Ciliate Gene Unscrambling with Fewer Templates

One of the theoretical models proposed for the mechanism of gene unscrambling in some species of ciliates is the template-guided recombination (TGR) system by Prescott, Ehrenfeucht and Rozenberg which has been generalized by Daley and McQuillan from a formal language theory perspective. In this paper, we propose a refinement of this model that generates regular languages using the iterated TGR system with a finite initial language and a finite set of templates, using fewer templates and a smaller alphabet compared to that of the Daley-McQuillan model. To achieve Turing completeness using only finite components, i.e., a finite initial language and a finite set of templates, we also propose an extension of the contextual template-guided recombination system (CTGR system) by Daley and McQuillan, by adding an extra control called permitting contexts on the usage of templates.Comment: In Proceedings DCFS 2010, arXiv:1008.127

Analysis of DIE5 and LIA5 reveals the importance of DNA repair in programmed DNA rearrangement of Tetrahymena thermophila

During its somatic nuclear differentiation, the single cell eukaryote Tetrahymena thermophila undergoes genome-wide programmed DNA rearrangement to eliminate transposon-like elements from its future soma. This process involves small RNA-directed heterochromatin formation followed by extensive nuclear reorganization to form subnuclear domains. While more has been known about small RNAs and heterochromatin, the mechanisms and players involved in the process of nuclear reorganization and the subsequent removal of transposon-like elements from the somatic genome are just starting to unravel. My thesis work centers on the study of two novel nuclear proteins Die5p: Chapter 2) and Lia5p: Chapter 3) and their roles in DNA rearrangement. These essential proteins function downstream of small RNA targeted heterochromatin establishment. While Lia5p is essential for nuclear reorganization to form distinct subnuclear structures, Die5p is a protein conserved across ciliate species and appears to be important for the integrity of the differentiating genome. Maintaining genome integrity during somatic nuclear differentiation has proven to be an active process. Similar to V(D)J recombination during mammalian B and T cell maturation, programmed DNA rearrangement in Tetrahymena induces global DNA damage that requires proper response and repair. Through the study of LIA5 and DIE5, we show that nuclear reorganization during Tetrahymena DNA rearrangement is intimately associated with the response to DNA damage. Furthermore, we implicate a chromodomain protein Pdd1 as a component of the DNA damage response system, thus providing evidence to support the link between heterochromatin and DNA repair during the reprogramming of Tetrahymena somatic genome

A highly condensed genome without heterochromatin : orchestration of gene expression and epigenomics in Paramecium tetraurelia

Epigenetic regulation in unicellular ciliates can be as complex as in metazoans and is well described regarding small RNA (sRNA) mediated effects. The ciliate Paramecium harbors several copies of sRNA-biogenesis related proteins involved in genome rearrangements resulting in chromatin alterations. The global chromatin organization thereby is poorly understood, and unusual characteristics of the somatic nucleus, like high polyploidy, high genome coding density, and absence of heterochromatin, ought to call for complex regulation to orchestrate gene expression. The present study characterized the nucleosomal organization required for gene regulation and proper Polymerase II activity. Histone marks reveal broad domains in gene bodies, whereas intergenic regions are nucleosome free. Low occupancy in silent genes suggests that gene inactivation does not involve nucleosome recruitment. Thus, Paramecium gene regulation counteracts the current understanding of chromatin biology. Apart from global nucleosome studies, two sRNA binding proteins (Ptiwis) classically associated with transposon silencing were investigated in the background of transgene-induced silencing. Surprisingly, both Ptiwis also load sRNAs from endogenous loci in vegetative growth, revealing a broad diversity of Ptiwi functions. Together, the studies enlighten epigenetic mechanisms that regulate gene expression in a condensed genome, with Ptiwis contributing to transcriptome and chromatin dynamics.Epigenetische Regulation kann in einzelligen Ciliaten so komplex sein wie in Vielzellern und wurde umfassend angesichts kleiner RNA (sRNA)-vermittelter Effekte untersucht. Der Ciliat Paramecium besitzt mehrere Kopien sRNA-Biogenese assoziierter Proteine, die an Genomprozessierungen und resultierenden Chromatinänderungen beteiligt sind. Die globale Organisation des Chromatins ist dabei kaum verstanden und obskure Eigenschaften des somatischen Kerns, wie hohe Polyploidie, Kodierungsdichte und Fehlen von Heterochromatin, sollten eine komplexe Regulation zur Steuerung der Genexpression erfordern. Die vorliegende Studie charakterisiert die Chromatinorganisation, die für die Genregulation und Polymerase II Aktivität notwendig ist. Histonmodifikationen zeigen breite Verteilungen in Genen, während intergenische Regionen Nukleosomen-frei sind. Ein Stilllegen von Genen scheint ohne die Rekrutierung von Nukleosomen zu erfolgen, womit die Genregulation in Paramecium dem aktuellen Verständnis der Chromatinbiologie widerspricht. Neben Nukleosomenstudien wurden zwei sRNA-bindende Proteine (Ptiwis), die klassisch mit Transposon-Silencing assoziiert sind, im Hintergrund des Transgeninduzierten Silencings untersucht. Überraschenderweise laden Ptiwis sRNAs von endogenen Loci im vegetativen Wachstum, was vielfältige Ptiwi-Funktionen offenbart. Die Studien zeigen epigenetische Mechanismen zur Genregulation in einem kompakten Genom, wobei Ptiwis zur Transkriptom- und Chromatindynamik beitragen

