Publications by authors named "Robert S Coyne"

16 Publications

  • Page 1 of 1

Hidden genomic evolution in a morphospecies-The landscape of rapidly evolving genes in Tetrahymena.

PLoS Biol 2019 06 3;17(6):e3000294. Epub 2019 Jun 3.

Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.

A morphospecies is defined as a taxonomic species based wholly on morphology, but often morphospecies consist of clusters of cryptic species that can be identified genetically or molecularly. The nature of the evolutionary novelty that accompanies speciation in a morphospecies is an intriguing question. Morphospecies are particularly common among ciliates, a group of unicellular eukaryotes that separates 2 kinds of nuclei-the silenced germline nucleus (micronucleus [MIC]) and the actively expressed somatic nucleus (macronucleus [MAC])-within a common cytoplasm. Because of their very similar morphologies, members of the Tetrahymena genus are considered a morphospecies. We explored the hidden genomic evolution within this genus by performing a comprehensive comparative analysis of the somatic genomes of 10 species and the germline genomes of 2 species of Tetrahymena. These species show high genetic divergence; phylogenomic analysis suggests that the genus originated about 300 million years ago (Mya). Seven universal protein domains are preferentially included among the species-specific (i.e., the youngest) Tetrahymena genes. In particular, leucine-rich repeat (LRR) genes make the largest contribution to the high level of genome divergence of the 10 species. LRR genes can be sorted into 3 different age groups. Parallel evolutionary trajectories have independently occurred among LRR genes in the different Tetrahymena species. Thousands of young LRR genes contain tandem arrays of exactly 90-bp exons. The introns separating these exons show a unique, extreme phase 2 bias, suggesting a clonal origin and successive expansions of 90-bp-exon LRR genes. Identifying LRR gene age groups allowed us to document a Tetrahymena intron length cycle. The youngest 90-bp exon LRR genes in T. thermophila are concentrated in pericentromeric and subtelomeric regions of the 5 micronuclear chromosomes, suggesting that these regions act as genome innovation centers. Copies of a Tetrahymena Long interspersed element (LINE)-like retrotransposon are very frequently found physically adjacent to 90-bp exon/intron repeat units of the youngest LRR genes. We propose that Tetrahymena species have used a massive exon-shuffling mechanism, involving unequal crossing over possibly in concert with retrotransposition, to create the unique 90-bp exon array LRR genes.
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http://dx.doi.org/10.1371/journal.pbio.3000294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6564038PMC
June 2019

RNAi-dependent repression controls transposable elements in .

Genes Dev 2019 03 26;33(5-6):348-364. Epub 2019 Feb 26.

Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA.

RNAi and repression play evolutionarily conserved and often coordinated roles in transcriptional silencing. Here, we show that, in the protozoan , germline-specific internally eliminated sequences (IESs)-many related to transposable elements (TEs)-become transcriptionally activated in mutants deficient in the RNAi-dependent repression pathway. Germline TE mobilization also dramatically increases in these mutants. The transition from noncoding RNA (ncRNA) to mRNA production accompanies transcriptional activation of TE-related sequences and vice versa for transcriptional silencing. The balance between ncRNA and mRNA production is potentially affected by cotranscriptional processing as well as RNAi and repression. We posit that interplay between RNAi and repression is a widely conserved phenomenon, whose ancestral role is epigenetic silencing of TEs.
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http://dx.doi.org/10.1101/gad.320796.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411011PMC
March 2019

Genomics: Stentor's Trumpet Sounds Anew.

Authors:
Robert S Coyne

Curr Biol 2017 02;27(4):R146-R148

J. Craig Venter Institute, 9714 Medical Center Drive, Rockville, MD 20850, USA. Electronic address:

The amazing regenerative abilities of the giant ciliate Stentor coeruleus made it a favorite subject for classical embryologists. Now, its genome has been sequenced, enabling renewed experimental study and revealing unexpected surprises in mRNA splicing and the genetic code.
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http://dx.doi.org/10.1016/j.cub.2016.12.059DOI Listing
February 2017

Structure of the germline genome of and relationship to the massively rearranged somatic genome.

Elife 2016 11 28;5. Epub 2016 Nov 28.

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, United States.

The germline genome of the binucleated ciliate 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 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 '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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http://dx.doi.org/10.7554/eLife.19090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5182062PMC
November 2016

Depletion of UBC9 Causes Nuclear Defects during the Vegetative and Sexual Life Cycles in Tetrahymena thermophila.

Eukaryot Cell 2015 Dec 9;14(12):1240-52. Epub 2015 Oct 9.

