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


Autoři: Augustin de Vanssay aff001;  Amandine Touzeau aff001;  Olivier Arnaiz aff002;  Andrea Frapporti aff001;  Jamie Phipps aff001;  Sandra Duharcourt aff001
Působiště autorů: Université de Paris, Institut Jacques Monod, CNRS, Paris, France aff001;  Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France aff002
Vyšlo v časopise: The Paramecium histone chaperone Spt16-1 is required for Pgm endonuclease function in programmed genome rearrangements. PLoS Genet 16(7): e32767. doi:10.1371/journal.pgen.1008949
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008949

Souhrn

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.

Klíčová slova:

DNA cleavage – DNA repair – Genomics – Histones – Chromatin – Paramecium – Protein domains – RNA interference


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