Junk DNA contains numerous transposable elements that are able to move from one genome position to another and are therefore potentially harmful to the integrity of genomes. On the other hand, many junk DNAs are not really junk but perform important functions by way of proper chromosome segregation, recombination, and gene expression. Several organisms dispense with junk DNAs during the development of their somatic lineage.

This developmentally programmed DNA elimination event presumably reflects two aspects of junk DNA: its harmfulness by the actions of transposons, and its usefulness in maintaining genome integrity in the germline lineage. The ciliated protozoan Tetrahymena (Figure 1) possesses a somatic macronucleus (Mac) and a germline micronucleus (Mic) in each cell. Mac is polyploid and transcriptionally active, whereas Mic is diploid and transcriptionally inert during vegetative growth. In the sexual process of conjugation, Mic gives rise to a new Mac and a Mic, and the parental Mac is destroyed. During the development of the new Mac, ~6000 Internal Eliminated Sequences (IESs) are removed (DNA elimination), and the remaining Mac-destined sequences are re-ligated. Most IESs are moderately repeated in the Mic and many of them are related to transposable elements (Figure 1).

Heterochromatin formation is involved in the IES elimination process (Figure 2). In Tetrahymena, heterochromatin components, including histone H3 methylated on lysine 9 (H3K9me) and on lysine 27 (H3K27me), and the chromodomain protein Pdd1p are specifically associated with eliminated IES sequences and are essential for DNA elimination. A mechanism related to RNA interference (RNAi) is also essential for DNA elimination (Figure 2).

Small (~28-29 nt) RNAs are produced by the Dicer protein Dcl1p and associate with the Argonaute protein Twi1p. Dcl1p and Twi1p are required for accumulation and/or targeting of H3K9me/H3K27me/Pdd1p as well as for DNA elimination. Thus, heterochromatin formation occurs downstream of the RNAi-related mechanism in the DNA elimination pathway. As recent studies in animals and plants have shown that transposable elements are silenced by a heterochromatin and/or RNAi-related mechanism, transposon silencing by RNAi-directed formation of heterochromatin has probably arisen in an ancestral eukaryote. Therefore, further study of the programmed DNA elimination process in Tetrahymena should yield basic and important data about transposon silencing by RNAi-directed formation of heterochromatin in eukaryotes.

In contrast to the evolutionary conservation of RNAi-directed formation of heterochromatin in transposon silencing, the DNA excision process is unique in some limited eukaryotic lineages; including ciliates. It has long been thought that a transposase-like endonuclease activity is involved in DNA elimination while the enzyme responsible for this activity had remained unknown. We recently identified a PiggyBac transposase-like enzyme (Tpb2p) which is essential for DNA elimination in vivo. Because recombinantly expressed Tpb2p can produce double-strand breaks in vitro (Figure 3), we believe Tpb2p is the enzyme catalyzing DNA elimination.

This discovery enables us to analyze how heterochromatin serves as a binding platform to recruit the DNA excision machinery, how specificity of the boundary of DNA elimination is determined, and how DNA elimination is linked to the subsequent DNA repair process. TPB2 appears to have gone through a domestication process to become a host gene and be maintained in the macronucleus. Although several domesticated transposon-derived proteins are known to be involved in host genome regulation in other eukaryotes, their evolutionary histories are not clear. Studies of Tpb2p may show how a transposon can be domesticated to regulate a eukaryotic genome.