Publications by authors named "Douglas L Chalker"

34 Publications

translational machinery condones polyadenosine repeats.

Elife 2020 05 29;9. Epub 2020 May 29.

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, United States.

is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or , an organism with similar AT-content. Finally, using stalling reporters we show that cells evolved not to have a fully functional NGD pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.57799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295572PMC
May 2020

Boundaries of eliminated heterochromatin of Tetrahymena are positioned by the DNA-binding protein Ltl1.

Nucleic Acids Res 2019 08;47(14):7348-7362

Biology Department, Washington University in St. Louis, St. Louis, MO 63130, USA.

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 each side of the D IES (Kd ∼ 43 nM). Together these data reveal that Ltl1 and Lia3 control different subsets of IESs and that their mechanisms for flanking sequence recognition are distinct.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698652PMC
August 2019

Transgenerational Inheritance: Parental Guidance Suggested.

Curr Biol 2018 06;28(12):R702-R704

Biology Department, Washington University in St. Louis, One Brookings Drive, St. Louis, MO 63130, USA. Electronic address:

RNAs have been attractive candidates to transmit epigenetic information over multiple generations. In Tetrahymena, a new study demonstrates that the selective degradation of small RNAs that occurs by interaction with the parental genome can communicate altered patterns of heterochromatin formation and DNA elimination in offspring.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2018.05.041DOI Listing
June 2018

Diversification of HP1-like Chromo Domain Proteins in Tetrahymena thermophila.

J Eukaryot Microbiol 2018 01 3;65(1):104-116. Epub 2017 Aug 3.

Department of Biology, Washington University, St. Louis, Missouri, 63130.

Proteins that possess a chromo domain are well-known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy-terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi-directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jeu.12443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762428PMC
January 2018

Rapid generation of hypomorphic mutations.

Nat Commun 2017 01 20;8:14112. Epub 2017 Jan 20.

Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms14112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5263891PMC
January 2017

One genome's junk is another's garbage.

Elife 2016 12 23;5. Epub 2016 Dec 23.

Biology Department, Washington University in St. Louis, St. Louis, United States.

Experiments on a single-celled ciliate reveal how mobile genetic elements can shape a genome, even one which is not transcriptionally active.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.23447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5182058PMC
December 2016

DRH1, a p68-related RNA helicase gene, is required for chromosome breakage in Tetrahymena.

Biol Open 2016 Dec 15;5(12):1790-1798. Epub 2016 Dec 15.

Department of Biology, Washington University in St. Louis, St Louis, MO 63130, USA

The p68 DEAD box helicases comprise a widely conserved protein family involved in a large range of biological processes including transcription, splicing and translation. The genome of the ciliate Tetrahymena thermophile encodes two p68-like helicases, Drh1p and Lia2p. We show that DRH1 is essential for growth and completion of development. In growing cells, Drh1p is excluded from the nucleus and accumulates near cortical basal bodies. In contrast, during sexual reproduction, this protein localizes to meiotic micronuclei, initially in punctate foci in regions where centromeres and telomeres are known to reside and later in post-zygotic differentiating somatic macronuclei. Differentiation of the macronuclear genome involves extensive DNA rearrangements including fragmentation of the five pairs of germline-derived chromosomes into 180 chromosomal sub-fragments that are stabilized by de novo telomere deletion. In addition, thousands of internal eliminated sequences (IESs) are excised from loci dispersed throughout the genome. Strains with DRH1 deleted from the germline nuclei, which do not express the protein during post-zygotic development, fail to fragment the developing macronuclear chromosomes. IES excision still occurs in the absence of DRH1 zygotic expression; thus, Drh1p is the first protein found to be specifically required for chromosome breakage but not DNA elimination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/bio.021576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5200911PMC
December 2016

A Parallel G Quadruplex-Binding Protein Regulates the Boundaries of DNA Elimination Events of Tetrahymena thermophila.

PLoS Genet 2016 Mar 7;12(3):e1005842. Epub 2016 Mar 7.

Department of Biology, Washington University in St. Louis, St. Louis, Missouri, United States of America.