The Role of dsRNA in Nuclear Differentiation and Remodeling in the Ciliate, Tetrahymena thermophila

The ciliate, Tetrahymena thermophila, like a handful of other eukaryotes, engages in massive genome reorganization known collectively as chromatin diminution. Part of this process involves large-scale DNA excision known as DNA elimination. Recent data has shown DNA elimination to be dependent on RNA interference: RNAi). Using T. thermophila, I have sought to determine the role of non-coding RNA: ncRNA) in RNAi-dependent DNA elimination through studies of DNA sequences that are to be eliminated called internal eliminated sequences: IESs) and through a conjugation-specific Dicer protein and its putative tandem dsRNA-binding motif: DSRM) protein partners. Studies of the R IES revealed the requirement of IES DNA for production of long, bidirectional ncRNA early in conjugation. This ncRNA is essential for IES excision in zygotic nuclei later in conjugation. The conjugation-specific Dicer homologue, DCL1, was shown to be required for production of a species of sRNA called scnRNAs from the long, bidirectional ncRNA from IESs. Knockouts of DCL1 displayed a loss of these scnRNAs as well as an increase in the long, bidirectional ncRNA precursors. A deficiency in these scnRNAs was sufficient to block modification of chromatin associated with IESs and prevent their rearrangement later in conjugation. Failure of DNA elimination caused DCL1 knockout cells to arrest before completion of conjugation. Further studies of the tandem DSRM-containing proteins, DRB2 and DRB1, revealed that neither are solely partners for DCL1 or any other Dicer protein but play other important roles during conjugation. Zygotic expression of DRB2 was shown to be essential for DNA elimination and completion of conjugation. Interaction with the chromo-domain containing protein, Pdd1p, by Drb2p implicates ncRNA or sRNA in later stages of conjugation after scnRNA production. Knockouts of the tandem DSRM-containing DRB1 caused higher numbers of cells to abort conjugation and therefore produce fewer progeny. Localization of this protein to the crescent micronucleus during prophase of meiosis I links DRB1 to a probable role in ensuring proper recombination during meiosis for haploid gamete production. All these studies suggest that ncRNA has many roles in conjugation-specific processes including RNAi-directed DNA elimination

