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

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.

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Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

eLife ◽

10.7554/elife.19090 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 64

Author(s):

Eileen P Hamilton ◽

Aurélie Kapusta ◽

Piroska E Huvos ◽

Shelby L Bidwell ◽

Nikhat Zafar ◽

...

Keyword(s):

Genome Rearrangement ◽

Tetrahymena Thermophila ◽

Chromosome Breakage ◽

Complete Sequence ◽

Mobile Dna ◽

Protein Coding ◽

Dna Elimination ◽

Dna Elements ◽

History Of ◽

Somatic Genome

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

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

Eukaryotic Cell ◽

10.1128/ec.4.2.421-431.2005 ◽

2005 ◽

Vol 4 (2) ◽

pp. 421-431 ◽

Cited By ~ 21

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.

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

Eukaryotic Cell ◽

10.1128/ec.00125-14 ◽

2014 ◽

Vol 13 (10) ◽

pp. 1300-1311 ◽

Cited By ~ 7

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.

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Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.1092-1099.1989 ◽

1989 ◽

Vol 9 (3) ◽

pp. 1092-1099

Author(s):

M C Yao ◽

C H Yao

Keyword(s):

Developmental Stages ◽

Transformation Efficiency ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Rrna Genes ◽

Somatic Nucleus ◽

Dna Breakage ◽

Cis Acting ◽

Palindromic Structure ◽

Rdna Amplification

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.

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RNA Polymerase II Localizes in Tetrahymena thermophila Meiotic Micronuclei When Micronuclear Transcription Associated with Genome Rearrangement Occurs

Eukaryotic Cell ◽

10.1128/ec.3.5.1233-1240.2004 ◽

2004 ◽

Vol 3 (5) ◽

pp. 1233-1240 ◽

Cited By ~ 34

Author(s):

Kazufumi Mochizuki ◽

Martin A. Gorovsky

Keyword(s):

Life Cycle ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genome Rearrangement ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Essential Gene ◽

Gene Encoding ◽

Sexual Process ◽

Rnap Ii

ABSTRACT The germ line micronucleus in Tetrahymena thermophila is transcriptionally silent in vegetatively growing cells. However, micronuclear transcription has been observed in the early (“crescent”) stages of the sexual process, conjugation. This transcription is proposed to play a central role in identifying sites for subsequent genome rearrangements that accompany development of the somatic macronucleus from the micronucleus. RPB3 (cnjC), a gene encoding a protein homologous to the third largest subunit of RNA polymerase II (RNAP II), was previously reported to be expressed specifically during conjugation, suggesting a role in micronucleus-specific transcription. Rpb3p localized in the micronucleus only during the meiotic prophase, when micronuclear transcription occurs, and its intranuclear distribution is strikingly similar to that for previously described sites of micronuclear RNA synthesis. By contrast, Rpc5p, the homologous subunit shared by RNAPs I and III, was not detectable in the micronucleus at any stage of the life cycle. However, Rpb3p is not specific to the transcribing micronucleus. Like Rpc5p, it also localizes to macronuclei in all stages of the life cycle. Rpb3p is encoded by a unique, essential gene in Tetrahymena. Thus, RNAP II is associated with both somatic transcription and crescent transcription and probably has an important role in genome rearrangement.

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Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.1092 ◽

1989 ◽

Vol 9 (3) ◽

pp. 1092-1099 ◽

Cited By ~ 36

Author(s):

M C Yao ◽

C H Yao

Keyword(s):

Developmental Stages ◽

Transformation Efficiency ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Rrna Genes ◽

Somatic Nucleus ◽

Dna Breakage ◽

Cis Acting ◽

Palindromic Structure ◽

Rdna Amplification

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

10.1101/2022.01.04.474918 ◽

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.

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Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in Tetrahymena thermophila

Eukaryotic Cell ◽

10.1128/ec.2.4.678-689.2003 ◽

2003 ◽

Vol 2 (4) ◽

pp. 678-689 ◽

Cited By ~ 14

Author(s):

Jeffrey D. Wuitschick ◽

Kathleen M. Karrer

Keyword(s):

Family Member ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Dna Rearrangement ◽

Developmentally Regulated ◽

Elimination Process ◽

Dna Elimination ◽

Flanking Sequences ◽

Foreign Dna

ABSTRACT Tlr elements are a novel family of ∼30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleus-retained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ∼23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA.

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Lia1p, a Novel Protein Required during Nuclear Differentiation for Genome-Wide DNA Rearrangements in Tetrahymena thermophila

Eukaryotic Cell ◽

10.1128/ec.00157-07 ◽

2007 ◽

Vol 6 (8) ◽

pp. 1320-1329 ◽

Cited By ~ 27

Author(s):

Charles H. Rexer ◽

Douglas L. Chalker

Keyword(s):

Genome Rearrangement ◽

Fluorescent Protein ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Chromatin Modification ◽

Striking Similarity ◽

Macronuclear Development ◽

Genome Wide ◽

Green Fluorescent ◽

Novel Protein

ABSTRACT Extensive genome-wide rearrangements occur during somatic macronuclear development in Tetrahymena thermophila. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one, LIA1, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of LIA1 (ΔLIA1) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the Tetrahymena genome.

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Zygotic Expression of the Double-Stranded RNA Binding Motif Protein Drb2p Is Required for DNA Elimination in the Ciliate Tetrahymena thermophila

Eukaryotic Cell ◽

10.1128/ec.05216-11 ◽

2011 ◽

Vol 10 (12) ◽

pp. 1648-1659 ◽

Cited By ~ 14

Author(s):

Jason A. Motl ◽

Douglas L. Chalker

Keyword(s):

Rna Binding ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Rna Binding Proteins ◽

Chromosome Breakage ◽

Binding Motif ◽

Double Stranded Rna ◽

Content Type ◽

Dna Elimination ◽

Genes Encoding

ABSTRACTDouble-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1andDRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliateTetrahymena thermophila.Both proteins are expressed throughout growth and development but exhibit distinct peaks of expression, suggesting different biological roles. In support of this, we show that expression ofDRB2is essential for vegetative growth whileDRB1expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lacking allDRB1copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. In contrast, cells lacking germ lineDRB2copies, which thus cannot express Drb2p zygotically, fail to produce progeny, arresting late into conjugation. This arrest phenotype is accompanied by a failure to organize the essential DNA rearrangement protein Pdd1p into DNA elimination bodies and execute DNA elimination and chromosome breakage. These results implicate zygotically expressed Drb2p in the maturation of these nuclear structures, which are necessary for reorganization of the somatic genome.

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