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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Microarray mRNA expression profiling of E16.5 foetal gonocytes

ABSTRACT: Gonocytes were FACS sorted from testes of E16.5 Miwi2 Tom/+ embryos using the tdTomato reporter knocked into the Miwi2 locus. RNA was isolated from sorted cells and transcript expression analyzed by microarray measurement.

ORGANISM(S): Mus musculus

SUBMITTER: Ansgar Zoch

PROVIDER: E-MTAB-7985 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation.

Zoch Ansgar A Auchynnikava Tania T Berrens Rebecca V RV Kabayama Yuka Y Schöpp Theresa T Heep Madeleine M Vasiliauskaitė Lina L Pérez-Rico Yuvia A YA Cook Atlanta G AG Shkumatava Alena A Rappsilber Juri J Allshire Robin C RC O'Carroll Dónal D

Nature 20200716 7822

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2-4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element tran ...[more]

PMID: 32674113

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Affymetrix gene expression array (mouse 2.0) of sorted undifferentiated spermatogonia in inducible Miwi2 knock-out model

Project description:Undifferentiated spermatogonia were sorted from Miwi2Tom/FL; ERT-Cre/+ (Miwi2-iKO) and Miwi2Tom/+; ERT-Cre/+ (Miwi2-CTL) mice injected with tamoxifen two weeks after last tamoxifen injection to understand an impact of acute Miwi2 deletion on spermatogenic homeostasis.

2018-03-21 | E-MTAB-6612 | biostudies-arrayexpress

Profiling of Mili and Miwi2 associated piRNAs in WT, MiliDAH, Miwi2DAH and Miwi2KO from E16.5 gonadocytes.

Project description:Piwi proteins and piRNAs have conserved functions in transposonM- silencing. The murine Piwi proteins Mili and Miwi2 direct epigeneticM- LINE1 (L1) and intracisternal A particle (IAP) transposon silencingM- during genome reprogramming in the embryonic male germline. PiwiM- proteins are proposed to be piRNA-guided endonucleases that initiateM- secondary piRNA biogenesis . However the actual contribution of theirM- endonuclease activities to piRNA biogenesis and transposon silencingM- remain unknown. To investigate the role of Piwi-catalyzedM- endonucleolytic activity, we engineered point mutations in the mouseM- that substitute the second D to an A in the catalytic triad (DDH) ofM- Mili and Miwi2, generating the MiliDAH and Miwi2DAH alleles,M- M- respectively. Analysis of Mili-bound piRNAs from homozygous MiliDAHM- fetal gonadocytes revealed the failure of transposon piRNA amplification resulting in the stark reduction of piRNA bound withinM- Miwi2 ribonuclear particles (RNPs). We find that Mili-mediated piRNA amplification is selectively required for L1 but not IAP silencing.M- The defective piRNA pathway in MiliDAH mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2DAH mice areM- fertile, transposon silencing is established normally and no defectsM- in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. WeM- conclude that cycles of intra-Mili secondary piRNA biogenesis fuelM- piRNA amplification that is selectively required for L1 silencing.M-

2011-10-28 | E-MTAB-730 | biostudies-arrayexpress

Single-cell RNA-seq for undifferentiated spermatogonia from the testes of control, aged and busulfan-treated C57BL/6 mice

Project description:GFRa1-GFP positive, Miwi2-tdTom negative and GFRa1-GFP negative, Miwi2-tdTom positive single cells from the testes of 2 adult, 2 aged and 3 busulfan-treated mice were sorted with FACS and cDNA libraries prepared with the SMARTseq2 protocol. Single-cell libraries were sequenced

2022-02-28 | E-MTAB-9239 | biostudies-arrayexpress

SPOCD1 is a novel executor of piRNA-directed DNA methylation that links MIWI2 to the de novo DNA methylation machiner

Project description:In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge, the necessity to erase and reset genomic methylation. In the male germline RNA-directed DNA methylation silences young active transposable elements (TEs). The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) are proposed to tether MIWI2 to nascent TE transcripts and instruct DNA methylation. The mechanism by which MIWI2 directs de novo TE methylation is poorly understood but central to the immortality of the germline. Here, we define the interactome of MIWI2 in foetal gonocytes that are undergoing de novo genome methylation and identify a novel MIWI2-associated factor, SPOCD1, that is essential for young TE methylation and silencing. The loss of Spocd1 in mice results in male specific infertility and does not impact on piRNA biogenesis nor localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein and its expression is restricted to the period of de novo genome methylation. We found SPOCD1 co-purified in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery as well as constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent TE transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through its association with SPOCD1. In summary, we have identified a novel and essential executor of mammalian piRNA-directed DNA methylation.

2020-06-22 | PXD016701 | Pride

Bulk RNA-seq for undifferentiated spermatogonia from the testes of control, aged and busulfan-treated C57BL/6 mice

Project description:Samples were prepared from 100 GFRa1-GFP positive, Miwi2-tdTom negative and 100 GFRa1-GFP negative, Miwi2-tdTom positive cells that were sorted with FACS. cDNA libraries were prepared with the SMARTseq2 protocol. Adult, control samples had four biological replicates while the aged and busulfan-treated samples were in triplicate. There were 2 technical replicates per biological sample.

2022-02-28 | E-MTAB-9238 | biostudies-arrayexpress

A transit amplifying population underpins the efficient regenerative capacity of the testis

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2017-04-01 | E-MTAB-4828 | biostudies-arrayexpress

TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing.

Project description:The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. Here we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation.

2020-06-17 | E-MTAB-9090 | biostudies-arrayexpress

Isolation of Mex3a / Lgr5 cell population in mouse small intestine

Project description:Proximal mouse small intestine from mice bearing the Lgr5 GFP/+ and Mex3a Tom/+ alleles were used to obtain single cell preparations. Cells were selected for GFP expression and different levels of tdTomato were defined. Sorted cells were lysed and processed for transcriptomic analysis

2017-06-01 | GSE90629 | GEO

A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice

Project description:We examined the construction of the piRNA system in the restricted developmental window in which methylation patterns are set during mammalian embryogenesis. We find robust expression of two Piwi family proteins, MIWI2 and MILI. Their associated piRNA profiles reveal differences from Drosophila wherein large piRNA clusters act as master regulators of silencing. Instead, in mammals, dispersed transposon copies initiate the pathway, producing primary piRNAs, which predominantly join MILI in the cytoplasm. MIWI2, whose nuclear localization and association with piRNAs depend upon MILI, is enriched for secondary piRNAs antisense to the elements that it controls. The Piwi pathway lies upstream of known mediators of DNA methylation, since piRNAs are still produced in Dnmt3L mutants, which fail to methylate transposons. This implicates piRNAs as specificity determinants of DNA methylation in germ cells. Examination of total small RNA and MILI associated piRNAs in embryonic and post-birth mouse testes, MIWI2-associated piRNAs in embryonic testes.

2008-10-01 | E-GEOD-12757 | biostudies-arrayexpress

TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing.

2020-06-18 | PXD019087 | Pride

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