Guanine (G)-rich DNA readily forms four-stranded quadruplexes in vitro, but evidence for their participation in genome regulation is limited. We have identified a quadruplex-binding protein, Lia3, that controls the boundaries of germline-limited, internal eliminated sequences (IESs) of Tetrahymena thermophila. Differentiation of this ciliate's somatic genome requires excision of thousands of IESs, targeted for removal by small-RNA-directed heterochromatin formation. In cells lacking LIA3 (ΔLIA3), the excision of IESs bounded by specific G-rich polypurine tracts was impaired and imprecise, whereas the removal of IESs without such controlling sequences was unaffected. We found that oligonucleotides containing these polypurine tracts formed parallel G-quadruplex structures that are specifically bound by Lia3. The discovery that Lia3 binds G-quadruplex DNA and controls the accuracy of DNA elimination at loci with specific G-tracts uncovers an unrecognized potential of quadruplex structures to regulate chromosome organization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1005842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780704PMC
March 2016

SUMOylation is developmentally regulated and required for cell pairing during conjugation in Tetrahymena thermophila.

Eukaryot Cell 2015 Feb 19;14(2):170-81. Epub 2014 Dec 19.

Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena, including a dynamic regulation associated with the sexual life cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00252-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311922PMC
February 2015

Morphogenesis: a Mob rules from the rear.

Curr Biol 2014 Aug;24(15):R700-2

Department of Biology, University of Iowa, 129 East Jefferson Street, Iowa City, Iowa 52242, USA.

The giant ciliated protozoan Stentor coeruleus is re-emerging as a model organism for morphogenesis and patterning in unicellular organisms. A new study provides evidence that cytokinesis and morphogenesis are mechanistically linked through the Mob1 protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2014.06.042DOI Listing
August 2014

LIA4 encodes a chromoshadow domain protein required for genomewide DNA rearrangements in Tetrahymena thermophila.

Eukaryot Cell 2014 Oct 1;13(10):1300-11. Epub 2014 Aug 1.

Department of Biology, Washington University, St. Louis, Missouri, USA

Extensive DNA elimination occurs as part of macronuclear differentiation during Tetrahymena sexual 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. LIA4 knockout (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, without LIA4, 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 during Tetrahymena macronuclear differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00125-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187650PMC
October 2014

Epigenetics: Keeping one's sex.

Nature 2014 May 7;509(7501):430-1. Epub 2014 May 7.

Department of Biology, Washington University in St. Louis, St Louis, Missouri 63130, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature13333DOI Listing
May 2014

Mutations in Pdd1 reveal distinct requirements for its chromodomain and chromoshadow domain in directing histone methylation and heterochromatin elimination.

Eukaryot Cell 2014 Feb 2;13(2):190-201. Epub 2013 Dec 2.

Department of Biology, Washington University, Saint Louis, Missouri, USA.

Pdd1, a specialized HP1-like protein, is required for genome-wide DNA rearrangements that restructure a previously silent germ line genome into an active somatic genome during macronuclear differentiation of Tetrahymena thermophila. We deleted or otherwise mutated conserved regions of the protein to investigate how its different domains promote the excision of thousands of internal eliminated sequences (IESs). Previous studies revealed that Pdd1 contributes to recognition of IES loci after they are targeted by small-RNA-guided methylation of histone H3 on lysine 27 (H3K27), subsequently aids the establishment of H3K9 methylation, and recruits proteins that lead to excision. The phenotypes we observed for different Pdd1 alleles showed that each of the two chromodomains and the chromoshadow domain (CSD) have distinct contributions during somatic genome differentiation. Chromodomain 1 (CD1) is essential for conjugation as either its deletion or the substitution of two key aromatic amino acid residues (the W97A W100A mutant) is lethal. These mutations caused mislocalization of a cyan fluorescent protein (CFP)-tagged protein, prevented the establishment of histone H3 dimethylated on K9 (H3K9me2), and abolished IES excision. Nevertheless, the requirement for CD1 could be bypassed by recruiting Pdd1 directly to an IES by addition of a specific DNA binding domain. Chromodomain 2 (CD2) was necessary for producing viable progeny, but low levels of H3K9me2 and IES excision still occurred. A mutation in the chromoshadow domain (CSD) prevented Pdd1 focus formation but still permitted ∼17% of conjugants to produce viable progeny. However, this mutant was unable to stimulate excision when recruited to an ectopic IES, indicating that this domain is important for recruitment of excision factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00219-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910979PMC
February 2014

Epigenetics of ciliates.

Cold Spring Harb Perspect Biol 2013 Dec 1;5(12):a017764. Epub 2013 Dec 1.

Department of Biology, Washington University, St. Louis, Missouri 63130.

