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Systematic Perturbation of Thousands of Retroviral LTRs in Mouse Embryos [RNA-Seq]

ABSTRACT: In mammals, many retrotransposons are de-repressed during zygotic genome activation (ZGA). However, their functions in early development remain elusive largely due to the challenge to simultaneously manipulate thousands of retrotransposon insertions in embryos. Here, we employed epigenome editing to perturb long terminal repeat (LTR) MT2_Mm, a well-known ZGA and totipotency marker that exists in ~2667 insertions throughout the mouse genome. CRISPR interference (CRISPRi) robustly targeted and repressed 2485 (~93%) MT2_Mm insertions and 1090 (~55%) insertions of the closely related MT2C_Mm in 2-cell embryos. Remarkably, such perturbation caused down-regulation of hundreds of ZGA genes at 2C stage and embryonic arrest mostly at morula stage. Mechanistically, MT2_Mm/MT2C_Mm primarily served as alternative ZGA promoters activated by Obox proteins. Thus, through large-scale epigenome editing, we addressed to what extent MT2_Mm/MT2C_Mm regulates ZGA and preimplantation development. Our approach could be adapted to systematically perturb retrotransposons in other mammalian embryos as it doesn’t require transgenic animals.

ORGANISM(S): Mus musculus

PROVIDER: GSE242121 | GEO | 2023/10/19

REPOSITORIES: GEO

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Systematic Perturbation of Thousands of Retroviral LTRs in Mouse Embryos [ChIP-Seq]

Project description:In mammals, many retrotransposons are de-repressed during zygotic genome activation (ZGA). However, their functions in early development remain elusive largely due to the challenge to simultaneously manipulate thousands of retrotransposon insertions in embryos. Here, we employed epigenome editing to perturb long terminal repeat (LTR) MT2_Mm, a well-known ZGA and totipotency marker that exists in ~2667 insertions throughout the mouse genome. CRISPR interference (CRISPRi) robustly targeted and repressed 2485 (~93%) MT2_Mm insertions and 1090 (~55%) insertions of the closely related MT2C_Mm in 2-cell embryos. Remarkably, such perturbation caused down-regulation of hundreds of ZGA genes at 2C stage and embryonic arrest mostly at morula stage. Mechanistically, MT2_Mm/MT2C_Mm primarily served as alternative ZGA promoters activated by Obox proteins. Thus, through large-scale epigenome editing, we addressed to what extent MT2_Mm/MT2C_Mm regulates ZGA and preimplantation development. Our approach could be adapted to systematically perturb retrotransposons in other mammalian embryos as it doesn’t require transgenic animals.

2023-10-19 | GSE242122 | GEO

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites

Project description:CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2,500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

2022-06-07 | GSE178440 | GEO

Selective binding of retrotransposons by ZFP352 facilitates the timely dissolution of totipotency network

Project description:Acquisition of new stem cell fates relies on the dissolution of the prior regulatory network sustaining the existing cell fates. Currently, extensive insights have been revealed for the totipotency regulatory network around the zygotic genome activation (ZGA) period. However, how the dissolution of the totipotency network is triggered to ensure the timely embryonic development following ZGA is largely unknown. In this study, we identified the unexpected role of a highly expressed 2C embryo specific transcription factor, ZFP352, in facilitating the dissolution of the totipotency network. We found that ZFP352 has bifurcated binding and regulation towards two different retrotransposon sub-families. ZFP352 coordinates with DUX to bind and regulate the 2C specific MT2_Mm sub-family. On the other hand, without DUX, ZFP352 switched affinity to bind extensively onto SINE_B1/Alu sub-family. This leads to the activation of later developmental programs like ubiquitination pathways, to facilitate the dissolution of the 2C state. Correspondingly, depleting ZFP352 in mouse embryos impaired the 2C to morula transition process. Thus, through differential regulation of MT2_Mm and SINE_B1/Alu, ZFP352 can trigger spontaneous dissolution of the totipotency network. Our study highlights the importance of different retrotransposons sub-families in facilitating the timely and programmed cell fates transition during early embryogenesis.

