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

2022 ◽  
Author(s):  
Aditi Singh ◽  
Xyrus X. Maurer-Alcalá ◽  
Therese Solberg ◽  
Silvan Gisler ◽  
Michael Ignarski ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Chromatin Remodeling ◽  
Dna Cleavage ◽  
Small Rnas ◽  
Heterochromatin Formation ◽  
Dna Accessibility ◽  
Dna Elimination ◽  
Nucleosome Density ◽  
Somatic Genome ◽  
Genomic Regions

Small RNAs are known to mediate silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. Post-zygotic development of the Paramecium somatic genome requires elimination of thousands of transposon remnants (IESs) and transposable elements that are scattered throughout the germline genome (Garnier et al. 2004). The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries (Bouhouche et al. 2011; Furrer et al. 2017). Previous research suggests that small RNAs induce heterochromatin formation within IESs, which, in turn, is required for DNA elimination (Liu et al. 2007). Here we show that IES recognition and precise excision is facilitated by recruitment of a homolog of a chromatin remodeler ISWI, which depletes target genomic regions of nucleosomes, making the chromatin accessible for DNA cleavage. ISWI knockdown in Paramecium leads to pronounced inhibition of DNA elimination. Furthermore, nucleosome profiling indicates that ISWI is required for IES elimination in nucleosome-dense genomic regions, while other IESs do not require small RNAs or ISWI for excision. ISWI silencing notably also reduces DNA elimination precision, resulting in aberrant excision at alternative IES boundaries. In summary, we demonstrate that chromatin remodeling that increases DNA accessibility together with small RNAs are necessary for efficient and precise DNA elimination in Paramecium.

scholarly journals Silencing of Euchromatic Transposable Elements as a Consequence of Nuclear Lamina Dysfunction

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 625
Author(s):  
Valeria Cavaliere ◽  
Giovanna Lattanzi ◽  
Davide Andrenacci
Keyword(s):  
Transposable Elements ◽  
Genome Instability ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Rna Degradation ◽  
Loss Of Function ◽  
Heterochromatin Formation ◽  
Repressive Effect ◽  
Regulatory Effects ◽  
Genomic Regions

Transposable elements (TEs) are mobile genomic sequences that are normally repressed to avoid proliferation and genome instability. Gene silencing mechanisms repress TEs by RNA degradation or heterochromatin formation. Heterochromatin maintenance is therefore important to keep TEs silent. Loss of heterochromatic domains has been linked to lamin mutations, which have also been associated with derepression of TEs. In fact, lamins are structural components of the nuclear lamina (NL), which is considered a pivotal structure in the maintenance of heterochromatin domains at the nuclear periphery in a silent state. Here, we show that a lethal phenotype associated with Lamin loss-of-function mutations is influenced by Drosophila gypsy retrotransposons located in euchromatic regions, suggesting that NL dysfunction has also effects on active TEs located in euchromatic loci. In fact, expression analysis of different long terminal repeat (LTR) retrotransposons and of one non-LTR retrotransposon located near active genes shows that Lamin inactivation determines the silencing of euchromatic TEs. Furthermore, we show that the silencing effect on euchromatic TEs spreads to the neighboring genomic regions, with a repressive effect on nearby genes. We propose that NL dysfunction may have opposed regulatory effects on TEs that depend on their localization in active or repressed regions of the genome.

scholarly journals LIA4Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

2014 ◽  
Vol 13 (10) ◽  
pp. 1300-1311 ◽  
Author(s):  
Scott A. Horrell ◽  
Douglas L. Chalker
Keyword(s):  
Critical Role ◽  
Dna Rearrangement ◽  
Rnai Machinery ◽  
Content Type ◽  
Heterochromatin Formation ◽  
H3k27 Methylation ◽  
Dna Elimination ◽  
Somatic Genome ◽  
Subsequent Elimination ◽  
Elimination Pathway

ABSTRACTExtensive DNA elimination occurs as part of macronuclear differentiation duringTetrahymenasexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway.LIA4knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, withoutLIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus duringTetrahymenamacronuclear differentiation.

scholarly journals Diversification of small RNA amplification mechanisms for targeting transposon-related sequences in ciliates

2019 ◽  
Vol 116 (29) ◽  
pp. 14639-14644 ◽  
Author(s):  
Masatoshi Mutazono ◽  
Tomoko Noto ◽  
Kazufumi Mochizuki
Keyword(s):  
Transposable Elements ◽  
Small Rna ◽  
Signal Amplification ◽  
Small Rnas ◽  
Ciliated Protozoa ◽  
Rna Amplification ◽  
Somatic Nucleus ◽  
Dna Elimination ◽  
Internal Eliminated Sequences ◽  
Rna Biogenesis

