Polycomb Repressive Complex in Paramecium: interaction with small RNA machinery and substrate specificity – POLYCHROME

To achieve our objectives, we combine genetic and cellular approaches, biochemical assays and genome-scale profiling of chromatin states and transcription. As a whole, our program provides a better understanding of the mechanisms that establish epigenetic marks in specific regions of the genome during development in Paramecium. We expect that the data collected during the course of our study will provide new ideas and a better understanding of how Polycomb complexes are regulated and tethered in eukaryotes.

We identified the protein partners of the catalytic subunit of the PRC2-Ezl1 complex. This complex is formed around 4 subunits which are very likely functional homologs of PRC2 in other organisms. Other subunits have been identified and do not resemble known PRC2 factors. The components of the complex are necessary to control transposable elements. Small RNAs are required for the deposition of H3K9me3 and H3K27me3 at TEs. We find that the physical interaction between PRC2 and the RNAi pathway is mediated by a RING finger protein and that small RNA recruitment of PRC2 to TEs is analogous to the small RNA recruitment of H3K9 methylation SU(VAR)3–9 enzymes.

We now want to reconstitute the activity of the recombinant complex and understand the role of the facultative subunits in the regulation of the complex activity.

1. Sellis S *, Gue´rin F*, Arnaiz O, Pett1 W, Lerat E, Boggetto N, Krenek S, Berendonk T, Couloux A, Aury J-M, Labadie K, Malinsky S, Bhullar S, Meyer E, Sperling L, Laurent Duret L*, Duharcourt S*. (2021). Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes. PLOS Biol. In press

2. Déléris A, Berger F, Duharcourt S*. (2021). Role of Polycomb in the control of transposable elements. Trends In Genetics. doi.org/10.1016/j.tig.2021.06.003
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3. Hardy A, Matelot M, Touzeau A, Klopp C, Lopez-Roques C, Duharcourt S*, Defrance M*. (2021). DNAModAnnot: a R toolbox for DNA modification filtering and annotation. Bioinformatics. 2021 Jan 20:btab032. doi: 10.1093/bioinformatics/btab032. PMID: 33471071

4. Vanssay A*, Touzeau A*, Arnaiz O, Frapporti A, Phipps J, Duharcourt S. (2020). The Paramecium histone chaperone Spt16-1 is required for Pgm endonuclease function in programmed genome rearrangements. PLoS Genet. 2020 Jul 23;16(7):e1008949. doi: 10.1371/journal.pgen.1008949.

The epigenetic landscape is established and maintained by different protein complexes in transcriptionally active versus silent regions of the genome. Polycomb Repressive Complex 2 (PRC2), a key component of the Polycomb machinery, trimethylates histone H3 on lysine 27 (H3K27me3), catalyzed by the Enhancer-of-zeste lysine methyltransferase subunit. Despite intensive research, the mechanisms that regulate the activity of the Polycomb complex and control its targeting remain largely enigmatic and are the focus of our proposal.

The ciliate Paramecium tetraurelia is an excellent unicellular model to unravel these mechanisms. In this organism, newly established chromatin regions with Polycomb signatures are removed from the somatic genome before cell division, during differentiation of distinct types of nuclei for germline and somatic functions. In Paramecium, two distinct nuclei coexist in the same cytoplasm. The diploid, transcriptionally silent micronuclei (MIC, 2n) contain the germline genome that is transmitted to sexual progeny, while a transcriptionally active somatic macronucleus (MAC, 800n) contains a reduced genome streamlined for gene expression. After fertilization, a new MAC differentiates from a copy of the zygotic nucleus. During MAC development, genome-wide rearrangements remove virtually all transposable elements (TEs), as well as 45,000 degenerate, single-copy remnants of ancient TE insertions (Internal Eliminated Sequences, IESs) in the germline. How such a large number of different germline sequences are specifically recognized and eliminated remains an intriguing question. Part of the answer comes from a piRNA-like class of small RNAs called scnRNAs. Initially produced from the MIC genome during meiosis by a specific RNA interference (RNAi) pathway, scnRNAs trigger the elimination of homologous germline-limited sequences in the developing zygotic MAC. This could in principle explain the recognition of all MIC-limited sequences despite the lack of a significant consensus. Nonetheless, it remains to be discovered how scnRNAs are linked to the elimination machinery.

We have recently demonstrated that chromatin modification is also involved in programmed DNA elimination. Enhancer-of-zeste like 1 (Ezl1), the Paramecium homolog of the lysine methyltransferase component of PRC2, is required for the elimination of repeated sequences and of the majority of IESs. We have shown that Ezl1 catalyzes H3K9me3 in vitro and in vivo, in addition to trimethylation of its conserved ad hoc substrate H3K27. We have further shown that these signatures co-occur on TE copies in an Ezl1-dependent manner. The data we collected so far thus indicate that scnRNA-directed TE inactivation is associated in Paramecium with H3K9me3 and H3K27me3 heterochromatin marks deposited by the Polycomb Ezl1 protein. The emerging picture is that TE inactivation depends on a prior step widely conserved in eukaryotes, the formation of heterochromatin via RNA-dependent mechanisms.

The POLYCHROME program aims at investigating the role of chromatin modification in the process bridging small RNA to DNA elimination. Our major objectives are to i) biochemically characterize the Paramecium PRC2-EZL1 complex; ii) gain insight into what targets the complex to specific chromatin sites and confers broad substrate specificity; iii) dissect the in vivo and in vitro function of novel identified components of the Paramecium PRC2-EZL1 complex. To achieve our objectives, we will combine genetic and cellular approaches, biochemical assays and genome-scale profiling of chromatin states and transcription. As a whole, our program will provide a better understanding of the mechanisms that establish epigenetic marks in specific regions of the genome during development in Paramecium. We expect that the data collected during the course of our study will provide new ideas and a better understanding of how Polycomb complexes are regulated and tethered in eukaryotes.