IMPACTS OF GENOME AND NUCLEAR ARCHITECTURE ON MOLECULAR EVOLUTION IN EUKARYOTES

The traditional view of genomes suggests that they are static entities changing slowly in sequence and structure through time (e.g. evolving over geological time-scales). This outdated view has been challenged as our understanding of the dynamic nature of genomes has increased. Changes in DNA content (i.e. polyploidy) are common to specific life-cycle stages in a variety of eukaryotes, as are changes in genome content itself. These dramatic genomic changes include chromosomal deletions (i.e. paternal chromosome deletion in insects; Goday and Esteban 2001; Ross, et al. 2010), developmentally regulated genome rearrangements (e.g. the V(D)J system in adaptive immunity in mammals; Schatz and Swanson 2011) and the specialization of a distinct somatic genome through epigenetically regulate DNA elimination during development (found in protists and some animals; Coyne, et al. 2012; Prescott 1994; Wang and Davis 2014; Wyngaard, et al. 2011). What likely allows genomes to be highly flexible is the separation of germline (i.e. ‘heritable’) and somatic (i.e. ‘functional’) material, even in the context of a single nucleus. Germline-soma distinctions have been best described (and most easily seen) in lineages of multicellular eukaryotes (e.g. plants, animals and fungi) due to obvious sexual structures. Germline genomes of these taxa are restricted to specialized cells (e.g. gametes; for example, pollen grains, eggs and spores) and remain undifferentiated (and often transcriptionally inactive), whereas the somatic cells (e.g. skin, leaves, hyphae) provide the basis for ensuring organismal survival to reproductive life-stages. Sequestered germline and somatic genomes are not restricted to these well-known multi-cellular lineages but are also well-described among ciliates (the focus of this dissertation) and some foraminifera. However, in these protists, germline and somatic genomes are not isolated into distinct cells and tissues but rather are isolated into distinct nuclei that share a common cytoplasm. Ciliates are a diverse and ancient clade of eukaryotes (~1-1.2 GYA old) and their study has led to the discovery of broad uniting eukaryotic features such as telomeres (Blackburn and Gall 1978) and self-splicing RNAs (Kruger, et al. 1982). As in the “macrobial” eukaryotes, the somatic genome (macronucleus; MAC) is transcriptionally active, transcribing all the genes necessary to maintain the cell, while the germline genome (micronucleus; MIC) remains transcriptionally inactive during the asexual portions of the life cycle. While the germline chromosomes in ciliates are physically similar to other ‘traditional’ eukaryotic chromosomes (e.g. being multi-Mbp with centromeres), the physical structure of the somatic chromosomes is highly variable. For example, in the model ciliate Tetrahymena thermophila, the somatic genome is composed of 225 unique chromosomes (most of them being ~200-400Kbp), with each at approximately 45 copies, whereas Oxytricha trifallax’s somatic genome is composed of ~16,000 gene-sized chromosomes (~2-3Kbp) with each chromosome at its own independent copy number (average copy number ~2,000). Despite dramatic differences in somatic genome architecture in ciliates, the development of a new somatic genome involves. For all ciliates studied to date, this metamorphosis from ‘traditional’ germline chromosomal architecture to the incredibly variable somatic genome architecture includes large-scale genome rearrangements and DNA elimination. This transformation involves the epigenetically-guided retention of somatically destined DNA from the background germline genome. While genomic rearrangements in most other eukaryotes are often fatal and are symptoms of well-known diseases (e.g. some cancers), this traditionally ‘catastrophic’ event is a fundamental part of ciliate life-cycles. Although studies of ciliate germline genomes have largely been restricted to only a few genera, there appear to be broad similarities in gene organization that may be phylogenetically conserved. Ciliate germline genome architecture has been categorized as either non-scrambled or scrambled, where non-scrambled architectures are often defined as possessing macronuclear destined sequences (MDSs; soma) that are separated by germline-limited DNA and remain in consecutive order (e.g. 1-2-3-4; Figure 3.1A and Figure 4.4A). Scrambled germline architectures are highly variable, but are broadly defined as MDSs being maintained in non-consecutive order (e.g. 1-3-4-2) and/or on opposing strands of DNA (Figure 3.1 B-D and Figure 4.4B). The germline genomes of Chilodonella uncinata (the main focus of this dissertation) possess a combination of scrambled and non-scrambled architectures. Before my thesis work, only those ciliates with gene-sized chromosomes have been demonstrated to have scrambled germline loci. Interestingly, previous work has implicated somatic genome architecture impacting the observable accelerated rates of protein evolution in ciliates, where the proteins of those ciliates possessing ‘gene-sized’ chromosomes experience the greatest evolutionary rates. These observations highlight the need for further work exploring the evolutionary impacts of different germline genome architectures, as the germline structure itself has direct impact on the development of the somatic genome. While this dissertation aims to elucidate some aspects of the evolution of germline-soma distinctions and the impact of genome and nuclear architecture (Chapters 2-4), there remain several fundamental questions that we can start addressing. For instance, in this work we observe that the most expanded gene families in Chilodonella uncinata are composed of genes that are disproportionately found at scrambled germline loci (Chapter 3). A major step future step will be to explore the functional implications of this increased paralog diversity through forward and reverse genetics techniques. Similarly, it will be incredibly valuable to better understand the nuclear architecture of the differing genomic contents of the three distinct nuclei present during ciliate development (i.e. the degrading parental MAC, the ‘new’ MIC, and the developing MAC). There may be observable compartmentalization that is exploitable or critical to the accurate rearrangement of the germline genome into a functional somatic genome. Finally, with the increasingly apparent utility of single-cell ‘omics techniques (which we use in Chapters 3 and 4), there is opportunity to probe into taxonomic groups where physical germline-soma separations exist, which will provide a far more expansive understanding of the evolutionary and functional impacts of harboring multiple distinct genomes inside of a single cell/organism

