield arrays of repeats larger and more irregular than the repeats produced by telomeras; nevertheless, these transpositions are, in principle, equivalent to the telomere-building action of telomerase. Both telomerase and transposition of HeT-A and TART extend chromosomes by RNA-templated addition of specific sequences. We have proposed that HeT-A has evolved from genes encoding telomerase components. Although both HeT-A and TART share some novel features, TART probably has a different origin from HeT-A. HeT-A and TART are clearly identifiable as non-long terminal repeat (non-LTR) retrotransposons. Both telomere elements transpose only to the ends of chromosomes (apparently to any chromosome end in D. melanogaster) and each contains a large segment of untranslated sequence. HeT-A and TART are the first examples of transposable elements with a clear role in chromosome structure. This has interesting implications for the evolution of both chromosomes and transposable elements. The finding also raises the possibility that other transposable elements with bona fide roles in the cell will be detected, not only in Drosophila, but also in other organisms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Return of the H-word (heterochromatin)

Recent advances in studies of yeast, Drosophila and humans have renewed interest in heterochromatin. These recent studies have demonstrated the interspersion and rapid spread of transposable elements into Drosophila heterochromatin; documented the requirement of heterochromatic genes for heterochromatin; identified heterochromatin-like regions in yeast chromosomes; confirmed an important role for satellite DNA in human centromere function; and suggested potential functions for heterochromatin-associated proteins.     

A new function for heterochromatin

Meiotic pairing has now been shown to require heterochromatic homology and to be sensitive to repeat number in both male and female Drosophila. The increased pairing ability of repetitive sequences could be one reason that most eukaryotes allow the accumulation of tandem repeated elements. This may well be a reflection of a general role for heterochromatin, and at least a partial explanation of the ubiquity of heterochromatin through the eukaryotes.     

The genomic organization of HeT-A retroposons inDrosophila melanogaster

Members of theDrosophila HeT-A family of transposable elements are LINE-like retroposons that are found at telomeres and in centric heterochromatin. We recently characterized an active HeT-A element that had transposed to a broken chromosome end fewer than mine generations before it was isolated. The sequence arerangement of this element, called 9D4, most likely represents the organization of an actively transposing member of the HeT-A family. Here we assess the degree of divergence among members of the HeT-A family and test a model of telomere length maintenance based on HeT-A transposition. The region containing the single open reading frame of this element appears to be more highly conserved than the non-coding regions. The HeT-A element has been implicated in theDrosophila telomere elongation process, because frequent transpositions to chromosome ends are sufficient to counter-balance nucleotide loss due to incomplete DNA replication. The proposed elongation model and the hypothetical mechanism of HeT-A transposition predict a predominant orientation of HeT-A elements with their oligo (A) tails facing proximally at chromosome ends, as well as the existence of irregular tandem arrays of HeT-A elements at chromosome ends resulting from transposition of new HeT-A elements onto chromosome ends with existing elements. Twenty-nine different HeT-A fragments were isolated from directional libraries that were enriched in terminal DNA fragments. Sequence analyses of these fragments and comparisons with the organization of the HeT-A element, 9D4, fit these two predictions and support the model ofDrosophila telomere elongation by transposition of HeT-A elements.     

Intragenomic Distribution and Stability of Transposable Elements in Euchromatin and Heterochromatin of Drosophila melanogaster: Elements with Inverted Repeats Bari 1, hobo, and pogo

The elements of the Bari 1, hobo, and pogo transposon families that are located in euchromatin, heterochromatin, and on the Y chromosome have been identified, and their stability has been assessed by Southern blot analysis. The fraction of heterochromatic elements appears to be distinctive of all transposon families tested, except for Bari 1. Evidence for instability of heterochromatic elements is described. The analysis of unstable elements in different Drosophila stocks suggests that the host genome contributes to the stability/instability of transposon families. Received: 21 August 1996 / Accepted: 24 March 1997     

Homolog pairing and sister chromatid cohesion in heterochromatin in <Emphasis Type="Italic">Drosophila</Emphasis> male meiosis I

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.     

Functional properties of the heterochromatic sequences inducing w m4 position-effect variegation in Drosophila melanogaster

In position-effect variegation euchromatic genes are brought into the vicinity of heterochromatic sequences as a result of chromosomal rearrangements. This results in the inactivation of these genes in a proportion of cells causing a variegated phenotype. Tartof et al. (1984) have shown that the flanking heterochromatin in the w ^m4 variegating rearrangement in Drosophila melanogaster is homologous to the Type I inserts found in some portions of the rDNA repeats. We have studied the functional properties of these sequences using 51 revertant chromosomes, several variant lines of w ^m4 , strong enhancer mutations of position-effect variegation and X heterochromatin deletions. Our results suggest an array of tandemly repeated sequences showing additive effects and probably subject to magnification and reduction in number. Since only 3 of the 51 revertants isolated do not show variegation if strong enhancer mutations are introduced, only a very short sequence must be essential for the induction of white gene inactivation in w ^m4 . This suggests that the heterochromatic junction itself is sufficient to initiate the variegation of an adjacent gene. Parental source as well as paternal effects on the activity of these sequences have been detected. Revertant chromosomes of w ^m4 can be found after P-directed mutagenesis in hybrid dysgenic crosses suggesting mobile genetic elements at the breakpoints of inversion w ^m4 . These results are discussed with respect to the structural basis of positioneffect variegation as well as the function of certain heterochromatic sequences.     

