Expanded View Figures

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1 The EMO Journal Dnmt regulates translational fidelity Francesca Tuorto et al Expanded View Figures x E3 cells/µl W g/l HG x e3 cells/µl PLT % of cell type 8 day 8 month % of cell type 8 day 8 month 8 day 8 month 8 day 8 month wild-type Dnmt / D recipients recipients 3 recipients nor derived P cells erived P cells % do % donor week 8 week week 6 week week nor derived P cells % do. w post transplant 8 7w post transplant w post transplant D4R D3 Db Ly6G erived P cells % donor d week 8 week week 7 week D4R D3 Db Ly6G nor derived P cells % do erived P cells % donor d week 8 week week * D4R D3 Db Ly6G Figure EV. Peripheral blood cell counts. Total blood cell counts of wild-type and Dnmt / mice showing no significant differences. Total white blood cell counts, hemoglobin concentration, and platelet cell counts of wild-type and Dnmt / mice (n = 3 mice per group). W: white blood cell counts; HG: hemoglobin; PLT: platelets. Lineage distribution of peripheral blood analyzed by the DVI hematology analyzer system. RETI: reticulocytes; NEUT: neutrophiles; LYMPH: lymphocytes; MONO: monocytes; EOS: eosinophiles; SO: basophiles; LU: large unstained cells. Giemsa staining of a representative femoral section from 4-month-old mice showing restoration of ossification defects. Scale bar: lm. D Stable engraftment (upper panels) and multi-lineage reconstitution (lower panels) in wild-type and Dnmt / recipient mice. Donor chimerism is indicated by D4. expression of peripheral blood (P) cells using flow cytometry. Peripheral blood was collected monthly and analyzed for engraftment and lineage distribution (Db: macrophages; Ly6G: granulocytes; D4R: cells; D3: T cells). Data information: Data are presented as mean SD. sterisks indicate statistically significant (P <., t-test) differences. EV The EMO Journal ª The uthors

2 Francesca Tuorto et al Dnmt regulates translational fidelity The EMO Journal Figure EV. nalysis of protein synthesis rates. nalysis of total protein synthesis in primary bone marrow stromal and haematopoietic cells by pulse-labeling with 3 H-Gly. s a negative control, protein synthesis was blocked in wild-type cells by treatment with lm sodium arsenite (so 3 ). Results were obtained from three independent experiments, and error bars represent standard deviations. Representative polysome profiles from Dnmt / and control primary bone marrow culture. Haematopoietic cells growing in suspension (ss) and attached stromal (st) cells were independently harvested and showed a higher proteins synthesis rate. s a measure for the global rate of translation, the fraction of polysomal ribosomes was quantified from polysome profiles. Results are shown for two independent biological replicates, and error bars represent standard deviations. ce at 4 nm (U) ss st - ss - st bsorba time on Polysomal fracti ss -ss st -st ª The uthors The EMO Journal EV

3 The EMO Journal Dnmt regulates translational fidelity Francesca Tuorto et al difference % codon frequency difference codon frequency Figure EV3. Regulation of codon fidelity. Heatmaps showing the significant codon biases in Dnmt down-regulated proteins in the DML dataset. n increase of a codon frequency relative to the average frequency of that codon in all identified proteins is displayed in red, whereas a reduction is depicted in green. odons showing no deviation from average values are displayed in white according to the scale provided in the figure. Heatmaps showing codon biases in all protein datasets. hanges of codon frequencies relative to the average frequency of the indicated codon are displayed according to the scale provided in the figure. EV3 The EMO Journal ª The uthors

4 Francesca Tuorto et al Dnmt regulates translational fidelity The EMO Journal Log footprint density (RPKM) Spearman correlation =.96 M SS Log footprint density (RPKM) Spearman correlation =.94 M SS Log footprint density (RPKM) replicate Log footprint density (RPKM) replicate Log footprint density (RPKM) Spearman correlation =.9 M ST Log footprint density (RPKM) replicate Log footprint density (RPKM) Spearman correlation =.9 M SS rrn aligned unaligned Log footprint density (RPKM) replicate replicate SS SS ST ST SS SS ST ST read counts [x 4 ] 3 9,, 8,, number of reads D read counts [x 4 ] position from start [nt] position from start [nt] Figure EV4. Reproducibility and quality control of ribosome profiling. orrelation between ribosome profiling dataset replicates. Haematopoietic cells growing in suspension (ss) and attached stromal cells (st) were independently harvested. Mapping results from the two independent ribosome profiling replicates. Representative metaplot of wild-type haematopoietic (ss) cells. 8-mer ribosome footprint reads were summed across all start codons. D Representative in-frame 8-mer ribosome footprint abundance in wild-type haematopoietic (ss) cells showing the correct periodicity. ª The uthors The EMO Journal EV4

5 The EMO Journal Dnmt regulates translational fidelity Francesca Tuorto et al k-s test P-value:.8E-3 GU Log codon occupancy at site k-s test P-value:4.9E- GU Log codon occupancy at site k-s test P-value:.7E-9 G k-s test P-value:3.E- G Log codon occupancy at site Log codon occupancy at site k-s test P-value:.E-6 GG k-s test P-value: 4.E-3 GG Log codon occupancy at site Log codon occupancy at site Fraction of codon ns k-s test P-value:.3E-8 GUU st Fraction of codon ns k-s test P-value:.6E- GUU Log codon occupancy at site Log codon occupancy at site ss P-values codon occupancy GG Glu G GU sp G GG -Gly GUU -Val others st P ss P Log / P P P ll Down Up Figure EV. Single-codon occupancy metric shows that differences in ribosome footprint occupancy at Dnmt target codons are statistically significant. Heatmap of P-values (Kolmogorov Smirnov test) for all sense codons in wild-type and Dnmt / ribosome and P sites. The two biological replicates of each cell type were pooled. : ribosomal -site; P: ribosomal P-site; st: stromal cells, ss: suspension cells. umulative distributions of single-codon occupancy for select codons in wild-type and Dnmt / stromal (st) and suspension (ss) cells. hanges in bulk codon occupancy in Dnmt / bone marrow suspension cells versus wild-type cells. Dnmt target codons and Glu codons are highlighted with the indicated colors. The two biological replicates of each cell type were pooled. : ribosomal -site; P: ribosomal P-site. EV The EMO Journal ª The uthors