Characterization of the macronuclear and micronuclear pheromone genes of Euplotes raikovi reveals the origin of the mating type genetic diversity.

Ciliates are a clade of diverse single-celled eukaryotic microorganisms that contain at least one somatic macronucleus (MAC) and germline micronucleus (MIC) within each cell/organism. Ciliates rely on complex genome rearrangements to generate somatic genomes from a zygotic nucleus. However, the development of somatic nuclei has only been documented for a few model ciliate genera, including Paramecium, Tetrahymena, and Oxytricha. Here, we study the MAC developmental process in the non-model ciliate, C. uncinata. We analyze both total DNA and the generation of gene-sized somatic chromosomes using a laser scanning confocal microscope to describe C. uncinata's nuclear life cycle. We show that DNA content changes dramatically during their life cycle and in a manner that differs from previous studies on model ciliates. Our study expands knowledge of genome dynamics in ciliates and among eukaryotes more broadly.

Ciliates are a model lineage for studies of genome architecture given their unusual genome structures. All ciliates have both somatic macronuclei (MAC) and germline micronuclei (MIC), both of which develop from a zygotic nucleus following sex (i.e., conjugation). Nuclear developmental stages are not as well explored among non-model ciliate genera, including Chilodonella uncinata (Class- Phyllopharyngea), the focus of our work. Here, we characterize nuclear architecture and genome dynamics in C. uncinata by combining DAPI (4′,6-diamidino-2-phenylindole) staining and fluorescence in situ hybridization (FISH) techniques with confocal microscopy. We developed a telomere probe for staining alongside DAPI, which allows for the identification of fragmented somatic chromosomes among the total DNA in the nuclei. We quantify both total DNA and telomere-bound signals to explore changes in DNA content and chromosome maturation across Chilodonella’s nuclear life cycle. Specifically, we find that MAC developmental stages in the ciliate C. uncinata are different than the data reported from other ciliate species. These data provide insights into nuclear dynamics during nuclear development and enrich our understanding of genome evolution in non-model ciliates.

ABSTRACTThe unicellular eukaryote Paramecium tetraurelia contains functionally distinct nuclei: germline micronuclei (MICs) and a somatic macronucleus (MAC). During sex, the MIC genome is reorganized into a new MAC genome and the old MAC is lost. Almost 45,000 unique internal eliminated sequences (IESs) distributed throughout the genome require precise excision to guarantee a functional new MAC genome. Here, we characterize a pair of paralogous PHD finger proteins involved in DNA elimination. DevPF1, the early-expressed paralog, is present in only some of the gametic and post-zygotic nuclei during meiosis. Both DevPF1 and DevPF2 localize in the new developing MACs, where IES excision occurs. Upon DevPF2 knockdown (KD), long IESs are preferentially retained and late-expressed small RNAs decrease; no length preference for retained IESs was observed in DevPF1-KD and development-specific small RNAs were abolished. The expression of at least two genes from the new MAC with roles in genome reorganization seems to be influenced by DevPF1- and DevPF2-KD. Thus, both PHD fingers are crucial for new MAC genome development, with distinct functions, potentially via regulation of non-coding and coding transcription in the MICs and new MACs.

Histone modification and nucleosome assembly play important roles in chromatin-related processes. Histone chaperones form different complexes and coordinate histone transportation and assembly. Various histone chaperone complexes have been identified in different organisms. The ciliate protozoa (ciliates) have various chromatin structures and different nuclear morphology. However, histone chaperone components and functions of different subunits remain unclear in ciliates. Tetrahymema thermophila contains a transcriptionally active macronucleus (MAC) and a transcriptionally inactive micronucleus (MIC) which exhibit multiple replication and various chromatin remodeling progresses during vegetative growth and sexual developmental stages. Here, we found histone chaperone RebL1 not only localized evenly in the transcriptionally active MAC but also dynamically changed in the MIC during vegetative growth and sexual developmental stages. REBL1 knockdown inhibited cellular proliferation. The macronuclear morphology became bigger in growing mutants. The abnormal macronuclear structure also occurred in the starvation stage. Furthermore, micronuclear meiosis was disturbed during sexual development, leading to a failure to generate new gametic nuclei. RebL1 potentially interacted with various factors involved in histone-modifying complexes and chromatin remodeling complexes in different developmental stages. REBL1 knockdown affected expression levels of the genes involved in chromatin organization and transcription. Taken together, RebL1 plays a vital role in maintaining macronuclear structure stability and gametogenesis in T. thermophila.

DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in E. coli or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. Tetrahymena undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6Tt localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6Tt only localized in MICs and newly developing MACs. MSH6Tt knockout led to aberrant nuclear division during vegetative growth. The MSH6TtKO mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. MSH6Tt knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6Tt interacted with Msh2Tt and MMR-independent factors. Downregulation of MSH2Tt expression affected the stability of Msh6Tt. In addition, MSH6Tt knockout led to the upregulated expression of several MSH6Tt homologs at different developmental stages. Msh6Tt is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in Tetrahymena.

Histones and DNA associate to form the nucleosomes of eukaryotic chromatin. Chromatin assembly factor 1 (CAF-1) complex and histone regulatory protein A (HIRA) complex mediate replication-couple (RC) and replication-independent (RI) nucleosome assembly, respectively. CHAF1B and HIRA share a similar domain but play different roles in nucleosome assembly by binding to the different interactors. At present, there is limited understanding for the similarities and differences in their respective functions. Tetrahymena thermophila contains transcriptionally active polyploid macronuclei (MAC) and transcriptionally silent diploid micronuclei (MIC). Here, the distribution patterns of Caf1b and Hir1 exhibited both similarities and distinctions. Both proteins localized to the MAC and MIC during growth, and to the MIC during conjugation. However, Hir1 exhibited additional signaling on parental MAC and new MAC during sexual reproduction and displayed a punctate signal on developing anlagen. Caf1b and Hir1 only co-localized in the MIC with Pcna1 during conjugation. Knockdown of CAF1B impeded cellular growth and arrested sexual reproductive development. Loss of HIR1 led to MIC chromosome defects and aborted sexual development. Co-interference of CAF1B and HIR1 led to a more severe phenotype. Moreover, CAF1B knockdown led to the up-regulation of HIR1 expression, while knockdown of HIR1 also led to an increase in CAF1B expression. Furthermore, Caf1b and Hir1 interacted with different interactors. These results showed that CAF-1 and Hir1 have independent and complementary functions for chromatin assembly in T. thermophila.

Multinucleate cells are found in many eukaryotes, but how multiple nuclei coordinate their functions is still poorly understood. In the cytoplasm of the ciliate Paramecium tetraurelia, two micronuclei (MIC) serving sexual reproduction coexist with a somatic macronucleus (MAC) dedicated to gene expression. During sexual processes, the MAC is progressively destroyed while still ensuring transcription, and new MACs develop from copies of the zygotic MIC. Several gene clusters are successively induced and switched off before vegetative growth resumes. Concomitantly, programmed genome rearrangement (PGR) removes transposons and their relics from the new MACs. Development of the new MACs is controlled by the old MAC, since the latter expresses genes involved in PGR, including the PGM gene encoding the essential PiggyMac endonuclease that cleaves the ends of eliminated sequences. Using RNA deep sequencing and transcriptome analysis, we show that impairing PGR upregulates key known PGR genes, together with ∼600 other genes possibly also involved in PGR. Among these genes, 42% are no longer induced when no new MACs are formed, including 180 genes that are co-expressed with PGM under all tested conditions. We propose that bi-directional crosstalk between the two coexisting generations of MACs links gene expression to the progression of MAC development.

