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NADPH levels affect cellular epigenetic state by inhibiting HDAC3–Ncor complex

Nature Metabolism volume 3, pages 75–89 (2021)Cite this article

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Abstract

NADPH has long been recognized as a key cofactor for antioxidant defence and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We find that the reduction of cellular NADPH levels, achieved by silencing malic enzyme or glucose-6-phosphate dehydrogenase, impairs global histone acetylation and transcription in both adipocytes and tumour cells. These effects can be reversed by supplementation with exogenous NADPH or by inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator nuclear receptor corepressor 2 (Ncor2; SMRT) or Ncor1, thereby impairing HDAC3 activation. Interestingly, NADPH and the inositol tetraphosphate molecule Ins(1,4,5,6)P4 appear to bind to the same domains on HDAC3, with NADPH having a higher affinity towards HDAC3 than Ins(1,4,5,6)P4. Thus, while Ins(1,4,5,6)P4 promotes formation of the HDAC3–Ncor complex, NADPH inhibits it. Collectively, our findings uncover a previously unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.

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Fig. 1: Malic enzyme silencing suppresses adipocytic gene transcription and global histone acetylation, resulting in a block to adipocyte differentiation.
Fig. 2: Malic enzymes regulate histone acetylation through HDAC3.
Fig. 3: HDAC3 mediates ME-regulated gene transcription.
Fig. 4: NADPH directly binds and inhibits HDAC3 in vitro.
Fig. 5: NADPH affects histone acetylation through inhibition of HDAC3.
Fig. 6: NADPH binds to HDAC3 and abrogates assembly of HDAC3–DAD complexes.
Fig. 7: NADPH change reprograms histone acetylation and transcription.

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Data availability

Uncropped versions of blots and source data are provided with this paper. The CHIP–seq data have been deposited in the Gene Expression Omnibus under accession GSE137694. The remaining data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank X. Wang and H. Liu for helping with analysis of ChIP–seq data. This work was supported by the National Key Research and Development Program of China (2019YFA0802600), CAMS Innovation Fund for Medical Sciences (2016-I2M-4-002), the National Natural Science Foundation of China (81672766), State Key Laboratory Special Fund (2060204), CAMS Basic Research Fund (2019-RC-HL-007, 2016ZX310186 and 2016RC310038) to W.D. and a postdoctoral fellowship at Peking-Tsinghua Center for Life Sciences and National Science Foundation for Young Scholars of China (31601154) and China Postdoctoral Science Foundation (2016M601005; to J.Q.)

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  1. These authors contributed equally: Wei Li, Junjie Kou, Junying Qin.

Authors and Affiliations

  1. State Key Laboratory of Medical Molecular Biology, Key Laboratory of RNA Regulation and Hematopoiesis, Department of Cell Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China

    Wei Li, Junjie Kou, Li Li, Zhenxi Zhang & Wenjing Du

  2. School of Life Sciences, Tsinghua University, Beijing, China

    Junying Qin

  3. School of Life Sciences, Tsinghua-Peking Joint Center for Life Sciences; Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China

    Ying Pan & Yi Xue

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Contributions

W.L., J.K. and J.Q. performed all experiments except those mentioned below. Y.P. and Y.X. performed the molecular docking calculation and MD simulations and interpreted the data. L.L. and Z.Z. helped with animal experiments. W.D. designed the experiments. W.D. and W.L. analysed the data. W.D. wrote the manuscript with the help of Y.X. All authors discussed the results and commented on the manuscript.

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Correspondence to Yi Xue or Wenjing Du.

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Peer review information Primary Handling Editor: Christoph Schmitt. Nature Metabolism thanks Michael Parker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ME silencing impairs adipocyte differentiation, histone acetylation and genes transcription.

