The Paramecium histone chaperone Spt16-1 is required for Pgm endonuclease function in programmed genome rearrangements

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. Author summary The genome is generally similar in all the cells of an organism. However, in the ciliate Paramecium tetraurelia, massive and reproducible programmed DNA elimination leads to a highly streamlined somatic genome. In eukaryotes, DNA is packaged into nucleosomes, which ensure genome integrity but act as a barrier to enzymes acting on DNA. How the endonuclease PiggyMac gains access to the genome to initiate DNA elimination remains elusive. Here, we identified four P. tetraurelia genes encoding homologs of the conserved histone chaperone Spt16, which can modulate acesss to DNA by promoting nucleosome assembly and disassembly. We demonstrated that the most divergent gene, SPT16-1, has a highly specialized expression pattern, similar to that of PiggyMac, and a specific role in programmed DNA elimination. We show that the Spt16-1 protein, like PiggyMac, is exclusively localized in the differentiating somatic nucleus, and is also required for the dramatic elimination of germline-limited sequences. We further show that Spt16-1 directs the correct nuclear localization of the PiggyMac endonuclease. Thus, Spt16-1 is essential for PiggyMac function during programmed DNA elimination. We propose that Spt16-1 mediates the interaction between PiggyMac and chromatin or DNA, facilitating endonuclease access to DNA cleavage sites.