http www tsbmi m utokyo ac jp Metabolic

－文部科学省イノベーションシステム整備事業「先端融合領域イノベーション創出拠点形成」－ システム疾患生命科学による先端医療技術開発 http: //www. tsbmi. m. u-tokyo. ac. jp/ Metabolic Regulation of Histone Demethylase KDM 4 A and Glycolytic Gene Expression by Isocitrate Dehydrogenase 3β during Adipogenesis Eko Fuji Ariyanto 1, Yoshihiro Matsumura 1, Tomoyoshi Soga 2, Takeshi Inagaki 1, Juro Sakai 1 Division of Metabolic Medicine, Research Center for Advanced Science and Technology, The University of Tokyo 1 , Institute for Advanced Biosciences, Keio University 2 Recent studies suggest that metabolic intermediates (e. g. α-ketoglutarate and acetyl-Co. A) potentially regulate activity of histone modification enzymes and contribute to epigenetic state. However, the mechanisms by which endogenous metabolites regulate histone modification remain largely unknown. To understand metabolic regulation of epigenome during adipogenesis, we performed metabolome analysis and found that metabolites on glycolysis and tricarboxylic acid (TCA) cycle dramatically change during adipogenesis. Notably, α-ketoglutarate, which is a co-factor of Jumonji-containing (Jmj. C) histone demethylases, showed 3 -fold elevation during adipogenesis. Microarray analysis showed m. RNA expressions of isocitrate dehydrogenase 3 enzymes (IDH 3α, IDH 3β, and IDH 3γ), which catalyze NAD+-dependent α-ketoglutarate production, were induced upon adipogenesis coincident with α-ketoglutarate elevation. Knockdown of IDH 3β inhibited the elevation of α-ketoglutarate, demethylation of repressive histone H 3 K 9 me 2 on glycolytic genes, their gene induction, and lipid accumulation. On the other hand, knockdown of NADP+-dependent IDH 1 and IDH 2 had minimal effects on expression of glycolytic genes and lipid accumulation. Screening by si. RNA revealed that KDM 4 A is responsible for demethylation of H 3 K 9 me 2 on glycolytic genes. These results reveal that IDH 3 -KDM 4 A axis transmits cellular metabolism to epigenome to characterize adipocytes. 1. Glucose is important for lipid accumulation 5. IDH 3β knockdown inhibits lipid accumulation 9. KDM 4 A is important for lipid and glycolytic gene induction in 3 T 3 -L 1 cells accumulation and glycolytic gene induction Day -4 0 2 4 6 8 -2 MDI Ins 25 m. M or 25 m. M 3 T 3 -L 1 1 m. M 0 -2 2 MDI 1 m. M glucose A si-Ctr 4 6 MDI : IBMX, Dexamethasone, Insulin Ins : Insulin Ins si-IDH 3α si-IDH 3β #1 #1 #2 Oil-Red-O staining (Day 8) 8 si-Ctr si-IDH 3γ #3 #2 si-Kdm 3 a si-Kdm 3 b #1 #1 #2 si-Kdm 4 a si-Ctr #2 #1 si-Ctr #3 si-Kdm 4 b si-Kdm 4 c #1 #1 si-Ctr 1000 B 25 m. M glucose 1 m. M glucose 500 si-IDH 1 si-IDH 3β #1 si-Ctr #2 KDM 3 A KDM 3 B si-IDH 2 #3 #1 #2 KDM 4 A KDM 4 B KDM 4 C 0 B (Microarray) D 0 D 4 D 8 3 T 3 -L 1 cells were induced with MDI on day 0 and cultured in 25 or 1 m. M glucose-containing medium. (A) Oil-Red O staining at day 8. (B) Microarray analysis shows m. RNA expression at day 0, 4 and 8. Expression 1 2 4 6 MDI : IBMX, Dexamethasone, Insulin Ins : Insulin Ins Acetyl-Co. A: acetyl donor for histone acetylation [fmol/cell] Acetyl-Co. A 3 T 3 -L 1 cells were transfected with control si-RNA or si-RNA against Idh 3 (A), Idh 1 or Idh 2 (B) on day -2 and day 0 and induced with MDI on day 0. Cells were stained with Oil-Red O at day 8. 