Research using ciliates revealed early examples of epigenetic phenomena and continues to provide novel findings. These protozoans maintain separate germline and somatic nuclei that carry transcriptionally silent and active genomes, respectively. Examining the differences in chromatin within distinct nuclei of Tetrahymena identified histone variants and established that transcriptional regulators act by modifying histones. Formation of somatic nuclei requires both transcriptional activation of silent chromatin and large-scale DNA elimination. This somatic genome remodeling is directed by homologous RNAs, acting with an RNA interference (RNAi)-related machinery. Furthermore, the content of the parental somatic genome provides a homologous template to guide this genome restructuring. The mechanisms regulating ciliate DNA rearrangements reveal the surprising power of homologous RNAs to remodel the genome and transmit information transgenerationally.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/cshperspect.a017764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839606PMC
December 2013

LIA5 is required for nuclear reorganization and programmed DNA rearrangements occurring during tetrahymena macronuclear differentiation.

PLoS One 2013 17;8(9):e75337. Epub 2013 Sep 17.

Biology Department, Washington University in St. Louis, St. Louis, Missouri, United States of America.

During macronuclear differentiation of the ciliate Tetrahymena thermophila, genome-wide DNA rearrangements eliminate nearly 50 Mbp of germline derived DNA, creating a streamlined somatic genome. The transposon-like and other repetitive sequences to be eliminated are identified using a piRNA pathway and packaged as heterochromatin prior to their removal. In this study, we show that LIA5, which encodes a zinc-finger protein likely of transposon origin, is required for both chromosome fragmentation and DNA elimination events. Lia5p acts after the establishment of RNAi-directed heterochromatin modifications, but prior to the excision of the modified sequences. In ∆LIA5 cells, DNA elimination foci, large nuclear sub-structures containing the sequences to be eliminated and the essential chromodomain protein Pdd1p, do not form. Lia5p, unlike Pdd1p, is not stably associated with these structures, but is required for their formation. In the absence of Lia5p, we could recover foci formation by ectopically inducing DNA damage by UV treatment. Foci in both wild-type or UV-treated ∆LIA5 cells contain dephosphorylated Pdd1p. These studies of LIA5 reveal that DNA elimination foci form after the excision of germ-line limited sequences occurs and indicate that Pdd1p reorganization is likely mediated through a DNA damage response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075337PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775806PMC
June 2014

Genome biology: the sleek and oh-so chic Oxytricha nanochromosomes.

Curr Biol 2013 Apr;23(7):R284-5

Biology Department, Washington University in St. Louis, One Brookings Drive, St. Louis, MO 63130, USA.

The somatic nucleus of Oxytricha trifallax contains over 15,000 different chromosomes, most containing a single gene. Analysis of this 50 Mb genome uncovers novel regulatory strategies and adaptive potential when gene copy number and allelic frequency are no longer constrained by genetic linkage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2013.02.041DOI Listing
April 2013

Transformation and strain engineering of Tetrahymena.

Methods Cell Biol 2012 ;109:327-45

Department of Biology, Washington University in St. Louis, St. Louis, MO, USA.

Transformation of Tetrahymena by microinjection of DNA was established 25 years ago. This rather labor-intensive technique has since been shelved, replaced by less time consuming and more efficient methods, electroporation and biolistics. Conjugative electroporation is the method of choice for introducing autonomously replicating, rDNA-based vectors into Tetrahymena. These are maintained as high-copy linear mini-chromosomes. Versatile expression cassettes in these vectors facilitate expression of most genes. Transformation efficiencies are sufficiently high to permit screens using expression libraries. Biolistic transformation is primarily used to introduce DNA for integration into the genome by homologous recombination. This technique has greatly enhanced strain engineering of Tetrahymena through facilitating the disruption of genes (creating targeted knockout cell lines) or epitope-tagging coding regions, allowing researchers to take full advantage of the sequenced genome. The presence of both germline and somatic nuclei in these cells requires different strategies to target DNA to the desired compartment. This presents challenges, including the need to engineer the polygenic macronuclear genome, which has nearly 50 copies of each gene. However, separate manipulation of functionally distinct genomes provides experimental opportunities, especially for the analysis of essential genes, by modifying the silent micronucleus then subsequently examining phenotypes in the next sexual generation. The flexibility to engineer strains as needed makes Tetrahymena a facile system with which to answer many biological questions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/B978-0-12-385967-9.00011-6DOI Listing
July 2012

Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila.

G3 (Bethesda) 2011 Nov 1;1(6):515-22. Epub 2011 Nov 1.

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 high-throughput 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 3' exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/g3.111.000927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3276166PMC
November 2011

Zygotic expression of the double-stranded RNA binding motif protein Drb2p is required for DNA elimination in the ciliate Tetrahymena thermophila.

