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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): Q Exactive HF

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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1 - 5 of 39

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

Similar Datasets

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

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

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

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

PRMT5 methyl proteomics in MiaPaca2 cells

Project description:Goal: To determine total PRMT5 methyl substrates and those that function through the novel substrate adaptor binding site

2022-06-13 | PXD019991 | Pride

A toolbox for systematic discovery of stable and transient protein interactors in baker’s yeast

Project description:Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation-based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin-ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation which we term ABOLISH (Auxin-induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full-genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries; some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.

2023-02-01 | PXD033348 | Pride

Developmental mRNA clearance by PIWI-bound endo-siRNAs in Paramecium

Project description:The clearance of untranslated mRNAs by Argonaute proteins is essential for embryonic development in metazoans. However, it is currently unknown whether a similar process exists in unicellular eukaryotes. The ciliate Paramecium tetraurelia harbors a vast array of Argonaute proteins of the Piwi-clade involved in various small RNA pathways, many of which have not yet been investigated. In this work, we investigated the function of a Piwi protein, Ptiwi08, whose expression is limited to a narrow time window during development, concomitant with the start of zygotic transcription. We show that Ptiwi08 acts in an endo-siRNA pathway involved in the clearance of a set of mRNAs at the post-transcriptional level. Based on their features, we hypothesized that these mRNAs may be untranslated. In this dataset, we used mass spectrometry to determine whether any protein products of these mRNAs can be found.

2023-05-10 | PXD038984 | Pride

HDACs act on ribosomal DNA to control the yeast replication program and the competition between origins for limiting initiation factors

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2014-05-14 | E-GEOD-57619 | biostudies-arrayexpress

iiCLIP protocol benchmarking with PTBP1

Project description:We describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions with 1 million HEK293 cells, and with public eCLIP and iCLIP PTBP1 data. There are 3 PTBP1 iiCLIP libraries, 1 input iiCLIP library and 1 PTBP1 iCLIP2 library produced in this study.

2022-08-01 | E-MTAB-10881 | biostudies-arrayexpress

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