Programmed DNA Elimination Research Articles

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

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. 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. We isolated a Mesorhabditis nematode and assembled its 108-Mb draft genome using both 6.3Gb (53 ×) of short-read and 3.0Gb (25 × , N50 = 5.7kb) 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. 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. 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.

Programmed DNA elimination

In most multicellular organisms, cells within an individual have essentially identical genomes. This principle underlies the ability to reprogram fibroblasts into induced pluripotent stem cells using defined transcription factors, clone a frog by transferring a nucleus from a tadpole somatic cell into an enucleated egg, and form totipotent callus cells by wounding plants. However, an exception to this one-body-one-genome principle exists in our blood cells. In developing lymphocytes in vertebrates, a process termed V(D)J recombination shuffles the antigen-binding regions of immunoglobulins and T-cell receptors.

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 human and pig parasitic nematode Ascaris to characterize the DSBs. Using END-seq, we demonstrate that DSBs 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 neotelomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends may be due to the sequestration of the eliminated DNA into micronuclei, preventing neotelomere formation at their ends. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for PDE. Overall, our data indicate that telomere healing of DSBs is specific to the break sites responsible 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 catalogue of chromosome counts for Phylum Nematoda.

Nematodes are important biological models in genetics and genomics, with research driven by basic biological as well as applied questions. The presence of holocentric chromosomes, clades with frequent polyploidy and the phenomenon of programmed DNA elimination make nematode karyotypic diversity of particular interest. Here we present a catalogue of published karyotypes of nematode species, rationalising and normalising descriptions from the previous 135 years. Karyotypes of 257 species are presented in taxonomic context. Nuclear chromosome counts range from 2 to 60. Tylenchina is identified as particularly diverse in karyotype. We highlight that Rhabditida and especially parasitic Rhabditina are well-represented, but there is a paucity of data from Enoplea, Dorylaimia, and from free-living marine groups in Chromadorea. The data have been uploaded to the Genomes on a Tree (GoaT) datasystem ( https://goat.genomehubs.org/) for integration with ongoing, large-scale genome sequencing efforts.

The hagfish genome and the evolution of vertebrates

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution1–3. Here we investigate the complex history, timing and functional role of genome-wide duplications4–7 and programmed DNA elimination8,9 in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1RV) that predates the early Cambrian cyclostome–gnathostome split, followed by a mid–late Cambrian allo-tetraploidization (2RJV) in gnathostomes and a prolonged Cambrian–Ordovician hexaploidization (2RCY) in cyclostomes. Subsequently, hagfishes underwent extensive genomic changes, with chromosomal fusions accompanied by the loss of genes that are essential for organ systems (for example, genes involved in the development of eyes and in the proliferation of osteoclasts); these changes account, in part, for the simplification of the hagfish body plan1,2. Finally, we characterize programmed DNA elimination in hagfish, identifying protein-coding genes and repetitive elements that are deleted from somatic cell lineages during early development. The elimination of these germline-specific genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline and pluripotency functions, paralleling findings in lampreys10,11. Reconstruction of the early genomic history of vertebrates provides a framework for further investigations of the evolution of cyclostomes and jawed vertebrates.

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.

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.

Rapid gene content turnover on the germline-restricted chromosome in songbirds

The germline-restricted chromosome (GRC) of songbirds represents a taxonomically widespread example of programmed DNA elimination. Despite its apparent indispensability, we still know very little about the GRC’s genetic composition, function, and evolutionary significance. Here we assemble the GRC in two closely related species, the common and thrush nightingale. In total we identify 192 genes across the two GRCs, with many of them present in multiple copies. Interestingly, the GRC appears to be under little selective pressure, with the genetic content differing dramatically between the two species and many GRC genes appearing to be pseudogenized fragments. Only one gene, cpeb1, has a complete coding region in all examined individuals of the two species and shows no copy number variation. The acquisition of this gene by the GRC corresponds with the earliest estimates of the GRC origin, making it a good candidate for the functional indispensability of the GRC in songbirds.

