The Role of piRNA-Mediated Epigenetic Silencing in the Population Dynamics of Transposable Elements in Drosophila melanogaster

The piwi-interacting RNAs (piRNA) are small RNAs that target selfish transposable elements (TEs) in many animal genomes. Until now, piRNAs’ role in TE population dynamics has only been discussed in the context of their suppression of TE transposition, which alone is not sufficient to account for the skewed frequency spectrum and stable containment of TEs. On the other hand, euchromatic TEs can be epigenetically silenced via piRNA-dependent heterochromatin formation and, similar to the widely known “Position-effect variegation”, heterochromatin induced by TEs can “spread” into nearby genes. We hypothesized that the piRNA-mediated spread of heterochromatin from TEs into adjacent genes has deleterious functional effects and leads to selection against individual TEs. Unlike previously identified deleterious effects of TEs due to the physical disruption of DNA, the functional effect we investigated here is mediated through the epigenetic influences of TEs. We found that the repressive chromatin mark, H3K9me, is elevated in sequences adjacent to euchromatic TEs at multiple developmental stages in Drosophila melanogaster. Furthermore, the heterochromatic states of genes depend not only on the number of and distance from adjacent TEs, but also on the likelihood that their nearest TEs are targeted by piRNAs. These variations in chromatin status probably have functional consequences, causing genes near TEs to have lower expression. Importantly, we found stronger selection against TEs that lead to higher H3K9me enrichment of adjacent genes, demonstrating the pervasive evolutionary consequences of TE-induced epigenetic silencing. Because of the intrinsic biological mechanism of piRNA amplification, spread of TE heterochromatin could result in the theoretically required synergistic deleterious effects of TE insertions for stable containment of TE copy number. The indirect deleterious impact of piRNA-mediated epigenetic silencing of TEs is a previously unexplored, yet important, element for the evolutionary dynamics of TEs.     

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.”     

Size matters: non-LTR retrotransposable elements and ectopic recombination in Drosophila

The Drosophila melanogaster genome contains approximately 100 distinct families of transposable elements (TEs). In the euchromatic part of the genome, each family is present in a small number of copies (5-150 copies), with individual copies of TEs often present at very low frequencies in populations. This pattern is likely to reflect a balance between the inflow of TEs by transposition and the removal of TEs by natural selection. The nature of natural selection acting against TEs remains controversial. We provide evidence that selection against chromosome abnormalities caused by ectopic recombination limits the spread of some TEs. We also demonstrate for the first time that some TE families in the Drosophila euchromatin appear to be only marginally affected by purifying selection and contain many copies at high population frequencies. We argue that TEs in these families attain high population frequencies and even reach fixation as a result of low family-wide transposition rates leading to low TE copy numbers and consequently reduced strength of selection acting on individual TE copies. Fixation of TEs in these families should provide an upward pressure on the size of intergenic sequences counterbalancing rapid DNA loss through small deletions. Copy-number-dependent selection on TE families caused by ectopic recombination may also promote diversity among TEs in the Drosophila genome.     

Conservation of gene order, structure and sequence between three closely linked genes in Drosophila melanogaster and Drosophila virilis

In Drosophila melanogaster, the apparently unrelated genes anon-66Da, RpL14, and anon-66Db (from telomere to centromere) are located on a 5547 bp genomic fragment on chromosome arm 3L at cytological position 66D8. The three genes are tightly linked, and flanked by two relatively large genes with unknown function. We have taken a comparative genomic approach to investigate the evolutionary history of the three genes. To this end we isolated a Drosophila virilis 7.3 kb genomic fragment which is homologous to a 5.5 kb genomic region of D. melanogaster. Both fragments map to Muller's element D, namely to section 66D in D. melanogaster and to section 32E in D. virilis, and harbor the genes anon-66Da, RpL14, and anon-66Db. We demonstrate that the three genes exhibit a high conservation of gene topography in general and in detail. While most introns and intergenic regions reveal sequence divergences, there are, however, a number of interspersed conserved sequence motifs. In particular, two introns of the RpL14 gene contain a short, highly conserved 60 nt long sequence located at corresponding positions. This sequence represents a novel Drosophila small nucleolar RNA, which is homologous to human U49. Whereas DNA flanking the three genes shows no significant interspecies homologies, the 3'-flanking region in D. virilis contains sequences from the transposable element Penelope. The Penelope family of transposable elements has been shown to promote chromosomal rearrangements in the D. virilis species group. The presence of Penelope sequences in the D. virilis 7.3 kb genomic fragment may be indicative for a transposon-induced event of transposition which did not yet scramble the order of the three genes but led to the breakdown of sequence identity of the flanking DNA.     

