Extended data figure 8. DNMT1 deletion delays oncogenesis in TET-deficient mice.

a) Diagrammatic representation of the strategy used to confirm G-quadruplex binding. Nuclear lysates of activated B cells were incubated with biotin-conjugated single stranded G4- or non-G4-forming control oligonucleotides (Oligos) captured using streptavidin beads. b) Immunoblots showing flag-tagged BG4 (positive control), ATRX, BLM and DNMT1 proteins. Left lane, 1/10^th input from nuclear lysates (1/10^th Input); middle lane, proteins pulled down with G4 forming oligonucleotides; and right lane, proteins pulled down with non-G4 control oligonucleotides. The data is representative of at least 2 independent experiments. c) - d) Quantification of c) cell numbers, d) spleen weights, of 10 week-old Tfl (grey), CD19 Dnmt1 KO (purple), CD19 DKO (red) and CD19 TKO (brown) mice from 5 independent experiments. e) Enlarged spleen of 75-week-old CD19 TKO mice compared with Tfl control mice. f) Flow cytometric detection of G-quadruplexes with BG4-Ig antibody or isotype IgG controls in B cells from Tfl (YFP⁻, grey), CD19 Dnmt1 KO (YFP⁺, purple), CD19 DKO (YFP⁺, red) and CD19 TKO (YFP⁺, brown) mice. g) – h) Quantification of median fluorescence intensity (MFI) of g) BG4-Ig signal, h) γH2AX signal from Tfl (YFP⁻), CD19 Dnmt1 KO (YFP⁺), CD19 DKO (YFP⁺) and CD19 TKO (YFP⁺) B cells from 4 independent experiments. i), k) Flow cytometric detection of i) G-quadruplexes with BG4-Ig antibody or isotype IgG controls and k) R-loops using V5-epitope-tagged recombinant RNASE H1 (rRNASE H1) or IgG controls in GC B cells (Fas⁺) from Tfl (YFP⁻, grey), CD19 DKO (YFP⁺, red) and CD19 TKO (YFP⁺, brown) mice. j), l) Quantification of median fluorescence intensity (MFI) of j) BG4-Ig signal and l) R-loops (rRNASE H1) signal in GC B cells (Fas⁺) from Tfl (YFP⁻, grey), CD19 DKO (YFP⁺, red) and CD19 TKO (YFP⁺, brown) mice from 3 independent experiments. m) Model proposing functional interplay between TET and DNMT activities to limit GC B cell expansion. TET deficiency in B cells leads to increased G4 and R-loop structures and is associated with altered gene expression, DNA damage and development of B cell lymphoma. Statistical significance is calculated using one-way ANOVA in c), d), g), h), j) and l). Error bars represent mean +/− standard deviation, * p value ≤0.05, ** p value ≤0.01, *** p value ≤0.0005 **** p value <0.0001.