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. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Furthermore, the endoribonuclease (Slicer) activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated small interfering RNAs (siRNAs), leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.

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. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

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. In growing cells, H2A.Y is incorporated into micronuclei only during S phase, which occurs immediately after micronuclear mitosis. Depletion of H2A.Y causes prolonged retention of mitosis-associated histone H3-S10 phosphorylation and mitotic abnormalities that mimic S10E mutation. In cells where H2A.Y is depleted, an inducible chimeric gene, in which the H2A.Y N terminus is attached to H2A.X, is shown to regulate micronuclear H3-S10 phosphorylation. H2A.Y can also be specifically coimmunoprecipitated with a Tetrahymena PP1 ortholog (Ppo1p). Taken together, these results argue that the N terminus of H2A.Y functions to regulate H3-S10 dephosphorylation. This striking in vivo case of "cross-talk" between a H2A variant and a specific post-translational modification of another histone demonstrates a novel function for a histone variant.

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). Here we present a proof-of-concept study demonstrating that crosstalk among PTMs and their functional significance can be revealed via systematic bioinformatic and proteomic analysis of steady-state histone PTM levels from cells under various perturbations. Using high resolution tandem MS, we quantified 53 modification states from all core histones and their conserved variants in the unicellular eukaryotic model organism Tetrahymena. By correlating histone PTM patterns across 15 different conditions, including various physiological states and mutations of key histone modifying enzymes, we identified 5 specific chromatin states with characteristic covarying histone PTMs and associated them with distinctive functions in replication, transcription, and DNA repair. In addition to providing a detailed picture on histone PTM crosstalk at global levels, this work has established a novel bioinformatic and proteomic approach, which can be adapted to other organisms and readily scaled up to allow increased resolution of chromatin states.

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. As in other organisms, the E2 ubiquitin-conjugating enzyme Ubc2 and the E3 ubiquitin ligase Bre1 were required for H2B ubiquitylation. However, neither enzyme was required for H3K4 methylation. These studies argue that, in T. thermophila, the histone ubiquitylation mechanism is not required for H3K4 methylation, demonstrating that different organisms can speak different languages in the "cross-talk" among post-translational modifications on different histones.

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., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and K. Oegema. 2004. Dev. Cell. 7:815-829; La Terra, S., C.N. English, P. Hergert, B.F. McEwen, G. Sluder, and A. Khodjakov. 2005. J. Cell Biol. 168:713-722), suggest that to achieve the precise temporal and spatial regulation of BB/centriole assembly, the initiation activity of gamma-tubulin is normally suppressed by a negative regulatory mechanism that acts through its NBD.

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. Unphosphorylated H1 is specifically enriched in a region of the CDC2 promoter when the gene's expression is reduced during starvation but not when CDC2 is highly active in growing cells. The region of unphosphorylated H1 coincides with a region that is essential for CDC2 expression. These studies are the first in vivo demonstration that the phosphorylation of H1 is being regulated at a fine level and that unphosphorylated H1 can be specifically targeted to a promoter, where it likely regulates transcription in a gene-specific manner.

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.X is one of two similarly expressed major H2As, thereby differing both from mammals, where H2A.X is a quantitatively minor component, and from Saccharomyces cerevisiae where it is the only type of major H2A. Tetrahymena H2A.X is phosphorylated in the SQ motif in both the mitotic micronucleus and the amitotic macronucleus in response to DSBs induced by chemical agents and in the micronucleus during prophase of meiosis, which occurs in the absence of a synaptonemal complex. H2A.X is phosphorylated when programmed DNA rearrangements occur in developing macronuclei, as for immunoglobulin gene rearrangements in mammals, but not during the DNA fragmentation that accompanies breakdown of the parental macronucleus during conjugation, correcting the previous interpretation that this process is apoptosis-like. Using strains containing a mutated (S134A) SQ motif, we demonstrate that phosphorylation of this motif is important for Tetrahymena cells to recover from exogenous DNA damage and is required for normal micronuclear meiosis and mitosis and, to a lesser extent, for normal amitotic macronuclear division; its absence, while not lethal, leads to the accumulation of DSBs in both micro- and macronuclei. These results demonstrate multiple roles of H2A.X phosphorylation in maintaining genomic integrity in different phases of the Tetrahymena life cycle.

Tetrahymena thermophila contains a conventional gamma-tubulin that is differentially required for the maintenance of different microtubule-organizing centers.

The gene (GTU1) encoding Tetrahymena thermophila gamma-tubulin was cloned and analyzed. GTU1 is a single-copy, essential gene encoding a conventional gamma-tubulin. HA-tagged GTU1p localizes to four microtubule-organizing centers (MTOCs) in vegetative cells: basal bodies (BBs), macronuclear envelopes, micronuclear envelopes, and contractile vacuole pores. gamma-Tubulin function was studied by placing the GTU1 gene under control of an inducible-repressible promoter. Overexpression of GTU1 had no detectable effect on cell growth or morphology. Depletion of gamma-tubulin resulted in marked changes in cell morphology and in MT bundling. MTOCs showed different sensitivities to gamma-tubulin depletion, with BBs being the most sensitive. gamma-Tubulin was required not only for the formation of new BBs but also for maintenance of mature BBs. BBs disappeared in stages, first losing gamma-tubulin and then centrin and glutamylated tubulin. When GTU1 expression was reinduced in depleted cells, BBs reformed rapidly, and the normal, highly organized structure of the Tetrahymena cell cortex was reestablished, indicating that the precise patterning of the cortex can be formed de novo.

Phosphorylation and an ATP-dependent process increase the dynamic exchange of H1 in chromatin.

In Tetrahymena cells, phosphorylation of linker histone H1 regulates transcription of specific genes. Phosphorylation acts by creating a localized negative charge patch and phenocopies the loss of H1 from chromatin, suggesting that it affects transcription by regulating the dissociation of H1 from chromatin. To test this hypothesis, we used FRAP of GFP-tagged H1 to analyze the effects of mutations that either eliminate or mimic phosphorylation on the binding of H1 to chromatin both in vivo and in vitro. We demonstrate that phosphorylation can increase the rate of dissociation of H1 from chromatin, providing a mechanism by which it can affect H1 function in vivo. We also demonstrate a previously undescribed ATP-dependent process that has a global effect on the dynamic binding of linker histone to chromatin.

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. 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.

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. Newly synthesized Cna1p-GFP enters micronuclei in mitosis and accumulates in the nucleoplasm. Its deposition at centromeres starts at early S phase and continues through most of S phase. CNA1 is required for vegetative cell growth. Knockdown of CNA1 genes in the somatic macronucleus results in micronuclear DNA loss and delayed chromosome segregation during mitosis. During conjugation, Cna1p-GFP disappears from the centromeres in the developing macronucleus, consistent with centromeric sequences being internal eliminated sequences. Surprisingly, zygotic CNA1 is required for efficient elimination of germ line-specific sequences during development of the new macronuclei but not for the RNA interference pathway, through which sequences are targeted for elimination. Zygotically expressed Cna1p localizes in the spherical structures in which the later stages of DNA elimination occur, and these structures cannot be formed in the absence of zygotic CNA1, suggesting that, in addition to functioning in centromeres, Cna1p may also play a role in organizing the formation of the DNA elimination structures.

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. Green fluorescent protein (GFP)-tagged H3 is deposited onto chromatin through a DNA replication-coupled (RC) pathway. GFP-tagged H3.3 and H3.4 can be deposited both by a transcription-associated, replication-independent (RI) pathway and also weakly by an RC pathway. Although both types of H3s can be deposited by the RC pathway, DNA repair synthesis associated with meiotic recombination utilizes H3 specifically. The regions distinguishing H3 and H3.3 for their deposition pathways were identified. RC major H3 is not essential. Cells can grow without major H3 if the minor H3s are expressed at high levels. Surprisingly, cells lacking RI H3s are also viable and maintain normal nucleosome density at a highly transcribed region. The RC H3 is not detectably deposited by the RI pathway, even when there are no RI H3s available, indicating that transcription-associated RI H3 deposition is not essential for transcription. Minor H3s are also required to produce viable sexual progeny and play an unexpected role in the germ line micronuclei late in conjugation that is unrelated to transcription.

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. We focused our efforts on the function of glutamylation on the alpha-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of alpha-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as alpha-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on alpha-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.

Small RNAs in genome rearrangement in Tetrahymena.

Small RNAs produced by an RNAi-related mechanism are involved in DNA elimination during development of the somatic macronucleus from the germline micronucleus in Tetrahymena. The properties of these small RNAs can explain how the primary sequence of the parental macronucleus epigenetically controls genome rearrangement in the new macronucleus and provide the first demonstration of an RNAi-mediated process that directly alters DNA sequence organization. Methylation of histone H3 on lysine 9 and accumulation of chromodomain proteins, hallmarks of heterochromatin, also occur specifically on sequences undergoing elimination and are dependent on the small RNAs. These findings contribute to a new paradigm of chromatin biology: regulation of heterochromatin formation by RNAi-related mechanisms in eukaryotes.

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. Sequences enriched in A5 cells included genes encoding proteases. Sequences enriched in E5 cells included genes encoding cdc2 kinase and a Ser/Thr kinase. These results indicate that H1 phosphorylation plays an important role in regulating the pattern of gene expression during the starvation response and that its role in transcription regulation can be either positive or negative. Treatment of starved cells with a phosphatase inhibitor caused CDC2 gene overexpression. Expression of the E5 version of H1 in starved cells containing endogenous, wild-type H1 caused the wild-type H1 to remain highly phosphorylated. These results argue that Cdc2p is the kinase that phosphorylates Tetrahymena H1, establish a positive feedback mechanism between H1 phosphorylation and CDC2 expression, and indicate that CDC2 gene expression is regulated by an H1 phosphatase.

Both carboxy-terminal tails of alpha- and beta-tubulin are essential, but either one will suffice.

Microtubules (MTs) are organized into distinct systems essential for cell shape, movement, intracellular transport, and division. Electron crystallographic analyses provide little information about how MTs produce diverse structures and functions, perhaps because they failed to visualize the last 10 residues of the alpha- and the last 18 of the beta-tubulin C-terminal tails (CTTs), which likely play a role in MT diversity. CTTs define conserved, nonallelic isotypes in mammals, are major sites of posttranslational modifications (PTMs), are binding sites for microtubule-associated proteins (MAPs), and determine MT motor processivity. Using mutagenesis and homologous gene replacement in Tetrahymena thermophila, we analyzed mutations, deletions, tail switches, and tail duplications of alpha- and beta-tubulin CTTs. We demonstrate that a tail is required for the essential function of both alpha- and beta-tubulin. However, the two tails are interchangeable, and cells grow normally with either an alpha or a beta tail on both tubulins. In addition, an alpha gene containing a duplicated alpha C terminus rescues a lethal mutant lacking all known posttranslational modification sites on the beta C terminus but cannot rescue deletion of the beta tail. Thus, tubulin tails have a second essential function that is not associated with posttranslational modification.

The nonessential H2A N-terminal tail can function as an essential charge patch on the H2A.Z variant N-terminal tail.

Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up approximately 80% of total H2A, and a conserved variant, H2A.Z. We showed previously that acetylation of H2A.Z was essential (Q. Ren and M. A. Gorovsky, Mol. Cell 7:1329-1335, 2001). Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo. Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein. Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential. Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail. Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

Cell context-specific effects of the beta-tubulin glycylation domain on assembly and size of microtubular organelles.

Tubulin glycylation is a posttranslational modification found in cells with cilia or flagella. The ciliate Tetrahymena has glycylation on ciliary and cortical microtubules. We showed previously that mutating three glycylation sites on beta-tubulin produces immotile 9 + 0 axonemes and inhibits cytokinesis. Here, we use an inducible glycylation domain mutation and epitope tagging to evaluate the potential of glycylation-deficient tubulin for assembly and maintenance of microtubular systems. In axonemes, the major defects, including lack of the central pair, occurred during assembly, and newly made cilia were abnormally short. The glycylation domain also was required for maintenance of the length of already assembled cilia. In contrast to the aberrant assembly of cilia, several types of cortical organelles showed an abnormally high number of microtubules in the same mutant cells. Thus, the consequences of deficiency in tubulin glycylation are organelle type specific and lead to either insufficient assembly (cilia) or excessive assembly (basal bodies and cortical microtubules). We suggest that the diverse functions of the beta-tubulin glycylation domain are executed by spatially restricted microtubule-associated proteins.