The silencing of repetitive transposable elements (TEs) is ensured by signal amplification of the initial small RNA trigger, which occurs at distinct steps of TE silencing in different eukaryotes. How such a variety of secondary small RNA biogenesis mechanisms has evolved has not been thoroughly elucidated. Ciliated protozoa perform small RNA-directed programmed DNA elimination of thousands of TE-related internal eliminated sequences (IESs) in the newly developed somatic nucleus. In the ciliate Paramecium, secondary small RNAs are produced after the excision of IESs. In this study, we show that in another ciliate, Tetrahymena, secondary small RNAs accumulate at least a few hours before their derived IESs are excised. We also demonstrate that DNA excision is dispensable for their biogenesis in this ciliate. Therefore, unlike in Paramecium, small RNA amplification occurs before IES excision in Tetrahymena. This study reveals the remarkable diversity of secondary small RNA biogenesis mechanisms, even among ciliates with similar DNA elimination processes, and thus raises the possibility that the evolution of TE-targeting small RNA amplification can be traced by investigating the DNA elimination mechanisms of ciliates.

scholarly journals Two GW Repeat Proteins Interact with Tetrahymena thermophila Argonaute and Promote Genome Rearrangement

2009 ◽  
Vol 29 (18) ◽  
pp. 5020-5030 ◽  
Author(s):  
Janna Bednenko ◽  
Tomoko Noto ◽  
Leroi V. DeSouza ◽  
K. W. Michael Siu ◽  
Ronald E. Pearlman ◽  
...  
Keyword(s):  
Genome Rearrangement ◽  
Developmental Stages ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Nuclear Bodies ◽  
Interacting Proteins ◽  
Heterochromatin Formation ◽  
Dna Elimination ◽  
Somatic Genome ◽  
Argonaute Family

ABSTRACT In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, ∼28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp. Both proteins contain GW (glycine and tryptophan) repeats, which are characteristic of several Argonaute-interacting proteins in other organisms. Wag1p and CnjBp colocalize with Twi1p in the parental macronucleus early in conjugation and in the new developing macronucleus during later developmental stages. Around the time DNA elimination occurs, Wag1p forms multiple nuclear bodies in the developing macronuclei that do not colocalize with heterochromatic DNA elimination structures. Analyses of ΔWAG1, ΔCnjB, and double ΔWAG1 ΔCnjB knockout strains revealed that WAG1 and CnjB genes need to be deleted together to inhibit the downregulation of specific scnRNAs, the formation of DNA elimination structures, and DNA excision. Thus, Wag1p and CnjBp are two novel players with overlapping functions in RNA interference-mediated genome rearrangement in Tetrahymena.

scholarly journals Elimination of Foreign DNA during Somatic Differentiation in Tetrahymena thermophila Shows Position Effect and Is Dosage Dependent

2005 ◽  
Vol 4 (2) ◽  
pp. 421-431 ◽  
Author(s):  
Yifan Liu ◽  
Xiaoyuan Song ◽  
Martin A. Gorovsky ◽  
Kathleen M. Karrer
Keyword(s):  
Transposable Elements ◽  
Dna Sequences ◽  
Copy Number ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Position Effect ◽  
Dna Elimination ◽  
Internal Eliminated Sequences ◽  
Somatic Genome ◽  
Foreign Dna

ABSTRACT In the ciliate Tetrahymena thermophila, approximately 15% of the germ line micronuclear DNA sequences are eliminated during formation of the somatic macronucleus. The vast majority of the internal eliminated sequences (IESs) are repeated in the micronuclear genome, and several of them resemble transposable elements. Thus, it has been suggested that DNA elimination evolved as a means for removing invading DNAs. In the present study, bacterial neo genes introduced into the germ line micronuclei were eliminated from the somatic genome. The efficiency of elimination from two different loci increased dramatically with the copy number of the neo genes in the micronuclei. The timing of neo elimination is similar to that of endogenous IESs, and they both produce bidirectional transcripts of the eliminated element, suggesting that the deletion of neo occurred by the same mechanism as elimination of endogenous IESs. These results indicate that repetition of an element in the micronucleus enhances the efficiency of its elimination from the newly formed somatic genome of Tetrahymena thermophila. The implications of these data in relation to the function and mechanism of IES elimination are discussed.

scholarly journals Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Julien Bischerour ◽  
Simran Bhullar ◽  
Cyril Denby Wilkes ◽  
Vinciane Régnier ◽  
Nathalie Mathy ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Nuclear Localization ◽  
Dna Cleavage ◽  
Biological Process ◽  
Genome Rearrangements ◽  
Catalytic Site ◽  
Dna Rearrangements ◽  
Precise Excision ◽  
Dna Elimination ◽  
Genome Wide

