Throwing away DNA: programmed downsizing in somatic nuclei.

Programmed elimination of DNA during development yields somatic cell nuclei with dramatically different DNA sequence and content relative to germline nuclei, profoundly influencing genome architecture and stability. Whole-genome sequencing has significantly expanded the list of taxa known to exhibit this trait and has revealed the identity of excised genes and transposable elements (TEs) in certain taxa. Here, we compare the diverse mechanisms employed by ciliates, nematodes, copepods, and lamprey to downsize their genomes during development and propose tests of hypotheses about the evolution and/or maintenance of this trait. We explore possible functional roles that programmed DNA elimination (PDE) could play in genomic defense (especially against TEs), regulation of development, sex determination, co-option, and modulating nucleotypic effects, which together argue for a place in the mainstream investigation of genome evolution.

Natural genetic engineering: A programmed chromosome/DNA elimination.

Typically, all cells of a given organism have the same set of chromosomes. However, there are exceptions to this rule, and in many organisms, the somatic cells and germ cells, various types of somatic cells or organs, or females and males, have different genomes. One of the sources of such differences is chromosome/DNA elimination/chromatin diminution that is a naturally programmed phenomenon. We describe chromosome/DNA elimination in various organisms and present the current hypotheses on its origin, mechanisms, significance, and consequences.

Germline-restricted chromosome shows remarkable variation in size among closely related passerine species.

Passerine birds have a supernumerary chromosome in their germ cells called the germline-restricted chromosome (GRC). The GRC was first discovered more than two decades ago in zebra finch but recent studies have suggested that it is likely present in all passerines, the most species rich avian order, encompassing more than half of all modern bird species. Despite its wide taxonomic distribution, studies on this chromosome are still scarce and limited to a few species. Here, we cytogenetically analyzed the GRC in five closely related estrildid finch species of the genus Lonchura. We show that the GRC varies enormously in size, ranging from a tiny micro-chromosome to one of the largest macro-chromosomes in the cell, not only among recently diverged species but also within species and sometimes even between germ cells of a single individual. In Lonchura atricapilla, we also observed variation in GRC copy number among male germ cells of a single individual. Finally, our analysis of hybrids between two Lonchura species with noticeably different GRC size directly supported maternal inheritance of the GRC. Our results reveal the extraordinarily dynamic nature of the GRC, which might be caused by frequent gains and losses of sequences on this chromosome leading to substantial differences in genetic composition of the GRC between and even within species. Such differences might theoretically contribute to reproductive isolation between species and thus accelerate the speciation rate of passerine birds compared to other bird lineages.

Germline-restricted chromosome (GRC) is widespread among songbirds.

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages.

Programmed DNA elimination: silencing genes and repetitive sequences in somatic cells.

In a multicellular organism, the genomes of all cells are in general the same. Programmed DNA elimination is a notable exception to this genome constancy rule. DNA elimination removes genes and repetitive elements in the germline genome to form a reduced somatic genome in various organisms. The process of DNA elimination within an organism is highly accurate and reproducible; it typically occurs during early embryogenesis, coincident with germline-soma differentiation. DNA elimination provides a mechanism to silence selected genes and repeats in somatic cells. Recent studies in nematodes suggest that DNA elimination removes all chromosome ends, resolves sex chromosome fusions, and may also promote the birth of novel genes. Programmed DNA elimination processes are diverse among species, suggesting DNA elimination likely has evolved multiple times in different taxa. The growing list of organisms that undergo DNA elimination indicates that DNA elimination may be more widespread than previously appreciated. These various organisms will serve as complementary and comparative models to study the function, mechanism, and evolution of programmed DNA elimination in metazoans.