Nuclear events during conjugation in the poorly studied model ciliate Paramecium jenningsi

Mismatch repair (MMR) is a conserved mechanism that is primarily responsible for the repair of DNA mismatches during DNA replication. Msh2 forms MutS heterodimer complexes that initiate the MMR in eukaryotes. The function of Msh2 is less clear under different chromatin structures. Tetrahymena thermophila contains a transcriptionally active macronucleus (MAC) and a transcriptionally silent micronucleus (MIC) in the same cytoplasm. Msh2 is localized in the MAC and MIC during vegetative growth. Msh2 is localized in the perinuclear region around the MIC and forms a spindle-like structure as the MIC divides. During the early conjugation stage, Msh2 is localized in the MIC and disappears from the parental MAC. Msh2 is localized in the new MAC and new MIC during the late conjugation stage. Msh2 also forms a spindle-like structure with a meiotic MIC and mitotic gametic nucleus. MSH2 knockdown inhibits the division of MAC and MIC during vegetative growth and affects cellular proliferation. MSH2 knockdown mutants are sensitive to cisplatin treatment. MSH2 knockdown also affects micronuclear meiosis and gametogenesis during sexual development. Furthermore, Msh2 interacts with MMR-dependent and MMR-independent factors. Therefore, Msh2 is necessary for macronuclear stability, as well as micronuclear mitosis and meiosis in Tetrahymena.

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists

Amitosis is widespread among eukaryotes, but the underlying mechanisms are poorly understood. The polyploid macronucleus (MAC) of unicellular ciliates divides by amitosis, making ciliates a potentially valuable model system to study this process. However, a method to accurately quantify the copy number of MAC chromosomes has not yet been established. Here, we used droplet digital PCR (ddPCR) to quantify the absolute copy number of the MAC chromosomes in Tetrahymena thermophila. We first confirmed that ddPCR is a sensitive and reproducible method to determine accurate chromosome copy numbers at the single-cell level. We then used ddPCR to determine the copy number of different MAC chromosomes by analyzing individual T.thermophila cells in the G1 and the amitotic (AM) phases. The average copy number of MAC chromosomes was 90.9 at G1 phase, approximately half the number at AM phase (189.8). The copy number of each MAC chromosome varied among individual cells in G1 phase and correlated with cell size, suggesting that amitosis accompanied by unequal cytokinesis causes copy number variability. Furthermore, the fact that MAC chromosome copy number is less variable among AM-phase cells suggests that the copy number is standardized by regulating DNA replication. We also demonstrated that copy numbers differ among different MAC chromosomes and that interchromosomal variations in copy number are consistent across individual cells. Our findings demonstrate that ddPCR can be used to model amitosis in T.thermophila and possibly in other ciliates.

Developmental DNA elimination in Paramecium tetraurelia occurs through a trans-nuclear comparison of the genomes of two distinct types of nuclei: the germline micronucleus (MIC) and the somatic macronucleus (MAC). During sexual reproduction, which starts with meiosis of the germline nuclei, MIC-limited sequences including Internal Eliminated Sequences (IESs) and transposons are eliminated from the developing MAC in a process guided by noncoding RNAs (scnRNAs and iesRNAs). However, our current understanding of this mechanism is still very limited. Therefore, studying both genetic and epigenetic aspects of these processes is a crucial step to understand this phenomenon in more detail. Here, we describe the involvement of homologs of classical meiotic proteins, Spo11, Msh4-1, and Msh5 in this phenomenon. Based on our analyses, we propose that proper functioning of Spo11, Msh4-1, and Msh5 during Paramecium sexual reproduction are necessary for genome reorganization and viable progeny. Also, we show that double-strand breaks (DSBs) in DNA induced during meiosis by Spo11 are crucial for proper IESs excision. In summary, our investigations show that early sexual reproduction processes may significantly influence later somatic genome integrity.