6. IDH 3β knockdown prevents α-KG elevation during adipogenesis D 0: pre-adipocyte D 6: adipocyte 15 20 15 10 WCL: whole cell lysate IDH Enzymatic Activity Assay IDH 3 Isocitrate + NAD+ Isocitrate + NADP+ 60 5 20 0 0 Fluorescence D 0 B D 0 D 6 Activity α-KG + NADH IDH 1 IDH 2 80 40 Fluorescence α-KG + NADPH IDH 1 NADP+ dependent IDH 2 NADP+ dependent IDH 3α, β, γ NAD+ dependent (A) Immunoblot analysis shows IDH 1, IDH 2 and IDH 3 proteins in preadipocytes (D 0) and adipocytes (D 6). (B) Whole cell lysate of 3 T 3 -L 1 preadipocytes (D 0) and adipocytes (D 6) was subjected to NAD+-IDH (IDH 3) or NADP+-IDH (IDH 1 and IDH 2) activity assay. IDH activity was measured by monitoring time-dependent increase in fluorescence of NADH or NADPH at 440 nm after excitation at 340 nm. 20 18 16 14 12 10 8 6 4 2 0 D 6 0 si -C -ID tr si H 3 -ID a si H 3 -ID b H 3 g Microarray (Day 8) si si 20 15 15 5 0 D 6 D 0 20 si-Ctr 15 15 15 10 10 5 5 0 D 4 D 8 D 0: pre-adipocyte D 6: adipocyte 2 1, 6 1, 2 0, 8 0, 4 0 0 Hk 2 T Slc 2 a 4 4 A (q. PCR) Hk 2 is IDH 3β-dependent. • KDM 4 A facilitates Slc 2 a 4 and Hk 2 induction and lipid accumulation. Glucose Glycolysis TCA cycle D 6 si-IDH 3 b Hk 2 +3. 1 kb 10 DNA purification IDH 3β α-KG Incubation with magnetic Dynabeads Incubation of eluent with Pronase to reverse the crosslinks Isocitrate KDM 4 A H 3 K 9 me 2 Me Me Me Glycolytic gene 5 0 D 0 Sonication 0 si-IDH 3 b Slc 2 a 4 +3. 1 kb 2 • IDH 3β elevates α-KG level during adipogenesis. • Removal of repressive H 3 K 9 me 2 from Slc 2 a 4 and 5 20 si-Ctr Actbsi-IDH 3 b 8 Nuclear pellet preparation 10 D 0 DI 6 DNA-protein crosslinking 25 Hk 2+3. 1 kb 20 D 6 DI 4 3 g 2000 10 D 0 2 H 3 b -ID H H tr 4000 25 Slc 2 a 4+3. 1 kb Actb 0 -W KDM 4 A-V 5 - KDM 4 A-V 5 Empty WT H 188 A si Adipoq 8000 6000 % Input 15 NADP+-IDH Activity (IDH 1, IDH 2) DNA % Input 25 20 18 16 14 12 10 8 6 4 2 0 % Input 37 100 Cd 36 % Input 50 H 3 30 A -2 Conclusions 8. Repressive H 3 K 9 me 2 is retained on glycolytic genes upon IDH 3β knockdown % Input H 3 37 37 120 NAD+-IDH Activity (IDH 3) 10000 3 T 3 -L 1 cells were transfected with control si-RNA or si-RNA against Idh 3 on day -5 and day 0 and induced with MDI on day 0. On day 8, RNA was collected and subjected to microarray analysis. % Input PPARγ 2 PPARγ 1 37 IDH 2 35 Specific activity (nmol/min/mg) IDH 3γ IDH 1 Specific activity (nmol/min/mg) IDH 3β 37 k. Da 3 T 3 -L 1 WCL (in vitro) Fabp 4 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -C -ID tr H si 3 a -ID H si 3 b -ID H 3 g 14000 12000 10000 8000 6000 4000 2000 0 si Pparg % Input 4. IDH 3 proteins and NAD+-IDH activity increase in adipocytes B -ID H tr si 3000 2500 2000 1500 1000 500 0 si 0 h 6 h 24 h 48 h 8 d (Microarray) (A) IDH enzymes play role in α-KG production in TCA cycle. (B) Subcellular localization and activity properties of IDH enzymes. (C) Metabolome analysis by CE-TOFMS shows α-ketoglutarate level during adipogenesis. (D) Microarray analysis shows m. RNA expression of IDHs during adipogenesis. D 0 D 6 -ID 0 3 a 0 (aa 709 -767)(aa 829 -885)(aa 895 -1011) (A) m. KDM 4 A-H 188 A (catalytic mutant) were overexpressed in 3 T 3 -L 1 cells by retroviral transduction. (B) Cells were induced with DI (Dexamethasone + Insulin) and stained with Oil-Red O at day 8. Slc 2 a 4 and Hk 2 expression was measured by q. PCR. si 200 500 Jmj. C (aa 142 -308) p 1000 400 PHD Tudor Jmj. N (aa 13 -55) DI: Dexamethasone + Insulin ORO Day 8 1500 600 C IB: α-H 3 Hk 2 2000 800 si -C Idh 3γ 3000 2500 2000 1500 1000 500 0 α-KG: co-factor for histone demethylation reaction 3 T 3 -L 1 WCL 2500 Slc 2 a 4 1000 si -C -ID tr si H 3 a -ID si H 3 b -ID H 3 g Expression score 0 h 1 h 6 h 12 h 24 h 36 h 48 h 4 d 8 d 0 h 6 h 24 h 48 h 8 d Expression score 0 h 1 h 6 h 12 h 24 h 36 h 48 h 4 d 8 d Expression score [fmol/cell] Expression score Wnt 3 a IB: α-V 5 (Collaboration with Prof. Soga) * Day -5 L 1/Empty or L 1/KDM 4 A-V 5 -WT or L 1/KDM 4 A-V 5 -H 188 A Adipogenic markers Idh 3β MDI IB: α-KDM 4 A si-IDH 3 b si NAD+ dependent 2000 1500 1000 500 0 8 [Day] 6 m. KDM 4 A N (1064 aa) Em 1200 Idh 2 Idh 3α 4 si -C IDH 3α, β, γ Complex? Mitochondrial? 