On the Roles of Heterochromatin and Euchromatin in Meiosis in Drosophila: Mapping Chromosomal Pairing Sites and Testing Candidate Mutations for Effects on X–Y Nondisjunction and Meiotic Drive in Male Meiosis

Mapping of pairing sites involved in meiotic homolog disjunction in Drosophilahas led to conflicting hypotheses about the nature of such sites and the role of heterochromatin in meiotic pairing. In the female-specific distributive system, pairing regions appear to be exclusively heterochromatic and map to broad regions encompassing many different sequences. In male meiosis, autosomal pairing sites appear to be distributed broadly within euchromatin but to be absent from heterochromatin, whereas the X-pairing site maps in the centric heterochromatin. The X site has been shown to coincide with the intergenic spacer (IGS) repeats within the rDNA arrays shared between the X and Y. It has not been clear whether the heterochromatic location of this pairing site has any significance. A novel assay for genic modifiers of X–Y chromosome pairing was developed based on the intermediate nondisjunction levels observed in males whose X chromosome lacks the native pairing site but contains two transgenic insertions of single rDNA genes. This assay was used to test several mutations in Su(var)(Suppressor of position effect variegation), PcG(Polycomb-Group) recombination defective, and repair-defective genes. No strong effects on disjunction were seen. However, the tests did uncover several mutations that suppress or enhance the meiotic drive (distorted X-Y recovery ratio) that accompanies X–Y pairing failure. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heterochromatic sequences in a Drosophila whole-genome shotgun assembly

Background

Most eukaryotic genomes include a substantial repeat-rich fraction termed heterochromatin, which is concentrated in centric and telomeric regions. The repetitive nature of heterochromatic sequence makes it difficult to assemble and analyze. To better understand the heterochromatic component of the Drosophila melanogaster genome, we characterized and annotated portions of a whole-genome shotgun sequence assembly.

Results

WGS3, an improved whole-genome shotgun assembly, includes 20.7 Mb of draft-quality sequence not represented in the Release 3 sequence spanning the euchromatin. We annotated this sequence using the methods employed in the re-annotation of the Release 3 euchromatic sequence. This analysis predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Bacterial artificial chromosome (BAC)-based fluorescence in situ hybridization analysis was used to correlate the genomic sequence with the cytogenetic map in order to refine the genomic definition of the centric heterochromatin; on the basis of our cytological definition, the annotated Release 3 euchromatic sequence extends into the centric heterochromatin on each chromosome arm.

Conclusions

Whole-genome shotgun assembly produced a reliable draft-quality sequence of a significant part of the Drosophila heterochromatin. Annotation of this sequence defined the intron-exon structures of 30 known protein-coding genes and 267 protein-coding gene models. The cytogenetic mapping suggests that an additional 150 predicted genes are located in heterochromatin at the base of the Release 3 euchromatic sequence. Our analysis suggests strategies for improving the sequence and annotation of the heterochromatic portions of the Drosophila and other complex genomes.     

Heterochromatic regions in different Drosophila melanogaster stocks contain similar arrangements of moderate repeats with inserted copia-like elements (MDG1)

Seven out of twenty 30–50 kb genome fragments with an MDG1 copia-like element cloned in cosmids were found to carry homologous sequences which belong to a new family of non-mobile heterochromatic moderate repeats (the HMR family). These repeats along with the MDG1 copies inserted in them are under-replicated in polytene chromosomes. Such repeats may also be located in the intercalary heterochromatin site 12E of the X chromosome. Chromosomal heterochromatic regions are enriched with one of the two main genomic variants of MDG1, MDG1^het, identifiable by EcoRI restriction. From Southern DNA blot analysis the number of MDG1^het copies and their sites within the heterochromatin are invariant in all the stocks examined, while there is not a single MDG1 site along the polytene chromosomes shared by all the stocks in question.     

A comparative study of the heterochromatin of Apodemus sylvaticus and Apodemus flavicollis

The two closely related species Apodemus sylvaticus and Apodemus flavicollis (Muridae) differ in the distribution of their heterochromatin. Two major repetitive sequences known to occur in both species were isolated from A. flavicollis after digestion of total nuclear DNA with the restriction enzymes HindIII and EcoRI respectively and characterized in both species by filter hybridisation and in situ hybridisation to metaphase chromosomes. The EcoRI clone detects a dispersed repetitive sequence family in the genome of both species. Southern blot hybridisation with the HindIII satellite DNA probe reveals major similarities and minor differences in the two species. In situ hybridisation with the HindIII probe labels all chromosomes of A. flavicollis exclusively in the centromeric heterochromatin, whereas in A. sylvaticus several autosomes are also labelled distally. The labelling patterns correspond to the distribution of heterochromatin in the two species. It is concluded that the additional distal heterochromatin of A. sylvaticus contains similar sequences to those of the centromeric heterochromatin of both species. The distal heterochromatin in A. sylvaticus most likely evolved by transposition and amplification of centromeric satellite DNA elements, after the separation of the two species.     