Eukaryot Cell 2011 Dec 21;10(12):1648-59. Epub 2011 Oct 21.

Department of Biology, Washington University in St. Louis, St. Louis, MO 63130-4899, USA.

Double-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 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena 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 of DRB2 is essential for vegetative growth while DRB1 expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lacking all DRB1 copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. In contrast, cells lacking germ line DRB2 copies, 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.05216-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3232721PMC
December 2011

DNA elimination in ciliates: transposon domestication and genome surveillance.

Annu Rev Genet 2011 9;45:227-46. Epub 2011 Sep 9.

Department of Biology, Washington University, St. Louis, Missouri 63130, USA.

Ciliated protozoa extensively remodel their somatic genomes during nuclear development, fragmenting their chromosomes and removing large numbers of internal eliminated sequences (IESs). The sequences eliminated are unique and repetitive DNAs, including transposons. Recent studies have identified transposase proteins that appear to have been domesticated and are used by these cells to eliminate DNA not wanted in the somatic macronucleus. This DNA elimination process is guided by meiotically produced small RNAs, generated in the germline nucleus, that recognize homologous sequences leading to their removal. These scan RNAs are found in complexes with PIWI proteins. Before they search the developing genome for IESs to eliminate, they scan the parental somatic nucleus and are removed from the pool if they match homologous sequences in that previously reorganized genome. In Tetrahymena, the scan RNAs target heterochromatin modifications to mark IESs for elimination. This DNA elimination pathway in ciliates shares extensive similarity with piRNA-mediated silencing of metazoans and highlights the remarkable ability of homologous RNAs to shape developing genomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-genet-110410-132432DOI Listing
February 2012

The conjugation-specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena.

Eukaryot Cell 2010 Jul 21;9(7):1087-99. Epub 2010 May 21.

Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47907-2063, USA.

Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena DeltaDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00379-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901671PMC
July 2010

Subtraction by addition: domesticated transposases in programmed DNA elimination.

Genes Dev 2009 Nov;23(21):2455-60

Biology Department, Washington University in St. Louis, St. Louis, Missouri 63130, USA.

The ciliate Paramecium tetraurelia must eliminate approximately 60,000 short sequences from its genome to generate uninterrupted coding sequences in its somatic macronucleus. In this issue of Genes & Development, Baudry and colleagues (pp. 2478-2483) identify the protein that excises these noncoding sequences: a domesticated piggyBac transposase that has been adapted to remove what are likely the remnants of transposon insertions. This new study reveals how addition of a transposase to small RNA-directed silencing machinery can guide major genome reorganization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.1864609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779744PMC
November 2009

Transposons that clean up after themselves.

Genome Biol 2009 15;10(6):224. Epub 2009 Jun 15.

Biology Department, Washington University in St, Louis, One Brookings Drive, St, Louis, MO 63130, USA.

A transposon in the germline genome of the ciliate Oxytricha uses its transposase to remove itself, as well as other germline-limited DNA, from the differentiating somatic genome during development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/gb-2009-10-6-224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2718489PMC
September 2009

Ciliate biology: dynamin goes nuclear.

Curr Biol 2008 Oct;18(19):R923-5

Biology Department, Washington University, St. Louis, Missouri 63130, USA.

Dynamin and dynamin-related proteins (DRPs) mediate an array of membrane fission processes. A Tetrahymena DRP has adopted a new role, assisting in nuclear differentiation, a finding that further highlights these proteins - and this ciliate - as biological innovators.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2008.07.080DOI Listing
October 2008

Dynamic nuclear reorganization during genome remodeling of Tetrahymena.

Biochim Biophys Acta 2008 Nov 28;1783(11):2130-6. Epub 2008 Jul 28.

Department of Biology, Washington University, St Louis, Missouri 63130, USA.

The single-celled ciliate Tetrahymena thermophila possesses two versions of its genome, one germline, one somatic, contained within functionally distinct nuclei (called the micronucleus and macronucleus, respectively). These two genomes differentiate from identical zygotic copies. The development of the somatic nucleus involves large-scale DNA rearrangements that eliminate 15 to 20 Mbp of their germline-derived DNA. The genomic regions excised are dispersed throughout the genome and are largely composed of repetitive sequences. These germline-limited sequences are targeted for removal from the genome by a RNA interference (RNAi)-related machinery that directs histone H3 lysine 9 and 27 methylation to their associated chromatin. The targeting small RNAs are generated in the micronucleus during meiosis and then compared against the parental macronucleus to further enrich for germline-limited sequences and ensure that only non-genic DNA segments are eliminated. Once the small RNAs direct these chromatin modifications, the DNA rearrangement machinery, including the chromodomain proteins Pdd1p and Pdd3p, assembles on these dispersed chromosomal sequences, which are then partitioned into nuclear foci where the excision events occur. This DNA rearrangement mechanism is Tetrahymena's equivalent to the silencing of repetitive sequences by the formation of heterochromatin. The dynamic nuclear reorganization that occurs offers an intriguing glimpse into mechanisms that shape nuclear architecture during eukaryotic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamcr.2008.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2588417PMC
November 2008