The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide. Unlike Pgm, PgmLs lack a conserved catalytic site, suggesting that they rather have an architectural function within a multi-component excision complex embedding Pgm. PgmL depletion can increase erroneous targeting of residual Pgm-mediated DNA cleavage, indicating that PgmLs contribute to accurately position the complex on IES ends. DNA rearrangements in Paramecium constitute a rare example of a biological process jointly managed by six distinct domesticated transposases.

scholarly journals Identification and analysis of functional associations among natural eukaryotic genome editing components

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1374 ◽  
Author(s):  
Estienne C. Swart ◽  
Cyril Denby Wilkes ◽  
Pamela Y. Sandoval ◽  
Cristina Hoehener ◽  
Aditi Singh ◽  
...  
Keyword(s):  
Genome Editing ◽  
Small Rnas ◽  
Rna Binding ◽  
Elongation Factor ◽  
Chromatin Remodelling ◽  
Eukaryotic Genome ◽  
Functional Relationships ◽  
Dna Elimination ◽  
Somatic Genome ◽  
Main Components

During development in the ciliate Paramecium, excess DNA interspersed throughout the germline genome is deleted to generate a new somatic genome. In this process, most of the intervening DNA is excised by a Piggybac-derived transposase, assisted by small RNAs (scnRNAs and iesRNAs) and chromatin remodelling. As the list of genes involved in DNA elimination has been growing, a need for a general approach to discover functional relationships among these genes now exists. We show that deep sequencing-based comparisons of experimentally-induced DNA retention provide a sensitive, quantitative approach to identify and analyze functional associations among genes involved in native genome editing. This reveals two functional molecular groups: (i) iesRNAs/scnRNAs, the putative Piwi- and RNA-binding Nowa1/2 proteins, and the transcription elongation factor TFIIS4; and (ii) PtCAF1 and Ezl1, two proteins involved in chromatin remodelling. Comparative analyses of silencing effects upon the largely unstudied regions comprising most developmentally eliminated DNA in Paramecium suggests a continuum between precise and imprecise DNA elimination. These findings show there is now a way forward to systematically elucidate the main components of natural eukaryotic genome editing systems.

scholarly journals Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

2021 ◽  
Author(s):  
Nour J Abdulhay ◽  
Laura J Hsieh ◽  
Colin P McNally ◽  
Mythili Ketavarapu ◽  
Sivakanthan Kasinathan ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Single Molecule ◽  
Single Fiber ◽  
Chromatin Remodelers ◽  
Linker Length ◽  
Dna Accessibility ◽  
Nucleosome Density ◽  
Regulatory Effects

ABSTRACTATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.

scholarly journals Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

2021 ◽  
Vol 7 (11) ◽  
pp. eabd6030
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
Bryan A. Gibson ◽  
Michael K. Rosen ◽  
Rick Russell ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Eukaryotic Cells ◽  
Loop Formation ◽  
Chromatin Compaction ◽  
Dna Targeting ◽  
Genomic Regions ◽  
Dna Bendability ◽  
Nucleosome Arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

scholarly journals Distinct Responses of Arabidopsis Telomeres and Transposable Elements to Zebularine Exposure

2021 ◽  
Vol 22 (1) ◽  
pp. 468
Author(s):  
Klára Konečná ◽  
Pavla Polanská Sováková ◽  
Karin Anteková ◽  
Jiří Fajkus ◽  
Miloslava Fojtová
Keyword(s):  
Transposable Elements ◽  
Transcriptional Activation ◽  
Genome Stability ◽  
Cytosine Methylation ◽  
Individual Variability ◽  
Correlated Response ◽  
Telomere Shortening ◽  
Treatment Changes ◽  
Telomere Homeostasis ◽  
Genomic Regions

Involvement of epigenetic mechanisms in the regulation of telomeres and transposable elements (TEs), genomic regions with the protective and potentially detrimental function, respectively, has been frequently studied. Here, we analyzed telomere lengths in Arabidopsis thaliana plants of Columbia, Landsberg erecta and Wassilevskija ecotypes exposed repeatedly to the hypomethylation drug zebularine during germination. Shorter telomeres were detected in plants growing from seedlings germinated in the presence of zebularine with a progression in telomeric phenotype across generations, relatively high inter-individual variability, and diverse responses among ecotypes. Interestingly, the extent of telomere shortening in zebularine Columbia and Wassilevskija plants corresponded to the transcriptional activation of TEs, suggesting a correlated response of these genomic elements to the zebularine treatment. Changes in lengths of telomeres and levels of TE transcripts in leaves were not always correlated with a hypomethylation of cytosines located in these regions, indicating a cytosine methylation-independent level of their regulation. These observations, including differences among ecotypes together with distinct dynamics of the reversal of the disruption of telomere homeostasis and TEs transcriptional activation, reflect a complex involvement of epigenetic processes in the regulation of crucial genomic regions. Our results further demonstrate the ability of plant cells to cope with these changes without a critical loss of the genome stability.

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