With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

Genome-wide DNA rearrangements occur in many eukaryotes but are most exaggerated in ciliates, making them ideal model systems for epigenetic phenomena. During development of the somatic macronucleus, Oxytricha trifallax destroys 95% of its germ line, severely fragmenting its chromosomes, and then unscrambles hundreds of thousands of remaining fragments by permutation or inversion. Here we demonstrate that DNA or RNA templates can orchestrate these genome rearrangements in Oxytricha, supporting an epigenetic model for sequence-dependent comparison between germline and somatic genomes. A complete RNA cache of the maternal somatic genome may be available at a specific stage during development to provide a template for correct and precise DNA rearrangement. We show the existence of maternal RNA templates that could guide DNA assembly, and that disruption of specific RNA molecules disables rearrangement of the corresponding gene. Injection of artificial templates reprogrammes the DNA rearrangement pathway, suggesting that RNA molecules guide genome rearrangement.Parental RNA transcripts and microRNAs are critical for programming development in metazoa 1-4 , raising the possibility that altered RNA molecules can reprogramme patterning on a developmental or evolutionary timescale 5 . Despite the suggestion of template-directed events involving "an ancestral RNA-sequence cache" 6 there has been limited evidence for a direct role of RNA as a template of information across generations 7,8 . Information transfer from RNA to DNA usually involves polymerization 9 . Here we show that RNA molecules can also organize DNA rearrangements, expanding the epigenetic influence of RNA beyond gene expression and priming or directing DNA and RNA synthesis, editing, modification or repair 9-11 .O. trifallax is a unicellular eukaryote harbouring two kinds of nuclei: germline micronuclei and somatic macronuclei. Diploid micronuclei are transcriptionally inert during vegetative growth but they transmit the germline genome through subsequent generations. Effectively polyploid macronuclei provide all vegetative gene expression, but degrade after fertilization, when new micronuclei and macronuclei develop. DNA differentiation in ciliates such as Oxytricha (also called Sterkiella) involves massive chromosome fragmentation and deletion of transposons and internally eliminated sequences (IESs), accomplishing 95% genome Author Information TEBPα and TEBPβ macronucleus and micronucleus sequences have been submitted to GenBank under accession numbers EU047938-EU047941. Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to L. RNAi against putative templates disrupts rearrangementTo test the hypothesis that putative maternal RNA templates influence rearrangement, we induced RNA interference (RNAi) to target homologous RNA degradation. Oxytricha cells, before and during conjugation, were fed Escherichia coli producing double stranded RNA fragments of two m...

BackgroundSupernumerary chromosomes have been found in many organisms. In fungi, these “accessory” or “dispensable” chromosomes are present at different frequencies in populations and are usually characterized by higher repetitive DNA content and lower gene density when compared to the core chromosomes. In the reference strain of the wheat pathogen, Zymoseptoria tritici, eight discrete accessory chromosomes have been found. So far, no functional role has been assigned to these chromosomes; however, they have existed as separate entities in the karyotypes of Zymoseptoria species over evolutionary time. In this study, we addressed what—if anything—distinguishes the chromatin of accessory chromosomes from core chromosomes. We used chromatin immunoprecipitation combined with high-throughput sequencing (“ChIP-seq”) of DNA associated with the centromere-specific histone H3, CENP-A (CenH3), to identify centromeric DNA, and ChIP-seq with antibodies against dimethylated H3K4, trimethylated H3K9 and trimethylated H3K27 to determine the relative distribution and proportion of euchromatin, obligate and facultative heterochromatin, respectively.ResultsCentromeres of the eight accessory chromosomes have the same sequence composition and structure as centromeres of the 13 core chromosomes and they are of similar length. Unlike those of most other fungi, Z. tritici centromeres are not composed entirely of repetitive DNA; some centromeres contain only unique DNA sequences, and bona fide expressed genes are located in regions enriched with CenH3. By fluorescence microscopy, we showed that centromeres of Z. tritici do not cluster into a single chromocenter during interphase. We found dramatically higher enrichment of H3K9me3 and H3K27me3 on the accessory chromosomes, consistent with the twofold higher proportion of repetitive DNA and poorly transcribed genes. In contrast, no single histone modification tested here correlated with the distribution of centromeric nucleosomes.ConclusionsAll centromeres are similar in length and composed of a mixture of unique and repeat DNA, and most contain actively transcribed genes. Centromeres, subtelomeric regions or telomere repeat length cannot account for the differences in transfer fidelity between core and accessory chromosomes, but accessory chromosomes are greatly enriched in nucleosomes with H3K27 trimethylation. Genes on accessory chromosomes appear to be silenced by trimethylation of H3K9 and H3K27.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0033-5) contains supplementary material, which is available to authorized users.