Tissue-Specific Transcriptome Analysis Reveals Candidate Transcripts Associated with the Process of Programmed B Chromosome Elimination in Aegilops speltoides.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from programmed elimination of chromosomes. Here, we present the first transcriptome analysis of programmed chromosome elimination using laser capture microdissection (LCM)-based isolation of the central meristematic region of Aegilops speltoides embryos where B chromosome (B) elimination occurs. The comparative RNA-seq analysis of meristematic cells of embryos with (Bplus) and without Bs (B0) allowed the identification of 14,578 transcript isoforms (35% out of 41,615 analyzed transcript isoforms) that are differentially expressed during the elimination of Bs. A total of 2908 annotated unigenes were found to be up-regulated in Bplus condition. These genes are either associated with the process of B chromosome elimination or with the presence of B chromosomes themselves. GO enrichment analysis categorized up-regulated transcript isoforms into 27 overrepresented terms related to the biological process, nine terms of the molecular function aspect and three terms of the cellular component category. A total of 2726 annotated unigenes were down-regulated in Bplus condition. Based on strict filtering criteria, 341 B-unique transcript isoforms could be identified in central meristematic cells, of which 70 were functionally annotated. Beside others, genes associated with chromosome segregation, kinetochore function and spindle checkpoint activity were retrieved as promising candidates involved in the process of B chromosome elimination.

Chromatin Diminution in Cyclops kolensis Lill. (Copepoda, Crustacea) as a Radical Way to Inactivate Redundant Genome in Somatic Cells.

Chromatin diminution (CD) is a phenomenon of programmed DNA elimination which takes place in early embryogenesis in some eukaryotes. The mechanism and biological role of CD remain largely unknown. During CD in the freshwater copepod Cyclops kolensis, the genome of cells of the somatic lineage is reorganized and reduced in size by more than 90% without affecting the genome of germline cells. Although the diploid chromosome number is unchanged, chromosome size is dramatically reduced by CD. The eliminated DNA consists primarily of repetitive sequences and localizes within granules during the elimination process. In this review, we provide an overview of CD in C. kolensis including both cytological and molecular studies.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

BACKGROUND: DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. RESULTS: We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. CONCLUSIONS: We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.

The macronuclear genomic landscape within Tetrahymena thermophila.

The extent of intraspecific genomic variation is key to understanding species evolutionary history, including recent adaptive shifts. Intraspecific genomic variation remains poorly explored in eukaryotic micro-organisms, especially in the nuclear dimorphic ciliates, despite their fundamental role as laboratory model systems and their ecological importance in many ecosystems. We sequenced the macronuclear genome of 22 laboratory strains of the oligohymenophoran Tetrahymena thermophila, a model species in both cellular biology and evolutionary ecology. We explored polymorphisms at the junctions of programmed eliminated sequences, and reveal their utility to barcode very closely related cells. As for other species of the genus Tetrahymena, we confirm micronuclear centromeres as gene diversification centres in T. thermophila, but also reveal a two-speed evolution in these regions. In the rest of the genome, we highlight recent diversification of genes coding for extracellular proteins and cell adhesion. We discuss all these findings in relation to this ciliate's ecology and cellular characteristics.

Units containing telomeric repeats are prevalent in subtelomeric regions of a Mesorhabditis isolate collected from the Republic of Korea.

BACKGROUND: Mesorhabditis is known for its somatic genome being only a small portion of the germline genome due to programmed DNA elimination. This phenotype may be associated with the maintenance of telomeres at the ends of fragmented somatic chromosomes. OBJECTIVE: To comprehensively investigate the telomeric regions of Mesorhabditis nematodes at the sequence level, we endeavored to collect a Mesorhabditis nematode in the Republic of Korea and acquire its highly contiguous genome sequences. METHODS: We isolated a Mesorhabditis nematode and assembled its 108-Mb draft genome using both 6.3 Gb (53 ×) of short-read and 3.0 Gb (25 × , N50 = 5.7 kb) of nanopore-based long-read sequencing data. Our genome assembly exhibits comparable quality to the public genome of Mesorhabditis belari in terms of contiguity and evolutionary conserved genes. RESULTS: Unexpectedly, our Mesorhabditis genome has many more interstitial telomeric sequences (ITSs), specifically subtelomeric ones, compared to the genomes of Caenorhabditis elegans and M. belari. Moreover, several subtelomeric sequences containing ITSs had 4-26 homologous sequences, implying they are highly repetitive. Based on this highly repetitive nature, we hypothesize that subtelomeric ITSs might have accumulated through the action of transposable elements containing ITSs. CONCLUSIONS: It still remains elusive whether these ITS-containing units are associated with programmed DNA elimination, but they may facilitate new telomere formation after DNA elimination. Our genomic resources for Mesorhabditis can aid in understanding how its distinct phenotypes have evolved.

