Extended Data Fig. 11: Linking asymmetric H3K9me3 segregation at HO Ls to their silencing during S phase.
From: Asymmetric distribution of parental H3K9me3 in S phase silences L1 elements
a. Isolation of mES cells at G1, S and G2 phases based on the expression of Cdt1-mKO2 and Geminin-mAG1 by flow cytometry. Cells were sorted based on the expression of these two cell cycle indicators. G1 phase cells only express Cdt1, but not Geminin. S phase cells express medium levels of Geminin, but not Cdt1, and G2 phase cells express the highest levels of Geminin. To increase the purity of S and G2 phase cells, we used a stringent gating strategy as shown with isolated G1, S and G2 phase of cells accounting for ~10%, ~15% and ~10% of total cells, respectively. b. Heatmaps of differentially expressed L1 elements in Mpp8 KO, Pole3 KO and Pole4 KO versus WT mES cells based on GRO-seq analysis. Numbers of total differentially expressed L1s in each mutant were shown. Please note that all L1 elements were used in the analysis. c. Relative expression of representative repetitive elements in Mpp8 KO, MPP8 W80A or TASOR M3 mutant cells compared to WT mES cells by RT-qPCR. The expression was normalized against WT. Data were plotted as mean ± s.e.m., n = 5–9. d. Snapshots of H3K9me3 signals (both ChIP-seq and CUT&Tag), H3K9me3 eSPAN bias and GRO-seq signals at three L1 elements in WT or mutant mES cells. The three up-regulated L1s were highlighted. e. Relative expression of representative repetitive elements in POLE3 KD and POLE4 KD cells compared to WT HeLa cells detected by RT-qPCR. The expression was normalized against WT. Data were plotted as mean ± s.e.m., n = 3. f. The expression of HO L1s (n = 2,662) in WT, Pole3 KO and Pole4 KO mES cells detected by GRO-seq after excluding the ones located within the transcribed regions of up-regulated genes in Pole3 KO or Pole4 KO mES cells defined by RNA-seq. g. The expression of HO L1s (n = 2,309) in WT, Pole3 KO and Pole4 KO mES cells detected by GRO-seq after excluding the ones located within any actively transcribed genes from analysis (cutoff: TPM > 0.5). h. The expression of full-length (≥ 6 kb) HO L1s (n = 703) with their own promoters in WT, Pole3 KO and Pole4 KO mES cells. See Materials and Methods section for more details for panels f-h. c, e, Two-sided Student’s t test. ****, p < 0.0001. ***, p < 0.001. **, p < 0.01. *, p < 0.05. Box plots (f-h) show the median, 25% and 75% quartiles and minimal and maximal values with p values by two-sided Mann–Whitney–Wilcoxon tests, and Bonferroni correction for multiple comparisons. Each panel is a representative of at least two independent experiments. See Materials and Methods for more details.
