Laura F. Landweber

The ciliate Oxytricha undergoes massive genome rearrangements during development to produce a functional nucleus from an encrypted zygotic genome. PIWI-interacting small RNAs protect DNA regions against deletion, but how that protective mark is established has been a mystery. Recently our lab discovered MTA1, a methyltransferase that catalyzes DNA N6-adenine (6mA) methylation. Both MTA1 and the Oxytricha Piwi protein, Otiwi1, are required for development, and Otiwi1 mutation eliminates 6mA signal. To examine the role of 6mA, we analyzed its genome-wide distribution across development in wild-type and MTA1 mutant backcrossed cells. We find specific and abundant enrichment on retained sequences, suggesting a protective role for this epigenetic mark. Furthermore, programmed retention of a DNA region that is normally deleted leads to accumulation of new 6mA marks on the ectopically retained DNA sequence. Together, these results suggest that piRNA-guided 6mA DNA methylation leads to protection of DNA sequences against deletion during nuclear differentiation. Highlights: DNA N6-methyladenine accumulates on retained DNA regions during Oxytricha development. mta1 mutant backcrosses have disrupted DNA methylation and a developmental delay. DNA N6-adenine methylation during genome rearrangement requires the presence of Otiwi1. Programmed retention of a germline-limited region leads to developmental methylation.

During post-zygotic development, the ciliate Oxytricha trifallax undergoes massive programmed genome rearrangement that involves over 225 000 DNA cleavage and joining events. An Oxytricha family of Tc1/mariner transposons, known as telomere-bearing elements (TBEs), encodes a transposase that has been implicated in rearrangement, but its high copy number (>34 000 paralogs) has precluded genetic strategies to investigate its DNA recognition properties directly in Oxytricha. Here, we developed a heterologous strategy to assay TBE transposase expression and activity in Escherichia coli, revealing highly promiscuous DNA cleavage properties. Systematic ChIP-seq experiments allowed us to define the DNA binding specificities of multiple distinct transposase subfamilies, which exhibited a binding and cleavage preference for short, degenerate sequence motifs that resemble features present within the TBE transposon ends. The relaxed sequence preference is striking for autonomous transposases, which typically recognize their end sequences with strict specificity to avoid compromising host fitness. Finally, we developed a custom antibody to investigate TBE transposases in their native environment and found that they precisely localize to the developing nucleus exclusively during the rearrangement process. Collectively, this work establishes a robust heterologous workflow for the biochemical investigation of enzymes that have been repurposed for large-scale genome rearrangements.

During post-zygotic development, the ciliate Oxytricha trifallax undergoes massive programmed genome rearrangement that involves over 225,000 DNA cleavage and joining events. An Oxytricha family of Tc 1/mariner transposons, known as Telomere-Bearing Elements (TBEs), encodes a transposase that has been implicated in rearrangement, but its high copy number (>34,000 paralogs) has precluded genetic strategies to investigate its DNA recognition properties directly in Oxytricha . Here, we developed a heterologous strategy to assay TBE transposase expression and activity in E. coli , revealing highly promiscuous DNA cleavage properties. Systematic ChIP-seq experiments allowed us to define the DNA binding specificities of multiple distinct transposase subfamilies, which exhibited a binding and cleavage preference for short, degenerate sequence motifs that resemble features present within the TBE transposon ends. The relaxed sequence preference is striking for autonomous transposases, which typically recognize their end sequences with strict specificity to avoid compromising host fitness. Finally, we developed a custom antibody to investigate TBE transposases in their native environment and found that they precisely localize to the developing nucleus exclusively during the rearrangement process. Collectively, this work establishes a robust heterologous workflow for the biochemical investigation of enzymes that have been repurposed for large-scale genome rearrangements.

Genome organization recapitulates function, yet ciliates like Oxytricha trifallax possess highly-specialized germline genomes, which are largely transcriptionally silent. During post-zygotic development, Oxytricha ’s germline undergoes large-scale genome editing, rearranging precursor genome elements into a transcriptionally-active genome with thousands of gene-sized nanochromosomes. Transgenerationally-inherited RNAs, derived from the parental somatic genome, program the retention and reordering of germline fragments. Retained and eliminated DNA must be distinguished and processed separately, but the role of chromatin organization in this process is unknown. We developed tools for studying Oxytricha nuclei and apply them to map the 3D organization of precursor and developmental states using Hi-C. We find that the precursor conformation primes the germline for development, while a massive spatial reorganization during development differentiates retained from eliminated regions before DNA rearrangement. Further experiments suggest a role for RNA-DNA interactions and chromatin remodeling in this process, implying a critical role for 3D architecture in programmed genome rearrangement.

Genomes sometimes undergo large-scale rearrangements. Programmed genome rearrangements in ciliates offer an extreme example, making them a compelling model system to study DNA rearrangements. Currently, available methods for genome annotation are not adequate for highly scrambled genomes. We present a theoretical framework and software implementation for the systematic extraction and analysis of DNA rearrangement annotations from pairs of genome assemblies corresponding to precursor and product versions. The software makes no assumptions about the structure of the rearrangements, and permits the user to select parameters to suit the data. Compared to previous approaches, this work achieves more complete precursor-product mappings, allows for full transparency and reproducibility, and can be adapted to genomic data from different sources.

