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Abstract
In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements.
Highlights
In the unicellular eukaryote Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and subsequent DNA excision and repair
We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination
Given human SPT16 has been shown to play a role in chromatin reorganization at the sites of DNA damage [27], we wanted to understand whether SPT16 in organisms that show programmed genome rearrangements might use SPT16 to facilitate this process
Summary
In the unicellular eukaryote Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and subsequent DNA excision and repair. The P. tetraurelia endonuclease PiggyMac (Pgm) must gain access to the genome to initiate excision of DNA for it to be eliminated. DNA is packaged into nucleosomes, which ensure genome integrity and represent a barrier to enzymes acting on DNA. Histone chaperones can modulate this barrier by promoting nucleosome assembly and disassembly (reviewed in [1]). FACT engages in multiple interactions with nucleosomal DNA and histones to reorganize nucleosomes and allow access to DNA [4,5,6,7,8,9]
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