End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well-characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the nematode Ascaris to study the timing of PDE breaks and examine the DSBs and their end processing. Using END-seq, we characterize the DSB ends and demonstrate that DNA breaks are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3' overhangs before the addition of telomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends in Ascaris may be due to the sequestration of the eliminated DNA into micronuclei, preventing their ends from telomere healing. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for nematode PDE.

Chromosome fusion and programmed DNA elimination shape karyotypes of nematodes.

An increasing number of metazoans undergo programmed DNA elimination (PDE), where a significant amount of DNA is selectively lost from the somatic genome during development. In some nematodes, PDE leads to the removal and remodeling of the ends of all germline chromosomes. In several species, PDE also generates internal breaks that lead to sequence loss and increased numbers of somatic chromosomes. The biological significance of these karyotype changes associated with PDE and the origin and evolution of nematode PDE remain largely unknown. Here, we assembled the single germline chromosome of the nematode Parascaris univalens and compared the karyotypes, chromosomal gene organization, and PDE features among other nematodes. We show that PDE in Parascaris converts an XX/XY sex-determination system in the germline into an XX/XO system in the somatic cells. Comparisons of Ascaris, Parascaris, and Baylisascaris ascarid chromosomes suggest that PDE existed in the ancestor of these nematodes, and their current distinct germline karyotypes were derived from fusion events of smaller ancestral chromosomes. The DNA breaks involved in PDE resolve these fused germline chromosomes into their pre-fusion karyotypes. These karyotype changes may lead to alterations in genome architecture and gene expression in the somatic cells. Cytological and genomic analyses further suggest that satellite DNA and the heterochromatic chromosome arms are dynamic and may play a role during meiosis. Overall, our results show that chromosome fusion and PDE have been harnessed in these ascarids to sculpt their karyotypes, altering the genome organization and serving specific functions in the germline and somatic cells.

A SUMO E3 ligase promotes long non-coding RNA transcription to regulate small RNA-directed DNA elimination.

Small RNAs target their complementary chromatin regions for gene silencing through nascent long non-coding RNAs (lncRNAs). In the ciliated protozoan Tetrahymena, the interaction between Piwi-associated small RNAs (scnRNAs) and the nascent lncRNA transcripts from the somatic genome has been proposed to induce target-directed small RNA degradation (TDSD), and scnRNAs not targeted for TDSD later target the germline-limited sequences for programmed DNA elimination. In this study, we show that the SUMO E3 ligase Ema2 is required for the accumulation of lncRNAs from the somatic genome and thus for TDSD and completing DNA elimination to make viable sexual progeny. Ema2 interacts with the SUMO E2 conjugating enzyme Ubc9 and enhances SUMOylation of the transcription regulator Spt6. We further show that Ema2 promotes the association of Spt6 and RNA polymerase II with chromatin. These results suggest that Ema2-directed SUMOylation actively promotes lncRNA transcription, which is a prerequisite for communication between the genome and small RNAs.

Germline-restricted chromosomes of the songbirds.

Germline-restricted chromosomes (GRCs) are present in the genomes of germline cells and absent from somatic cells. A GRC is found in all species of the songbirds (Passeri) and in none of the other bird orders studied to date. This indicates that GRC originated in the common ancestor of the songbirds. The germline-restricted chromosome is permanently absent from somatic cells of the songbird, while female germline cells usually contain two copies of GRC and male ones have one copy. In females, GRCs undergo synapsis and restricted recombination in their terminal regions during meiotic prophase. In males, it is almost always eliminated from spermatocytes. Thus, GRC is inherited almost exclusively through the maternal lineage. The germline-restricted chromosome is a necessary genomic element in the germline cells of songbirds. To date, the GRC genetic composition has been studied in four species only. Some GRC genes are actively expressed in female and male gonads, controlling the development of germline cells and synthesis of the proteins involved in the organization of meiotic chromosomes. Songbird species vary in GRC size and genetic composition. The GRC of each bird species consists of amplified and modified copies of genes from the basic genome of that species. The level of homology between GRCs of different species is relatively low, indicating a high rate of genetic evolution of this chromosome. Transmission through the maternal lineage and suppression of the recombination contribute significantly to the accelerated evolution of GRCs. One may suggest that the rapid coordinated evolution between the GRC genes and the genes of the basic genome in the songbirds might be responsible for the explosive speciation and adaptive radiation of this most species-rich and diverse infraorder of birds.

