A domesticated piggyBac transposase plays key roles in heterochromatin dynamics and DNA cleavage during programmed DNA deletion in Tetrahymena thermophila (original) (raw)
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Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila
G3: Genes, Genomes, Genetics, 2011
Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first highthroughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the 25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (,500 bp) IES with precise elimination boundaries that can contribute the 39 exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner. KEYWORDS Tetrahymena ciliate nuclear dualism community sequencing project genome rearrangement DNA breakage and joining Regulated genome rearrangements are an evolutionarily widespread mechanism for affecting changes in gene expression, for example, switching cell mating type, alternating surface protein presentation, or expanding the repertoire of antibody production (Rusche and Rine 2010). More extreme cases of genome remodeling by large-scale chromatin diminution have been demonstrated in diverse eukaryotes, including ciliated protozoa, parasitic nematodes, and hagfish (Kloc and Zagrodzinska 2001). In the entire phylum Ciliata, a transcriptionally active MAC is differentiated from a copy of the germline MIC by elimination of repetitive DNA (Karrer 2000; Duharcourt et al. 2009). This process is proposed to accomplish a defense of the phenotypically expressed genome from the influence of foreign DNA. Consistent with this hypothesis, repetitive DNA elimination in ciliates involves the same process of RNA-guided heterochromatin formation required for transposon silencing in other eukaryotes (Mochizuki and
Nucleic Acids Research, 2019
Eukaryotic cells pack their genomic DNA into euchromatin and heterochromatin. Boundaries between these domains have been shown to be set by boundary elements. In Tetrahymena, heterochromatin domains are targeted for deletion from the somatic nuclei through a sophisticated programmed DNA rearrangement mechanism, resulting in the elimination of 34% of the germline genome in ∼10,000 dispersed segments. Here we showed that most of these deletions occur consistently with very limited variations in their boundaries among inbred lines. We identified several potential flanking regulatory sequences, each associated with a subset of deletions, using a genome-wide motif finding approach. These flanking sequences are inverted repeats with the copies located at nearly identical distances from the opposite ends of the deleted regions, suggesting potential roles in boundary determination. By removing and testing two such inverted repeats in vivo, we found that the ability for boundary maintenance of the associated deletion were lost. Furthermore, we analyzed the deletion boundaries in mutants of a known boundary-determining protein, Lia3p and found that the subset of deletions that are affected by LIA3 knockout contained common features of flanking regulatory sequences. This study suggests a common mechanism for setting deletion boundaries by flanking inverted repeats in Tetrahymena thermophila.
Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila
Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first highthroughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the 25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (,500 bp) IES with precise elimination boundaries that can contribute the 39 exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.
Nucleic Acids Research, 2019
During differentiation of the Tetrahymena thermophila somatic nucleus, its germline-derived DNA undergoes extensive reorganization including the removal of ∼50 Mb from thousands of loci called internal eliminated sequences (IESs). IES-associated chromatin is methylated on lysines 9 and 27 of histone H3, marking newly formed heterochromatin for elimination. To ensure that this reorganized genome maintains essential coding and regulatory sequences, the boundaries of IESs must be accurately defined. In this study, we show that the developmentally expressed protein encoded by Lia3-Like 1 (LTL1) (Ttherm_00499370) is necessary to direct the excision boundaries of particular IESs. In ΔLTL1 cells, boundaries of eliminated loci are aberrant and heterogeneous. The IESs regulated by Ltl1 are distinct from those regulated by the guanine-quadruplex binding Lia3 protein. Ltl1 has a general affinity for double stranded DNA (Kd ∼ 350 nM) and binds specifically to a 50 bp A+T rich sequence flanking ...