Nuclear dimorphism is a fundamental feature of ciliated protozoa, which have separate somatic and germline genomes in two distinct organelles within a single cell. The transcriptionally active somatic genome, contained within the physically larger macronucleus, is both structurally and functionally different from the silent germline genome housed in the smaller micronucleus. This difference in genome architecture is particularly exaggerated in Oxytricha trifallax, in which the somatic genome comprises tens of thousands of gene-sized nanochromosomes maintained at a high and variable ploidy, while the germline has a diploid set of megabase-scale chromosomes. To examine the compositional differences between the nuclear structures housing the genomes, we performed a proteomic survey of both types of nuclei and of macronuclear histones using quantitative mass spectrometry. We note distinct differences between the somatic and germline nuclei, with many functional proteins being highly enriched in one of the two nuclei. To validate our conclusions and the efficacy of nuclear separation, we used protein localization through a combination of transformations and immunofluorescence. We also note that the macronuclear histones strikingly display only activating marks, consistent with the conclusion that the macronucleus is the hub of transcription. These observations suggest that the compartmentalization of different genome features into separate structures has been accompanied by a similar specialization of nuclear components that maintain and facilitate the functions of the genomes specific to each nucleus.

Ciliates are microbial eukaryotes that undergo extensive programmed genome rearrangement, a natural genome editing process that converts long germline chromosomes into smaller gene-rich somatic chromosomes. Three well-studied ciliates include Oxytricha trifallax, Tetrahymena thermophila, and Paramecium tetraurelia, but only the Oxytricha lineage has a massively scrambled genome, whose assembly during development requires hundreds of thousands of precisely programmed DNA joining events, representing the most complex genome dynamics of any known organism. Here we study the emergence of such complex genomes by examining the origin and evolution of discontinuous and scrambled genes in the Oxytricha lineage. This study compares six genomes from three species, the germline and somatic genomes for Euplotes woodruffi, Tetmemena sp., and the model ciliate O. trifallax. We sequenced, assembled, and annotated the germline and somatic genomes of E. woodruffi, which provides an outgroup, and the germline genome of Tetmemena sp. We find that the germline genome of Tetmemena is as massively scrambled and interrupted as Oxytricha’s: 13.6% of its gene loci require programmed translocations and/or inversions, with some genes requiring hundreds of precise gene editing events during development. This study revealed that the earlier diverged spirotrich, E. woodruffi, also has a scrambled genome, but only roughly half as many loci (7.3%) are scrambled. Furthermore, its scrambled genes are less complex, together supporting the position of Euplotes as a possible evolutionary intermediate in this lineage, in the process of accumulating complex evolutionary genome rearrangements, all of which require extensive repair to assemble functional coding regions. Comparative analysis also reveals that scrambled loci are often associated with local duplications, supporting a gradual model for the origin of complex, scrambled genomes via many small events of DNA duplication and decay.

DNA rearrangements are important in various contexts, such as in vertebrate immunity, and cancer genome instability. The single-celled eukaryote Oxytricha trifallax undergoes massive and reproducible genome rearrangement during post-zygotic development, making it a compelling model organism to study DNA rearrangements. To date, computational tools for the extraction and analysis of rearrangement annotations lack transparency and rely on assumptions that may not hold for all analyzed data, leading to irreproducibility of results and loss of information through data filtering or misrepresentation. An implementation of a procedure for the annotation and analysis of DNA rearrangement as a web application is discussed and tested. The resulting annotations provide an improvement over previous annotations in the following manner. (a) SDRAP achieves more complete precursor-product mappings than previous software (b) the software allows for full transparency of all parameters used during the annotation and therefore facilitates reproducible results, and (c) this parameter transparency makes SDRAP suitable for comparison of genomic data from different sources, including cancer genomes. This work introduces a theoretical framework and software to systematically extract and analyze annotations from pairs of genome assemblies corresponding to precursor and product rearrangement data. The software makes no assumptions about the structure of the rearrangements, and permits the user to select parameters to suit the data.

Summary: Ciliates are microbial eukaryotes that undergo extensive programmed genome rearrangement that converts long germline chromosomes into smaller gene-rich somatic chromosomes. Three well-studied ciliates include Oxytricha trifallax , Tetrahymena thermophila and Paramecium tetraurelia , but only the Oxytricha lineage has a massively scrambled genome whose assembly requires hundreds of thousands of precise DNA joining events. Here we study the emergence of genome complexity by examining the origin and evolution of discontinuous and scrambled genes in the Oxytricha lineage. We sequenced, assembled and annotated the germline and somatic genomes of Euplotes woodruffi and the germline genome of Tetmemena sp. , and compared their genome rearrangement features to that of the model ciliate Oxytricha trifallax . The germline genome of Tetmemena is as massively scrambled and interrupted as Oxytricha ’s: 13.6% of its gene loci rearrange via translocations and/or inversions. This study revealed that the earlier-diverged spirotrich, E. woodruffi , also has a scrambled genome, but approximately half as many loci (7.3%) are scrambled, supporting its position as a possible evolutionary intermediate in this lineage, in the process of accumulating complex genome rearrangements. Scrambled loci are more often associated with local duplications, supporting a simple model for the origin of scrambled genes via DNA duplication and decay.

The germline genomes of ciliated protists are replete with “junk” DNA insertions that need to be removed for gene expression. Unlike introns, these are spliced as DNA. What is their source, and why are they so abundant? A new study in PLOS Biology supports a classic model of transposon origins.