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Nucleosome mapping of Tetrahymena thermophila chromatin in vivo and in vitro

ABSTRACT: A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream of transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from subsets of nucleosomes, rather than the whole array, being present in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes, and affects codon usage and amino acid composition in genes. We propose that these ‘seed’ nucleosomes may aid the AT-rich Tetrahymena genome – which is intrinsically unfavorable for nucleosome formation – in establishing nucleosome arrays in vivo in concert with trans-acting factors, while minimizing changes to the coding sequences they are embedded within.

ORGANISM(S): Tetrahymena thermophila

PROVIDER: GSE64061 | GEO | 2015/07/30

SECONDARY ACCESSION(S): PRJNA269988

REPOSITORIES: GEO

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Nucleosome mapping of Tetrahymena thermophila chromatin in vivo and in vitro

Project description:A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream of transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from subsets of nucleosomes, rather than the whole array, being present in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes, and affects codon usage and amino acid composition in genes. We propose that these ‘seed’ nucleosomes may aid the AT-rich Tetrahymena genome – which is intrinsically unfavorable for nucleosome formation – in establishing nucleosome arrays in vivo in concert with trans-acting factors, while minimizing changes to the coding sequences they are embedded within. All data are from the macronuclear genome. Datasets: 1) Log-phase cells, fixed chromatin, light MNase digest; 2) Log-phase cells, native chromatin, heavy MNase digest; 3) Starved cells, fixed chromatin, light MNase digest; 4) Starved cells, native chromatin, heavy MNase digest; 5) in vitro reconstituted chromatin, 50ul reaction, 4:10 histone:DNA ratio, light MNase digest; 6) in vitro reconstituted chromatin, 50ul reaction, 7:10 histone:DNA ratio, light MNase digest; 7) in vitro reconstituted chromatin, 150ul reaction, 4:10 histone:DNA ratio, light MNase digest; 8) in vitro reconstituted chromatin, 150ul reaction, 4:10 histone:DNA ratio, heavy MNase digest; Control dataset: 9): MNase-digested naked DNA

2015-07-30 | E-GEOD-64061 | biostudies-arrayexpress

Characterization of the CORVET complexes of Tetrahymena thermophila

Project description:In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The ciliate Tetrahymena thermophila is a unicellular organism with elaborate endolysosomal pathways. Curiously, Tetrahymena and related protozoa have lost HOPS. Tetrahymena encodes multiple paralogs of most CORVET subunits, assembled into six distinct hexameric complexes, including a distinct Vps8 subunit. Here we analyze the composition of these complexes immunoisolated by affinity capture using the Vps8-FLAG paralogs as bait.

2019-12-19 | PXD014895 | Pride

RNA-seq analysis of Tetrahymena thermophila

Project description:We present a comprehensive transcriptome of ciliate T. thermophila using the Illumina RNA-seq platform. The data was generated from the six mRNA samples of growth, starvation and conjugation of Tetrahymena. Despite an AT rich genome, there are about 124.6 million reads mapped to T. thermophila genome. Using these mapped reads, we have significantly improved the previous genome annotation and investigated the gene expression. Besides, our result also provided a comprehensive understanding of the alternative splicing in T. thermophila, and suggested the existence of the regulated unproductive splicing and translation (RUST) in the single-celled eukaryote. RNA-seq for six samples of Tetrahymena growth, starvation and conjugation.

2012-03-15 | E-GEOD-27971 | biostudies-arrayexpress

Tetrahymena thermophila

Project description:Nucleosome mapping of Tetrahymena thermophila chromatin in vivo and in vitro