Nucleic Acids Research, 2017
Developmentally programmed genome rearrangement accompanies differentiation of the silent germline micronucleus into the transcriptionally active somatic macronucleus in the ciliated protozoan Tetrahymena thermophila. Internal eliminated sequences (IES) are excised, followed by rejoining of MAC-destined sequences, while fragmentation occurs at conserved chromosome breakage sequences, generating macronuclear chromosomes. Some macronuclear chromosomes, referred to as non-maintained chromosomes (NMC), are lost soon after differentiation. Large NMC contain genes implicated in development-specific roles. One such gene encodes the domesticated piggyBac transposase TPB6, required for heterochromatin-dependent precise excision of IES residing within exons of functionally important genes. These conserved exonic IES determine alternative transcription products in the developing macronucleus; some even contain freestanding genes. Examples of precise loss of some exonic IES in the micronucleus and retention of others in the macronucleus of related species suggest an evolutionary analogy to introns. Our results reveal that germline-limited sequences can encode genes with specific expression patterns and developmentrelated functions, which may be a recurring theme in eukaryotic organisms experiencing programmed genome rearrangement during germline to soma differentiation.
RNA-mediated nucleosome depletion is required for elimination of transposon-derived DNA
2022
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 DN...
A large number of DNA segments are excised from the chromosomes of the somatic nucleus during development of Tetrahymena thermophila. How these germline-limited sequences are recognized and excised is still poorly understood. We have found that many of these noncoding DNAs are transcribed during nuclear development. Transcription of the germline-limited M element occurs from both DNA strands and results in heterogeneous transcripts of < 200 b to > 1 kb. Transcripts are most abundant when developing micro-and macronuclei begin their differentiation. Transcription is normally restricted to unrearranged DNA of micronuclei and/or developing nuclei, but germline-limited DNAs can induce their own transcription when placed into somatic macronuclei. Brief actinomycin D treatment of conjugating cells blocked M-element excision, providing evidence that transcription is important for efficient DNA rearrangement. We propose that transcription targets these germline-limited sequences for elimination by altering chromatin to ensure their accessibility to the excision machinery. ; FAX (314) 935-4432. Article and publication are at www.genesdev.org/cgi/doi/10.1101/ gad.884601.
Cell, 1996
an undifferentiated zygotic nucleus gives rise to two Meng-Chao Yao, † ‡ and C. David Allis* lineages represented by two functionally distinct nuclei, *Department of Biology a germline micronucleus, and a somatic macronucleus. University of Rochester In Tetrahymena thermophila, during the differentiation of Rochester, New York 14627 new macronuclei, or anlagen, from zygotic micronuclei, † Division of Basic Sciences extensive removal of micronuclear-limited DNA seg-‡ Molecular and Cellular Biology Program ments occurs %51ف( of the genome; Yao and Gorovsky, Fred Hutchinson Cancer Research Center 1974) from an estimated 6000 internal chromosomal Seattle, Washington 98104 sites (Callahan et al., 1984; Yao et al., 1984). During this period, the original, or parental, macronucleus becomes pycnotic and degenerates in a manner resembling Summary apoptosis (Davis et al., 1992). The significance of these highly coordinated DNA elimination events is unclear. It During Tetrahymena conjugation, programmed DNA has been suggested (Yao, 1996; Coyne et al., 1996) that degradation occurs in two separate nuclei. Thousands they may be linked to the segregation of chromosomal of germline-specific deletion elements are removed functions that exists in ciliates, in which faithful transfrom the genome of the developing somatic macronumission of the genetic material is restricted to the microcleus, and the old parental macronucleus is degraded nucleus, whereas its expression is restricted to the macby an apoptotic mechanism. An abundant polypeptide, ronucleus. Pdd1p (formerly p65), localizes to both of these nuclei In Tetrahymena, the programmed DNA degradations at the time of DNA degradation. Here we report that, in in the old and newly developing macronucleus occur developing macronuclei, Pdd1p localizes to electronduring approximately the same period of development dense, heterochromatic structures that contain germ-(Austerberry et al., 1984; Yokoyama and Yao, 1982; line-specific deletion elements. Pdd1p also associates Davis et al., 1992). The large-scale nature of these events with parental macronuclei during terminal stages of suggests the possible involvement of stage-specific, apoptosis. Sequencing of the PDD1 gene reveals it to potentially abundant polypeptides. Although studies on be a member of the chromodomain family, suggesting transacting factors mediating these events are in their a molecular link between heterochromatin assembly infancy, an abundant protein (originally referred to as and programmed DNA degradation. p65) has recently been described that preferentially localizes to developing new macronuclei during the time