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1 doi: /nature10860 Supplementary Discussion It remains unclear why H3K9 demethylation appeared to be more sensitive to suppression than at least some other histone methylation marks as a result of IDH mutation. This may be because H3K9 demethylases are more sensitive to inhibition by 2HG in comparison to other JHDM family members 14, 15. Alternatively, H3K9 methylation is a histone mark that positively correlates with DNA methylation and participates in repressive heterochromatin formation associated with HP1 recruitment 13. H3K9 methylation is also mutually exclusive with H3K9 acetylation, a mark that positively correlates with increased gene expression and the promotion of H3K4 methylation at promoters 31. The fact that H3K27me3 can also significantly increase in response to IDH mutation suggests that there is a potential crosstalk between H3K9 methylation and Trithorax and Polycomb group proteins that regulate H3K4 and H3K27 methylation, respectively. In addition, given the delayed kinetics of other alterations in histone marks following introduction of mutant IDH, some of these changes may result from a mutant IDHinduced block in differentiation rather than direct inhibition of the relevant demethylases. It should be noted that KDM4C is not the only H3K9 demethylase and the fact that H3K9 methylation is increased in mutant IDH-infected fibroblasts prior to the induction of adipocytic differentiation suggests that 2HG is affecting additional H3K9 demethylases which have been shown to share a structurally similar binding pocket 32, 33 but can exhibit tissue- and developmentspecific variations in their expression. Our data contribute to the mounting evidence that dysregulation of covalent histone modifications can contribute to tumorigenesis through blocking cellular differentiation. For example, the MLL gene, which encodes a H3K4 methyltransferase, is involved in chromosomal rearrangement in ~80% of infant leukemia and 5-10% of adult AML 19. KDM3B (also known as JMJD1B) is an H3K9 demethylase which is located at chromosome 5q31, a region frequently deleted in AML and myelodysplastic syndrome 20. More recently, systematic mutational screens of the coding genome identified inactivating mutations of KDM6A (H3K27 demethylase, also known as UTX) in a large array of cancers 21. A direct role of histone methylation in stem cell maintenance and differentiation has also been shown. For example, loss of KMT1E (also known as SETDB1), a H3K9 methyltransferase, resulted in impaired stem cell renewal 22. Recently, KMT1E was found to be recurrently amplified in melanoma and accelerated melanoma onset 1

2 RESEARCH SUPPLEMENTARY INFORMATION after BRAF mutation 23. The present data suggest that 2HG-producing IDH mutations represent an additional mechanism to impair the function of histone demethylases and thus block cellular differentiation. Additional References: 31. Latham, J.A. & Dent, S.Y. Cross-regulation of histone modifications. Nat. Struct. Mol. Biol. 14, (2007). 32. Chen, Z. et al. Structural insights into histone demethylation by JMJD2 family members. Cell 125, (2006). 33. Ng, S.S. et al. Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity. Nature 448, (2007). 2

3 RESEARCH a Band int tensity normalized to total H3 b Vec IDH1 IDH1 IDH2 IDH2 WT R132H WT R172K Histone methylation marks Correlation coefficient H3K9me H3K9me H3K4me H3K27me H3K36me H3K79me H3K9me2 H3K9me3 H3K4me3 H3K27me3 H3k36me33 H3K79me2 Supplementary Figure 1: Band intensity quantification and correlation analysis of Western blot shown in Fig. 1b. a, Western blot band intensity was quantified by Image J software and normalized to levels of total H3. Error bars represent standard deviation from three independent experiments. b, correlation coefficient of histone methylation levels and intracellular 2HG levels shown in Fig. 1b. 3

4 RESEARCH SUPPLEMENTARY INFORMATION a b Supplementary Figure 2: Synthesis of Octyl-2HG a Synthesis route of D-2HG-1-octyl ester Supplementary Figure 2: Synthesis of Octyl-2HG. a, Synthesis route of D-2HG-1-octyl ester. TFAA, trifluoroacetic anhydride; TFH, tetrahydrofuran. b, 3T3-L1 cells were treated with 2mM octyl-2hg for 7 hours and intracellular 2HG level was measured by GC-MS. Metabolite abundance refers to GC-MS signal intensity. 4

5 RESEARCH MA coverage Vector #1 Vector #2 Vector #3 IDH2 WT #1 IDH2 WT #2 IDH2 WT #3 IDH2 Mut #1 IDH2 Mut #2 IDH2 Mut #3 Adipoq % Methylation MA coverage Vector #1 Vector #2 Vector #3 IDH2 WT #1 IDH2 WT #2 IDH2 WT #3 IDH2 Mut #1 IDH2 Mut #2 IDH2 Mut #3 Cebpa Supplementary Figure 3: DNA methylation levels at promoters of adipogenesis genes were not affected by IDH mutation. 3T3-L1 cells stably expressing empty vector, wild-type, or R172K mutant IDH2 were induced to differentiate into mature adipocytes for 4 days. DNA was extracted from cells and bisulfite-converted. Promoter DNA methylation levels were assessed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry using EpiTyper by MassARRAY (Sequenom). MassARRAY coverage for each promoter was indicated. Percentage of methylation was shown for triplicate samples from each condition. 5

6 RESEARCH SUPPLEMENTARY INFORMATION Signal intensity normalized to tota al H H3K9me3 H3K9me2 Ac H3 0 d0 d4 vec IDH2 IDH2 vec IDH2 IDH2 WT R172K WT R172K Supplementary Figure 4: Band intensity quantification of Western blot shown in Fig. 2g. Western blot band intensity was quantified by Image J software and normalized to levels of total H3. Error bars represent standard deviation from three independent experiments. 6