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

Ubc9p is the sole E2-conjugating enzyme for SUMOylation, and its proper function is required for regulating key nuclear events such as transcription, DNA repair, and mitosis. In Tetrahymena thermophila, the genome is separated into a diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid somatic macronucleus (MAC) that divides amitotically. This unusual nuclear organization provides novel opportunities for the study of SUMOylation and Ubc9p function. We identified the UBC9 gene and demonstrated that its complete deletion from both MIC and MAC genomes is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with CdCl2-dependent expression of green fluorescent protein (GFP)-Ubc9p. Depletion of Ubc9p in vegetative cells resulted in the loss of MICs, but MACs continued to divide. In contrast, expression of catalytically inactive Ubc9p resulted in the accumulation of multiple MICs. Critical roles for Ubc9p were also identified during the sexual life cycle of Tetrahymena. Cell lines that were depleted for Ubc9p did not form mating pairs and therefore could not complete any of the subsequent stages of conjugation, including meiosis and macronuclear development. Mating between cells expressing catalytically inactive Ubc9p resulted in arrest during macronuclear development, consistent with our observation that Ubc9p accumulates in the developing macronucleus.
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http://dx.doi.org/10.1128/EC.00115-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664876PMC
December 2015

Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis.

Elife 2014 Aug 26;3:e02996. Epub 2014 Aug 26.

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, United States Center for Epigenetics, The Johns Hopkins University School of Medicine, Baltimore, United States

Despite the well-established role of heterochromatin in protecting chromosomal integrity during meiosis and mitosis, the contribution and extent of heterochromatic histone posttranslational modifications (PTMs) remain poorly defined. Here, we gained novel functional insight about heterochromatic PTMs by analyzing histone H3 purified from the heterochromatic germline micronucleus of the model organism Tetrahymena thermophila. Mass spectrometric sequencing of micronuclear H3 identified H3K23 trimethylation (H3K23me3), a previously uncharacterized PTM. H3K23me3 became particularly enriched during meiotic leptotene and zygotene in germline chromatin of Tetrahymena and C. elegans. Loss of H3K23me3 in Tetrahymena through deletion of the methyltransferase Ezl3p caused mislocalization of meiosis-induced DNA double-strand breaks (DSBs) to heterochromatin, and a decrease in progeny viability. These results show that an evolutionarily conserved developmental pathway regulates H3K23me3 during meiosis, and our studies in Tetrahymena suggest this pathway may function to protect heterochromatin from DSBs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4141274PMC
http://dx.doi.org/10.7554/eLife.02996DOI Listing
August 2014

Whole genome studies of Tetrahymena.

Methods Cell Biol 2012 ;109:53-81

Department of Genomic Medicine, J. Craig Venter Institute, Rockville, MD, USA.

Within the past decade, genomic studies have emerged as essential and highly productive tools to explore the biology of Tetrahymena thermophila. The current major resources, which have been extensively mined by the research community, are the annotated macronuclear genome assembly, transcriptomic data and the databases that house this information. Efforts in progress will soon improve these data sources and expand their scope, including providing annotated micronuclear and comparative genomic sequences. Future studies of Tetrahymena cell and molecular biology, development, physiology, evolution and ecology will benefit greatly from these resources and the advanced genomic technologies they enable.
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http://dx.doi.org/10.1016/B978-0-12-385967-9.00004-9DOI 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.
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http://dx.doi.org/10.1534/g3.111.000927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3276166PMC
November 2011

RNA-guided DNA rearrangements in ciliates: is the best genome defence a good offence?

Biol Cell 2012 Jun 18;104(6):309-25. Epub 2012 Apr 18.

J. Craig Venter Institute, Rockville, MD 20850, USA.

Genomes, like crazy patchwork quilts, are stitched together over evolutionary time from diverse elements, including some unwelcome invaders. To deal with parasitic mobile elements, most eukaryotes employ a genome self-defensive manoeuvre to recognise and silence such elements by homology-dependent interactions with RNA-protein complexes that alter chromatin. Ciliated protozoa employ more 'offensive' tactics by actually unstitching and reassembling their somatic genomes at every sexual generation to eliminate transposons and their remnants, using as patterns the maternal genomes that were rearranged in the previous cycle. Genetic and genomic studies of the distant relatives Paramecium and Tetrahymena have begun to reveal how such events are carried out with remarkable precision. Whole genome, non-coding transcripts from the maternal genome are compared with transcripts from the zygotic genome that are processed through an RNA interference (RNAi)-related process. Sequences found only in the latter are targeted for elimination by the resulting short 'scanRNAs' in many thousand DNA splicing reactions initiated by a domesticated transposase. The involvement of widely conserved mechanisms and protein factors clearly shows the relatedness of these phenomena to RNAi-mediated heterochromatic gene silencing. Such malleability of the genome on a generational time scale also has profound evolutionary implications, possibly including the epigenetic inheritance of acquired adaptive traits.
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http://dx.doi.org/10.1111/boc.201100057DOI Listing
June 2012

Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control.

Genome Biol 2011 Oct 17;12(10):R100. Epub 2011 Oct 17.

Genomic Medicine, J Craig Venter Institute, Rockville, MD 20850, USA.

Background: Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism.

