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-1787

+1. hMLH1. -1787. +19. B. C. SW480. RKO. SW480. RKO. Acetylated H3 Enrichment. +. +. -. -. +. +. -. -. Ab. SW480. SW480. RKO. RKO. B. I. B. I. B. I. B. I. hMLH1. GAPDH. Position from +1. D. E. RKO. SW480. +. +. -. -. +. +. -. -. Ab. Methyl-H3-K4

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-1787

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  1. +1 hMLH1 -1787 +19 B C SW480 RKO SW480 RKO Acetylated H3 Enrichment + + - - + + - - Ab SW480 SW480 RKO RKO B I B I B I B I hMLH1 GAPDH Position from +1 D E RKO SW480 + + - - + + - - Ab Methyl-H3-K4 enrichment B I B I B I B I hMLH1 GAPDH Position from +1 F G RKO SW480 + + - - + + - - Ab Methyl-H3-K9 enrichment B I B I B I B I hMLH1 GAPDH Position from +1 Figure 1 A 25

  2. A B +TSA +TSA +TSA -TSA -TSA -TSA - - - - + + + + TSA B I B I B I B I Acetylated H3 enrichment + + - - + + - - Ab hMLH1 GAPDH Position from +1 C D - - - - + + + + TSA + + - - + + - - Ab Methyl-H3-K4 enrichment B I B I B I B I hMLH1 GAPDH Position from +1 E F - - - - + + + + TSA + + - - + + - - Ab B I B I B I B I Methyl-H3-K9 enrichment hMLH1 GAPDH Position from +1 Figure 2 27

  3. A B +5-Aza-dC +5-Aza-dC +5-Aza-dC Acetylated H3 enrichment -5-Aza-dC -5-Aza-dC -5-Aza-dC - - - - + + + + 5-Aza-dC + + - - + + - - Ab B I B I B I B I hMLH1 GAPDH Position from +1 C - - - - + + + + D + + - - + + - - B I B I B I B I Methyl-H3-K4 enrichment 5-Aza-dC Ab hMLH1 GAPDH Position from +1 E F - - - - + + + + 5-Aza-dC Methyl-H3-K9 enrichment + + - - + + - - Ab B I B I B I B I hMLH1 GAPDH Position from +1 Figure 3 29

  4. Figure 4 31

  5. A 12 h 24 h 48 h 5 d Bisulfite dH2O PCR dH2O 5-Aza-dC 5-Aza-dC 5-Aza-dC 5-Aza-dC SW480 mock mock mock mock RKO U M U M U M U M U M U M U M U M U M U M U M U M B 12 h 24 h 48 h 5 d 5-Aza-dC 5-Aza-dC 5-Aza-dC 5-Aza-dC dH2O mock mock mock mock RT + - + - + - + - + - + - + - + - GAPDH hMLH1 Figure 5 33

  6. H3.3 3xHA 408 bp 90 bp 18 bp linker WT 1 2 3 4 5 8 9 10 11 12 14 15 β-Actin H3.3-HA WT 16 17 18 19 20 21 22 23 24 β-Actin H3.3-HA Anti-HA DAPI Merge Clone 10 Clone 16 WT RKO 2 1 2 3 4 6 8 9 10 11 12 13 β-Actin H3.3-HA Figure 6 A B C D 53

  7. RKO 2 RKO 2 WT 1 2 3 4 6 8 9 10 11 12 β-Actin H3.3-HA RKO 2 WT 1 2 3 4 5 6 7 8 9 10 β-Actin H3.3-HA 11 12 13 14 15 16 17 18 19 20 β-Actin H3.3-HA RKO Clones SW480 clone 20 SW480 WT RKO WT 1 3 4 11 18 β-Actin H3.3-HA Figure 7 A B C 55

  8. RKO Clones RKO WT 18 IgG 1 3 4 11 18 SW480 clone 20 RKO Clones SW480 WT RKO WT 1 3 4 11 18 Figure 8 Figure 8. H3.3-HA can be immunoprecipitated with anti-HA. Whole cell (top panel) or nuclear (bottom panel) lysates were immunoprecipitated with an anti-HA antibody. Numbers across the top indicate clone number. Whole cell lysate from RKO clone 18 was incubated with normal rabbit IgG as a negative control. 56

  9. H3.3 Enrichment Position from +1 SW480 clone SW480 clone SW480 WT SW480 WT RKO clone RKO clone RKO WT RKO WT Methyl-H3-K4 Enrichment Position from +1 Figure 9 A B 58

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