Chromosome Descrambling Order Analysis in ciliates

Ciliates are a type of unicellular eukaryotic organism that has two types of nuclei within each cell; one is called the macronucleus (MAC) and the other is known as the micronucleus (MIC). During mating, ciliates exchange their MIC, destroy their own MAC, and create a new MAC from the genetic material of their new MIC. The process of developing a new MAC from the exchanged new MIC is known as gene assembly in ciliates, and it consists of a massive amount of DNA excision from the micronucleus, and the rearrangement of the rest of the DNA sequences. During the gene assembly process, the DNA segments that get eliminated are known as internal eliminated segments (IESs), and the remaining DNA segments that are rearranged in an order that is correct for creating proteins, are called macronuclear destined segments (MDSs). A topic of interest is to predict the correct order to descramble a gene or chromosomal segment. A prediction can be made based on the principle of parsimony, whereby the smallest sequence of operations is likely close to the actual number of operations that occurred. Interestingly, the order of MDSs in the newly assembled 22,354 Oxytricha trifallax MIC chromosome fragments provides evidence that multiple parallel recombinations occur, where the structure of the chromosomes allows for interleaving between two sections of the developing macronuclear chromosome in a manner that can be captured with a common string operation called the shuffle operation (the shuffle operation on two strings results in a new string by weaving together the first two, while preserving the order within each string). Thus, we studied four similar systems involving applications of shuffle to see how the minimum number of operations needed to assemble differs between the types. Two algorithms for each of the first two systems have been implemented that are both shown to be optimal. And, for the third and fourth systems, four and two heuristic algorithms, respectively, have been implemented. The results from these algorithms revealed that, in most cases, the third system gives the minimum number of applications of shuffle to descramble, but whether the best implemented algorithm for the third system is optimal or not remains an open question. The best implemented algorithm for the third system showed that 96.63% of the scrambled micronuclear chromosome fragments of Oxytricha trifallax can be descrambled by only 1 or 2 applications of shuffle. This small number of steps lends theoretical evidence that some structural component is enforcing an alignment of segments in a shuffle-like fashion, and then parallel recombination is taking place to enable MDS rearrangement and IES elimination. Another problem of interest is to classify segments of the MIC into MDSs and IESs; this is the second topic of the thesis, and is a matter of determining the right "class label", i.e. MDS or IES, on each nucleotide. Thus, training data of labelled input sequences was used with hidden Markov models (HMMs), which is a well-known supervised machine learning classification algorithm. HMMs of first-, second-, third-, fourth-, and fifth-order have been implemented. The accuracy of the classification was verified through 10-fold cross validation. Results from this work show that an HMM is more likely to fail to accurately classify micronuclear chromosomes without having some additional knowledge

