Role of histone deacetylation in developmentally programmed DNA rearrangements in Tetrahymena thermophila

In Tetrahymena, as in other ciliates, development of the somatic macronucleus during conjugation involves extensive and reproducible rearrangements of the germ line genome, including chromosome fragmentation and excision of internal eliminated sequences (IESs). The molecular mechanisms controlling these events are poorly understood. To investigate the role that histone acetylation may play in the regulation of these processes, we treated Tetrahymena cells during conjugation with the histone deacetylase inhibitor trichostatin A (TSA). We show that TSA treatment induces developmental arrests in the early stages of conjugation but does not significantly affect the progression of conjugation once the mitotic divisions of the zygotic nucleus have occurred. Progeny produced from TSA-treated cells were examined for effects on IES excision and chromosome breakage. We found that TSA treatment caused partial inhibition of excision of five out of the six IESs analyzed but did not affect chromosome breakage at four different sites. TSA treatment greatly delayed in some cells and inhibited in most the excision events in the developing macronucleus. It also led to loss of the specialized subnuclear localization of the chromodomain protein Pdd1p that is normally associated with DNA elimination. We propose a model in which underacetylated nucleosomes mark germ line-limited sequences for excision.     

Deletion of the centromere as a mechanism for achieving stability of a dicentric chromosome

A patient with primary amenorrhea and a terminal rearrangement of chromosomes 13 and 20 is described. The stability of the dicentric translocation chromosome was achieved by suppression of the activity of the centromere of chromosome 20 and actual deletion of the centromere of chromosome 13. These two mechanisms may be fundamentally similar if centromere inactivation results from a visually undetectable deletion.     

Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila.

DNA deletion by site-specific chromosome breakage and rejoining occurs extensively during macronuclear development in the ciliate Tetrahymena thermophila. We have sequenced both the micronuclear (germ line) and rearranged macronuclear (somatic) forms of one region from which 1.1 kilobases of micronuclear DNA are reproducibly deleted during macronuclear development. The deletion junctions lie within a pair of 6-base-pair direct repeats. The termini of the deleted sequence are not inverted repeats. The precision of deletion at the nucleotide level was also characterized by hybridization with a synthetic oligonucleotide matching the determined macronuclear (rejoined) junction sequence. This deletion occurs in a remarkably sequence-specific manner. However, a very minor degree of variability in the macronuclear junction sequences was detected and was shown to be inherent in the mechanism of deletion itself. These results suggest that DNA deletion during macronuclear development in T. thermophila may constitute a novel type of DNA recombination and that it can create sequence heterogeneity on the order of a few base pairs at rejoining junctions.     

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.     

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.     

The nematode Oscheius tipulae as a genetic model for programmed DNA elimination

1. Download : Download high-res image (197KB)
2. Download : Download full-size image

     

Protozoan parasites: programmed cell death as a mechanism of parasitism

Programmed cell death (PCD) is a potent mechanism to remove parasitized cells, but it has also been shown that protozoan parasites can induce or inhibit apoptosis in host cells. In recent years, it has become clear that unicellular parasites can also undergo PCD, meaning that they commit suicide in response to various stimuli. This review focuses on the role of protozoan PCD and on the interaction between protozoan parasites and the host cell death machinery from the perspective of parasite survival strategies.     

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.     

Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila.

Deletions of specific DNA sequences are known to occur in Tetrahymena thermophila as a developmentally regulated process. Deletions of a particular region (region M) were previously shown to be of two alternative sizes, 0.6 or 0.9 kilobases (kb) (C.F. Austerberry, C.D. Allis, and M.-C. Yao, Proc. Natl. Acad. Sci. USA 81: 7383-7387). In this study, the nucleotide sequences for both deletions were determined. These two deletions share the same right junction, but their left junctions are 0.3 kb apart. An 8-base-pair (bp) sequence is present at both junctions of the 0.6-kb deletion, but only 5 bp of this direct repeat are present at the left junction of the 0.9-kb deletion. Further comparison revealed a common 10-bp sequence near each of the two left junctions and a similar sequence in inverted orientation near the right junction. These sequences may play a role in the developmental regulation of the deletion process.     

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Approximately 6000 specific DNA deletion events occur during development of the somatic macronucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis-acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetrahymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis-acting sequences outside the deleted DNA.     

The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5).     

Developing a programmed restriction endonuclease for highly specific DNA cleavage

Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4-8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-(gamma-maleimidobutryloxy) succinimide ester to a TFO (5'-NH2-[CH2](6 or 12)-MPMPMPMPMPPPPPPT-3', with M being 5-methyl-2'-deoxycytidine and P being 5-[1-propynyl]-2'-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO ('addressed' site: 5'-TTTTTTTCTCTCTCTCN(approximately 10)CAGCTG-3'), leaving 'unaddressed' PvuII sites intact. The preference for cleavage of an 'addressed' compared to an 'unaddressed' site is >1000-fold, if the cleavage reaction is initiated by addition of Mg2+ ions after preincubation of scPvuII-TFO and substrate in the absence of Mg2+ ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO.     

Physiological scenarios of programmed loss of mitochondrial DNA function and death of yeast

Recently it was convincingly shown that the yeast Saccharomyces cerevisiae does possess the basic modules of programmed cell death machinery. As programmed cell death is suicide for a unicellular organism, it is reasonable to assume that they trigger the program when the death is beneficial for the rest of the population. Not surprisingly, most of the scenarios of physiological death of S. cerevisiae, i.e. cell death in stationary culture, during meiosis, during mating, and driven by viruses are dependent on quorum sensing, meaning that they depend on the cell density. Here we also discuss possible mechanisms that govern fitness decline during replicative aging of S. cerevisiae cells. We argue that loss of mitochondrial DNA function that occurs during replicative aging is programmed and adaptive. Indeed, yeast cells with nonfunctional mitochondrial DNA are known to be extremely stress-resistant, and also the presence of a subpopulation of such cells might protect the culture from degeneration by preventing the fixation of opportunistic mutations.     

An essential role for the caspase dronc in developmentally programmed cell death in Drosophila

Dronc is a caspase recruitment domain-containing Drosophila caspase that is expressed in a temporally and spatially restricted fashion during development. Dronc is the only fly caspase known to be regulated by the hormone ecdysone. Here we show that ectopic expression of dronc in the developing fly eye leads to increased cell death and an ablated eye phenotype that can be suppressed by halving the dosage of the genes in the H99 complex (reaper, hid, and grim) and enhanced by mutations in diap1. In contrast to previous reports, we show that the dronc eye ablation phenotype can be suppressed by coexpression of the baculoviral caspase inhibitor p35. Dronc also interacts, both genetically and biochemically, with the CED-4/Apaf-1 fly homolog, Dark. Furthermore, extracts made from Dark homozygous mutant flies have reduced ability to process Dronc, showing that Dark is required for Dronc processing. Finally, using the RNA interference technique, we show that loss of Dronc function in early Drosophila embryos results in a dramatic decrease in cell death, indicating that Dronc is important for programmed cell death during embryogenesis. These results suggest that Dronc is a key caspase mediating programmed cell death in Drosophila.     

Role of micronucleus-limited DNA in programmed deletion of mse2.9 during macronuclear development of Tetrahymena thermophila

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.     

Latent mitochondrial DNA deletion mutations drive muscle fiber loss at old age

With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation‐containing electron transport chain‐deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.