DNA Methylation. (B-Value) 3BKO. (B-Value) Supplementary Figure BKO HCT116 DKO8

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1 Supplementary Figure 1 a 1 DNA Methylation 0 3BKO DKO8 b DNA Methylation 0.5 3BKO DKO8 Supplementary Fig. 1. 3BKO and DKO8 cells compared to the parental cell line. (a) Heatmap showing the 10% (n=39,605) most differentially methylated CpG sites between the 3 cell lines. Each row is an individual CpG site. Color scale ranges from cold to warm (β-value 0-1, 0-100% methylated). (b) Boxplot of all CpG sites from the 450K showing DNA methylation distribution of, 3BKO and DKO8 cell lines. 1

2 Supplementary Figure 2 a Relative Expression to PCNA Days 56 Days DKO8 DNMT3A Δ3B2 Δ3B2-M Δ Relative Expression to PCNA Day 0 Day 42 3BKO DNMT3A b Relative Expression to PCNA DKO8 Exogenous DNMT3B or DNMT 21 Days 56 Days Δ3B2 Δ3B2-M Δ Relative Expression to PCNA BKO Exogenous DNMT3B or DNMT Day 0 Day 42 Supplementary Fig. 2. DNMT3A and DNMT3B mrna expression levels in DKO8 and 3BKO cells. (a) DNMT3A mrna expression levels at days 21 and 56 post transfection in DKO8 cells, expressing exogenous DNMT3B or DNMT normalised to PCNA (left). DNMT3A mrna expression levels at days 0 and 42 post 5-Aza-CdR treatment in 3BKO cells expressing exogenous DNMT3B or DNMT normalised to PCNA (right). (b) mrna expression level of endogenous DNMT3B in cells, exogenous DNMT3B isoforms and DNMT at days 21 and 56 post transfection, normalised to PCNA in DKO8 cells (left). mrna expression level of endogenous DNMT3B in cells, exogenous DNMT3B isoforms and DNMT at days 0 and 42 post 5-Aza-CdR treatment in 3BKO cells, normalised to PCNA (right). 2

3 Supplementary Figure 3 a DNA Methylation 0.5 b Endogenous Methylation Δ3B2 Δ3B2-M Δ Methylation Changes [Construct-] Day 56 Location CGI 54,911 CpG Sites DKO8 Δ3B2 Δ3B2-M Δ Others Promoter Methylation B-Value Methylation Difference Value Gene Body CGI NON-CGI Accessibility 3

4 Supplementary Fig. 3. DNMT3B isoforms and DNMT restore DNA methylation at specific CpG sites. (a) Boxplots showing the distribution of DNA methylation levels of 385,826 CpG sites for each indicated cell line. cells global methylation level is included for comparison to the derivative cell line DKO8. (b) Heatmap showing 54,911 CpG sites in DKO8 cells, expressing the indicated DNMT isoforms. CpG sites in genomic locations targeted by DNMT3Bs and with respect to promoter (maroon), gene body (orange) and other regions, excluding promoters and gene bodies (gray), are shown in the left panel. CpG sites in CpG islands are represented in green in the left panel. Endogenous methylation levels in and DKO8 cells are represented by a cold to warm color scale (β-value 0-1, 0-100% methylated), where every row represents one CpG site in the middle panel. DNA methylation differences between DNMT isoforms and empty vector () are shown in the right panel on a green to red color scale (0=no change). 4

5 Supplementary Figure 4 a ρ=0.99 b ρ=0.98 Rep 2 Rep 2 c ρ=0.98 Rep 1 d ρ=0.98 Rep 1 Rep 2 Rep 2 Rep 1 Rep 1 Supplementary Fig. 4. DNA methylation density scatterplots between replicates of DKO8 cells transfected for 56 days with a specific DNMT. Dashed white lines indicate 1:1 line and ± Beta- Value differences. Regions of high density are red, low density are blue and black areas represent no density. Spearman correlation values are indicted for each comparison. (a-d) Scatterplots of 450K array replicates for the selected constructs, DNMT, DNMT and DNMT constructs, respectively. 5

6 Supplementary Figure 5 a ρ=0.97 b ρ=0.91 Rep 2 Rep 2 c ρ=0.90 Rep 1 d ρ=0.90 Rep 1 Rep 2 Rep 2 Rep 1 Rep 1 Supplementary Fig. 5. DNA methylation density scatterplots between replicates of DKO8 cells transfected for 56 days with a specific DNMT for targeted CpG sites. Dashed white lines indicate 1:1 line and ± Beta-Value differences. Regions of high density are red, low density are blue and black areas represent no density. Spearman correlation values are indicted for each comparison. (a-d) Scatterplots of the selected 54,911 CpG sites from the 450K array replicates of, DNMT, DNMT and DNMT constructs, respectively. 6

7 Supplementary Figure 6 Rep 2 Rep 1 a Rep 1 ρ=9 Rep 2 Rep 1 Rep 2 ρ=6 Rep 1 c Rep 2 Rep 2 Rep 1 b Rep 1 ρ=0.92 Rep 2 7

8 Supplementary Fig. 6. DNA methylation density scatterplots of replicates between and transfected cells with a specific DNMT. Dashed white lines indicate 1:1 line and ± Beta-Value differences. Regions of high density are red, low density are blue and black areas represent no density. (a-c) Scatterplots of the selected 54,911 CpG sites from the 450K array comparing to DNMT, DNMT and DNMT with the first array replicate on the left and the second array replicate on the right, respectively. 8