Simple Quantitative PCR Approach to Reveal Naturally Occurring and Mutation-Induced Repetitive Sequence Variation on the Drosophila Y Chromosome

Heterochromatin is a significant component of the human genome and the genomes of most model organisms. Although heterochromatin is thought to be largely non-coding, it is clear that it plays an important role in chromosome structure and gene regulation. Despite a growing awareness of its functional significance, the repetitive sequences underlying some heterochromatin remain relatively uncharacterized. We have developed a real-time quantitative PCR-based method for quantifying simple repetitive satellite sequences and have used this technique to characterize the heterochromatic Y chromosome of Drosophila melanogaster. In this report, we validate the approach, identify previously unknown satellite sequence copy number polymorphisms in Y chromosomes from different geographic sources, and show that a defect in heterochromatin formation can induce similar copy number polymorphisms in a laboratory strain. These findings provide a simple method to investigate the dynamic nature of repetitive sequences and characterize conditions which might give rise to long-lasting alterations in DNA sequence.     

Sex Differences in Drosophila melanogaster Heterochromatin Are Regulated by Non-Sex Specific Factors

The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. We adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulates autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also binds to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also makes autosomal heterochromatin in XX flies dependent on roX and enhances the effect of Top2 mutations, suggesting a combinatorial action. We postulate that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.     

The effect of distamycin A on heterochromatin condensation of Drosophila chromosomes

Larval neuroblasts of four species of Drosophila (melanogaster, hydei, virilis and funebris) were treated with distamycin A, a DNA ligand which induces distinct undercondensation in AT-rich heterochromatin. For each species the patterns of undercondensation were correlated with distribution of quinacrine-bright regions and of satellite DNAs. An overlapping of distamycin A-sensitive and quinacrine-bright heterochromatic regions was demonstrated for D. melanogaster and D. virilis, but not for D. hydei and D. funebris. Distamycin A undercondensation is thus a further criterion for resolving heterochromatin into different parts and enables identification of the steps of the condensation process within heterochromatic regions.     

New families of site-specific repetitive DNA sequences that comprise constitutive heterochromatin of the Syrian hamster (Mesocricetus auratus, Cricetinae, Rodentia)

We molecularly cloned new families of site-specific repetitive DNA sequences from BglII- and EcoRI-digested genomic DNA of the Syrian hamster (Mesocricetus auratus, Cricetrinae, Rodentia) and characterized them by chromosome in situ hybridization and filter hybridization. They were classified into six different types of repetitive DNA sequence families according to chromosomal distribution and genome organization. The hybridization patterns of the sequences were consistent with the distribution of C-positive bands and/or Hoechst-stained heterochromatin. The centromeric major satellite DNA and sex chromosome-specific and telomeric region-specific repetitive sequences were conserved in the same genus (Mesocricetus) but divergent in different genera. The chromosome-2-specific sequence was conserved in two genera, Mesocricetus and Cricetulus, and a low copy number of repetitive sequences on the heterochromatic chromosome arms were conserved in the subfamily Cricetinae but not in the subfamily Calomyscinae. By contrast, the other type of repetitive sequences on the heterochromatic chromosome arms, which had sequence similarities to a LINE sequence of rodents, was conserved through the three subfamilies, Cricetinae, Calomyscinae and Murinae. The nucleotide divergence of the repetitive sequences of heterochromatin was well correlated with the phylogenetic relationships of the Cricetinae species, and each sequence has been independently amplified and diverged in the same genome.     

Evolution of the large genome in Capsicum annuum occurred through accumulation of single-type long terminal repeat retrotransposons and their derivatives

Although plant genome sizes are extremely diverse, the mechanism underlying the expansion of huge genomes that did not experience whole‐genome duplication has not been elucidated. The pepper, Capsicum annuum, is an excellent model for studies of genome expansion due to its large genome size (2700 Mb) and the absence of whole genome duplication. As most of the pepper genome structure has been identified as constitutive heterochromatin, we investigated the evolution of this region in detail. Our findings show that the constitutive heterochromatin in pepper was actively expanded 20.0–7.5 million years ago through a massive accumulation of single‐type Ty3/Gypsy‐like elements that belong to the Del subgroup. Interestingly, derivatives of the Del elements, such as non‐autonomous long terminal repeat retrotransposons and long‐unit tandem repeats, played important roles in the expansion of constitutive heterochromatic regions. This expansion occurred not only in the existing heterochromatic regions but also into the euchromatic regions. Furthermore, our results revealed a repeat of unit length 18–24 kb. This repeat was found not only in the pepper genome but also in the other solanaceous species, such as potato and tomato. These results represent a characteristic mechanism for large genome evolution in plants.