On the abundance and distribution of transposable elements in the genome of Drosophila melanogaster

The abundance and distribution of transposable elements (TEs) in a representative part of the euchromatic genome of Drosophila melanogaster were studied by analyzing the sizes and locations of TEs of all known families in the genomic sequences of chromosomes 2R, X, and 4. TEs contribute to up to 2% of the sequenced DNA, which corresponds roughly to the euchromatin of these chromosomes. This estimate is lower than that previously available from in situ data and suggests that TEs accumulate in the heterochromatin more intensively than was previously thought. We have also found that TEs are not distributed at random in the chromosomes and that their abundance is more strongly associated with local recombination rates, rather than with gene density. The results are compatible with the ectopic exchange model, which proposes that selection against deleterious effects of chromosomal rearrangements is a major force opposing element spread in the genome of this species. Selection against insertional mutations also influences the observed patterns, such as an absence of insertions in coding regions. The results of the analyses are discussed in the light of recent findings on the distribution of TEs in other species.     

The elevated GC content at exonic third sites is not evidence against neutralist models of isochore evolution

The human genome is divided into isochores, large stretches (>300 kb) of genomic DNA with more or less consistent GC content. Mutational/neutralist and selectionist models have been put forward to explain their existence. A major criticism of the mutational models is that they cannot account for the higher GC content at fourfold-redundant silent sites within exons (GC4) than in flanking introns (GCi). Indeed, it has been asserted that it is hard to envisage a mutational bias explanation, as it is difficult to see how repair enzymes might act differently in exons and their flanking introns. However, this rejection, we note, ignores the effects of transposable elements (TEs), which are a major component of introns and tend to cause them to have a GC content different from (usually lower than) that dictated by point mutational processes alone. As TEs tend not to insert at the extremities of introns, this model predicts that GC content at the extremities of introns should be more like that at GC4 than are the intronic interiors. This we show to be true. The model also correctly predicts that small introns should have a composition more like that at GC4 than large introns. We conclude that the logic of the previous rejection of neutralist models is unsafe.     

Constitutive heterochromatin and transposable elements in Drosophila melanogaster

Several families of transposable elements (TEs), most of them belonging to the retrotransposon catagory, are particularly enriched in Drosophila melanogaster constitutive heterochromatin. The enrichment of TE-homologous sequences into heterochromatin is not a peculiar feature of the Drosophila genome, but appears to be widespread among higher eukaryotes. The constitutive heterochromatin of D. melanogaster contains several genetically active domains; this raises the possibility that TE-homologous sequences inserted into functional heterochromatin compartments may be expressed. In this review, I present available data on the genetic and molecular organization of D. melanogaster constitutive heterochromatin and its relationship with transposable elements. The implications of these findings on the possible impact of heterochromatic TEs on the function and evolution of the host genome are also discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

