Table S1.Cell surface marker expression in splenocytes of Dnmt3a+/+ and Dnmt3a-/- mice.Expression of surface markers incells isolated from spleens of 21 day oldDnmt3a+/+ (n=4), Dnmt3a-/- (n=4), Dnmt3a-/-;Dnmt3b-/- (n=4) mice as well as Dnmt3a-/-mice with splenic tumors (n=10). The average percentage of EGFP+ cells expressing each marker is shown with + SEM.
Figure S1. Analysis of EGFP and cell surface marker expression inDnmt3a+/+ and Dnmt3a-/- mice. (a) FACS analysis of EGFP expression in fetal liver cells isolated from 15.5 day embryos with indicated genotypes.EGFP-positive cells were further analyzed for expression of B220, CD3, CD5, CD8, Gr-1, Ter119lineage markers to define lineage-positive cells. Lineage-negative cells were analyzed for expression of Thy1.1, CD11b and Sca-1. Lineage-negative, Thy1.1lo, CD11b+, Sca-1+represent fetal liver HSCs.The percentage of positive cells is indicated in the FACS diagrams.(b) Representative FACS analysis of EGFP+ LSK cells in the bone marrow of 21days oldDnmt3a+/+and Dnmt3a-/-mice.Representative FACS diagrams show percentage of EGFP+LSK cells identified as Lineage-negative,Sca1+,c-kit+. Lineage-negative populations were identified byCD4, CD8, CD11b, B220, CD3, TER119 lineage marker expression. Average percentages of LSK cells are shown in the FACS diagrams. (c) Total percentage of EGFP+ LSK cells in the bone marrow of 21 days old Dnmt3a+/+(n=4) and Dnmt3a-/-(n=5) mice. Error bars represent the SEM. (d) Representative FACS diagrams showing expression of EGFP, Ter119, B220, CD11b, CD4 and CD8 in spleens (SP) and thymi (Th) of Dnmt3a+/+ mice. Percentages of positive cellsfor each quadrant are shown in top right corner.
Figure S2. Cellularity of Dnmt3a and Dnmt3a/Dnmt3b knockout mice. The average cell counts of aged-matched Dnmt3a+/+ (black), Dnmt3a-/-(red) Dnmt3a-/-;Dnmt3b-/-(green)spleens and bone marrow at age 21 days (21d). Error bars represent the SEM.
Figure S3. Deletion efficiency of Dnmt3a conditional knockout allele in Dnmt3a-/- tumors. PCR-based analysis of deletion efficiency of the Dnmt3a conditional knockout allele in DNA from splenic cells isolated from Dnmt3a-/- mice with CLL. Fragments derived from floxed (F) and knockout (KO) alleles are shown. Dnmt3aF/F genomic DNA served as a control.
Figure S4. Histological analysis of Dnmt3a-/- splenic tumors. (a)A spleen from a Dnmt3a+/+mouse with normal architecture.White pulp (long arrow) and red pulp (short arrow) are indicated (H&E 100x). (b) Dnmt3a-/- tumors showing diffuse effacement of the splenic architecture with expansion of the white pulp (H&E 100x). (c) Dnmt3a-/-tumor showing neoplastic cell populations (long arrow), resting lymphocytes (short arrow) and prolymphocytes (block arrow) (H&E 400x).
Figure S5. Dnmt3a-/- splenic tumor cells induce splenomegaly and CLL disease in recipient mice.(a) A picture showing spleens from aDnmt3a+/+ control and a Dnmt3a-/-recipient at 90 days post intraperitoneal injection. (b) A bar graph representing the average spleen weight in grams from Dnmt3a+/+ controls and Dnmt3a-/- recipients at 90 day post i.p. injection. n denotes the number of mice. Error bars represent the SEM. (c) A representative flow diagram depicting B220 and CD5 expression in splenic tumor cells from Dnmt3a-/- recipient at 90 day post i.p. injection.
Figure S6. Methylation analysis of gene bodies and repetitive elements. Bar graphs depictingin silico analysis of relative methylation of gene body, Line/L1, Line/L2, and Sine/Alu repeats in Dnmt3a+/+ B1 cells and Dnmt3a-/- tumors (CLL) as determined by MSCC.The total number of tags obtained from next generation sequencing of MSCC libraries (HpaII and HpyCh4IV) generated from Dnmt3a+/+ B1 cells (n=2) and Dnmt3a-/- CLL samples (n=3) specific to genebody (defined as +500 base pairs from transcription start site to the end of the gene) or repeat elements were summed. Averages of B1 tags were used for normalization of the data. As the number of MSCC tags inversely correlates with methylation levels the inverse values represent relative methylation levels.Error bars represent ± SEM (* denotes P<0.05).
Figure S7. Schematic representation of the genetic setting to delete conditional alleles of Dnmt3a and Dnmt3b in hematopoietic system.