2023-06-01 | GSE222636 | GEO

Zfp352 and Zfp352+Dux over expression chip-seq result

Project description:Our data delineated bifurcated binding and regulation of ZFP352 towards two distinct retrotransposons, MT2_Mm and SINE_B1/Alu,further ChIP-seq experiments validated that over-expression of Dux displaced ZFP352 from SINE_B1/Alu and DUX-independent ZFP352 binding sites, but promoted ZFP352 binding onto MT2_Mm and DUX_ZFP352 co-bound sites

2023-06-01 | GSE222634 | GEO

Capture of a functionally active Methyl-CpG Binding Domain by an arthropod retrotransposon family

Project description:The repressive capacity of cytosine DNA methylation is mediated by recruitment of silencing complexes by methyl-CpG binding domain (MBD) proteins. Unexpectedly, we discovered that a family of arthropod Copia retrotransposons have incorporated a host-derived MBD domain. We functionally demonstrate how retrotransposon encoded MBDs preferentially bind to CpG-dense methylated regions, which correspond to transposable element regions of the host genome, in the myriapod Strigamia maritima. Consistently, young MBD-encoding Copia retrotransposons (CopiaMBD) accumulate in regions with higher CpG-densities than other LTR-retrotransposons also present in the genome. This would suggest that retrotransposons use MBDs to integrate into heterochromatic regions in Strigamia, avoiding potentially harmful insertions into host genes. In contrast, CopiaMBD insertions in the spider Stegodyphus dumicola genome disproportionately accumulate in methylated gene bodies when compared to other spider LTR-retrotransposons. Given that transposons are not actively targeted by DNA methylation in the spider genome, this distribution bias would also support a role for MBDs in the integration process. Together, these data demonstrate that retrotransposons can co-opt host-derived epigenome readers, potentially harnessing the host epigenome landscape to advantageously tune the retrotransposition process.

2019-06-17 | GSE118012 | GEO

Hijacking of transcriptional condensates by endogenous retroviruses

Project description:Heterochromatin plays essential roles in repressing retrotransposons, e.g. endogenous retroviruses (ERVs) during mammalian development, but the contribution of retrotransposition to lethality observed in embryonic cells deficient for heterochromatin-mediated ERV repression is poorly understood. Here we report that selective degradation of the TRIM28 heterochromatin adapter protein leads to reduced association of transcriptional condensates with loci encoding super-enhancer -driven pluripotency genes in embryonic stem cells, a collapse of the pluripotency transcriptional circuit, and a pre-lethal restriction of pluripotent lineages in mouse embryos. De-repressed ERVs recruit transcriptional condensates in the absence of TRIM28, and ERV RNA facilitates condensation of RNA Polymerase II in vitro. We propose that retrotransposons contribute to the genomic distribution of nuclear condensates, and that RNA species may facilitate “hijacking” of transcriptional condensates in various developmental and disease contexts.

2022-07-08 | PXD021895 | Pride

Zfp352 over expression ATAC-seq result

Project description:Our data delineated bifurcated binding and regulation of ZFP352 towards two distinct retrotransposons, MT2_Mm and SINE_B1/Alu,and whether ZFP352 can regulate mouse genome openness remains unknown,here we conduct ATAC-seq of Zfp352 over expression sample to detect ZFP352's role in regulating chromatin openness.

2023-06-01 | GSE222635 | GEO

The landscape of RNA Pol II binding reveals a step-wise transition during ZGA

Project description:Zygotic genome activation (ZGA) is the first transcription event in life. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. Here, by developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc–seq), we investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. We found that Pol II undergoes ‘loading’, ‘pre-configuration’, and ‘production’ during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, step-wise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation.

2020-10-28 | GSE135457 | GEO

Oryzias latipes strain:d-rR

Project description:Targeted in vivo epigenome editing using medaka embryos

| PRJDB7919 | ENA

Quantitative proteomics reveals the dynamic proteome landscape of zebrafish embryos during maternal-to-zygotic transition and its relationship to zygotic genome activation

Project description:Upon fertilization, the embryonic genome remains transcriptionally inactive until the mid-blastula transition. Zygotic genome activation (ZGA) of vertebrate embryos has been extensively studied using nucleic acid-based strategies, but proteomics data are still scarce, impeding the full mechanistic understanding of how ZGA is executed during the maternal-to-zygotic transition (MZT). Here, we performed quantitative proteomics to decipher the proteome landscape of zebrafish embryos during the MZT, quantifying nearly 5,000 proteins across four embryonic stages. The stage-specific clustering based on protein expression pattern revealed that helicases (i.e., eif4a2 and ruvbl1) facilitate pluripotency factors (i.e., nanog, pou5f3, ctcf, and hmga1) triggering ZGA in zebrafish, accompanied by the maternal product decay with P-bodies and ubiquitin dependent proteolytic pathway. Dozens of transcription factors show wave-like expression patterns during MZT, implying their diverse functions in triggering the ZGA and modulating differentiation for organ development. The combination of morpholino knockdown and quantitative proteomics demonstrated that maternal Nanog is required for proper embryogenesis by regulating 1) interactions with other pluripotency factors, 2) F-actin band formation, 3) cell cycle checkpoints and 4) maternal product degradation. This study represents the most systematic proteomics survey of developmentally regulated proteins and their expression profiles accompanying MZT in zebrafish, which is a valuable proteome resource for understanding ZGA.

2024-05-01 | PXD036678 | Pride

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