Programmed DNA elimination in Mesorhabditis nematodes.

Some species undergo programmed DNA elimination (PDE), whereby portions of the genome are systematically destroyed in somatic cells. PDE has emerged independently in several phyla, but its function is unknown. Although the mechanisms are partially solved in ciliates, PDE remains mysterious in metazoans because the study species were not yet amenable to functional approaches. We fortuitously discovered massive PDE in the free-living nematode genus Mesorhabditis, from the same family as C. elegans. As such, these species offer many experimental advantages to start elucidating the PDE mechanisms in an animal. Here, we used cytology to describe the dynamics of chromosome fragmentation and destruction in early embryos. Elimination occurs once in development, at the third embryonic cell division in the somatic blastomeres. Chromosomes are first fragmented during S phase. Next, some of the fragments fail to align on the mitotic spindle and remain outside the re-assembled nuclei after mitosis. These fragments are gradually lost after a few cell cycles. The retained fragments form new mini chromosomes, which are properly segregated in the subsequent cell divisions. With genomic approaches, we found that Mesorhabditis mainly eliminate repeated regions and also about a hundred genes. Importantly, none of the eliminated protein-coding genes are shared between closely related Mesorhabditis species. Our results strongly suggest PDE has not been selected for regulating genes with important biological functions in Mesorhabditis but rather mainly to irreversibly remove repeated sequences in the soma. We propose that PDE may target genes, provided their elimination in the soma is invisible to selection.

Chromosome fusion and programmed DNA elimination shape karyotypes of parasitic nematodes.

A growing list of metazoans undergo programmed DNA elimination (PDE), where a significant amount of DNA is selectively lost from the somatic genome during development. In some nematodes, PDE leads to the removal and remodeling of the ends of all germline chromosomes. In several species, PDE also generates internal breaks that lead to sequence loss and an increased number of somatic chromosomes. The biological significance of these karyotype changes associated with PDE and the origin and evolution of nematode PDE remain largely unknown. Here, we assembled the single germline chromosome of the horse parasite Parascaris univalens and compared the karyotypes, chromosomal gene organization, and PDE features among ascarid nematodes. We show that PDE in Parascaris converts an XX/XY sex-determination system in the germline into an XX/XO system in the somatic cells. Comparisons of Ascaris, Parascaris, and Baylisascaris ascarid chromosomes suggest that PDE existed in the ancestor of these parasites, and their current distinct germline karyotypes were derived from fusion events of smaller ancestral chromosomes. The DNA breaks involved in PDE resolve these fused germline chromosomes into their pre-fusion karyotypes, leading to alterations in genome architecture and gene expression in the somatic cells. Cytological and genomic analyses further suggest that satellite DNA and the heterochromatic chromosome arms play a dynamic role in the Parascaris germline chromosome during meiosis. Overall, our results show that chromosome fusion and PDE have been harnessed in these ascarids to sculpt their karyotypes, altering the genome organization and serving specific functions in the germline and somatic cells.

Nematode chromosomes.

The nematode Caenorhabditis elegans has shed light on many aspects of eukaryotic biology, including genetics, development, cell biology, and genomics. A major factor in the success of C. elegans as a model organism has been the availability, since the late 1990s, of an essentially gap-free and well-annotated nuclear genome sequence, divided among 6 chromosomes. In this review, we discuss the structure, function, and biology of C. elegans chromosomes and then provide a general perspective on chromosome biology in other diverse nematode species. We highlight malleable chromosome features including centromeres, telomeres, and repetitive elements, as well as the remarkable process of programmed DNA elimination (historically described as chromatin diminution) that induces loss of portions of the genome in somatic cells of a handful of nematode species. An exciting future prospect is that nematode species may enable experimental approaches to study chromosome features and to test models of chromosome evolution. In the long term, fundamental insights regarding how speciation is integrated with chromosome biology may be revealed.