Results: We report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. thermophila, the Ich genome is reduced approximately two-fold in length and gene density and three-fold in gene content. We analyzed in detail several gene classes with diverse functions in behavior, cellular function and host immunogenicity, including protein kinases, membrane transporters, proteases, surface antigens and cytoskeletal components and regulators. We also mapped by orthology Ich's metabolic pathways in comparison with other ciliates and a potential host organism, the zebrafish Danio rerio.

Conclusions: Knowledge of the complete protein-coding and metabolic potential of Ich opens avenues for rational testing of therapeutic drugs that target functions essential to this parasite but not to its fish hosts. Also, a catalog of surface protein-encoding genes will facilitate development of more effective vaccines. The potential to use T. thermophila as a surrogate model offers promise toward controlling 'white spot' disease and understanding the adaptation to a parasitic lifestyle.
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http://dx.doi.org/10.1186/gb-2011-12-10-r100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3341644PMC
October 2011

Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure.

BMC Genomics 2008 Nov 26;9:562. Epub 2008 Nov 26.

J. Craig Venter Institute (formerly The Institute for Genomic Research), 9704 Medical Center Dr., Rockville, MD, USA.

Background: Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.

Results: We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.

Conclusion: We report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.
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http://dx.doi.org/10.1186/1471-2164-9-562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612030PMC
November 2008

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

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote.

PLoS Biol 2006 Sep;4(9):e286

The Institute for Genomic Research, Rockville, Maryland, United States of America.

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.
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http://dx.doi.org/10.1371/journal.pbio.0040286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1557398PMC
September 2006

The condensin complex is essential for amitotic segregation of bulk chromosomes, but not nucleoli, in the ciliate Tetrahymena thermophila.

Mol Cell Biol 2006 Jun;26(12):4690-700

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

The macronucleus of the binucleate ciliate Tetrahymena thermophila contains fragmented and amplified chromosomes that do not have centromeres, eliminating the possibility of mitotic nuclear division. Instead, the macronucleus divides by amitosis with random segregation of these chromosomes without detectable chromatin condensation. This amitotic division provides a special opportunity for studying the roles of mitotic proteins in segregating acentric chromatin. The Smc4 protein is a core component of the condensin complex that plays a role in chromatin condensation and has also been associated with nucleolar segregation, DNA repair, and maintenance of the chromatin scaffold. Mutants of Tetrahymena SMC4 have remarkable characteristics during amitosis. They do not form microtubules inside the macronucleus as normal cells do, and there is little or no bulk DNA segregation during cell division. Nevertheless, segregation of nucleoli to daughter cells still occurs, indicating the independence of this process and bulk DNA segregation in ciliate amitosis.
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http://dx.doi.org/10.1128/MCB.02315-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489118PMC
June 2006

Functional characterization of BRCA1 sequence variants using a yeast small colony phenotype assay.

Cancer Biol Ther 2004 May 18;3(5):453-7. Epub 2004 May 18.

Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia, USA.

Germline mutations that inactivate the tumor suppressor gene BRCA1 are associated with an increased risk of cancers of the breast and other tissues, but the functional consequence of many missense variants found in the human population is uncertain. Several predictive methods have been proposed to distinguish cancer-predisposing missense mutations from harmless polymorphisms, including a small colony phenotype (SCP) assay performed in the model organism, yeast (Saccharomyces cerevisiae). The goal of this study was to further evaluate this colony size assay. We constructed 28 missense mutations throughout the C-terminal 305 amino acid residues of BRCA1. Mutated proteins were expressed in yeast and evaluated using the SCP assay. We conclude there is as yet no evidence the assay can identify inactivating mutations upstream of the BRCT repeats. However, within and between the BRCT repeats, results of the assay are in general agreement with predictions based on structural modeling, other in vitro and in vivo assays, and cross-species sequence conservation. Thus, the yeast assay appears to provide confirmatory in vivo evidence to aid in characterizing some BRCA1 missense variants.
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http://dx.doi.org/10.4161/cbt.3.5.809DOI Listing
May 2004

Methylation of histone h3 at lysine 9 targets programmed DNA elimination in tetrahymena.

Cell 2002 Sep;110(6):701-11

Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA 22908, USA.

Histone H3 lysine 9 methylation [Me(Lys9)H3] is an epigenetic mark for heterochromatin-dependent gene silencing, mediated by direct binding to chromodomain-containing proteins such as Heterochromatin Protein 1. In the ciliate Tetrahymena, two chromodomain proteins, Pdd1p and Pdd3p, are involved in the massive programmed DNA elimination that accompanies macronuclear development. We report that both proteins bind H3(Lys9)Me in vitro. In vivo, H3(Lys9)Me is confined to the time period and location where DNA elimination occurs, and associates with eliminated sequences. Loss of parental Pdd1p expression drastically reduces H3(Lys9)Me. Finally, tethering Pdd1p is sufficient to promote DNA excision. These results extend the range of H3(Lys9)Me involvement in chromatin activities outside transcriptional regulation and also strengthen the link between heterochromatin formation and programmed DNA elimination.
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http://dx.doi.org/10.1016/s0092-8674(02)00941-8DOI Listing
September 2002