Nucleus-specific importin alpha proteins and nucleoporins regulate protein import and nuclear division in the binucleate Tetrahymena thermophila.

Eukaryot Cell 2008 Sep 1;7(9):1487-99. Epub 2008 Aug 1.

Biology Department, Washington University, One Brookings Drive, Campus Box 1137, St. Louis, MO 63130, USA.

The ciliate Tetrahymena thermophila, having both germ line micronuclei and somatic macronuclei, must possess a specialized nucleocytoplasmic transport system to import proteins into the correct nucleus. To understand how Tetrahymena can target proteins to distinct nuclei, we first characterized FG repeat-containing nucleoporins and found that micro- and macronuclei utilize unique subsets of these proteins. This finding implicates these proteins in the differential permeability of the two nuclei and implies that nuclear pores with discrete specificities are assembled within a single cell. To identify the import machineries that interact with these different pores, we characterized the large families of karyopherin homologs encoded within the genome. Localization studies of 13 putative importin (imp) alpha- and 11 imp beta-like proteins revealed that imp alpha-like proteins are nucleus specific--nine localized to the germ line micronucleus--but that most imp beta-like proteins localized to both types of nuclei. These data suggest that micronucleus-specific proteins are transported by specific imp alpha adapters. The different imp alpha proteins exhibit substantial sequence divergence and do not appear to be simply redundant in function. Disruption of the IMA10 gene encoding an imp alpha-like protein that accumulates in dividing micronuclei results in nuclear division defects and lethality. Thus, nucleus-specific protein import and nuclear function in Tetrahymena are regulated by diverse, specialized karyopherins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00193-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2547059PMC
September 2008

Lia1p, a novel protein required during nuclear differentiation for genome-wide DNA rearrangements in Tetrahymena thermophila.

Eukaryot Cell 2007 Aug 22;6(8):1320-9. Epub 2007 Jun 22.

Department of Biology, Campus Box 1137, Washington University, St. Louis, MO 63130, USA.

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 (DeltaLIA1) 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00157-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951122PMC
August 2007

Identification of novel chromatin-associated proteins involved in programmed genome rearrangements in Tetrahymena.

J Cell Sci 2007 Jun 22;120(Pt 12):1978-89. Epub 2007 May 22.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, and Department of Biology, Washington University, St. Louis, MO 63130, USA.

Extensive DNA rearrangements occur during the differentiation of the developing somatic macronuclear genome from the germ line micronuclear genome of Tetrahymena thermophila. To identify genes encoding proteins likely to be involved in this process, we devised a cytological screen to find proteins that specifically localize in macronuclear anlagen (Lia proteins) at the stage when rearrangements occur. We compared the localization of these with that of the chromodomain protein, Pdd1p, which is the most abundant known participant in this genome reorganization. We show that in live cells, Pdd1p exhibits dynamic localization, apparently shuttling from the parental to the developing nuclei through cytoplasmic bodies called conjusomes. Visualization of GFP-tagged Pdd1p also highlights the substantial three-dimensional nuclear reorganization in the formation of nuclear foci that occur coincident with DNA rearrangements. We found that late in macronuclear differentiation, four of the newly identified proteins are organized into nuclear foci that also contain Pdd1p. These Lia proteins are encoded by primarily novel genes expressed at the beginning of macronuclear differentiation and have properties or recognizable domains that implicate them in chromatin or nucleic acid binding. Three of the Lia proteins also localize to conjusomes, a result that further implicates this structure in the regulation of DNA rearrangement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.006502DOI Listing
June 2007

Genome evolution: a double take for Paramecium.

Curr Biol 2007 Feb;17(3):R97-9

Department of Biology, Washington University in St Louis, St. Louis, Missouri 63130, USA.

The surprising discovery of a whole-genome duplication in the otherwise compact genome of Paramecium tetraurelia displays the early forces driving gene retention and loss.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2006.12.002DOI Listing
February 2007