a, b, 3T3-L1 preadipocytes transfected with control siRNA, or different sets of ME1 (a) or ME2 siRNAs (b) as indicated were induced to differentiate for 7 days. The accumulation of differentiation-induced lipid droplets was visualized by Oil Red O staining (left). Oil Red O staining was quantified (right, top panel). The protein expression is shown below. c, d, mRNA expression of 3T3-L1 preadipocytes transfected with control siRNA, or different sets of ME1 (c) or ME2 siRNAs (d) as indicated before (Day 0) and after differentiating for 7 days (Day 7). e, mRNA expression of control or ME knockout 3T3-L1 cells using sgRNA CRISPR/Cas9. f–h, 3T3-L1 preadipocytes transfected with control, ME1, ME2 and/or p53 siRNAs as indicated were induced to differentiate for 7 days. Cells were stained with Oil Red O (f, top). Oil Red O staining was quantified (f, bottom). Protein expression (g) and mRNA levels (h) are shown. i, Western blots of 3T3-L1 preadipocytes transfected with control siRNA, or different sets of ME1 (left) or ME2 siRNAs (right) as indicated after 7 days of differentiation. j, Protein expression of control or ME knockout 3T3-L1 cells using sgRNA CRISPR/Cas9 after differentiating for 7 days. In a, b, f, n = 3 biological replicates; In c, d, e, h, n = 3 technical replicates of one out of three independent experiments with similar results. Data are mean ± s.d. Statistical significance was determined by two-tailed unpaired t-test. In a, b, f, g, i, j, Oil Red O staining images and western blots represent three independent experiments. For all western blot data, histone expression was analyzed using acid-extracted cell lysates, total H3 was used as loading control; all other protein expression was assessed with total cell lysates, Actin was used as loading control. The samples derived from the same experiment and the blots were processed in parallel.

Source data

Extended Data Fig. 2 MEs inhibition resulted in decreased histone acetylation and glycolytic genes expression.

U2OS cells were transfected with control, ME1 or ME2 siRNA for 48 hours. a, RNA was extracted and expressions of glucose metabolism genes were analyzed by qRT-PCR. b, Acid extracts were analyzed by western blot with antibodies against indicated acetylated histones. Expressions of actin and total H3 were determined as loading controls. Western blots represent three independent experiments. c, Relative glucose consumption and lactate production were determined by YSI2700 Biochemistry Analyzer. In a, n = 3 technical replicates of one out of three independent experiments with similar results; In c, n = 3 biological replicates. Data are mean ± s.d.. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 3 HDAC3 mediates ME-regulated histone acetylation and genes expression in adipocytes and tumour cells.

a–c, 3T3-L1 preadipocytes transfected with control, ME1 or ME2 siRNA were induced to differentiate for 7 days with or without 0.1 μM TSA. (a) Cells were fixed and stained with Oil Red O (left). Oil Red O staining was quantified (right). (b) Relative gene expression was assessed by qRT-PCR. (c) Protein expression was analyzed by western blotting with specific antibodies. d, 3T3-L1 preadipocytes treated with control, ME1 or ME2 siRNA were induced to differentiate for 7 days in the absence or presence of 50 μM NAM. Protein expression was analyzed by western blot. e, Protein expression of U2OS cells transfected with control, ME1 or ME2 siRNA in presence of HDAC1-6 siRNAs respectively as indicated. f, i, 3T3-L1 preadipocytes treated with control (−), ME1 or ME2 siRNA were induced to differentiate for 7 days with or without 10 μM RGFP966. Protein expression (f) and mRNA levels (i) are shown. g, j, U2OS and HCT116 cells were transfected with control (−), ME1 or ME2 siRNA and 2 days later treated with 10 μM RGFP966 for another 24 hours. Protein expression (g) and relative Glut4 expression (j) are shown. h, 3T3-L1 preadipocytes transfected with control or HDAC3 siRNAs were induced to differentiate for 7 days. Relative mRNA expression was assessed by qRT-PCR. In a, n = 3 biological replicates; In b, h, i, j, n = 3 technical replicates of one out of three independent experiments with similar results. Data are mean ± s.d.. Statistical significance was determined by two-tailed unpaired t-test. NS, not significant. In a, c, d, e, f, g, Oil Red O staining images and western blots represent three independent experiments. For all western blot data, histone expression was analyzed using acid-extracted cell lysates, total H3 was used as loading control; all other protein expression was assessed with total cell lysates, Actin was used as loading control. The samples derived from the same experiment and the blots were processed in parallel.