A Yeast Artificial Chromosome Clone Map of the Drosophila Genome

We describe the mapping of 979 randomly selected large yeast artificial chromosome (YAC) clones of Drosophila DNA by in situ hybridization to polytene chromosomes. Eight hundred and fifty-five of the clones are euchromatic and have primary hybridization sites in the banded portions of the polytene chromosomes, whereas 124 are heterochromatic and label the chromocenter. The average euchromatic clone contains about 211 kb and, at its primary site, labels eight or nine contiguous polytene bands. Thus, the extent as well as chromosomal position of each clone has been determined. By direct band counts, we estimate our clones provide about 76% coverage of the euchromatin of the major autosomes, and 63% coverage of the X. When previously reported YAC mapping data are combined with ours, euchromatic coverage is extended to about 90% for the autosomes and 82% for the X. The distribution of gap sizes in our map and the coverage achieved are in good agreement with expectations based on the assumption of random coverage, indicating that euchromatic clones are essentially randomly distributed. However, certain gaps in coverage, including the entire fourth chromosome euchromatin, may be significant. Heterochromatic sequences are underrepresented among the YAC clones by two to three fold. This may result, at least in part, from underrepresentation of heterochromatic sequences in adult DNA (the source of most of the clones analyzed), or from clone instability.     

Give-and-take: interactions between DNA transposons and their host plant genomes

Recent genome sequencing efforts have revealed how extensively transposable elements (TEs) have contributed to the shaping of present day plant genomes. DNA transposons associate preferentially with the euchromatic or genic component of plant genomes and have had the opportunity to interact intimately with the genes of the plant host. These interactions have resulted in TEs acquiring host sequences, forming chimeric genes through exon shuffling, replacing regulatory sequences, mobilizing genes around the genome, and contributing genes to the host. The close interaction of transposons with genes has also led to the evolution of intricate cellular mechanisms for silencing transposon activity. Transposons have thus become important subjects of study in understanding epigenetic regulation and, in cases where transposons have amplified to high numbers, how to escape that regulation.     

白菜和甘蓝基因组转座子表达及其对基因调控的潜在影响

转座子或转座元件是大多数真核生物基因组的主要组成成分。甘蓝(Brassica oleracea)基因组比白菜(B. rapa)大主要是转座子的扩增差异造成的。然而, 这两个芸薹属近缘物种转座子表达水平以及对基因的调控和功能的影响目前还不清楚。文章对白菜和甘蓝叶、根、茎3个器官的转录组数据进行了初步分析。结果显示, 转座子的表达量很低, 转录组reads中有1%来自转座子的转录本; 转座子的表达存在器官差异, 且不同类别和家族的转座子表达量相差很大, 相同类别和同一家族的转座子在白菜和甘蓝基因组中的表达活性也不相同。进一步鉴定到转录读出的LTR反转座子, 其与下游基因距离小于2 kb的有41个, 小于100 bp的有9个, 这些LTR的转录读出很可能通过正义或反义的转录本激活或干扰下游基因的表达。同时, 具有转录读出的intact LTR比solo LTR具有更强的读出活性。通过深入分析转座子的插入位点发现, 白菜基因组中转座子插入基因内部的频率比甘蓝基因组中的高; 与反转座子相比, DNA转座子更偏向于插入或保留在基因的内含子当中。这些结果为认识转座子对其他蛋白编码基因的影响提供了基础。     

A Physical Map of the X Chromosome of Drosophila Melanogaster: Cosmid Contigs and Sequence Tagged Sites

A physical map of the euchromatic X chromosome of Drosophila melanogaster has been constructed by assembling contiguous arrays of cosmids that were selected by screening a library with DNA isolated from microamplified chromosomal divisions. This map, consisting of 893 cosmids, covers ~64% of the euchromatic part of the chromosome. In addition, 568 sequence tagged sites (STS), in aggregate representing 120 kb of sequenced DNA, were derived from selected cosmids. Most of these STSs, spaced at an average distance of ~35 kb along the euchromatic region of the chromosome, represent DNA tags that can be used as entry points to the fruitfly genome. Furthermore, 42 genes have been placed on the physical map, either through the hybridization of specific probes to the cosmids or through the fact that they were represented among the STSs. These provide a link between the physical and the genetic maps of D. melanogaster. Nine novel genes have been tentatively identified in Drosophila on the basis of matches between STS sequences and sequences from other species.     