Abstract 1385: Oncogenic regulation of autophagy and DNA emission

Abstract Background: Cancer cells release higher levels of cell-free DNA (cfDNA) into the circulatory system than normal cells. However, the molecular mechanisms underlying the process of programmed DNA elimination remain poorly understood. Emerging evidence has indicated that oncogenes such as HRAS can be released through extracellular vesicles (EVs). Moreover, studies have shown an active mechanism of DNA emission through autophagy. Autophagy is an evolutionarily conserved process that degrades and recycles cytoplasmic materials such as organelles and proteins in the lysosomes. Interestingly, Microtubule-associated protein 1 light chain-3 (LC3B) is known to degrade nuclear lamina components in HRAS-induced cells, potentially producing a pool of cytoplasmic chromatin. Cytoplasmic chromatin is ultimately degraded in lysosomes or re-routed outside of the cells. The aim of our study was to evaluate the role of autophagy proteins in circulating tumor (ctDNA) and EV-associated DNA emission in cancer cell lines harboring oncogenes (RAS and BRAF) as this aspect of the process is relatively understudied. Methods: We have assessed protein and RNA expression of autophagy genes (LC3B, ATG5 and ATG7) in IEC-18 cells that have been transduced with the HRAS oncogene (RAS3 cells) and human colorectal cancer cell lines SW620 and HCT116 using RT-qPCR and western blot (WB). All conditions were compared to their respective control cells (IEC-18, CCD-841 CoN and CCD18-Co cells. To determine the role of LC3 in nuclear membrane degradation, we employed transmission electron microscopy (TEM). We have further validated the role of autophagy genes in EV-DNA emission by shRNA knockdown using shRNA against highly expressed autophagy genes, such as LC3B in RAS3 cells. EV-DNA was measured using droplet digital PCR (ddPCR). Results: To determine whether autophagy expels oncogenic DNA via EVs in KRAS-mutant CRC cells, we focused on the role of ATG5, LC3B and ATG7, as this aspect of the process is understudied. Our data shows higher LC3B and ATG7 expression in transformed RAS3 cells, compared to control cell lines (IEC18). Moreover, ATG5 is overexpressed in SW620 and HT29 colorectal cancer cells compared to control cell lines. LC3 is known to degrade the nuclear membrane in HRAS-activated cells. Indeed, we observed degradation of the nuclear membrane in RAS3 cells leading to the accumulation of cytosolic DNA shown by TEM. Furthermore, overexpression of LC3B in IEC18 cells using GFP-tagged LC3B resulted in increased EV emission. These findings suggest that the autophagy axis may contribute to oncogenic DNA emission via EVs. Lastly, shRNA knockdown of LC3 resulted in reduction of EV-DNA emission (ddPCR). Impact: EV biogenesis and autophagy pathways play pivotal and interconnected roles during DNA processing and extracellular emission. Therefore, understanding the detailed mechanisms is critical for biomarker discovery and potential therapeutic strategies targeting this process. Citation Format: Thupten Tsering, Kyle Dickinson, Laura Montermini, Janusz Rak, Julia Burnier. Oncogenic regulation of autophagy and DNA emission [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1385.

Programmed DNA elimination in the parasitic nematode Ascaris.

In most organisms, the whole genome is maintained throughout the life span. However, exceptions occur in some species where the genome is reduced during development through a process known as programmed DNA elimination (PDE). In the human and pig parasite Ascaris, PDE occurs during the 4 to 16 cell stages of embryogenesis, when germline chromosomes are fragmented and specific DNA sequences are reproducibly lost in all somatic cells. PDE was identified in Ascaris over 120 years ago, but little was known about its molecular details until recently. Genome sequencing revealed that approximately 1,000 germline-expressed genes are eliminated in Ascaris, suggesting PDE is a gene silencing mechanism. All germline chromosome ends are removed and remodeled during PDE. In addition, PDE increases the number of chromosomes in the somatic genome by splitting many germline chromosomes. Comparative genomics indicates that these germline chromosomes arose from fusion events. PDE separates these chromosomes at the fusion sites. These observations indicate that PDE plays a role in chromosome karyotype and evolution. Furthermore, comparative analysis of PDE in other parasitic and free-living nematodes illustrates conserved features of PDE, suggesting it has important biological significance. We summarize what is known about PDE in Ascaris and its relatives. We also discuss other potential functions, mechanisms, and the evolution of PDE in these parasites of humans and animals of veterinary importance.

Programmed DNA elimination: New metazoan models

Programmed DNA elimination (PDE) occurs in various metazoans. Parasitic nematodes have long been the major experimental model for PDE investigation. New studies have reported that some genetically tractable free-living nematodes also undergo PDE, paving the way for understanding the molecular mechanisms of PDE in metazoans.

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.

Developmental timing of programmed DNA elimination in Paramecium tetraurelia recapitulates germline transposon evolutionary dynamics.

With its nuclear dualism, the ciliate Paramecium constitutes a unique model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MICs) and a polyploid somatic macronucleus (MAC) that develops from one MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of approximately 45,000 TE-derived internal eliminated sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by noncoding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with high-throughput sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), whereas TEs are eliminated at a later stage. We show that DNA elimination proceeds independently of replication. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends, and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision by acquiring stronger sequence determinants and escaping epigenetic control.

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.

The transient Spt4-Spt5 complex as an upstream regulator of non-coding RNAs during development.

The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline and somatic nuclei, massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.

PIWI-Directed DNA Elimination for Tetrahymena Genetics.

Piwi-bound small RNAs induce programmed DNA elimination in the ciliated protozoan Tetrahymena. Using the phenomenon called codeletion, this process can be reprogrammed to induce ectopic DNA elimination at basically any given genomic location. Here, we describe the usage of codeletion for genetic studies in Tetrahymena and for investigations of the molecular mechanism of Piwi-directed programmed DNA elimination.

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.

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

ABSTRACTGenome 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.

New Perspectives on the Evolution of Within-Individual Genome Variation and Germline/Soma Distinction.

Genomes can vary significantly even within the same individual. The underlying mechanisms are manifold, ranging from somatic mutation and recombination, development-associated ploidy changes and genetic bottlenecks, over to programmed DNA elimination during germline/soma differentiation. In this perspective piece, we briefly review recent developments in the study of within-individual genome variation in eukaryotes and prokaryotes. We highlight a Society for Molecular Biology and Evolution 2020 virtual symposium entitled “Within-individual genome variation and germline/soma distinction” and the present Special Section of the same name in Genome Biology and Evolution, together fostering cross-taxon synergies in the field to identify and tackle key open questions in the understanding of within-individual genome variation.