Abundant ϳ28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (⌬DCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These ⌬DCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation ⌬DCL1 cells die without eliminating the germ linelimited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicerrelated gene is required for programmed DNA rearrangements. Surprisingly, ⌬DCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.RNA interference (RNAi) describes an array of related mechanisms involved in diverse biological processes including defense against RNA viruses, specification of centromeric heterochromatin structure, and developmental control of gene expression (reviewed in reference 25). These mechanisms share the use of small RNAs to target specific effector protein complexes to homologous sequences via base-pairing interactions. The use of small, homologous RNAs as specificity factors imparts tremendous flexibility of targets on a single protein complex. These targeting RNAs are generated by RNase III enzymes, collectively called Dicer ribonucleases, that cleave longer, double-stranded RNA (dsRNA) into ϳ20-to 26-nucleotide (nt) species that are incorporated into the effector complexes (3, 24, 27, 30; reviewed in reference 6). The genomes of many eukaryotes encode multiple Dicer-related proteins, and the specific Dicer used to generate the small RNAs can determine the downstream pathway that they enter. For instance, in Arabidopsis thaliana, the Dicer-like 3 (Dcl3) gene product is required to produce endogenous short interfering RNAs (siRNAs), A. thaliana Dcl2 is necessary for accumulation of siRNAs in response to RNA virus infections, and A. thaliana Dcl1 is necessary to generate micro-RNAs (miRNAs) involved in the control of flower development (31,53). Similarly, th...

Over 190 independent insertions into target plasmids of the retro virus-like element Ty3 were recovered and mapped. Ty3 was shown to insert upstream of tRNA, 5S, and U6 genes, all of which are transcribed by RNA polymerase III. Integration sites were within 1-4 nucleotides of the position of transcription initiation, even for one mutant gene where the polymerase III initiation site was shifted to a completely new context. Mutagenesis of a SUP2 tRNA gene target showed that integration required functional promoter elements but that it did not correlate in a simple way with target transcription. This is the first report directly linking a discrete genomic function with preferential insertion of a retrotransposon.

A large number of DNA segments are excised from the chromosomes of the somatic nucleus during development of Tetrahymena thermophila. How these germline-limited sequences are recognized and excised is still poorly understood. We have found that many of these noncoding DNAs are transcribed during nuclear development. Transcription of the germline-limited M element occurs from both DNA strands and results in heterogeneous transcripts of < 200 b to > 1 kb. Transcripts are most abundant when developing micro-and macronuclei begin their differentiation. Transcription is normally restricted to unrearranged DNA of micronuclei and/or developing nuclei, but germline-limited DNAs can induce their own transcription when placed into somatic macronuclei. Brief actinomycin D treatment of conjugating cells blocked M-element excision, providing evidence that transcription is important for efficient DNA rearrangement. We propose that transcription targets these germline-limited sequences for elimination by altering chromatin to ensure their accessibility to the excision machinery. A remarkable process of DNA rearrangement occurs during development of Tetrahymena thermophila in which ∼ 6000 DNA segments are coordinately excised from the newly forming somatic nucleus (reviewed in Coyne et al. 1996;Yao et al. 2001). Like other ciliated protozoa, Tetrahymena contain two types of nuclei, a somatic macronucleus and a germline micronucleus. During vegetative growth, all gene expression occurs from the macronucleus, whereas the micronucleus remains transcriptionally silent (Gorovsky and Woodard 1969). In each sexual generation the macronucleus is destroyed, and a new micronucleus and macronucleus are created, each deriving its genome from micronuclear DNA. In new micronuclei the five pairs of chromosomes remain unaltered, whereas in the macronuclear precursors the chromosomes are fragmented and ∼ 15% of the germline DNA is eliminated by site-specific DNA deletion. After these DNA rearrangements, the macronuclear chromosome fragments are amplified to ∼ 50 copies/nucleus.The DNA segments eliminated from the developing macronucleus, known as deletion elements, consist of either unique or moderately repetitive sequences (Yao and Gorovsky 1974). These predominantly noncoding DNAs vary greatly in size, ranging from about 600 bp to greater than 13 kbp. Nine of the estimated 6000 deletion elements have been sequenced and found to have little or no sequence homology (for review see Yao et al. 2001). Extensive analyses of two of these, the M and R elements, have revealed that the boundaries of DNA deletion are delimited by pairs of cis-acting sequences located a short distance outside of the ends of each element (Chalker et al. 1999;Godiska et al. 1993;Godiska and Yao 1990). Notably, the exact sequence of these functionally similar boundary determinants is different for each element; a fact that further highlights the extraordinary diversity of these germline-limited sequences.Three proteins, named Programmed DNA degradation proteins (Pddps) have...

Ciliated protozoa extensively remodel their somatic genomes during nuclear development, fragmenting their chromosomes and removing large numbers of internal eliminated sequences (IESs). The sequences eliminated are unique and repetitive DNAs, including transposons. Recent studies have identified transposase proteins that appear to have been domesticated and are used by these cells to eliminate DNA not wanted in the somatic macronucleus. This DNA elimination process is guided by meiotically produced small RNAs, generated in the germline nucleus, that recognize homologous sequences leading to their removal. These scan RNAs are found in complexes with PIWI proteins. Before they search the developing genome for IESs to eliminate, they scan the parental somatic nucleus and are removed from the pool if they match homologous sequences in that previously reorganized genome. In Tetrahymena, the scan RNAs target heterochromatin modifications to mark IESs for elimination. This DNA elimination pathway in ciliates shares extensive similarity with piRNA-mediated silencing of metazoans and highlights the remarkable ability of homologous RNAs to shape developing genomes.

SUMMARYResearch using ciliates revealed early examples of epigenetic phenomena and continues to provide novel findings. These protozoans maintain separate germline and somatic nuclei that carry transcriptionally silent and active genomes, respectively. Examining the differences in chromatin within distinct nuclei of Tetrahymena identified histone variants and established that transcriptional regulators act by modifying histones. Formation of somatic nuclei requires both transcriptional activation of silent chromatin and large-scale DNA elimination. This somatic genome remodeling is directed by homologous RNAs, acting with an RNA interference (RNAi)-related machinery. Furthermore, the content of the parental somatic genome provides a homologous template to guide this genome restructuring. The mechanisms regulating ciliate DNA rearrangements reveal the surprising power of homologous RNAs to remodel the genome and transmit information transgenerationally.Outline