Histone modification and nucleosome assembly are mainly regulated by various histone-modifying enzymes and chaperones. The roles of histone-modification enzymes have been well analyzed, but the molecular mechanism of histone chaperones in histone modification and nucleosome assembly is incompletely understood. We previously found that the histone chaperone Nrp1 is localized in the micronucleus (MIC) and the macronucleus (MAC) and involved in the chromatin stability and nuclear division of Tetrahymena thermophila. In the present work, we found that truncated C-terminal mutant HA-Nrp1TrC abnormally localizes in the cytoplasm. The truncated-signal-peptide mutants HA-Nrp1TrNLS1 and HA-Nrp1TrNLS2 are localized in the MIC and MAC. Overexpression of Nrp1TrNLS1 inhibited cellular proliferation and disrupted micronuclear mitosis during the vegetative growth stage. During sexual development, Nrp1TrNLS1 overexpression led to abnormal bouquet structures and meiosis arrest. Furthermore, Histone H3 was not transported into the nucleus; instead, it formed an abnormal speckled cytoplastic distribution in the Nrp1TrNLS1 mutants. The acetylation level of H3K56 in the mutants also decreased, leading to significant changes in the transcription of the genome of the Nrp1TrNLS1 mutants. The histone chaperone Nrp1 regulates the H3 nuclear import and acetylation modification of H3K56 and affects chromatin stability and genome transcription in Tetrahymena.

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.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

Histone chaperones facilitate DNA replication and repair by promoting chromatin assembly, disassembly and histone exchange. Following histones synthesis and nucleosome assembly, the histones undergo posttranslational modification by different enzymes and are deposited onto chromatins by various histone chaperones. In Tetrahymena thermophila, histones from macronucleus (MAC) and micronucleus (MIC) have been comprehensively investigated, but the function of histone chaperones remains unclear. Histone chaperone Nrp1 in Tetrahymena contains four conserved tetratricopepeptide repeat (TPR) domains and one C-terminal nuclear localization signal. TPR2 is typically interrupted by a large acidic motif. Immunofluorescence staining showed that Nrp1 is located in the MAC and MICs, but disappeared in the apoptotic parental MAC and the degraded MICs during the conjugation stage. Nrp1 was also colocalized with α-tubulin around the spindle structure. NRP1 knockdown inhibited cellular proliferation and led to the loss of chromosome, abnormal macronuclear amitosis, and disorganized micronuclear mitosis during the vegetative growth stage. During sexual developmental stage, the gametic nuclei failed to be selected and abnormally degraded in NRP1 knockdown mutants. Affinity purification combined with mass spectrometry analysis indicated that Nrp1 is co-purified with core histones, heat shock proteins, histone chaperones, and DNA damage repair proteins. The physical direct interaction of Nrp1 and Asf1 was also confirmed by pull-down analysis in vitro. The results show that histone chaperone Nrp1 is involved in micronuclear mitosis and macronuclear amitosis in the vegetative growth stage and maintains gametic nuclei formation during the sexual developmental stage. Nrp1 is required for chromatin stability and nuclear division in Tetrahymena thermophila.

New contribution to epigenetic studies: Isolation of micronuclei with high purity and DNA integrity in the model ciliated protist, Tetrahymena thermophila

The germline-soma divide is a fundamental distinction in developmental biology, and different genes are expressed in germline and somatic cells throughout metazoan life cycles. Ciliates, a group of microbial eukaryotes, exhibit germline-somatic nuclear dimorphism within a single cell with two different genomes. The ciliate Oxytricha trifallax undergoes massive RNA-guided DNA elimination and genome rearrangement to produce a new somatic macronucleus (MAC) from a copy of the germline micronucleus (MIC). This process eliminates noncoding DNA sequences that interrupt genes and also deletes hundreds of germline-limited open reading frames (ORFs) that are transcribed during genome rearrangement. Here, we update the set of transcribed germline-limited ORFs (TGLOs) in O. trifallax. We show that TGLOs tend to be expressed during nuclear development and then are absent from the somatic MAC. We also demonstrate that exposure to synthetic RNA can reprogram TGLO retention in the somatic MAC and that TGLO retention leads to transcription outside the normal developmental program. These data suggest that TGLOs represent a group of developmentally regulated protein-coding sequences whose gene expression is terminated by DNA elimination.