Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination.

Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena.

Bioinformatic and proteomic analysis of bulk histones reveals PTM crosstalk and chromatin features.

Systems analysis of chromatin has been constrained by complex patterns and dynamics of histone post-translational modifications (PTMs), which represent major challenges for both mass spectrometry (MS) and immuno-based approaches (e.g., chromatin immuno-precipitation, ChIP).

The nonhistone, N-terminal tail of an essential, chimeric H2A variant regulates mitotic H3-S10 dephosphorylation.

H2A.Y is an essential, divergent Tetrahymena thermophila histone variant. It has a long nonhistone N terminus that contains leucine-rich repeats (LRR) and an LRR cap domain with similarity to Sds22p, a regulator of yeast protein phosphatase 1 (PP1) activity in the nucleus.

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 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.

Tetrahymena Gene Expression Database (TGED): a resource of microarray data and co-expression analyses for Tetrahymena.

Tetrahymena thermophila is a model eukaryotic organism. Functional genomic analyses in Tetrahymena present rich opportunities to address fundamental questions of cell and molecular biology. The Tetrahymena Gene Expression Database (TGED; available at http://tged.

The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus.

Emerging evidence suggests that RNA interference (RNAi)-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination.

Histone H2B ubiquitylation is not required for histone H3 methylation at lysine 4 in tetrahymena.

Ubiquitylation of histone H2B and/or a component of the system that ubiquitylates H2B is required for methylation of histone H3 at lysine 4 (H3K4) in yeasts and probably in humans. In this study, the single ubiquitylation site was mapped to conserved lysine 115 of the C-terminal region of histone H2B in the single-cell model organism Tetrahymena thermophila. In strains lacking H2B ubiquitylation, H3K4 methylation was not detectably affected.

Two GW repeat proteins interact with Tetrahymena thermophila argonaute and promote genome rearrangement.

In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, approximately 28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp.

Nuclear dimorphism: two peas in a pod.

The macro- and micronuclei of Tetrahymena reside in the same cytoplasm but are about as different as night and day. This extreme case of nuclear dimorphism can now be partially attributed to differences in the subunit compositions of their nuclear pore complexes.

Microarray analyses of gene expression during the Tetrahymena thermophila life cycle.

Background: The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression.

Methodology/principal Findings: A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation.

Study of an RNA helicase implicates small RNA-noncoding RNA interactions in programmed DNA elimination in Tetrahymena.

Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction. Homology between the sequences to be eliminated and approximately 28-nucleotide small RNAs (scnRNAs) associated with an Argonaute family protein Twi1p likely underlies this elimination process. However, the mechanism by which Twi1p-scnRNA complexes identify micronuclear-limited sequences is not well understood.

Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.

Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules.

Tetrahymena IFT122A is not essential for cilia assembly but plays a role in returning IFT proteins from the ciliary tip to the cell body.

Intraflagellar transport (IFT) moves multiple protein particles composed of two biochemically distinct complexes, IFT-A and IFT-B, bi-directionally within cilia and is essential for cilia assembly and maintenance. We identified an ORF from the Tetrahymena macronuclear genome sequence, encoding IFT122A, an ortholog of an IFT-A complex protein. Tetrahymena IFT122A is induced during cilia regeneration, and epitope-tagged Ift122Ap could be detected in isolated cilia.

Different effects of Tetrahymena IFT172 domains on anterograde and retrograde intraflagellar transport.

Intraflagellar transport (IFT) particles are multiprotein complexes that move bidirectionally along the cilium/flagellum. The Tetrahymena IFT172 gene encodes a protein with an N-terminal WD domain (WDD) and a C-terminal repeat domain (RPD). Epitope-tagged Ift172p localized to the basal body and in cilia along the axoneme, and IFT172 knockout cells lost cilia and motility.

Phosphorylation of the SQ H2A.X motif is required for proper meiosis and mitosis in Tetrahymena thermophila.

Phosphorylation of the C terminus SQ motif that defines H2A.X variants is required for efficient DNA double-strand break (DSB) repair in diverse organisms but has not been studied in ciliated protozoa. Tetrahymena H2A.

Unphosphorylated H1 is enriched in a specific region of the promoter when CDC2 is down-regulated during starvation.

Tetrahymena thermophila macronuclear histone H1 is phosphorylated by a cdc2 kinase, and H1 phosphorylation regulates CDC2 expression by a positive feedback mechanism. In starved wild-type cells, decreased expression of the CDC2 gene is correlated with a global reduction in the phosphorylation of H1 and reduced phosphorylation of H1 in the region upstream of the CDC2 gene. To determine whether the reduced H1 phosphorylation upstream of the CDC2 gene is merely a reflection of global dephosphorylation or is due to specific targeting of dephosphorylation of H1 to the CDC2 promoter during starvation, the CDC2 promoter was mapped, and the distributions of phosphorylated and unphosphorylated H1 across the CDC2 gene were determined using chromatin immunoprecipitation.

Endoplasmic reticulum retention signal-dependent glycylation of the Hsp70/Grp170-related Pgp1p in Tetrahymena.

Glycylation is an uncommon posttranslational modification. It has been found that tubulin glycylation is essential for cell survival in Tetrahymena. Here we describe PGP1, a Tetrahymena gene encoding an Hsp70 homologue that is a novel glycylated protein.

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

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.

Deposition and function of histone H3 variants in Tetrahymena thermophila.

In Tetrahymena, HHT1 and HHT2 genes encode the same major histone H3; HHT3 and HHT4 encode similar minor H3 variants (H3s), H3.3 and H3.4.

Centromeric histone H3 is essential for vegetative cell division and for DNA elimination during conjugation in Tetrahymena thermophila.

The Tetrahymena thermophila CNA1 gene encodes the centromeric H3, Cna1p. Green fluorescent protein (GFP)-tagged Cna1p localizes in micronuclei in dots whose number and behavior during mitosis and conjugation are consistent with centromeres. During interphase, Cna1p-GFP localizes in peripheral dots, suggesting centromeres are associated with the nuclear envelope.

Mutational analyses reveal a novel function of the nucleotide-binding domain of gamma-tubulin in the regulation of basal body biogenesis.

We have used in vitro mutagenesis and gene replacement to study the function of the nucleotide-binding domain (NBD) of gamma-tubulin in Tetrahymena thermophila. In this study, we show that the NBD has an essential function and that point mutations in two conserved residues lead to over-production and mislocalization of basal body (BB) assembly. These results, coupled with previous studies (Dammermann, A.

The H1 phosphorylation state regulates expression of CDC2 and other genes in response to starvation in Tetrahymena thermophila.

In Tetrahymena thermophila, highly phosphorylated histone H1 of growing cells becomes partially dephosphorylated when cells are starved in preparation for conjugation. To determine the effects of H1 phosphorylation on gene expression, PCR-based subtractive hybridization was used to clone cDNAs that were differentially expressed during starvation in two otherwise-isogenic strains differing only in their H1s. H1 in A5 mutant cells lacked phosphorylation, and H1 in E5 cells mimicked constitutive H1 phosphorylation.

Elimination of foreign DNA during somatic differentiation in Tetrahymena thermophila shows position effect and is dosage dependent.

In the ciliate Tetrahymena thermophila, approximately 15% of the germ line micronuclear DNA sequences are eliminated during formation of the somatic macronucleus. The vast majority of the internal eliminated sequences (IESs) are repeated in the micronuclear genome, and several of them resemble transposable elements. Thus, it has been suggested that DNA elimination evolved as a means for removing invading DNAs.