DNA elimination in the ciliate Tetrahymena

Während der sexuellen Fortpflanzung finden im neu entstehenden Makronukleus von Tetrahymena weitreichende Umgestaltungen des Genoms statt, bei denen mehr als 30 % des Genoms eliminiert werden. Dieser Prozess beinhaltet die spezifische Erkennung interner eliminierter Sequenzen durch einen Argonaut-scanRNA Komplex und die darauf folgende Heterochromatinbildung. Diese führt zur Akkumulierung der methylierten Lysine 9 und 27 des Histons H3 woraufhin das Chromodomänen Protein Pdd1p rekrutiert wird. Im Anschluss daran bilden sich sichtbar abgegrenzte Heterochromatinkörperchen von denen man davon ausgeht, dass in ihnen die DNS Eliminierung stattfindet. In meiner Doktorarbeit konnte ich zeigen, dass die piggyBac-ähnliche Transposase Tpb2p in diese Heterochromatinkörperchen rekrutiert wird und dass sie für die DNS Eliminierung unerlässlich ist. Zudem erkennt und schneidet das rekombinant in Bakterien hergestellte Tpb2p die Grenzen verschiedener interner eliminierter Sequenzen in vitro, wenn diese in der Mitte einer künstlich ausgearbeiteten und synthetisierten oligo DNS vorhanden sind. Daher führt Tpb2p wahrscheinlich auch den initialen Doppelstrangbruch während des Ausschneidens der internen eliminierten Sequenzen aus. Um einen Einblick zu erhalten wie die Präzission während des Ausschneidens erreicht wird, habe ich die Nuklease Aktivität des rekombinanten Tpb2p genauer untersucht. Dafür verwendete ich synthetisierte oligo DNS in der jede Position der linken Grenze des gut untersuchten R-Elements (Sequenz AGTGAT) individuell mutiert wurde. Ich konnte zeigen, dass die Positionen drei und vier wichtig für ein effektives Schneiden der Sequenz durch Tpb2p sind. Des Weiteren konnte ich veranschaulichen, dass diese Positionen auch für die präzise Eliminierung des R-Elements wichtig sind. Daher trägt die Präferenz von Tpb2p für bestimmte DNS Sequenzen sicherlich zur genauen Eliminierung der internen eliminierten Sequenzen bei. Andererseits lokalisiert Tpb2p in den Heterochromatinkörperchen und ist für deren Ausbildung essentiell. Daher ist es auch möglich, dass Tpb2p direkt mit dem Heterochromatin interagiert, und dass diese Interaktion die präzise Eliminierung ermöglicht. Tpb2p hat eine Endonuklease Domäne und eine Zink Finger Domäne. Ich habe herausgefunden, dass die Zink Finger Domäne, allerdings nicht die Endonuklease Domäne essentiell für die Ausbildung der Heterochromatinkörperchen ist. Außerdem konnte ich zeigen, dass die Zink Finger Domäne in vitro an Peptide des Histons H3 bindet, wenn diese an Lysin 9 oder 27 tri-methyliert sind. Dies lässt die Schlussfolgerung zu, dass diese Modifikationen, die spezifisch sind für interne eliminierte Sequenzen – zusammen mit der Sequenz Präferenz von Tpb2p – das präzise Ausschneiden der internen eliminierten Sequenzen vermittelt.During sexual reproduction the new developing macronucleus of Tetrahymena undergoes massive programmed DNA rearrangement, where over 30 % of the genome is eliminated. This process involves the sequence specific recognition of internal eliminated sequences by an Argonaute-scan RNA complex followed by heterochromatin formation including the accumulations of methylated histone H3 at lysine 9 and lysine 27 and the chromodomain protein Pdd1p. This heterochromatin formation eventually leads to the formation of distinct heterochromatin bodies in which DNA elimination is believed to occur. In my thesis I demonstrated that the piggyBac-like transposase Tpb2p is recruited to the heterochromatin bodies and that it is essential for DNA elimination. Furthermore, the recombinantly expressed Tpb2p from bacteria can recognize and cut boundaries of different internal eliminated sequences in vitro when they are placed in the middle of an artificially designed and synthesized oligo. Thus Tpb2p probably introduces the initial double strand break during DNA excision. To get insight into how the precision of excision is achieved, I first analyzed the nuclease activity of recombinant Tpb2p in more detail. Using synthesized oligo DNAs where every position of the reported left boundary of the well studied R element (sequence AGTGAT) was individually mutated, I found that the third and fourth positions in the boundary sequence are important for efficient cleavage by Tpb2p. Furthermore, an in vivo study confirmed that these two positions were crucial for the precise elimination of the R IES element. Therefore, some DNA sequence preference of Tpb2p clearly contributes to the precise elimination of internal eliminated sequences. On the other hand, because Tpb2p is a component of heterochromatin and is required for heterochromatin body formation, heterochromatin interaction with Tpb2p might also be involved in the precise DNA elimination. Tpb2p has an endonuclease domain and a zinc finger domain. I found that the zinc finger domain, but not the endonuclease domain, was essential for heterochromatin body formation. I could show in vitro that the zinc finger domain binds to histone H3 peptides that are tri-methylated at lysines 9 or 27 suggesting that these modifications specific to internal eliminated sequences are - together with the sequence preference of Tpb2p- specifying the precise IES excision

Hox3 duplication and divergence in the Lepidoptera

Using the Speckled Wood Butterfly Pararge aegeria as the model species, this thesis presents the possible evolutionary significance of a set of duplications found in the Hox cluster of the Lepidoptera, called the Special Homeobox genes. An annotation of this duplicated cluster across a wide number of Lepidoptera was performed in order to assess patterns of duplication and loss across the order. The sequences recovered revealed a large amount of variation associated with the duplicate genes, indicating these are evolving very rapidly in different lineages. Patterns of sequence variation were examined to ascertain whether the observed variation was maintained due to selection at three separate levels of divergence: within the Ditrysia, within the more recently diverged Heliconius genus, and at the intraspecific level by quantifying nucleotide polymorphism within Pararge aegeria. Selective pressures were found to be operating between paralogous and orthologous genes, suggesting these have evolved, in part, under positive selection. The potential function of the duplicates was examined by means of CRISPR/Cas9 geneome editing, but revealed inconclusive results. Genome editing, however, was shown to be largely applicable to P. aegeria, and resulted in consistent mutations associated with wing patterning genes. The potential significance of the duplications for Lepidopeteran biology are discussed, as well as future applications for genome editing techniques in P. aegeria