Chromosome and genome stability are important for normal cell function as instability often correlates with disease and dysfunction of DNA repair mechanisms. Many organisms maintain supernumerary or accessory chromosomes that deviate from standard chromosomes. The pathogenic fungus Zymoseptoria tritici has as many as eight accessory chromosomes, which are highly unstable during meiosis and mitosis, transcriptionally repressed, show enrichment of repetitive elements, and enrichment with heterochromatic histone methylation marks, e.g., trimethylation of H3 lysine 9 or lysine 27 (H3K9me3, H3K27me3). To elucidate the role of heterochromatin on genome stability in Z . tritici , we deleted the genes encoding the methyltransferases responsible for H3K9me3 and H3K27me3, kmt1 and kmt6 , respectively, and generated a double mutant. We combined experimental evolution and genomic analyses to determine the impact of these deletions on chromosome and genome stability, both in vitro and in planta . We used whole genome sequencing, ChIP-seq, and RNA-seq to compare changes in genome and chromatin structure, and differences in gene expression between mutant and wildtype strains. Analyses of genome and ChIP-seq data in H3K9me3-deficient strains revealed dramatic chromatin reorganization, where H3K27me3 is mostly relocalized into regions that are enriched with H3K9me3 in wild type. Many genome rearrangements and formation of new chromosomes were found in the absence of H3K9me3, accompanied by activation of transposable elements. In stark contrast, loss of H3K27me3 actually increased the stability of accessory chromosomes under normal growth conditions in vitro , even without large scale changes in gene activity. We conclude that H3K9me3 is important for the maintenance of genome stability because it disallows H3K27me3 in regions considered constitutive heterochromatin. In this system, H3K27me3 reduces the overall stability of accessory chromosomes, generating a “metastable” state for these quasi-essential regions of the genome.

27Chromosome and genome stability are important for normal cell function as instability often 28 correlates with disease and dysfunction of DNA repair mechanisms. Many organisms maintain 29 supernumerary or accessory chromosomes that deviate from standard chromosomes. The 30 pathogenic fungus Zymoseptoria tritici has as many as eight accessory chromosomes, which are 31 highly unstable during meiosis and mitosis, transcriptionally repressed, show enrichment of 32 repetitive elements, and enrichment with heterochromatic histone methylation marks, e.g., 33trimethylation of H3 lysine 9 or lysine 27 (H3K9me3, H3K27me3). To elucidate the role of 34 heterochromatin on genome stability in Z. tritici, we deleted the genes encoding the 35 methyltransferases responsible for H3K9me3 and H3K27me3, kmt1 and kmt6, respectively, and 36 generated a double mutant. We combined experimental evolution and genomic analyses to 37 determine the impact of these deletions on chromosome and genome stability, both in vitro and 38 in planta. We used whole genome sequencing, ChIP-seq, and RNA-seq to compare changes in 39 genome and chromatin structure, and differences in gene expression between mutant and 40 wildtype strains. Analyses of genome and ChIP-seq data in H3K9me3-deficient strains revealed 41 dramatic chromatin reorganization, where H3K27me3 is mostly relocalized into regions that are 42 enriched with H3K9me3 in wild type. Many genome rearrangements and formation of new 43 chromosomes were found in the absence of H3K9me3, accompanied by activation of transposable 44 elements. In stark contrast, loss of H3K27me3 actually increased the stability of accessory 45 chromosomes under normal growth conditions in vitro, even without large scale changes in gene 46 activity. We conclude that H3K9me3 is important for the maintenance of genome stability 47 because it disallows H3K27me3 in these regions. In this system, H3K27me3 reduces the overall 48 stability of accessory chromosomes, generating a "metastable" state for these quasi-essential 49 regions of the genome. 50 3 Author Summary 51 Genome and chromosome stability are essential to maintain normal cell function and viability. 52However, differences in genome and chromosome structure are frequently found in organisms 53 that undergo rapid adaptation to changing environmental conditions, and in humans are often 54 found in cancer cells. We study genome instability in a fungal pathogen that exhibits a high degree 55 of genetic diversity. Regions that show extraordinary diversity in this pathogen are the 56 transposon-rich accessory chromosomes, which contain few genes that are of unknown benefit 57 to the organism but maintained in the population and thus considered "quasi essential". 58Accessory chromosomes in all fungi studied so far are enriched with markers for 59 heterochromatin, namely trimethylation of H3 lysine 9 and 27 (H3K9me3, H3K27me3). We show 60 that loss of these heterochromatin marks has strong but opposing effects on genome stability. 61While loss of the transposon-associate...

