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. Author manuscript; available in PMC: 2024 Apr 22.
Published in final edited form as: Nature. 2023 Nov 8;623(7987):643–651. doi: 10.1038/s41586-023-06711-3

Extended Data Figure 9. Effects of POLE3 or POLE4 deletion/depletion on H3K9me3 density and H3K9me3 eSPAN bias in mES and HeLa cells.

Extended Data Figure 9.

a. Immunoblots of POLE3 and POLE4 to confirm their deletion in mES and depletion in HeLa cells. Note that cloned ES cells (KO) and pooled HeLa cells (KD) were used for analysis and that H3K9me3 levels remained largely unaffected in the mutant cells. n = 3.

b, c. H3K9me3 CUT&Tag (b) or CUT&RUN (c) signals at HO L1s in WT, POLE3 KO and POLE4 KO mES cells. Heatmaps (left) were sorted by the average H3K9me3 signals of each row in WT cells, with average density shown at the bottom. Note that very little changes of H3K9me3 levels were observed in the mutants.

d. H3K9me3 CUT&Tag signals at HO L1s in WT, POLE3 KD and POLE4 KD HeLa cells, with the heatmaps (top, sorted as in b) and average density at HO L1s (bottom) shown. HO L1s were grouped as long and short, as defined in Extended Data Figure 7e.

e, f. H3K9me3 CUT&Tag signals at HO L1s in WT, POLE3 KO (e) and POLE4 KO (f) mES cells. Heatmaps (left, sorted as in b) and average density (right) were shown. HO L1s were separated into two groups based on the effects POLE3 or POLE4 KO on H3K9me3 levels at HO L1s, with a reduction of more than 1.5-fold defined as the Down group and the rest of L1s within this cutoff being grouped as No-diff group. Note that less than 50 (~0.4%) HO L1s showed a marked reduction of H3K9me3 density and therefore the eSPAN bias was not calculated at this group separately.

g. H3K9me3 eSPAN bias around replication origins (top) and at HO L1s (bottom, n = 11,268) in WT, POLE3 KD and POLE4 KD HeLa cells. Long and short HO L1 elements were defined as in Extended Data Figure 7.

Box plots show (g) 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.

For gel source data, see Supplementary Figure 1.