Eukaryotic chromosomes from the structural basis of inheritance, and contain key
components such as centromeres and telomeres to maintain stability and facilitate
transmission. Loss of chromosome integrity causes severe problems including
tumorigenesis (1-3). In order to maintain chromosome stability, cells initiate several
responses to DNA damage, such as cell cycle arrest, apoptosis, and DNA repair (4, 5).
Double-stranded DNA breakage (DSB) is a major form of DNA damage, which is often
repaired by homologous recombination (HR) and nonhomologous end-joining (NHEJ).
In addition, previous study has also revealed another possibility: end-healing (de novo
telomere addition). End-healing is mutagenic but, interestingly, organisms have evolved
mechanisms to utilize chromosome breakage and end-healing to alter their genome
structures during differentiation (6). Previous studies have shown that programmed
genome alterations occur in more than 100 species of diverse organisms including ciliates
and vertebrates (7, 8). Programmed genome alterations have been link to gene silencing,
dosage compensation and/or sex determination in these organisms (8).
Programmed DNA elimination is a prominent form of genome alteration and was
first discovered as chromatin diminution in the nematode Parascaris univalens (8, 9). In
Ascaris suum and P. univalens, 13% and 88% of genomic DNA, respectively, is
16
eliminated during somatic cell differentiation (10-13). This process removes all
detectable heterochromatin from the somatic progenitor cells during early embryonic
cleavages (8, 10). In the pre-somatic cells of A. suum, telomere repeats are added de novo
to all broken ends within a 4 to 6-kb region after chromosome breakage, including those
destined for elimination (14, 15). These fragments fail to attach to microtubules of the
mitotic spindle during anaphase, and remain in the cytoplasm where they are degraded
after cell division (10, 12, 16). Transcriptome analysis has revealed that at least 685
germline-expressed genes are eliminated from somatic cells (13), with only a few of them
encoding proteins with known functions (17-19).
Programmed DNA elimination has also been found in ciliated protozoa, including
Tetrahymena, Paramecium (20, 21), Stylonychia (22-24), Euplotes (25) and Oxytricha
(26). Like all ciliates, Tetrahymena thermophila, displays nuclear dualism and contains a
somatic nucleus (macronucleus, MAC) and a germline nucleus (micronucleus, MIC) in
the same cell. During the growth phase, the MAC undergoes amitotic division and the
MIC divides by typical mitosis. During conjugation, the MIC goes through meiosis,
mitosis and cross-fertilization to generate zygotic nuclei, which further divide and
develop into new MAC and MIC. The developing new MAC undergoes a series of
dramatic programmed DNA rearrangements, including the elimination of ~34% of the
genome (from 157 Mb to 104 Mb) and the fragmentation of the five MIC chromosomes
17
into about 225 minichromosomes that are retained in the MAC (Fig 1A) (27-29).
Two globally occurring processes have been found: IES (internal elimination
sequence) deletion and chromosome breakage. In IES deletion, several lines of evidence
have provided an RNA-guided DNA deletion model indicating that small RNAs mediate
histone modifications for programmed DNA rearrangements (28). During conjugation,
bidirectional transcripts are generated from the micronuclear genome and processed into
small RNAs (30, 31). These small RNAs target the homologous sequences in the
developing macronucleus to trigger the histone H3K9 and H3K27 methylation (32, 33).
Methylations of H3K27 and H3K9 are recognized by programmed DNA degradation 1
protein, Pdd1p, which shares homology with the chromodomain of HP1 (34-36). A
Pdd1p-containing complex recruits Tpb2p, a domesticated piggyBac transposase (37).
Tpb2p executes the excision of the deletion elements, the so-called IESs (internal
eliminated sequences), with sequence microheterogeneity at the deletion boundary in
different lines (38, 39). The flanking DNAs are rejoined through a nonhomologous
end-joining (NHEJ) pathway (40). Furthermore, a recent study discovered the two other
domesticated piggyBac transposases, TPB1 and TPB6, which also participate in IES
eliminations. Remarkably, the junctions at the TPB1-dependent IESs contain
piggyBac-like terminal repeats that are necessary for precise excision (41). These studies
reveal that there are several regulatory systems to controlling IES eliminations.
18
In chromosome breakage, approximately 200 specific sites are broken and telomeres
are added to both ends after limited nucleotide loss (42-45), which produces chromosome
fragments (minichromosomes) averaging 462 kb in size (46). The molecular structure of
a chromosome breakage site was first characterized at the rDNA (ribosomal RNA gene)
locus of Tetrahymena. The rDNA exists as a single copy gene in the MIC genome (47).
During new MAC development, it is amplified to become ~9,000 copies of linear
minichromosomes, each containing two copies of the gene as a head-to-head dimer
(48-51). Further studies have revealed that specific chromosome breakage occurs at both
ends of the rDNA, which share a 20-nucleotide sequence motif (51). By searching for
additional copies of similar sequences in the genome, more breakage sites have been
discovered and these have been shown to contain a chromosome breakage sequence
(Cbs), a 15-bp AAAGAGGTTGGTTTA element, which was later shown to be necessary
and sufficient for chromosome breakage to occur (42, 44). Breakage is coupled with de
novo telomere addition within 30 bp of the Cbs (45). Moreover, the Cbs sequence is very
well conserved. Copies of the Cbs at the rDNA locus are nearly identical among six
additional Tetrahymena and two related species. This finding suggests that the Cbs also
serves as the chromosome breakage signal in these species (52). Subsequently, Hamilton
et al. (2006) examined 40 additional Cbs sites in T. thermophila and found that a 10-bp
core of this 15-bp sequence was completely conserved. The other positions showed
19 restricted one or two nucleotide variations (53).
In this study, we focused on the two main processes of DNA rearrangements. We
first tried to understand the mechanism of boundary determination of IES elimination.
Comparison of IESs in different strains indicated that IESs were conserved, but they have
different forms that showed boundary variations among strains. Moreover, the
distribution of boundary elements in the IES flanking regions shed light on the possible
regulatory mode of boundary determination. The involvement of domesticated
transposases further supports our findings. We searched globally for the Cbs globally and
determined the consensus sequence. However, when presumed MAC minichromosomes
were compared to the MIC genome, we found that some MAC minichromosomes had
been lost after conjugation. Detailed investigation showed that these eliminated
minichromosomes disappeared at a similar time period, revealing the regulatory
machinery responsible for the elimination of these minichromosomes. This study
provides a comprehensive overview of both IES elimination and chromosome breakage
and reveals more information on how cells globally regulate complex DNA
rearrangements.
20