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The EMBO Journal Nov 2022Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The...
Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.
Topics: Paramecium; DNA Transposable Elements; Chromatin Assembly and Disassembly; Nucleosomes; DNA, Protozoan
PubMed: 36221862
DOI: 10.15252/embj.2022111839 -
Molecular Biology and Evolution Apr 2022Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there...
Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there are two distinct nuclei in ciliates with separate germline and somatic cell functions. Here, we assembled a near-complete macronuclear genome of Fabrea salina, which belongs to one of the oldest clades of ciliates. Its extremely minimized genome (18.35 Mb) is the smallest among all free-living heterotrophic eukaryotes and exhibits typical streamlined genomic features, including high gene density, tiny introns, and shrinkage of gene paralogs. Gene families involved in hypersaline stress resistance, DNA replication proteins, and mitochondrial biogenesis are expanded, and the accumulation of phosphatidic acid may play an important role in resistance to high osmotic pressure. We further investigated the morphological and transcriptomic changes in the macronucleus during sexual reproduction and highlighted the potential contribution of macronuclear residuals to this process. We believe that the minimized genome generated in this study provides novel insights into the genome streamlining theory and will be an ideal model to study the evolution of eukaryotic heterotrophs.
Topics: Ciliophora; DNA, Protozoan; Genome, Protozoan; Introns; Macronucleus; Sequence Analysis, DNA
PubMed: 35325184
DOI: 10.1093/molbev/msac062 -
BioRxiv : the Preprint Server For... Jun 2024Ciliates are single-celled microbial eukaryotes that diverged from other eukaryotic lineages over a billion years ago. The extensive evolutionary timespan of ciliate has...
Ciliates are single-celled microbial eukaryotes that diverged from other eukaryotic lineages over a billion years ago. The extensive evolutionary timespan of ciliate has led to enormous genetic and phenotypic changes, contributing significantly to their high level of diversity. Recent analyses based on molecular data have revealed numerous cases of cryptic species complexes in different ciliate lineages, demonstrating the need for a robust approach to delimit species boundaries and elucidate phylogenetic relationships. Heterotrich ciliate species of the genus are abundant in freshwater and brackish environments and are commonly used as biological indicators for assessing water quality. However, some species are difficult to identify due to a lack of distinguishable morphological characteristics, and the existence of cryptic species in this genus remains largely unexplored. Previous phylogenetic studies have focused on only a few loci, namely the ribosomal RNA genes, alpha-tubulin, and mitochondrial CO1. In this study, we obtained single-cell transcriptome of 25 species populations (representing six morphospecies) sampled from South Korea and the USA, and used concatenation- and coalescent-based methods for species tree inference and delimitation. Phylogenomic analysis of 37 populations and 265 protein-coding genes provided a robustious insight into the evolutionary relationships among species and confirmed that species with moniliform and compact macronucleus each form a distinct monophyletic lineage. Furthermore, the multispecies coalescent (MSC) model suggests that there are at least nine cryptic species in the genus, three in , two in , and each. Overall, our fine sampling of closely related populations and wide scRNA-seq allowed us to demonstrate the hidden crypticity of species within the genus , and to resolve and provide much stronger support than hitherto to the phylogeny of this important ciliate genus.
PubMed: 38854132
DOI: 10.1101/2024.05.29.596006 -
Parasites & Vectors Mar 2023Chilodonella uncinata is an aerobic ciliate capable of switching between being free-living and parasitic on fish fins and gills, causing tissue damage and host...
BACKGROUND
Chilodonella uncinata is an aerobic ciliate capable of switching between being free-living and parasitic on fish fins and gills, causing tissue damage and host mortality. It is widely used as a model organism for genetic studies, but its mitochondrial metabolism has never been studied. Therefore, we aimed to describe the morphological features and metabolic characteristics of its mitochondria.