The 1.688 repetitive DNA of Drosophila: concerted evolution at different genomic scales and association with genes

Concerted evolution leading to homogenization of tandemly repeated DNA arrays is widespread and important for genome evolution. We investigated the range and nature of the process at chromosomal and array levels using the 1.688 tandem repeats of Drosophila melanogaster where large arrays are present in the heterochromatin of chromosomes 2, 3, and X, and short arrays are found in the euchromatin of the same chromosomes. Analysis of 326 euchromatic and heterochromatic repeats from 52 arrays showed that the homogenization of 1.688 repeats occurred differentially for distinct genomic regions, from euchromatin to heterochromatin and from local arrays to chromosomes. We further found that most euchromatic arrays are either close to, or are within introns of, genes. The short size of euchromatic arrays (one to five repeats) could be selectively constrained by their role as gene regulators, a situation similar to the so-called "tuning knobs."     

Evolution of gene sequence in response to chromosomal location

Evolutionary forces acting on the repetitive DNA of heterochromatin are not constrained by the same considerations that apply to protein-coding genes. Consequently, such sequences are subject to rapid evolutionary change. By examining the Troponin C gene family of Drosophila melanogaster, which has euchromatic and heterochromatic members, we find that protein-coding genes also evolve in response to their chromosomal location. The heterochromatic members of the family show a reduced CG content and increased variation in DNA sequence. We show that the CG reduction applies broadly to the protein-coding sequences of genes located at the heterochromatin:euchromatin interface, with a very strong correlation between CG content and the distance from centric heterochromatin. We also observe a similar trend in the transition from telomeric heterochromatin to euchromatin. We propose that the methylation of DNA is one of the forces driving this sequence evolution.     

果蝇转座因子对基因组进化的影响

真核生物基因组织有很多可移动ＤＮＡ片段为称转座因子,果蝇是大量系统研究的最好实验材料之一,其基因组的１０％－１２％是由转座因子组成,在宿主中,ＴＥs也许改变基因表达模型,也许改变ＯＲＦs编码序列,也许对细胞功能产生影响,这此因子遗传的可动性也可能使它们适于建造载体产生转基因生物。因此,对ＴＥs进化的动态研究以及对宿主基因组进化影响的探索将有助于TEs作为载体的细胞工程研究。     

Common Mechanisms of Y Chromosome Evolution

Y chromosome evolution is characterized by the expansion of genetic inertness along the Y chromosome and changes in the chromosome structure, especially the tendency of becoming heterochromatic. It is generally assumed that the sex chromosome pair has developed from a pair of homologues. In an evolutionary process the proto-Y-chromosome, with a very short differential segment, develops in its final stage into a completely heterochromatic and to a great extends genetically eroded Y chromosome. The constraints evolving the Y chromosome have been the objects of speculation since the discovery of sex chromosomes. Several models have been suggested. We use the exceptional situation of the in Drosophila mirandato analyze the molecular process in progress involved in Y chromosome evolution. We suggest that the first steps in the switch from a euchromatic proto-Y-chromosome into a completely heterochromatic Y chromosome are driven by the accumulation of transposable elements, especially retrotransposons inserted along the evolving nonrecombining part of the Y chromosome. In this evolutionary process trapping and accumulation of retrotransposons on the proto-Y-chromosome should lead to conformational changes that are responsible for successive silencing of euchromatic genes, both intact or already mutated ones and eventually transform functionally euchromatic domains into genetically inert heterochromatin. Accumulation of further mutations, deletions, and duplications followed by the evolution and expansion of tandem repetitive sequence motifs of high copy number (satellite sequences) together with a few vital genes for male fertility will then represent the final state of the degenerated Y chromosome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spreading of Heterochromatin Is Limited to Specific Families of Maize Retrotransposons

Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800–1,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences.