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

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.

SUMOylation is developmentally regulated and required for cell pairing during conjugation in Tetrahymena thermophila.

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena, including a dynamic regulation associated with the sexual life cycle.

Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.

The conjugation-specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena.

Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena DeltaDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway.

Coordinate regulation of a family of promastigote-enriched mRNAs by the 3'UTR PRE element in Leishmania mexicana.

Post-transcriptional regulation is a key feature controlling gene expression in the protozoan parasite Leishmania. The nine-nucleotide paraflagellar rod regulatory element (PRE) in the 3'UTR of Leishmania mexicana PFR2 is both necessary and sufficient for the observed 10-fold higher level of PFR2 mRNA in promastigotes compared to amastigotes. It is also found in the 3'UTRs of all known PFR genes. A search of the Leishmania major Friedlin genomic database revealed several genes that share this cis element including a homolog of a heterotrimeric kinesin II subunit, and a gene that shares identity to a homolog of a Plasmodium antigen. In this study, we show that genes that harbor the PRE display promastigote-enriched transcript accumulation ranging from 4- to 15-fold. Northern analysis on episomal block substitution constructs revealed that the regulatory element is necessary for the proper steady-state accumulation of mRNA in L. mexicana paraflagellar rod gene 4 (PFR4). Also we show that the PRE plays a major role in the proper steady-state mRNA accumulation of PFR1, but may not account for the full regulatory mechanism acting on this mRNA. Our evidence suggests that the PRE coordinately regulates the mRNA abundance of not only the PFR family of genes, but also in a larger group of genes that have unrelated functions. Although the PRE alone can regulate some mRNAs, it may also act in concert with additional elements to control other RNA transcripts.

Global gene expression in Leishmania.

The completion of the genomic sequences of many protozoan pathogens of humans, including species of Leishmania, Trypanosoma and Plasmodium, provide new approaches to study the pattern of gene expression during differentiation and development. Leishmania are a major public health risk in many countries and cause a wide spectrum of clinical disease referred to as leishmaniasis. The Leishmania life cycle consists of two morphologically distinct stages: intracellular amastigotes that reside in the phagolysosome of mammalian macrophages, and extracellular promastigotes that reside within the gut of the sandfly vector. DNA microarray analysis is a powerful method to study global gene expression in terms of quantitation of mRNA levels. This review discusses the application of DNA microarray technology to study the pattern of global gene expression of Leishmania promastigote and amastigote life stages. Results from several studies show that, overall, there is a surprisingly low level of differentially expressed genes, ranging from 0.2% to 5% of total genes, between the amastigote and promastigote life stages. Thus, the Leishmania genome can be considered to be constitutively expressed with a limited number of genes showing stage-specific expression. Comparative genomic analyses of gene expression levels between Leishmania major and Leishmania mexicana show that the majority of differentially expressed genes between amastigotes and promastigotes are species specific with relatively few differentially expressed genes in common between these two Leishmania species. Quantitative proteomic analysis of Leishmania relative protein expression shows there is a weak correlation to gene expression. Therefore, Leishmania protein expression levels are likely regulated at the level of translation or by post transcriptional mechanisms, and differential protein modifications may be more important in development than the regulation of gene expression.

The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia.

Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling.

Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana.

We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8156 predicted coding regions revealed 288 genes (3.5% of all genes) whose steady-state mRNA levels meet criteria for differential regulation between promastigotes and lesion-derived amastigotes. Interestingly, sample comparison of promastigotes to axenic amastigotes resulted in only 17 genes (0.2%) that meet the same statistical criteria for differential regulation. The reduced number of regulated genes is a consequence of an increase in the magnitude of the transcript levels in cells under axenic conditions. The expression data for a subset of genes was validated by quantitative PCR. Our studies show that interspecies hybridization on microarrays can be used to analyze closely related protozoan parasites, that axenic culture conditions may alter amastigote transcript abundance, and that there is only a relatively modest change in abundance of a few mRNAs between morphologically distinct promastigote and amastigote cultured cells. Leishmania may represent an alternative paradigm for eukaryotic differentiation with minimal contributions from changes in mRNA abundance.

Analysis of Paramecium tetraurelia A-51 surface antigen gene mutants reveals positive-feedback mechanisms for maintenance of expression and temperature-induced activation.

In Paramecium tetraurelia, variable surface antigen loci show mutually exclusive expression which is controlled primarily at the transcriptional level. Clonally stable expression of a single antigen has attracted models involving self-regulation by their gene products. However, direct demonstration of self-feedback at the molecular level has been complicated due to the inability to separate the functional gene from its product as well as copy number effects associated with injected extrachromosomal DNA in the polygenomic somatic nucleus. In this study, we exploited several germ line termination and frameshift mutations in the A-51 surface antigen gene to analyze variable surface antigen expression. These mutant alleles have the same copy number as the wild-type allele and therefore eliminate possible copy number effects. The mutant alleles were not transcribed at 27 degrees C, consistent with positive-feedback models for gene expression. However, further analysis showed that high temperatures (34 degrees C) induced transcription of the mutant A genes even in the presence of a different antigen on the cell surface. Thus, transcription was temperature dependent. Unlike wild-type cells, transcription of the mutant A genes at high temperatures was not maintained after temperature shift back to 27 degrees C in homozygous mutant cells. Importantly, transcription of the mutant allele was maintained at 27 degrees C in heterozygous cells with one copy of the wild-type allele. These results indicate that expression of the wild-type gene is required to stabilize its own transcriptional state at 27 degrees C.

The two cytoplasmic dynein-2 isoforms in Leishmania mexicana perform separate functions.

Eukaryotic organisms with cilia or flagella typically express two non-axonemal or "cytoplasmic" dyneins, dynein-1 and dynein-2. Interestingly, we find that Leishmania mexicana is unusual and contains two distinct cytoplasmic dynein-2 heavy chain genes (designated LmxDHC2.1 and LmxDHC2.2) along with a single dynein-1 heavy chain (LmxDHC1). Disruption of LmxDHC2.2 resulted in immotile parasites that had a rounded cell body. Although they assume amastigote morphology, immunoblot analysis of these cells demonstrates protein expression consistent with the promastigote stage. Ultrastructural analysis revealed non-emergent flagella that lacked the paraflagellar rod and an axoneme with deficiencies in several components. We confirmed the absence of paraflagellar rod proteins PFR1 and PFR2. These results show that LmxDHC2.2 is required for flagellar assembly and also participates in the maintenance of promastigote cell shape. In contrast to the results with LmxDHC2.2, we were unable to generate homologous disruptions of LmxDHC2.1. This result suggests that, unlike LmxDHC2.2, LmxDHC2.1 is an essential gene in Leishmania. Together, these findings demonstrate that the two dynein-2 heavy chain isoforms in Leishmania perform distinct functions. The observation that the genomes of Leishmania major, Leishmania infantum and Trypanosoma brucei also contain two dynein-2 isoforms suggests that this unusual aspect of cytoplasmic dynein is a conserved feature of the kinetoplastids.

Identification of a developmentally regulated translation elongation factor 2 in Tetrahymena thermophila.

Protein synthesis elongation factor 2 (eEF2) catalyzes the translocation of the peptidyl-tRNA from the A site to the P site of the ribosome. Most organisms encode a single EF2 protein and its activity is regulated by phosphorylation. We have identified a family of genes in Tetrahymena thermophila that encode proteins homologous to eEF2, yet are expressed only during sexual reproduction. These genes have been designated EFR for Elongation Factor 2 Related. EFR transcripts were not detected in vegetative cell cultures but rapidly increased about 6 h after the start of conjugation (mating). For comparison, we cloned, sequenced and analyzed the expression of the standard eEF2 gene from T. thermophila. Unlike EFR, transcripts from eEF2 were detected in vegetative cells but were present at lower concentrations during conjugation. Despite the high sequence identity between EFR and eEF2 from other organisms (about 42% at the amino acid level), key regulatory sequences that are involved in the regulation of eEF2 are altered in EFR. The sequence and expression data suggest that EFR is an eEF2 variant involved in a major translation regulatory mechanism that occurs during the formation of the macronuclear genome in conjugating cells.

Identification of single nucleotide mutations that prevent developmentally programmed DNA elimination in Paramecium tetraurelia.

The excision of internal eliminated sequences (IESs) occurs during the differentiation of a new somatic macronuclear genome in ciliated protozoa. In Paramecium tetraurelia, IESs show few conserved features with the exception of an invariant 5'-TA-3' dinucleotide that is part of an 8-bp inverted terminal repeat consensus sequence with similarity to the ends of mariner/Tc1 transposons. We have isolated and analyzed two mutant cell lines that are defective in excision of individual IESs in the A-51 surface antigen gene. Each cell line contains a mutation in the flanking 5'-TA-3' dinucleotide of IES6435 and IES1835 creating a 5'-CA-3' flanking sequence that prevents excision. The results demonstrate that the first position of the 5'-TA-3' is required IES excision just as previous mutants have shown that the second position (the A residue) is required. Combining these results with other Paramecium IES mutants suggests that there are few positions essential for IES excision in Paramecium. Analysis of many IESs reveals that there is a strong bias against particular nucleotides at some positions near the IES termini. Some of these strongly biased positions correspond to known IES mutations, others correlate with unusual features of excision.