Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation

Liu

et al. 2016

Sci. China Life Sci.

Self Cite

Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus (MIC) and macronucleus (MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease (MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of ~200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.

Section: Illumina Sequencing and Data Processingmentioning

confidence: 99%

Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila

Chen

Liu

et al. 2016

Sci. China Life Sci.

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“…More cryptic species were subsequently discovered and currently the T. pyriformis complex comprises 18 micronucleate and 4 amicronucleate species (Feng et al 1988;Jerome et al 1996;Nanney et al 1980;Nyberg 1981;Simon et al 1985). Species in the T. pyriformis complex, such as Tetrahymena thermophila, are now used as model organisms for research in a wide range of fields including cell biology, epigenetics, and molecular ecology (Been and Cech 1986;Cervantes et al 2015;Cherry and Blackburn 1985;Chung and Yao 2012;Gao et al 2013;Liu et al 2007;Mochizuki et al 2002;Nanney 1953). The former Syngen 11 of the T. pyriformis complex was designated Tetrahymena australis, mainly according to its geographical distribution (Elliott 1970(Elliott , 1973, although it was subsequently found to be globally distributed (Simon et al 1985(Simon et al , 2008.…”

Section: Introductionmentioning

confidence: 99%

Tetrahymena australis (Protozoa, Ciliophora): A Well‐Known But “Non‐Existing” Taxon – Consideration of Its Identification, Definition and Systematic Position

Liu

Fan

J Eukaryotic Microbiology

et al. 2016

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A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.A wide variety of protists, including hymenostome ciliates, have relatively simple body structures with few morphological characters for species circumscription, which makes them difficult to separate from each other (Borden et al. 1977;Simon et al. 2008). Early taxonomists identified and classified such organisms based only on morphological and cell cycle characters. As a consequence, independent species were sometimes misidentified as morphologically similar species within a morphospecies complex (for instance, Paramecium aurelia). The development of modern methods, especially gene sequencing and phylogenetic analyses, has improved this situation and the taxonomic positions of several such species have been clarified in recent years (Quintela-Alonso et al. 2013;Zhao et al. 2016).Cryptic species of ciliates were first discovered by Sonneborn (1939) for the model organism P. aurelia Ehrenberg, 1838. Cryptic species have since been found in a variety of ciliate genera including Aspidisca, Colpidium, Euplotes, Glaucoma, Oxytricha, Paramecium, Pseudourostyla, Stylonychia, Tetrahymena, Tokophrya, and Uronychia (Dini and Nyberg 1993;Przybos 1986;Simon et al. 2008). The probable wide occurrence of cryptic speciation has been cited as evidence for the underestimation of ciliate species diversity (Foissner et al. 2007).Tetrahymena pyriformis (Ehrenberg, 1830) Lwoff, 1947 is another example now known to be a species complex (Nanney and McCoy 1976). Species of Tetrahymena were previously assigned to the genera Leucophyrs or Glaucoma until the establishment of the genus Tetrahymena by Furgason (1940). Using mating type tests, Gruchy (1955) first discovered the heterogeneity of T. pyriformis by describin...

“…Construction of HMT Knockout Strains-HMT mutants, ⌬TXR1 and ⌬EZL2, were generated from Tetrahymena wild-type CU428 cells as described previously (18). Briefly, genomic sequences flanking TXR1 or EZL2 were PCR amplified and fused with the neo4 cassette, which confers paromomycin resistance (20).…”

Section: Methodsmentioning

confidence: 99%

“…Cell Culture, Core Histone Preparation, and HPLC PurificationMedia, cell culture conditions, and procedures used for nuclear preparation, acid extraction of histone, and histone purification were as described previously (17,18). The overall experimental design is illustrated in supplemental Fig.…”

Section: Methodsmentioning

confidence: 99%

“…In vegetatively growing cells, only TXR1 and EZL2 are significantly expressed. Our previous studies show that they jointly regulate H3K27 methylation at this physiological stage: TXR1 preferentially deposits H3K27me1 and affects replication elongation, whereas EZL2 is mainly responsible for H3K27me2/3 and transcription repression (18). Although H3K27 methylation is extensively studied, its relationship with other histone PTMs has not been systematically explored.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Quantitative Proteomics Reveals Histone Modifications in Crosstalk with H3 Lysine 27 Methylation

Zhang

Molecular & Cellular Proteomics

Molascon

et al. 2014

Self Cite

Methylation at histone H3 lysine 27 (H3K27me) is an evolutionarily conserved epigenetic mark associated with transcriptional repression and replication elongation. We have previously shown that in Tetrahymena thermophila, a unicellular eukaryote, the histone methyltransferases (HMTs) TXR1 and EZL2 are primarily responsible for H3K27 mono-methylation (H3K27me1) and di-/tri-methylation (H3K27me2/3), respectively. In eukaryotic cells, nuclear DNA wraps around an octamer of two copies each of the four core histones H2A, H2B, H3, and H4, to form the nucleosome, the basic unit of chromatin. Many post-translational modifications (PTMs) 1 , including methylation, acetylation, phosphorylation, biotinylation, citrullination, ADP-ribosylation, and ubiquitylation, occur at numerous sites on histones (1). Dynamic changes in the chromatin structure and architecture, including the switch between condensed and decondensed states as well as interactions with a wide range of protein complexes, are modulated by these PTMs, deposited by histone modifying enzymes in a combinatorial pattern that is still being actively deciphered (2, 3). Reflecting its crucial role in DNA-mediated transactions, the functionality of chromatin is relatively robust, tolerating substantial mutations in histone residues carrying PTMs as well as histone modifying enyzmes (4). The robustness is generally attributed to redundant or parallel roles played by many histone modifications, which often adapt compensatory changes to these perturbations. However, details of the crosstalk among histone PTMs and their physiological impacts are still incomplete (5). Deposited by histone methyltransferases (HMTs) and removed by demethylases, histone lysine methylation (mono-, di-, and tri-methylated forms) is a dynamic epigenetic mark (6 -8). Boosted by the identification and characterization of the enzymatic machinery, histone lysine methylation has been the target of considerable research efforts, reflecting its key role in modulating histone functions. Unlike acetylation, methylation does not directly affect histone/DNA interactions by altering the net charge of histones, and its physiological impacts depend on the exact form and site of modification, as well as the effector proteins recognizing them (9).The evolutionarily conserved H3K27 methylation has been traditionally associated with heterochromatin formation and transcriptional repression (10). Enhancer of zeste (E(z)), initially identified by its role in Polycomb repression, is the HMT required for H3K27 methylation in Drosophila (11-13). E(z) homologs are found in protozoa, metazoa, and plants, though they are conspicuously missing in fungi. All three forms of H3K27 methylation (H3K27me1/2/3) are catalyzed by E(z) homologs in metazoa (14). However, in protozoa and plants, a different family of HMTs are specific for H3K27me1, examplified by Arabidopsis ATXR5/ATXR6 (15).As a protozoan model organism, Tetrahymena thermophila features high levels of H3K27 methylation and several H3K27-