Source data

Extended Data Fig. 4 Malic enzymes affect cellular NADPH levels.

a, 3T3-L1 preadipocytes were induced to differentiate for 7 days. Before (day 0) and after differentiation induction (day 7), cells were fractionated and cytoplasmic (including mitochondria) and nuclear protein extracts were analyzed by western blot using indicated antibodies. Tubulin and total H3 were used as fraction and loading controls. Western blots represent three independent experiments. b, 3T3-L1 preadipocytes transfected with control, ME1 or ME2 siRNA were stimulated to differentiate for 7 days. Cellular NADPH levels were determined. Protein expression was determined by western blot. Actin was used as loading control. Western blots represent three independent experiments. c, Related to Figure. 2a and 2c. 3T3-L1 preadipocytes transfected with control siRNA, or two sets of ME1 or ME2 siRNAs as indicated, NADPH levels were determined. d, Adipose tissues from ME1 or ME2 knock-out mice (n = 3) were prepared for assaying NADPH levels. e, Tissues from ME1 or ME2 knock-out mice (n = 3) were analyzed NADPH levels using NADPH quantification kit. In b, c, d, e, n = 3 biological replicates; Data are mean ± s.d.. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 5 NADPH is an endogenous inhibitor of HDAC3, ROS and acetyl-CoA are not involved in ME-mediated histone acetylation modification.

a–c, 3T3-L1 preadipocytes were transfected with increased amount of NADPH using X-tremeGENE HP DNA Transfection reagent for 24h. (a) Cellular NADPH levels were examined. b, Cellular HDAC3 enzymatic activity were analyzed. HDAC3 expression were detected by western blotting. Actin was used as loading control. c, HAT activity of nucleic extracts was examined. d, Nucleic extracts from 3T3-L1 preadipocytes were incubated with increased amount of NADPH as indicated for 6h, and then HAT activity were determined. e, 3T3-L1 preadipocytes transfected with control (−), ME1 or ME2 siRNA were induced to differentiate for 7 days in the absence or presence of 5 mM NAC (N-acetyl-L-cysteine). Protein expression is shown (left). Cellular ROS levels were detected by FACS with DCF (2,7-dichlorodihydrofluorescein diacetate) staining (right). f, g, 3T3-L1 preadipocytes transfected with control (−), ME1 or ME2 siRNA were induced to differentiate for 7 days in the absence or presence of 10mM GSH as indicated. NADPH levels (f right panel) and GSH levels (g) were determined before differentiation (with 10mM GSH for 48h). Protein expression (f left panel) was analyzed after differentiation. h, 3T3-L1 preadipocytes transfected with control (−), ME1 or ME2 siRNA were induced to differentiate for 7 days in the absence or presence of 5 mM acetate combined with or without 10 μM NADPH as indicated. Protein expression was determined using western blot. In a, b, c, d, e, f, g, n = 3 biological replicates. Data are mean ± s.d.. Statistical significance was determined by two-tailed unpaired t-test. NS, not significant. For b (bottom panel), e (left panel), f (left panel), h, western blots represent three independent experiments. Histone expression was analyzed using acid-extracted cell lysates, total H3 was used as loading control; all other protein expression was assessed with total cell lysates, Actin was used as loading control. The samples derived from the same experiment and the blots were processed in parallel.

Source data

Extended Data Fig. 6 NADPH disrupts HDAC3-DAD association.

a, b, 3T3-L1 preadipocytes transfected with control, ME1 or ME2 siRNA as indicated were induced to differentiate for 7 days in the absence or presence of 20 μM NADPH (NADPH were transfected into cells using X-tremeGENE HP DNA Transfection reagent). Cells were then lysed and subjected to immunoprecipitation using anti-Ncor1 (a) or anti-Ncor2 (b) antibody. Input and bound proteins were analyzed by western blot. Actin was used as loading control. c, d, HA-DAD (Ncor1) (c), HA-DAD (Ncor2) (d) or Flag-HDAC3 was expressed in HEK293T cells individually. After 48 h, protein extracts from HA-DAD (Ncor1) (c) / HA-DAD (Ncor2) (d) and Flag-HDAC3 transfected cells were mixed at a ratio of 1:1 (Ncor1/Ncor2:HDAC3) in the presence or absence of NADPH and/or 20μM Ins(1,4,5,6)P4 (overnight) before immunoprecipitation using anti-IgG or anti-Flag antibody. e, f, HEK293T cells co-transfected with Flag-HDAC3 and HA-DAD(Ncor1) (e) or HA-DAD (Ncor2) (f) were treated with NADPH (NADPH were transfected into cells using X-tremeGENE HP DNA Transfection reagent) and/or Ins(1,4,5,6)P4 overnight as indicated. Cells were then lysed and subjected to immunoprecipitation using anti-Flag antibody. g–i, The dissociation constant (KD) for immobilized mutant HDAC3 (L1, L6 or Mut-all) from NADPH or Ins(1,4,5,6)P4 were determined by surface plasmon resonance (SPR). Shown are real-time graphs of response units against NADPH or Ins(1,4,5,6)P4 concentrations. In a–f, western blots represent three independent experiments. The samples derived from the same experiment and the blots were processed in parallel.

