Fig. 3

In vivo genetic complementation assay. a Life-cycle of Paramecium. Each cell contains a macronucleus (MAC) and two micronuclei (MICs), both of which divide and segregate to daughter cells during vegetative growth. Starvation triggers MIC meiosis, and one of the meiotic products divides mitotically to form two haploid nuclei. During autogamy, a self-fertilization process, the two haploid nuclei will fuse to produce the zygotic nucleus (karyogamy), which then divides twice to form two new MACs and two MICs. During development of the new MAC, the MIC genome undergoes massive programmed DNA elimination. Upon nutrient supply, post-autogamous cells will undergo the first cellular division and produce sexual progeny that will resume vegetative growth. The parental MAC is degraded into fragments that will eventually be diluted through cell divisions. b H3K27me3 and H3K9me3 immunostaining of non-injected cells (NI), and of cells transformed with GFP-EZL1^wt or GFP-EZL1^H526A RNAi-resistant transgenes upon control or EZL1 RNAi during MAC development, at ~25 h after the onset of sexual events. The non-essential ND7 gene was used as RNAi control. Representative images (percentage in cell population is indicated, n ≥ 100) are displayed. Overlay of Z-projections of magnified views of H3K27me3- or H3K9me3-specific antibodies (in cyan), of GFP (in green) and Hoechst (in red) are presented. Dashed white circles indicate the new developing MACs. The other Hoechst-stained nuclei are fragments from the old vegetative MAC. In control cells, H3K27me3 is detected in the fragments from the old vegetative MAC, while both H3K9me3 and H3K27me3 are detected in the new developing MACs. The scale bar is 10 μm. c Production of sexual progeny following control (CTL) or EZL1 gene silencing in non-injected (NI) cells, GFP-EZL1^wt and GFP-EZL1^H526A transformed cells. Cells were starved in each medium to induce autogamy and, following 3–4 days of starvation, post-autogamous cells were transferred individually to standard growth medium to assess the ability of sexual progeny to resume vegetative growth. The total number of cells analyzed for each RNAi and the number of independent experiments (in parenthesis) are indicated. Transgene copy number per haploid genome (see Methods): 6.9 (n = 30) and 76.4 (n = 58) for GFP-EZL1^wt, 3.4 (n = 30), and 5.8 (n = 60) for GFP-EZL1^H526A. d Quantification of the relative copy number of individual TEs (Anchois; RT12275; RT25765; RT31010; RT42890) as measured by qPCR from total genomic DNA extracted at the end of MAC development from non-injected (NI), GFP-EZL1^wt and GFP-EZL1^H526A transformed cells after control (CTL) or EZL1 RNAi. Values were normalized to a MIC-specific, single copy genomic locus. The horizontal bars represent mean of biological replicates. Circles indicate the individual data points. Source data are provided as a Source Data file