Delete and survive: strategies of programmed genetic material elimination in eukaryotes.

Genome stability is a crucial feature of eukaryotic organisms because its alteration drastically affects the normal development and survival of cells and the organism as a whole. Nevertheless, some organisms can selectively eliminate part of their genomes from certain cell types during specific stages of ontogenesis. This review aims to describe the phenomenon of programmed DNA elimination, which includes chromatin diminution (together with programmed genome rearrangement or DNA rearrangements), B and sex chromosome elimination, paternal genome elimination, parasitically induced genome elimination, and genome elimination in animal and plant hybrids. During programmed DNA elimination, individual chromosomal fragments, whole chromosomes, and even entire parental genomes can be selectively removed. Programmed DNA elimination occurs independently in different organisms, ranging from ciliate protozoa to mammals. Depending on the sequences destined for exclusion, programmed DNA elimination may serve as a radical mechanism of dosage compensation and inactivation of unnecessary or dangerous genetic entities. In hybrids, genome elimination results from competition between parental genomes. Despite the different consequences of DNA elimination, all genetic material destined for elimination must be first recognised, epigenetically marked, separated, and then removed and degraded.

The nematode Oscheius tipulae as a genetic model for programmed DNA elimination.

Programmed DNA elimination (PDE) is a notable exception to the paradigm of genome integrity. In metazoa, PDE often occurs coincident with germline to somatic cell differentiation. During PDE, portions of genomic DNA are lost, resulting in reduced somatic genomes. Prior studies have described the sequences lost, as well as chromosome behavior, during metazoan PDE. However, a system for studying the mechanisms and consequences of PDE in metazoa is lacking. Here, we present a functional and genetic model for PDE in the free-living Rhabditidae nematode Oscheius tipulae, a family that also includes Caenorhabditis elegans. O. tipulae was recently suggested to eliminate DNA. Using staged embryos and DNA FISH, we showed that O. tipulae PDE occurs during embryogenesis at the 8-16 cell stages. We identified a conserved motif, named Sequence For Elimination (SFE), for all 12 break sites on the six chromosomes at the junctions of retained and eliminated DNA. SFE mutants exhibited a "fail-to-eliminate" phenotype only at the modified sites. END-seq revealed that breaks can occur at multiple positions within the SFE, with extensive end resection followed by telomere addition to both retained and eliminated ends. We identified many functional SFEs at the chromosome ends through END-seq in the wild-type embryos, genome sequencing of SFE mutants, and comparative genomics of 23 wild isolates. We suggest that these alternative SFEs provide flexibility in the sequences eliminated and a fail-safe mechanism for PDE. These studies establish O. tipulae as a new, attractive model for studying the mechanisms and consequences of PDE in a metazoan.

Evolutionary Perspectives on Germline-Restricted Chromosomes in Flies (Diptera).

In some eukaryotes, germline soma differentiation involves elimination of parts of the genome from somatic cells. The portions of the genome restricted to the germline often contain genes that play a role in development and function of the germline. Lineages with germline-restricted DNA are taxonomically diverse, and the size of the germline-restricted genome varies substantially. Unfortunately, few of these lineages have been studied in detail. As a result, we understand little about the general evolutionary forces that drive the origin and maintenance of germline-restricted DNA. One of the taxonomic groups where germline-restricted DNA has been poorly studied are the flies (Diptera). In three Dipteran families, Chironomidae, Cecidomyiidae, and Sciaridae, entire chromosomes are eliminated from somatic cells early in embryonic development. Germline-restricted chromosomes are thought to have evolved independently in the Dipteran families and their size, number, and transmission patterns vary between families. Although there is a wealth of cytological studies on these chromosomes in flies, almost no genomic studies have been undertaken. As a result, very little is known about how and why they evolved and what genes they encode. This review summarizes the literature on germline-restricted chromosomes in Diptera, discusses hypotheses for their origin and function, and compares germline-restricted DNA in Diptera to other eukaryotes. Finally, we discuss why Dipteran lineages represent a promising system for the study of germline-restricted chromosomes and propose future avenues of research on this topic.