2 H 188 A Glycolytic genes IDH: isocitrate dehydrogenase 300 250 200 150 100 50 0 0 3 g NADP+ dependent 0 h 6 h 24 h 48 h 8 d 8 H Mitochondrial Wnt 3 a 6 3 b IDH 2 MDI A 7. IDH 3β knockdown inhibits glycolytic gene expression H NADP+ dependent 120 100 80 60 40 20 0 10. KDM 4 A catalytic activity is required for lipid accumulation and glycolytic gene induction B 3 a Cytosolic 4000 3000 2000 1000 0 MDI : IBMX, Dexamethasone, Insulin Ins : Insulin 3 T 3 -L 1 cells were transfected with control si-RNA or si-RNA against Idh 3 b on day -2 and day 0 and induced with MDI on day 0. Metabolome analysis by CE-TOFMS shows α-ketoglutarate level. -ID IDH 1 Idh 1 4 s si i-C -ID tr si H 3 -ID a si H 3 -ID b H 3 g α-KG IDH 3α 2 si Activity IDH 8 α-KG: co-factor for histone demethylation reaction Expression score Subcellular localization Isocitrate D 0 D 6 k. Da D 0 D 2 D 4 D 6 D 8 4 A 3. Isocitrate Dehydrogenase 3 enzymes are induced during adipogenesis A (q. PCR) (A) 3 T 3 -L 1 cells were transfected with control si-RNA or si-RNA against H 3 K 9 me 2 demethylases on day -2 and day 0 and induced with MDI on day 0. Cells were stained with Oil-Red O at day 8. (B) 3 T 3 -L 1 cells were transfected with control si-RNA or si-RNA against KDM 4 A on day -2 and day 0 and induced with MDI on day 0. Slc 2 a 4 and Hk 2 expression was measured by q. PCR. Expression Metabolome analysis by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) shows levels of TCA cycle metabolites during adipogenesis. B 6 Ins si-Ctr α-KG: co-factor for histone demethylation reaction (Collaboration with Prof. Soga) 0, 2 D 0 D 2 D 4 D 6 D 8 3 T 3 -L 1 Day 0 0. 25. 5 1 1. 5 2 4 6 8 (day) 0, 4 p α -KG 0 0. 25. 5 1 1. 5 2 4 6 8 D 0, 6 0 [fmol/cell] 0. 25. 5 1 1. 5 2 4 6 8 [fmol/cell] α-ketoglutarate (α -KG) Succinic acid Succinyl-Co. A 4 α-ketoglutarate IDH Fumaric acid 2 MDI Isocitrate TCA cycle α-KG 0 -2 Metabolome Citrate Malic acid C 0, 8 Hk 2 si-KDM 4 a Em Acetyl-Co. A TCA cycle 1, 2 si-Ctr 8 A 3 T 3 -L 1 0. 25. 5 1 1. 5 2 4 6 8 Oxaloacetic acid Day -4 -ID 0. 25. 5 1 1. 5 2 4 6 8 A Slc 2 a 4 si-KDM 4 a Empty KDM 4 A-V 5 -WT KDM 4 A-V 5 -H 188 A 0. 25. 5 1 1. 5 2 4 6 8 (day) 0. 25. 5 1 1. 5 2 4 6 8 1, 6 1, 4 1, 2 1 0, 8 0, 6 0, 4 0, 2 0 si-Ctr si. RNA si Metabolome 8 -ID 3 T 3 -L 1 0 1, 4 1 2. α-ketoglutarate elevates during adipogenesis Day -4 -2 #3 PHF 2 PHF 8 18 0 1500 -H 500 #1 H 3 K 9 me 2 histone demethylases: T 1000 Hk 2 2000 si-Ctr 4 A 1500 2500 Slc 2 a 4 si-Phf 8 #2 -W 350 300 250 200 150 100 50 0 Pparg 4 A Expression score 2000 #1 ORO (Day 8) Glycolytic genes #3 #3 si-Phf 2 B #3 8 A A 25 m. M glucose Day -4 18 Glucose concentration A si. RNA -H 3 T 3 -L 1 MDI : IBMX, Dexamethasone, Insulin Ins : Insulin 0 D 4 D 8 D 0 D 4 D 8 Gene-specific q. PCR analysis (H 3 K 9 me 2 Ch. IP-q. PCR) Quantitative Ch. IP-PCR shows H 3 K 9 me 2 level on Actb, Slc 2 a 4 and Hk 2 in 3 T 3 -L 1 preadipocytes (D 0) and adipocytes (D 6) (A) and during IDH 3β-knockdown 3 T 3 -L 1 differentiation (B). Division of Metabolic Medicine, RCAST URL: http: //www. mm. rcast. u-tokyo. ac. jp/ Acknowledgement We would thank Dr. Hiroshi Kimura for histone antibody. TSBMI Translational Systems Biology and Medicine Initiative