METHODS
Fluorescence staining and transmission electron microscopy (TEM) were used to observe the morphology of mitochondria. Single-cell transcriptome data of C. uncinata were annotated by the Clusters of Orthologous Genes (COG) database. Meanwhile, the metabolic pathways were constructed based on the transcriptomes. The phylogenetic analysis was also made based on the sequenced cytochrome c oxidase subunit 1 (COX1) gene.
RESULTS
Mitochondria were stained red using Mito-tracker Red staining and were stained slightly blue by DAPI dye. The cristae and double membrane structures of the mitochondria were observed by TEM. Besides, many lipid droplets were evenly distributed around the macronucleus. A total of 2594 unigenes were assigned to 23 functional classifications of COG. Mitochondrial metabolic pathways were depicted. The mitochondria contained enzymes for the complete tricarboxylic acid (TCA) cycle, fatty acid metabolism, amino acid metabolism, and cytochrome-based electron transport chain (ETC), but only partial enzymes involved in the iron-sulfur clusters (ISCs).
CONCLUSIONS
Our results showed that C. uncinata possess typical mitochondria. Stored lipid droplets inside mitochondria may be the energy storage of C. uncinata that helps its transmission from a free-living to a parasitic lifestyle. These findings also have improved our knowledge of the mitochondrial metabolism of C. uncinata and increased the volume of molecular data for future studies of this facultative parasite.
Topics: Animals; Alveolata; Parasites; Phylogeny; Ciliophora; Mitochondria
PubMed: 36882771
DOI: 10.1186/s13071-023-05695-3 -
Genome Research Mar 2020The somatic macronucleus (MAC) and germline micronucleus (MIC) of differ in chromosome numbers, sizes, functions, transcriptional activities, and cohesin complex...
The somatic macronucleus (MAC) and germline micronucleus (MIC) of differ in chromosome numbers, sizes, functions, transcriptional activities, and cohesin complex location. However, the higher-order chromatin organization in is still largely unknown. Here, we explored the higher-order chromatin organization in the two distinct nuclei of using the Hi-C and HiChIP methods. We found that the meiotic crescent MIC has a specific chromosome interaction pattern, with all the telomeres or centromeres on the five MIC chromosomes clustering together, respectively, which is also helpful to identify the midpoints of centromeres in the MIC. We revealed that the MAC chromosomes lack A/B compartments, topologically associating domains (TADs), and chromatin loops. The MIC chromosomes have TAD-like structures but not A/B compartments and chromatin loops. The boundaries of the TAD-like structures in the MIC are highly consistent with the chromatin breakage sequence (CBS) sites, suggesting that each TAD-like structure of the MIC chromosomes develops into one MAC chromosome during MAC development, which provides a mechanism of the formation of MAC chromosomes during conjugation. Overall, we demonstrated the distinct higher-order chromatin organization in the two nuclei of the and suggest that the higher-order chromatin structures may play important roles during the development of the MAC chromosomes.
Topics: Centromere; Chromatin; Chromosomes; Macronucleus; Meiosis; Micronucleus, Germline; Tetrahymena thermophila
PubMed: 32165395
DOI: 10.1101/gr.241687.118 -
Genome Research Apr 2022The unicellular ciliate contains a large vegetative macronucleus with several unusual characteristics, including an extremely high coding density and high polyploidy....
The unicellular ciliate contains a large vegetative macronucleus with several unusual characteristics, including an extremely high coding density and high polyploidy. As macronculear chromatin is devoid of heterochromatin, our study characterizes the functional epigenomic organization necessary for gene regulation and proper Pol II activity. Histone marks (H3K4me3, H3K9ac, H3K27me3) reveal no narrow peaks but broad domains along gene bodies, whereas intergenic regions are devoid of nucleosomes. Our data implicate H3K4me3 levels inside ORFs to be the main factor associated with gene expression, and H3K27me3 appears in association with H3K4me3 in plastic genes. Silent and lowly expressed genes show low nucleosome occupancy, suggesting that gene inactivation does not involve increased nucleosome occupancy and chromatin condensation. Because of a high occupancy of Pol II along highly expressed ORFs, transcriptional elongation appears to be quite different from that of other species. This is supported by missing heptameric repeats in the C-terminal domain of Pol II and a divergent elongation system. Our data imply that unoccupied DNA is the default state, whereas gene activation requires nucleosome recruitment together with broad domains of H3K4me3. In summary, gene activation and silencing in run counter to the current understanding of chromatin biology.