9 Supplementary Figure 7 Location CGI Others Promoter Gene Body CGI NON-CGI Rep-1 Rep-2 Rep-1 Rep-2 0 Rep-1 Rep-2 Rep-1 Methylation B-Value 1 Rep-2 Supplementary Fig. 7. Replicates of DNMT3B isoforms and DNMT show consistent target CpG sites. Heatmap showing 54,911 CpG sites in DKO8 cells, expressing the indicated DNMT isoforms. CpGs were selected because they were targeted by at least one construct. CpG sites in genomic locations targeted by DNMT3Bs and with respect to promoter (maroon), gene body (orange) and other regions, excluding promoters and gene bodies (gray), are shown in the left panel. CpG sites in CpG islands are represented in green in the left panel. Endogenous methylation level in cells are represented by a cold to warm colour scale (β-value 0-1, 0-100% methylated), where every row represents one CpG. DNA methylation levels of DKO8 cells expressing a DNMT3B isoforms or DNMT are shown in the right panel with side by side comparisons of biological array replicates. 9

10 Group I DNA Methylation DNA Methylation Group II Group 3 Group III HCT DNA Methylation a Group 2 DNA Methylation Supplementary Figure 8 Group 1 Group 4 Group IV HCT Days Post Treatment b Days Post TreatmentDays Post TreatmentDays Post Treatment Days Post Treatment Day 0 Day 5 Day 42 Group IV 10,907 CpG Sites Others Promoter Gene Body CGI NON-CGI 1 Methylation β-value 0 3BKO 3BKO 3BKO DNA Methylation B-Value HCT HCT 10

11 Supplementary Fig. 8. DNMT3B isoforms restore DNA methylation in a DNMT3B knock out cell line. (a) Previously described Groups I-IV were used to show average DNA methylation values for each 3BKO cell line expressing a specific DNMT3 isoform at days 0, 5, 10 and 42 post 24 hour 5-Aza- CdR treatment 1. (b) Heatmaps and boxplots showing non H3K36me3-enriched and slow rebounding Group IV CpG sites in cell lines expressing different DNMTs before 5-Aza-CdR treatment and at day 5 and day 42 post treatment 1. Individual CpG sites falling in genomic locations targeted by DNMT3Bs and in respect to promoter (maroon), gene body (orange) and other regions, excluding promoters and gene bodies (gray) are shown in the left panel. CpG sites in CpG islands are represented in green in the left panel. Endogenous methylation levels in cell lines are represented by a cold to warm color scale (β-value 0-1, 0-100% methylation) where every row represents one CpG site in the 3 right panels. 11

12 Supplementary Figure 9 a 3ABDKO HCT3ABKO DNMT3A DNMT3A FC Relative to TBP5 Day 0 Day 42 b FC Relative to TBP8 HCT3ABKO 3ABDKO Exogenous DNMT3B or or DNMT Day 0 Day 42 Supplementary Fig. 9. DNMT3A and DNMT3B mrna expression levels in 3ABDKO cells. (a) mrna expression level of endogenous DNMT3A in cells and mrna expression levels at days 0 and 42 post 5-Aza-CdR treatment in 3ABDKO cells expressing exogenous DNMT3B or DNMT, normalised to TBP. (b) mrna expression level of endogenous DNMT3B in cells, exogenous DNMT3B isoforms and DNMT at days 0 and 42 post 5-Aza-CdR treatment in 3ABDKO cells, normalised to TBP. 12

13 Others Promoter Gene Body CGI NON-CGI 1 Methylation β-value 0 13 Day 5 3ABKO Day 0 3ABKO 3ABKO DNA Methylation B-Value Group IV 10,907 CpG Sites Supplementary Figure 10 Day 42

14 Supplementary Fig. 10. DNMT3B isoforms do not restore DNA methylation in a DNMT3A knock out cell line. Heatmaps and boxplots showing non H3K36me3-enriched and slow rebounding Group IV CpG sites in cell lines expressing different DNMTs before 5-Aza-CdR treatment and at day 5 and day 42 post treatment 1. Individual CpG sites falling in genomic locations targeted by DNMT3Bs and in respect to promoter (maroon), gene body (orange) and other regions, excluding promoters and gene bodies (gray) are shown in the left panel. CpG sites in CpG islands are represented in green in the left panel. Endogenous methylation levels in cell lines are represented by a cold to warm color scale (β-value 0-1, 0-100% methylation) where every row represents one CpG site in the 3 right panels. 14

15 Supplementary Figure 11 Fig 1d. 250kDa 150kDa 100kDa 75kDa 50kDa 37kDa 250kDa 150kDa 100kDa 75kDa 50kDa 37kDa 50kDa 37kDa 50kDa 37kDa Fig 1e. 100kDa 75kDa 37kDa 50kDa 37kDa 100kDa 75kDa 37kDa 50kDa 37kDa Supplementary Fig. 11. The original full Western blots images used in Figure 1. 15

16 Supplementary References 1 Yang, X. et al. Gene Body Methylation Can Alter Gene Expression and Is a Therapeutic Target in Cancer. Cancer Cell 26, 1-14, doi: (2014). 16