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RNA-mediated nucleosome depletion is required for elimination of transposon-derived DNA

ABSTRACT: To study the role of chromatin remodeler during programmed genome reorganization in Paramecium tetraurelia. Paramecium ISWI1 was tagged with 3 FLAG and HA at its C-terminal. The recombinant plasmid was transformed into Paramecium and used for co-immunoprecipitation and Mass spectrometry studies to identify novel interacting partners of Paramecium ISWI1 protein that modulates the elimination of transposable elements.

INSTRUMENT(S):

ORGANISM(S): Paramecium Tetraurelia

TISSUE(S): Eukaryotic Cell, Cell Culture

SUBMITTER: Manfred Heller

LAB HEAD: Mariusz Nowacki

PROVIDER: PXD027206 | Pride | 2023-03-11

REPOSITORIES: Pride

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			Action	DRS
	20170424_ISWI1_AS_i01.raw	Raw
	20170424_ISWI1_AS_i02.raw	Raw
	20170424_ISWI1_AS_i03.raw	Raw
	20170424_ISWI2_AS_i01.raw	Raw
	20170424_ISWI2_AS_i02.raw	Raw

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Publications

Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium.

Singh Aditi A Maurer-Alcalá Xyrus X XX Solberg Therese T Häußermann Lilia L Gisler Silvan S Ignarski Michael M Swart Estienne C EC Nowacki Mariusz M

The EMBO journal 20221011 22

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 germl ...[more]

PMID: 36221862

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The Paramecium Ezl1 protein mediates dual methylation of H3 lysines 9 and 27

Project description:The goal of the present project was to determine which Paramecium histone H3 residues are methylated in an Ezl1-dependent manner.

2019-06-26 | PXD012170 | Pride

Paramecium tetraurelia autogamy series 4

Project description:In the ciliate Paramecium tetraurelia, autogamy is a self-fertilization process, during which the zygotic nucleus results from the fusion of two identical gametic nuclei. This phenomenon occurs in response to starvation. It starts with meiosis of the germline nuclei (micronuclei or MIC) and fragmentation of the parental somatic nucleus (macronucleus or MAC). This is followed by mitotic division of one haploid nucleus issued from meiosis to yield two identical gametic nuclei, then karyogamy takes place, followed by mitosis of zygotic nucleus and differentiation of new MICs and MACs from the resulting copies of the zygotic nucleus. Within the developing new MACs, developmentally programmed DNA amplification and extensive genome rearrangements (precise excision of short non coding Internal Eliminated Sequences and chromosome fragmentation associated with the imprecise elimination of repetitive DNA) give rise to the highly polyploid somatic genome. To gain further insight into the complex regulation of these successive steps, we used whole genome microarrays to study the different gene networks that become activated throughout autogamy. Total RNA was extracted before the begining of autogamy (reference sample) and at 3 different time points during the process of autogamy.

2010-07-07 | E-GEOD-17997 | biostudies-arrayexpress

Paramecium tetraurelia autogamy series 1

Project description:In the ciliate Paramecium tetraurelia, autogamy is a self-fertilization process, during which the zygotic nucleus results from the fusion of two identical gametic nuclei. This phenomenon occurs in response to starvation. It starts with meiosis of the germline nuclei (micronuclei or MIC) and fragmentation of the parental somatic nucleus (macronucleus or MAC). This is followed by mitotic division of one haploid nucleus issued from meiosis to yield two identical gametic nuclei, then karyogamy takes place, followed by mitosis of zygotic nucleus and differentiation of new MICs and MACs from the resulting copies of the zygotic nucleus. Within the developing new MACs, developmentally programmed DNA amplification and extensive genome rearrangements (precise excision of short non coding Internal Eliminated Sequences and chromosome fragmentation associated with the imprecise elimination of repetitive DNA) give rise to the highly polyploid somatic genome. To gain further insight into the complex regulation of these successive steps, we used whole genome microarrays to study the different gene networks that become activated throughout autogamy. Total RNA was extracted before the begining of autogamy (reference sample) and at 5 different time points during the process of autogamy. There are two biological replicates for each point.

2010-07-07 | E-GEOD-17996 | biostudies-arrayexpress

Paramecium tetraurelia autogamy series 3

Project description:In the ciliate Paramecium tetraurelia, autogamy is a self-fertilization process, during which the zygotic nucleus results from the fusion of two identical gametic nuclei. This phenomenon occurs in response to starvation. It starts with meiosis of the germline nuclei (micronuclei or MIC) and fragmentation of the parental somatic nucleus (macronucleus or MAC). This is followed by mitotic division of one haploid nucleus issued from meiosis to yield two identical gametic nuclei, then karyogamy takes place, followed by mitosis of zygotic nucleus and differentiation of new MICs and MACs from the resulting copies of the zygotic nucleus. Within the developing new MACs, developmentally programmed DNA amplification and extensive genome rearrangements (precise excision of short non coding Internal Eliminated Sequences and chromosome fragmentation associated with the imprecise elimination of repetitive DNA) give rise to the highly polyploid somatic genome. To gain further insight into the complex regulation of these successive steps, we used whole genome microarrays to study the different gene networks that become activated throughout autogamy. Total RNA was extracted before the begining of autogamy (c3_vegetative cells : reference sample) and at 5 different time points during the process of autogamy.