The Oxytricha trifallax mitochondrial genome contains the largest sequenced ciliate mitochondrial chromosome (∼70 kb) plus a ∼5-kb linear plasmid bearing mitochondrial telomeres. We identify two new ciliate split genes (rps3 and nad2) as well as four new mitochondrial genes (ribosomal small subunit protein genes: rps- 2, 7, 8, 10), previously undetected in ciliates due to their extreme divergence. The increased size of the Oxytricha mitochondrial genome relative to other ciliates is primarily a consequence of terminal expansions, rather than the retention of ancestral mitochondrial genes. Successive segmental duplications, visible in one of the two Oxytricha mitochondrial subterminal regions, appear to have contributed to the genome expansion. Consistent with pseudogene formation and decay, the subtermini possess shorter, more loosely packed open reading frames than the remainder of the genome. The mitochondrial plasmid shares a 251-bp region with 82% identity to the mitochondrial chromosome, suggesting that it most likely integrated into the chromosome at least once. This region on the chromosome is also close to the end of the most terminal member of a series of duplications, hinting at a possible association between the plasmid and the duplications. The presence of mitochondrial telomeres on the mitochondrial plasmid suggests that such plasmids may be a vehicle for lateral transfer of telomeric sequences between mitochondrial genomes. We conjecture that the extreme divergence observed in ciliate mitochondrial genomes may be due, in part, to repeated invasions by relatively error-prone DNA polymerase-bearing mobile elements.

The human fungal pathogen Cryptococcus deuterogattii is RNAi-deficient and lacks active transposons in its genome. C. deuterogattii has regional centromeres that contain only transposon relics. To investigate the impact of centromere loss on the C. deuterogattii genome, either centromere 9 or 10 was deleted. Deletion of either centromere resulted in neocentromere formation and interestingly, the genes covered by these neocentromeres maintained wild-type expression levels. In contrast to cen9∆ mutants, cen10∆ mutant strains exhibited growth defects and were aneuploid for chromosome 10. At an elevated growth temperature (37°C), the cen10∆ chromosome was found to have undergone fusion with another native chromosome in some isolates and this fusion restored wild-type growth. Following chromosomal fusion, the neocentromere was inactivated, and the native centromere of the fused chromosome served as the active centromere. The neocentromere formation and chromosomal fusion events observed in this study in C. deuterogattii may be similar to events that triggered genomic changes within the Cryptococcus/Kwoniella species complex and may contribute to speciation throughout the eukaryotic domain.

Background: Antagonistic co-evolution can drive rapid adaptation in pathogens and shape genome architecture. Comparative genome analyses of several fungal pathogens revealed highly variable genomes, for many species characterized by specific repeat-rich genome compartments with exceptionally high sequence variability. Dynamic genome structure may enable fast adaptation to host genetics. The wheat pathogen Zymoseptoria tritici with its highly variable genome, has emerged as a model organism to study genome evolution of plant pathogens. Here, we compared genomes of Z. tritici isolates and of sister species infecting wild grasses to address the evolution of genome composition and structure. Results: Using long-read technology, we sequenced and assembled genomes of Z. ardabiliae, Z. brevis, Z. pseudotritici and Z. passerinii, together with two isolates of Z. tritici. We report a high extent of genome collinearity among Zymoseptoria species and high conservation of genomic, transcriptomic and epigenomic signatures of compartmentalization. We identify high gene content variability both within and between species. In addition, such variability is mainly limited to the accessory chromosomes and accessory compartments. Despite strong host specificity and non-overlapping host-range between species, predicted effectors are mainly shared among Zymoseptoria species, yet exhibiting a high level of presence-absence polymorphism within Z. tritici. Using in planta transcriptomic data from Z. tritici, we suggest different roles for the shared orthologs and for the accessory genes during infection of their hosts. Conclusion: Despite previous reports of high genomic plasticity in Z. tritici, we describe here a high level of conservation in genomic, epigenomic and transcriptomic composition and structure across the genus Zymoseptoria. The compartmentalized genome allows the maintenance of a functional core genome co-occurring with a highly variable accessory genome.