Source data

Extended Data Fig. 7 The molecular docking result targeting HDAC3.

a, Electrostatic surface representation of the HDAC3-DAD complex onto which Ins(1,4,5,6)P4 docks. Ins(1,4,5,6)P4 molecules are shown in stick view either in green (the most energetically favored docking pose in the binding pocket) or in yellow (extracted from the crystal structure with PDB ID 4A69). b, The zoom-in structure of (a) showing details of the interaction between HDAC3-DAD complex and docked (green sticks) or native (yellow sticks) pose of Ins(1,4,5,6)P4. Hydrogen bonds and salt bridges are indicated by green or yellow dashed lines, respectively. HDAC3 (light blue) and DAD (magenta) are represented in cartoon view. c, The blind docking result of NADPH onto HDAC3. The pose in the binding interface is colored in green, other representative poses are colored in yellow. d, The blind docking result of Ins(1,4,5,6)P4 onto HDAC3. The pose in the binding interface is colored in green, other representative poses are colored in yellow. e, Clashes are found after adding DAD (magenta cartoon) into the docked structure of HDAC3-NADPH (left), while no clashes are observed when DAD is added into the docked structure of HDAC3-Ins(1,4,5,6)P4 (right). Residues from HDAC3-DAD binding interface are shown in sticks, clashed residues are highlighted by red circles. f, The 2D interaction diagrams are shown for the docking results of HDAC3-NADPH (left) and HDAC3-Ins(1,4,5,6)P4 (right). g, Statistics of binding energies for the docking results of NAD+, NADH, NADP+, NADPH and Ins(1,4,5,6)P4 targeting HDAC3. The objective docking was repeated for nine times, yielding 81 poses for each ligand.

Extended Data Fig. 8 The sidechain dynamics of Tyr 298 as revealed by MD simulations.

The dihedral angle (N-Cα-Cβ-Cγ) probability distributions of Tyr298 shown for MD simulations of HDAC3, HDAC3-Ins(1,4,5,6)P4, HDAC3-DAD-Ins(1,4,5,6)P4 and HDAC3-NADPH.

Extended Data Fig. 9 NADPH promotes adipocyte differentiation, and inhibition of oxidative PPP or folate metabolism reduces histone acetylation.

a, b, Oil Red O staining of 3T3-L1 cells transfected with control, different sets of ME1 (a) or ME2 siRNA (b) in the presence of increasing amounts of NADPH after 7 days of differentiation. Wells and higher magnification images are shown (left). Oil Red O staining was quantified (right). Protein expression is shown below. c, 3T3-L1 preadipocytes treated with or without 6-AN were induced to differentiate for 7 days. (left and middle) Differentiation-induced lipid droplet accumulation was visualized by Oil Red O staining and quantified. (right) Acid extracts were analyzed by western blot with antibodies against acetylated histones. Total H3 was used as loading control. d, 3T3-L1 preadipocytes transfected with control, G6PD or MTHFD2 siRNA were induced to differentiate for 7 days. Cellular NADPH levels were analyzed. e–g, NADP+/NADPH ratios in differentiating 3T3-L1 adipocytes (e) U2OS cells (f) and HCT116 cells (g) treated with control, ME1, ME2, G6PD or MTHFD2 siRNA as indicated were determined respectively. The protein expression was determined by western blot. Actin was used as loading control. h, Expressions of acetylated histones in U2OS cells and HCT116 cells treated with control, G6PD or MTHFD2 siRNA as indicated. Actin and total H3 were used as loading controls. In a, b, c, d, e, f, g, n = 3 biological replicates; Data are mean ± s.d.. Statistical significance was determined by two-tailed unpaired t-test. NS, not significant. In a,b (right, below panel), c (right panel), e–g (right panel), h, western blots represent three independent experiments. The samples derived from the same experiment and the blots were processed in parallel.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Video

The MD simulation movie for the docked HDAC3–NADPH structure. The length of the MD is 500 ns. Colour scheme: grey for HDAC3; red for NADPH; yellow for residues H17, G21, K25, R265 and R301 of HDAC3.

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Li, W., Kou, J., Qin, J. et al. NADPH levels affect cellular epigenetic state by inhibiting HDAC3–Ncor complex. Nat Metab 3, 75–89 (2021). https://doi.org/10.1038/s42255-020-00330-2

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