7 RESEARCH IDH1 WT IDH1 R132H P4 P7 P12 P17 P22 P27 Supplementary Figure 5: Representative FACS histograms of CpG methylation staining in NHA cells. NHA cells stably expressing wild-type or R132H mutant IDH1 were harvested at different passages and levels of CpG methylation were measured by FACS using 5- methylcytosine-specific antibody. Representative histograms from 1 of 3 independent experiments are shown. Measurement of CpG methylation using dot blot was also performed on DNA extracted from late passage NHA cells as in Fig. 3a. Quantification showed that IDH1 R132H mutant cells exhibited a ~1.8 fold increase compared to wild-type or parental cells. 7

8 RESEARCH SUPPLEMENTARY INFORMATION Vector IDH1 WT IDH1 R132H IDH1 IDH1 R132H β actin Supplementary Figure 6: Creating neurosphere culture stably expressing mutant IDH1. Primary neurosphere cultures established from the brains of p16/p19 -/- mice were retrovirally transduced with empty vector, wild-type or R132H mutant IDH1. Levels of protein expression of wild-type IDH1 or mutant IDH1 were analyzed by Western blotting with specific antibodies. 8

9 RESEARCH a Human GST KDM4C D 2HG (mm) L 2HG (mm) H3K9me3 Total H3 b Signal intensity no ormalized to total H KDM4C D 2HG (mm) L 2HG (mm) Supplementary Figure 7: Stereoisomer-specific inhibition of KDM4C by 2HG. a, Bulk histones were incubated with purified GST-tagged human KDM4C in the reaction mix with 2, 5 or 10 mm D-2HG or L-2HG. Levels of H3K9me3 3 were assessed by Western blotting with specific antibody. Total H3 was used as loading control. b, quantification of Western blot band intensity by Image J. 9

10 RESEARCH SUPPLEMENTARY INFORMATION a KDM4C sirna: Ctrl #1 #2 KDM4C β actin b Absorban nce 500nm Ctrl #1 #2 Supplementary Figure 8: Two additional sirna oligonucleotides suppress KDM4C expression and inhibit cell differentiation. a, 3T3-L1 cells were transfected with control sirna or two additional sirna specific for KDM4C. After three days, cells were lysed and assessed for expression levels of KDM4C by Western blotting. β-actin was used as loading control. b, Cells from the same treatment were induced to differentiate for 7 days. The accumulation of lipid droplets was assessed by Oil-red O staining. After washing, Oil-red O was dissolved in isopropanol o and quantified by measuring absorbance b at 500 nm. Error bars represent ese standard d deviation from three independent experiments. 10

11 RESEARCH Supplementary Table 1 Gene Symbol RefSeq q-value(%) Fold-Change (mut vs. WT) p-value (mut vs. WT) CHI3L1 NM_ E-05 PDPN NM_ E-05 WDR69 NM_ E-05 NEK10 NM_ E-05 DDR2 AY C10orf134 ENST GBP4 NM_ CHI3L2 NM_ RGS22 NM_ ST6GALNAC5 NM_ KIAA0746 NM_ GBP3 NM_ SLC9A11 NM_ LPAR3 NM_ CCDC30 NM_ GAPT NM_ DDR2 NM_ PTPN20A NM_

12 RESEARCH SUPPLEMENTARY INFORMATION NOG NM_ BMP2 NM_ FRMPD2L1 NM_ FRMPD2L1 NM_ MOXD1 NM_ PDYN NM_ PCDHB10 NM_ GALNT13 NM_ EFCAB1 NM_ FRZB NM_ C2orf67 NM_ CRTAC1 NM_ FAM110B NM_ GRIA4 NM_ ANKRD45 NM_ PTPLAD2 NM_ GNG12 NM_ C1orf173 BC CDH19 NM_ INA NM_ FAM114A1 BC

13 RESEARCH LPHN2 NM_ TIMP1 NM_ GRM7 NM_ C15orf51 ENST C15orf51 ENST CACNG2 NM_ TOX3 NM_ CALCRL NM_ FBP2 NM_ DNAH6 NM_ SPAG17 NM_ LOC ENST LOC ENST DCX NM_ KLRC3 NM_ FERMT1 NM_ C15orf51 AK EPHA5 NM_ DNAH6 NM_ SERPING1 NM_ DNAH6 NM_

14 RESEARCH SUPPLEMENTARY INFORMATION KIAA1324 NM_ C15orf51 ENST LOC ENST C15orf51 AK LRRN1 NM_ DLL3 NM_ KLRC2 NM_ C1orf201 BC CCDC39 NM_ TMEM100 NM_ ACCN4 NM_ KLRC4 NM_ HLA-DQA1 NM_ CHD5 NM_ B3GALT1 NM_ TTC6 BC ABCA5 NM_ Supplementary Table 1: List of differentially expressed genes between IDH1/2 wild-type vs. mutant primary glioma samples. 14

15 RESEARCH Supplementary Table 2 Genes upregulated in IDH1/2 mutant samples Gene Ontology category regulation of astrocyte differentiation regulation of glial cell differentiation regulation of gliogenesis ion channel activity P-Value 4.90E E E E-03 Genes downregulated in IDH1/2 mutant samples Gene Ontology category extracellular region part platelet alpha granule lumen cytoplasmic membrane-bounded vesicle lumen vesicle lumen P-Value 4.48E E E E-03 Supplementary Table 2: List of top four gene ontology categories enriched in differentially expressed genes between IDH1/2-mutant vs. wild-type glioma samples. 15