A Domesticated PiggyBac Transposase Interacts with Heterochromatin and Catalyzes Reproducible DNA Elimination in Tetrahymena

Abstract

The somatic genome of the ciliated protist Tetrahymena undergoes DNA elimination of defined sequences called internal eliminated sequences (IESs), which account for ∼30% of the germline genome. During DNA elimination, IES regions are heterochromatinized and assembled into heterochromatin bodies in the developing somatic nucleus. The domesticated piggyBac transposase Tpb2p is essential for the formation of heterochromatin bodies and DNA elimination. In this study, we demonstrate that the activities of Tpb2p involved in forming heterochromatin bodies and executing DNA elimination are genetically separable. The cysteine-rich domain of Tpb2p, which interacts with the heterochromatin-specific histone modifications, is necessary for both heterochromatin body formation and DNA elimination, whereas the endonuclease activity of Tpb2p is only necessary for DNA elimination. Furthermore, we demonstrate that the endonuclease activity of Tpb2p in vitro and the endonuclease activity that executes DNA elimination in vivo have similar substrate sequence preferences. These results strongly indicate that Tpb2p is the endonuclease that directly catalyzes the excision of IESs and that the boundaries of IESs are at least partially determined by the combination of Tpb2p-heterochromatin interaction and relaxed sequence preference of the endonuclease activity of Tpb2p.

Heterochromatin aggregation during DNA elimination in Tetrahymena is facilitated by a prion-like protein

“…These results indicate that the Pdd1p-containing bodies that form in the absence of Jub6p are still associated with heterochromatin and, thus, probably represent an abnormal type of heterochromatin body caused by disturbance of the subnuclear localization of heterochromatin within the new MAC. This abnormal heterochromatin body formation is unlikely to be due to blockage of DNA elimination, as such abnormal heterochromatin bodies have not been detected in our previous studies of other DNA-elimination-defective mutants by the same immunofluorescent staining procedures (Kataoka and Mochizuki, 2015;Kataoka et al, 2016;Vogt and Mochizuki, 2013).…”

“…Next, a neo4 cassette was inserted into the SmaI site. The JUB6 KO construct was obtained from the vector through digestion with XbaI and XhoI, and was introduced into conjugating UMPS strains (Vogt and Mochizuki, 2013) through biolistic transformation. Paromomycin-and 5-fluoroorotic acid (5-FOA)-resistant progeny cells were selected, and cells with the JUB6-KO locus in the MIC were selected following genomic PCR with PRN1-gKO_cFW2 and PRN1-gKO_cRV2 and EcoRI digestion (Fig.…”

“…The first group consists of proteins required for the formation of heterochromatin at IESs, including the RNAi machinery components Dcl1p, Twi1p, Giw1p and Ema1p, the histone methyltransferase Ezl1p and the HP1-like protein Pdd1p (Aronica et al, 2008;Coyne et al, 1999;Liu et al, 2004Liu et al, , 2007Malone et al, 2005;Mochizuki and Gorovsky, 2005;Noto et al, 2010). The second group includes proteins that are dispensable for heterochromatin formation but are required for its assembly into heterochromatin bodies: Jub1p, Lia1p, Lia4p, Lia5p, Pdd2p, Tku80p and Tpb2p Rexer and Chalker, 2007;Horrell and Chalker, 2014;Shieh and Chalker, 2013;Liu et al, 2007;Cheng et al, 2010;Lin et al, 2012;Vogt and Mochizuki, 2013;Kataoka and Mochizuki, 2015). Although some of the mechanisms responsible for the former RNAi-directed heterochromatin formation process have been elucidated at the biochemical level, the latter heterochromatin body formation process remains elusive.…”

LIA4 Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

“…⌬TKU80 cells are able conduct IES excision yet fail to form DNA elimination foci, which demonstrates that IES excision does not require formation of the largest versions of these structures (29). In contrast, mutational analysis of TPB2 has revealed that Pdd1 foci form in the presence of a non-catalytically active protein as long as the Tpb2 zinc finger domain is functional (59). Thus, DNA elimination structures form in the absence of cleavage.…”

Programmed Genome Rearrangements in Tetrahymena

“…Tpb2p probably lost its original specificity for the transposon terminal inverted repeats and became targeted to heterochromatin through the acquired C-terminal sequence. Site-directed mutagenesis studies showed that the zinc-finger domain in the C-terminal half was indeed important for Tpb2p localization (79). A TPB2 ortholog, PGM1, has been found in Paramecium and likely serves a similar role (80).…”