| PRJNA269988 | ENA

RNA-seq analysis of Tetrahymena thermophila

2012-03-15 | GSE27971 | GEO

A single fiber view of the nucleosome organization of eukaryotic chromatin

Project description:Nucleosomes arrange into extended arrays, much like beads on a string. They are often phased at genomic landmarks and are thought to be evenly spaced. Here we tested to what extent this stereotypic organization describes the nucleosome landscape in Saccharomyces cerevisiae using a long-read nucleosome-sequencing technique called Fiber-Seq. Fiber-Seq maps the nucleosome pattern on individual chromatin fibers. As such, it is ideally suited to measure the density of nucleosomes per read and quantitate the nucleosome occupancy throughout the genome. We document substantial deviations from the stereotypical nucleosome organization, with unexpectedly long linker DNAs between individual nucleosomes, genomic regions lacking entire nucleosomes, heterogeneous phasing of arrays, truly irregular spacing of arrays and read-to-read variation in nucleosome densities. We exploited the technology to test mechanistic models for the biogenesis of nucleosome arrays. We can rule out transcription elongation playing a decisive role in array formation and detect signatures for a clamping activity of remodelers of the ISWI and CHD1 families after acute nucleosome depletion in vivo. Given that nucleosomes are cis-regulatory elements, the cell-to-cell heterogeneity that Fiber-Seq uncovers provides much needed information to understand chromatin structure and function.

2024-01-17 | GSE224713 | GEO

N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA

Project description:N6-methyldeoxyadenosine (6mA or m6dA) was shown more than 40 years ago to exist in simple eukaryotes, yet functional studies have been limited. Recent investigations in multiple eukaryotes suggest 6mA as a potential DNA epigenetic mark that plays regulatory roles in gene regulation. Here we use Tetrahymena thermophila as a model to examine the effects of 6mA on nucleosome positioning. We have employed independent methods to identify genome-wide 6mA distribution, which revealed the enrichment after transcription start sites with a periodic pattern and a mutually exclusive relationship with the positions of nucleosomes. The exclusive distribution pattern of 6mA and nucleosome can be recapitulated by in vitro nucleosome assembly on native Tetrahymena genomic DNA, but not on DNA without 6mA. Model DNA containing artificially installed 6mA resists nucleosome assembling compared to unmodified DNA in vitro. Computational simulation revealed that 6mA increases dsDNA rigidity, which disfavors nucleosome wrapping. Knockout of a potential 6mA methyltransferase disturbs the nucleosome positioning in Tetrahymena, leading to the transcriptome-wide change of gene expression. These findings uncover a new mechanism by which DNA 6mA assists to shape the chromatin topology in order to stabilize gene expression.

2018-10-17 | GSE104699 | GEO

Sequence, Biogenesis, and Function of Diverse sRNA Classes Bound to the Piwi-family Proteins of Tetrahymena thermophila

Project description:PAZ/PIWI-domain (PPD) proteins carrying small RNAs (sRNAs) function in gene and genome regulation. The ciliate Tetrahymena thermophila encodes numerous PPD proteins exclusively of the Piwi clade. We show that the three Tetrahymena Piwi-family proteins (Twis) preferentially expressed in growing cells differ in their genetic essentiality and subcellular localization. Affinity purification of all eight distinct Twi proteins revealed unique properties of their bound sRNAs. Deep sequencing of Twi-bound and total sRNAs in strains disrupted for various silencing machinery uncovered an unanticipated diversity of 23-24 nt sRNA classes in growing cells, each with distinct genetic requirements for accumulation. Altogether Twis distinguish sRNAs derived from loci of pseudogene families, three types of DNA repeats, structured RNAs, and EST-supported loci with convergent or paralogous transcripts. Most surprisingly, Twi7 binds complementary strands of unequal length while Twi10 binds a specific permutation of the guanosine-rich telomeric repeat. These studies greatly expand the structural and functional repertoire of endogenous sRNAs and RNPs. 11 libraries were analyzed. 5 are total sRNA (from knockout strains or WT background strains), and 6 are Argonaute-bound sRNA

2009-11-23 | E-GEOD-17006 | biostudies-arrayexpress

Sequence, Biogenesis, and Function of Diverse sRNA Classes Bound to the Piwi-family Proteins of Tetrahymena thermophila

2009-11-23 | GSE17006 | GEO

Characterization of a novel membrane complex required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila

Project description:In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon termed regulated exocytosis. A forward genetic approach uncovered MDL1 (Mucocyst Discharge with a LamG domain) as essential gene for regulated exocytosis of mucocysts. The encoded protein, Mdl1p, is a 66 kD membrane-associated glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. Immunoisolation by affinity capture using Mdl1p-FLAG as bait, described here, revealed that Mdl1p forms a complex with 9 other proteins, all of them novel and largely restricted to Alveolates.

2022-04-06 | PXD028372 | Pride

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