Topics: Chromatin; Histone Code; Histones; Nucleosomes; Paramecium; RNA Polymerase II
PubMed: 35264449
DOI: 10.1101/gr.276126.121 -
Marine Life Science & Technology May 2024Histone modification and nucleosome assembly play important roles in chromatin-related processes. Histone chaperones form different complexes and coordinate histone...
UNLABELLED
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. 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. 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. 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 .
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s42995-024-00219-z.
PubMed: 38827131
DOI: 10.1007/s42995-024-00219-z -
Microorganisms Jan 2023Nuclear dimorphism is a fundamental feature of ciliated protozoa, which have separate somatic and germline genomes in two distinct organelles within a single cell. The...
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 , 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.
PubMed: 36838311
DOI: 10.3390/microorganisms11020343 -
Molecular Biology of the Cell Feb 2023harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the "cell cycles" of the diploid micronucleus and polyploid...
harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the "cell cycles" of the diploid micronucleus and polyploid macronucleus are offset. Micronuclear S phase initiates just before cytokinesis and is completed in daughter cells before onset of macronuclear DNA replication. Mitotic micronuclear division occurs mid-cell cycle, while macronuclear amitosis is coupled to cell division. Here we report the first RNA-seq cell cycle analysis of a binucleated ciliated protozoan. RNA was isolated across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the 26,000+ predicted genes were shown to be cell cycle regulated. Proteins present in both nuclei exhibit a single mRNA peak that always precedes their macronuclear function. Nucleus-limited genes, including nucleoporins and importins, are expressed before their respective nucleus-specific role. Cyclin D and A/B gene family members exhibit different expression patterns that suggest nucleus-restricted roles. Periodically expressed genes cluster into seven cyclic patterns. Four clusters have known PANTHER gene ontology terms associated with G1/S and G2/M phase. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication, and cell division.
Topics: Tetrahymena thermophila; Cell Nucleus; Cell Cycle; Mitosis; Ciliophora; Gene Expression Profiling; Tetrahymena
PubMed: 36475712
DOI: 10.1091/mbc.E22-08-0326 -
Marine Life Science & Technology Aug 2022Ciliated protists are ideal material for studying the origin and evolution of sex, because of their nuclear dimorphism (containing both germline micronucleus and somatic...
Ciliated protists are ideal material for studying the origin and evolution of sex, because of their nuclear dimorphism (containing both germline micronucleus and somatic macronucleus in the same cytoplasm), special sexual processes (conjugation and autogamy), and high diversity of mating-type systems. However, the study of sexual process is limited to only a few species, due to the difficulties in inducing or observing conjugation. In the present study, we investigate the conjugation process in : (1) of the three prezygotic divisions, all micronuclei undergo the first two divisions (meiosis I, II), while a variable number of nuclei undergo the third division (mitosis); (2) the synkaryon divides three times after fertilization, giving rise to eight products that differentiate into four macronuclear anlagen and four micronuclei; (3) cells restore the vegetative stage after two successive cell fissions during which the macronuclear anlagen are distributed into daughter cells without division, while micronuclei divide mitotically; (4) the parental macronucleus begins to fragment following the first meiotic division and finally degenerates completely; (5) the entire process takes about 110 h, of which about 85 h are required for macronuclear development. In addition, we describe for the first time the process of genomic exclusion occurring between amicronucleate and micronucleate cells of , during which the micronucleate cell contributes a pronucleus to the amicronucleate cell, resulting in both exconjugants being homozygotes. These results provide new insights into the diversity of sexual processes and lay an important cytological basis for future in-depth studies of mating systems in ciliates.
PubMed: 37073165
DOI: 10.1007/s42995-022-00137-y