2010-07-07 | E-GEOD-17998 | biostudies-arrayexpress

Transcriptome analysis upon TFIIS4 silencing during autogamy time course

Project description:At each sexual cycle, during development of the somatic macronucleus (MAC) from the germline micronucleus (MIC), the genome of the ciliate Paramecium tetraurelia is massively rearranged through the reproducible elimination of germline-specific sequences. It has been reported previously that targeting of these sequences is mediated by non-protein-coding RNAs including different classes of small RNAs and longer non-coding transcripts. Using RNA interference, we showed that TFIIS4 gene encoding development-specific TFIIS elongation factor is essential for the formation of a functional somatic genome. We demonstrated that genome rearrangements taking place during MAC development were inhibited in TFIIS4-depleted cells, which led to high lethality in the sexual progeny. The role of TFIIS4 elongation factor in coding transcription at the genome-wide level was studied by performing a microarray hybridization experiment using RNA samples extracted during vegetative growth and at five time points during the progression of the sexual cycle (autogamy). Total RNA samples were extracted during vegetative growth and at five different time points of autogamy from mass cultures fed with bacteria producing dsRNA to induce TFIIS4 or ICL7a silencing. TFIIS4 gene encodes TFIIS elongation factor and is strongly induced during autogamy, a self-fertilization process. RNAi against TFIIS4 leads to strong lethality in post-autogamous progeny. ICL7a is a non-essential gene; the loss of function of this gene by with dsRNA feeding results in mutant phenotypes: absence of intraciliary lattice and defect in calcium-induced cell contractility.

2015-06-12 | E-GEOD-64682 | biostudies-arrayexpress

A H3K27me3 reader complex couples H3K27me3 accumulation to nascent transcription of transposable elements in Paramecium

Project description:BackgroundThe ability to deposit histone H3K27-trimethyl (me3) marks is essential for transcriptional repression by Polycomb Repressive Complex 2 (PRC2). This is largely attributed to Polycomb repressive complex 1 (PRC1), whose recruitment is H3K27me3-dependent. Yet it is unclear how H3K27me3 contributes to transcription regulation independently of PRC1.ResultsTo address this question, we identified H3K27me3-binding proteins in the unicellular eukaryote Paramecium where PRC2 targets transposable elements (TEs) and PRC1 is absent. We show that the chromodomain protein Firefly is a H3K27me3 reader in vitro and accumulates onto TEs in a H3K27me3-dependent manner. We also identify Firefly interactors: Sleepy, a coiled-coil containing protein and TfIIs4, a transcription elongation factor. We show that Firefly, Sleepy and TfIIs4 are jointly required in vivo for correct H3K27me3 accumulation and nascent transcription at TEs.ConclusionsThis positive feedback loop to enrich H3K27me3 at TEs is analogous to self-reinforcing loops leading to H3K9 methylation at repeats in fungi, plants and animals. Our work reveals a unique association between H3K27me3 readers and active transcription.

2026-03-03 | PXD065449 | Pride

Structural maintenance of chromosomes (SMCs) proteins are required for developmental DNA elimination in Paramecium tetraurelia

Project description:To study the role of structural maintenance of chromosome (SMC)4-1 and SMC4-2 during programmed genome rearrangement in Paramecium tetraurelia. Paramecium SMC4-1 and SMC4-2 was tagged with 3 FLAG and HA at its C-terminal separately. The recombinant plasmid was microinjected into macronuclear and used for co-immunoprecipitation and Mass spectrometry studies to identify interacting proteins of SMC4-1 and SMC4-2 that indicates the different functions between Paramecium SMC4s.

2023-12-05 | PXD041826 | Pride

Identification of the Polycomb repressive complex 2 and its interacting proteins in Paramecium tetraurelia

Project description:Please provide an overall description of your study, think something similar in scope to the manuscript abstractn Paramecium tetraurelia, Ezl1 (Homolog of EZH2) and PtCAF1 (homolog of RbAp46/48) play important roles in genome rearrangement by disrupting the transposon and transposon-derived DNA elimination. Their influences on are very similar, which make us speculate they belong to the same complex. After verifying their interaction, we speculate the complex is polycomb repressive complex 2 (PRC2) and want to find the other components of this complex. We first tagged PtCAF1 with FlagHA and used anti-HA antibody to enrich for the interacting proteins, coupled with mass spectrometry analyses (previous submission). In this way, we identified the core components of PRC2, as well as some interacted proteins with this complex. In a follow up dataset, we tagged two other subunits of the PRC2 complex we identified, Ezl1 and Eed, with FlagHA,used an anti-HA antibody to enrich for the interacting proteins, coupled with mass spectrometry analyses, and verified the complex.

2023-05-10 | PXD028503 | Pride

Control of developmentally primed erythroid genes by combinatorial corepressor actions

Project description:How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2â??IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation. ChIP-Sequencing profiles of the IRF2BP2, GFI1B and LSD1 proteins were generated using mouse erythroleukemia (MEL) cells. RNA-seq experiments of Irf2bp2-WT, Irf2bp2-KD, Eto2-WT, Eto2-KD, Gfi1b-WT, Gfi1b-KD, Lsd1-WT, Lsd1-KD, MEL-non-induced, and MEL-induced stages were performed using standard RNA-seq protocol. Illumina HiSeq 2000 (standard TruSeq RNA sequencing protocol) was used for the sequencing.

2015-11-19 | E-GEOD-59859 | biostudies-arrayexpress

Two paralogous PHD finger proteins participate in Paramecium tetraurelia's natural genome editing

Project description:The unicellular eukaryote Paramecium tetraurelia contains functionally distinct nuclei: germline micronuclei (MICs) and a somatic macronucleus (MAC). During sexual reproduction, the MIC genome is reorganized into a new MAC genome and the old MAC is lost. Almost 45,000 unique Internal Eliminated Sequences (IESs) located 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 IESs excision occurs. In 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 seems to be influenced by DevPF1- and DevPF2-KD. Thus, both PHD fingers are crucial for new MAC genome development, with distinct functionalities, potentially via regulation of non-coding and coding transcription in the MICs and new MACs.

2024-08-30 | PXD046704 | Pride

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